KR20130032897A - Production of alcohol esters and in situ product removal during alcohol fermentation - Google Patents

Abstract

Esterification of a product (e.g., lipase) capable of esterifying a product alcohol and a carboxylic acid (e.g. fatty acid) and a product alcohol such as butanol to the carboxylic acid to produce an alcohol ester in a fermentation medium. Alcohol fermentation process and composition comprising the production of an alcohol ester. The alcohol ester can be extracted from the fermentation medium and the product alcohol can be recovered from the alcohol ester. The carboxylic acid can also serve as an extractant for removing the alcohol esters from the fermentation medium.

Description

Production of alcohol esters during alcohol fermentation and removal of product in situ {PRODUCTION OF ALCOHOL ESTERS AND IN SITU PRODUCT REMOVAL DURING ALCOHOL FERMENTATION}

This application is incorporated by reference in US Provisional Application No. 61 / 356,290, filed June 18, 2010, the entire contents of which are incorporated herein by reference; US Provisional Application No. 61 / 368,451, filed July 28, 2010; US Provisional Application No. 61 / 368,436, filed July 28, 2010; US Provisional Application No. 61 / 368,444, filed July 28, 2010; US Provisional Application No. 61 / 368,429, filed July 28, 2010; US Provisional Application No. 61 / 379,546, filed September 2, 2010; US Provisional Application No. 61 / 440,034, filed February 7, 2011; And US patent application Ser. No. 13 / 160,766, filed June 15, 2011.

Sequence listings associated with this application are submitted in electronic form via EFS-Web, which is incorporated herein by reference in its entirety.

The present invention relates to fermentation production of alcohols, including ethanol and butanol, and all related by-products, and methods of enhancing alcohol fermentation using in situ product removal methods.

Alcohols have a variety of uses in industry and science, such as beverages (ie ethanol), fuels, reagents, solvents, and preservatives. Butanol, for example, is an important industrial chemical with a variety of uses, including use as a renewable fuel additive, as a feedstock chemical in the plastics industry, and as a food grade extractant in the food and spice industries. And alcohols which are drop-in fuel components. Therefore, there is a strong demand for alcohols such as butanol, and an efficient and environmentally friendly production method.

The production of alcohol using fermentation by microorganisms is one such environmentally friendly production method. In butanol production, in particular, some microorganisms that produce butanol in high yields also have a low butanol toxicity threshold. As it is produced, removing butanol from fermentation vessels is a means to cope with this low butanol toxicity threshold. Thus, there remains a continuing need for effective methods and systems for producing butanol in high yield despite the low butanol toxicity threshold of butanol producing microorganisms in fermentation broth.

In-situ product removal (ISPR) (also referred to as extraction fermentation) is used to remove butanol (or other fermentation alcohol) from the fermentation vessel as it is produced so that the microorganisms can produce butanol in high yield. One ISPR method for removing fermented alcohols described in the art is liquid-liquid extraction (US Patent Application Publication 2009/0305370). In general, with regard to butanol fermentation, the fermentation medium containing the microorganism is contacted with the organic extractant at a point before the butanol concentration reaches the toxic level. The organic extractant and the fermentation medium form a biphasic mixture. Butanol is distributed into the organic extractant phase, whereby the concentration of butanol in the aqueous phase containing the microorganisms is reduced, thereby limiting the exposure of the microorganisms to inhibitory butanol. In order to be technically economically viable, liquid-liquid extraction can be achieved by contacting the extractant with the fermentation broth for efficient mass transfer of the product alcohol to the extractant; Phase separation of the extractant from the fermentation broth (on fermentation and / or after fermentation); Efficient recovery and recycling of extractant; And minimal reduction of the partition coefficient of the extractant over long periods of work.

The extractant may be contaminated over time at each recycle by, for example, lipid build-up present in the biomass fed to the fermentation vessel as a feedstock of hydrolysable starch. As an example, upon conversion of glucose to butanol, liquified corn mash loaded into the fermentation vessel with 30 wt% dry corn solids is subjected to simultaneous saccharification fermentation (addition of glucoamylase to produce glucose). Fermentation broth containing about 1.2 wt% corn oil produced by the saccharification of the liquefied corn mash occurring in Dissolution of corn oil lipids into oleyl alcohol (OA), which acts as an extractant at ISPR, increases the lipid concentration in the OA with each OA recycle, thereby reducing the lipid in each OA recycle, which reduces the partition coefficient of the product alcohol. May result in increased concentration.

In addition, the presence of insoluble solids in the extraction fermentation may adversely affect the alcohol production efficiency. For example, the presence of insoluble solids lowers the mass transfer coefficient in the fermentation vessel, inhibits phase separation in the fermentation vessel, and leads to a decrease in extraction efficiency over time by corn oil accumulation due to insoluble solids in the extractant, It can be trapped in solids and eventually removed as Dried Distillers' Grains with Solubles (DDGS), slowing the release of extractant drops from the fermentation broth and / or lowering the volumetric efficiency of the fermentation vessel.

Several approaches for reducing the partition coefficient reduction of extractant used in extractive fermentation include wet milling, fractionation, and removal of solids. Wet milling is an expensive, multi-step process that separates biomass (eg, corn) into its main components (embryo, rind fiber, starch, and gluten) to gain value from each by-product separately. This process provides a purified starch stream; It is expensive and involves the separation of biomass into its non-starchy components, which is unnecessary for the production of fermentation alcohol. By fractionation, embryos containing a large number of lipids present in fiber and ground whole corn are removed, resulting in fractionated corn with a high starch (endosperm) content. Dry fractionation does not separate the embryos from the fibers and, therefore, is less expensive than wet milling. However, fractionation does not remove the entire fiber or embryo and does not result in loss of solids. In addition, starch is slightly lost in fractionation. Wet milling of corn is more expensive than dry fractionation, but dry fractionation is more expensive than dry grinding of unfractionated corn. Removal of solids, including lipid containing embryos, from liquefied mash prior to use in fermentation, for example, in co-owned US Provisional Application No. 61 / 356,290, filed June 18, 2010, co-pending. As described, substantially insoluble solids can be removed. However, it would be advantageous if the reduction in the partition coefficient of the extractant due to contamination by lipids could be reduced, even without fractionation or substantially all insoluble solids removed. The conversion of lipids present in the liquefied mash into extractants that can be used in ISPR is, for example, co-pending US Provisional Application Nos. 61 / 368,436 and 61 /, all filed on July 28, 2010, for example. As described in 368,444, another method is to reduce the amount of lipids fed to the fermentation vessel.

Alternative extraction that does not require the distribution of product alcohol between the fermentation medium and the ISPR extractant as a means of reducing the toxic effects of product alcohols such as butanol on the microorganism, and can also reduce the drop in the partition coefficient of the fermentation product extractant. Fermentation methods continue to be required.

Summary of the Invention

The conversion of alcohols, such as butanol produced from microorganisms, into fermentation broth to substances that are less toxic to microorganisms, can increase the production of alcohols such as butanol for a given fermentation vessel volume. Alcohol esters may be produced by contacting an alcohol in a fermentation medium with a carboxylic acid (eg, a fatty acid) and a catalyst capable of esterifying the alcohol with a carboxylic acid. In addition, the carboxylic acid may act as an ISPR extractant to which the alcohol ester is dispensed. The carboxylic acid may be derived from a biomass that is fed into the fermentation vessel and / or feeds a fermentable carbon feed to the fermentation vessel. Lipids present in the feedstock can be catalytically hydrolyzed to carboxylic acids, and the same catalyst (eg, an enzyme) can esterify the carboxylic acid with an alcohol (eg, butanol); Lipids can also be transesterified directly with a catalyst to produce alcohol esters. The catalyst may be fed to the feedstock prior to fermentation, or may be fed to the fermentation vessel before or simultaneously with the feedstock feed. When the catalyst is fed to the fermentation vessel, the alcohol ester can be obtained by hydrolysis of the lipid to carboxylic acid and the simultaneous esterification of the carboxylic acid with butanol present in the fermentation vessel; Lipids may also be transesterified directly with butanol by means of catalysts to produce alcohol esters. Native oils that are not derived from carboxylic acids and / or feedstocks can also be supplied to fermentation vessels, which are hydrolyzed to carboxylic acids. Natural oils not derived from carboxylic acids and / or feedstock may be fed to the fermentation vessel in an amount sufficient to produce a biphasic mixture comprising an organic phase and an aqueous phase. Thus, in some embodiments, any carboxylic acid not esterified with alcohol may act as or as part of an ISPR extractant. Extractant containing alcohol esters can be separated from the fermentation medium, and alcohol can be recovered from the extractant. The extractant may be recycled to the fermentation vessel. Thus, in the case of butanol production, for example, conversion of butanol to esters reduces the free butanol concentration in the fermentation medium, protecting the microorganisms from the toxic effects of increasing butanol concentration. In addition, fine fractioned grains can be used as feedstock even if lipids are not separated therein, since the lipids can be catalytically hydrolyzed to carboxylic acids, thereby reducing the rate of lipid accumulation in the ISPR extractant.

The present invention includes contacting butanol produced in the fermentation process with at least one carboxylic acid and at least one catalyst capable of esterifying the carboxylic acid with butanol to produce butyl esters of carboxylic acids; A method for producing butyl esters wherein the carboxylic acid in the fermentation process is present at a concentration sufficient to produce a biphasic mixture. In one embodiment, butanol production and butyl ester production occur simultaneously or sequentially. In one embodiment, the feedstock in the fermentation process includes one or more fermentable sugars. In another embodiment, the feedstock in the fermentation process may include crop residues such as corn kernels, corn cobs, corn husks, corn cobs, grass, wheat, rye, straw, barley, barley straw, hay, rice straw, switchgrass. , Waste paper, sugarcane waste, sorghum, sugarcane, soybean, ingredients obtained from milling of cereals, cellulosic substances, lignocellulosic substances, trees, branches, roots, leaves, wood chips, sawdust, shrubs and bushes, vegetables, One or more fermentable sugars derived from fruit, flower, animal compost, and mixtures thereof. In one embodiment, the method further comprises providing a natural oil to contact the natural oil with one or more enzymes to convert at least some natural oils to carboxylic acids. In one embodiment, the carboxylic acid comprises a fatty acid. In another embodiment, the carboxylic acid comprises 12 to 22 carbons. In one embodiment, the carboxylic acid is a mixture of carboxylic acids. In another embodiment, the butyl ester of carboxylic acid is butyl ester of fatty acid. In one embodiment, the catalyst is an enzyme capable of esterifying a carboxylic acid with butanol to produce a butyl ester of carboxylic acid. In another embodiment, the enzyme is an esterase, lipase, phospholipase, or lysophospholipase.

The invention also provides a method of preparing a feedstock comprising the steps of: (a) providing a feedstock; (b) liquefying the feedstock to produce a liquefied biomass comprising oligosaccharides; (c) separating the feedstock slurry to produce a product comprising an aqueous stream comprising oligosaccharides, an oil stream, and a solid; (d) adding the aqueous stream to a fermentation vessel comprising fermentation broth; (e) saccharifying oligosaccharides in the aqueous stream; And (f) fermenting the oligosaccharide saccharification product present in the aqueous stream to produce butanol, while simultaneously contacting butanol with at least one carboxylic acid and at least one catalyst capable of esterifying the carboxylic acid with butanol, thereby carboxyl Producing a butyl ester of an acid, wherein the carboxylic acid is present at a concentration sufficient to produce a biphasic mixture; A process for producing butanol and butyl esters from a feedstock, wherein optionally steps (e) and (f) occur simultaneously. In one embodiment, the method further comprises obtaining an oil from the oil stream and converting at least some oil into carboxylic acid. In one embodiment, the feedstock slurry is decanter bowl centrifuge, tricant centrifuge, disk stack centrifuge, filtration centrifuge, decanter centrifuge, filtration, vacuum filtration, belt filter. Pressure filtration, screen filtration, screen separation, grating, porous grating, floatation, hydroclone, filter press, screw press, gravity settler, vortex separator, or Separated by a combination thereof. In another embodiment, the carboxylic acid comprises a fatty acid. In another embodiment, the carboxylic acid comprises 12 to 22 carbons. In one embodiment, the method further comprises adding the oil to the fermentation vessel before converting at least some oil into the carboxylic acid. In one embodiment, the method further comprises adding additional carboxylic acid to the fermentation vessel. In one embodiment, the oil is converted to carboxylic acid after the step of adding additional carboxylic acid. In another embodiment, the carboxylic acid is corn oil fatty acid, soybean oil fatty acid, or a mixture of corn oil fatty acid and soybean oil fatty acid. In one embodiment, the oil obtained from the oil stream comprises glycerides and the one or more catalysts hydrolyze the glycerides into fatty acids. In another embodiment, the butyl ester of carboxylic acid is butyl ester of fatty acid. In one embodiment, the catalyst is an enzyme capable of esterifying a carboxylic acid with butanol to produce a butyl ester of carboxylic acid. In another embodiment, the enzyme is an esterase, lipase, phospholipase, or lysophospholipase. In one embodiment, the method further comprises washing the solid with a solvent. In one embodiment, the solvent is selected from petroleum distillates such as hexane, isobutanol, isohexane, ethanol, petroleum ether, or mixtures thereof. In another embodiment, the solids are processed to produce animal feed products. In another embodiment, the solids are processed to produce animal feed products. In one embodiment, the animal feed product is one or more crude protein, crude fat, triglycerides, fatty acids, fatty acid isobutyl esters, lysine, neutral detergent insoluble fiber (NDF), and acid resistant fiber (ADF) It includes. In another embodiment, the animal feed product further comprises one or more vitamins, minerals, flavors, or colorants. In one embodiment, the animal feed product contains 20 to 35 wt% crude protein, 1 to 20 wt% crude fat, 0 to 5 wt% triglycerides, 4 to 10 wt% fatty acids, and 2 to 6 wt% fatty acid isobutyl esters. Include. In one embodiment, separating the solids from the feedstock slurry may increase the butanol production efficiency by increasing the liquid-liquid mass transfer coefficient of butanol from the fermentation broth to the extractant; Use of an extractant to increase butanol extraction efficiency to increase butanol production efficiency; Increasing the rate of phase separation between the fermentation broth and the extractant to increase butanol production efficiency; Increased recovery and recycle of the extractant to increase butanol production efficiency; Reducing the flow rate of the extractant increases the butanol production efficiency.

The invention also provides a method of preparing a feedstock comprising the steps of: (a) providing a feedstock; (b) liquefying the feedstock to produce a liquefied biomass comprising oligosaccharides; (c) separating the feedstock slurry to produce a stream comprising oligosaccharides and oil, and a solid; (d) adding the stream to a fermentation vessel comprising fermentation broth; (e) saccharifying oligosaccharides of the stream; And (f) fermenting the oligosaccharide saccharification product present in the stream to produce butanol, while simultaneously contacting butanol with at least one carboxylic acid and at least one catalyst capable of esterifying the carboxylic acid with butanol, thereby Producing a butyl ester of a carboxylic acid present in a concentration sufficient to produce a biphasic mixture; A process for producing butanol and butyl esters from a feedstock, wherein optionally steps (e) and (f) occur simultaneously. In one embodiment, the method further comprises converting at least some oil into carboxylic acid. In one embodiment, the feedstock slurry is decanter ball centrifugation, tricanter centrifugation, disk stack centrifugation, filtration centrifugation, decanter centrifugation, filtration, vacuum filtration, belt filter, pressure filtration, screen filtration, screen separation, grating , By means of pore grating, floatation, hydroclone, filter press, screw press, gravity settling tank, vortex separator, or a combination thereof. In another embodiment, the carboxylic acid comprises a fatty acid. In another embodiment, the carboxylic acid comprises 12 to 22 carbons. In one embodiment, the method further comprises adding oil to the fermentation vessel. In one embodiment, the method further comprises adding additional carboxylic acid to the fermentation vessel. In one embodiment, the oil is converted to carboxylic acid after the step of adding additional carboxylic acid. In one embodiment, the carboxylic acid is corn oil fatty acid, soybean oil fatty acid, or a mixture of corn oil fatty acid and soybean oil fatty acid. In one embodiment, the oil comprises glycerides and the one or more catalysts hydrolyze the glycerides into fatty acids. In one embodiment, the butyl ester of carboxylic acid is butyl ester of fatty acid. In one embodiment, the catalyst is an enzyme capable of esterifying a carboxylic acid with butanol to produce a butyl ester of carboxylic acid. In another embodiment, the enzyme is an esterase, lipase, phospholipase, or lysophospholipase. In one embodiment, the method further comprises washing the solid with a solvent. In one embodiment, the solvent is selected from petroleum distillates such as hexane, isobutanol, isohexane, ethanol, petroleum ether, or mixtures thereof. In another embodiment, the solids are processed to produce animal feed products. In another embodiment, the solids are processed to produce animal feed products. In some embodiments, the animal feed product includes one or more crude protein, crude fat, triglycerides, fatty acids, fatty acid isobutyl esters, lysine, neutral detergent insoluble fiber (NDF), and acid resistant fiber (ADF). In some embodiments, the animal feed product further comprises one or more vitamins, minerals, flavors, or colorants. In some embodiments, the animal feed product contains 20 to 35 wt% crude protein, 1 to 20 wt% crude fat, 0 to 5 wt% triglycerides, 4 to 10 wt% fatty acids, and 2 to 6 wt% fatty acid isobutyl esters. Include. In some embodiments, separating the solids from the feedstock slurry may increase the butanol production efficiency by increasing the liquid-liquid mass transfer coefficient of butanol from the fermentation broth to the extractant; Use of an extractant to increase butanol extraction efficiency to increase butanol production efficiency; Increasing the rate of phase separation between the fermentation broth and the extractant to increase butanol production efficiency; Increased recovery and recycle of the extractant to increase butanol production efficiency; Reducing the flow rate of the extractant increases the butanol production efficiency.

The invention also comprises the steps of (a) contacting butanol produced in the fermentation process with at least one carboxylic acid and at least one catalyst capable of esterifying the carboxylic acid with butanol to produce a butyl ester of carboxylic acid. The carboxylic acid in the fermentation process is present in a concentration sufficient to produce a biphasic mixture comprising an aqueous phase and a butyl ester containing organic phase; (b) separating the butyl ester containing organic phase from the aqueous phase; And (c) recovering butanol from the butyl ester. In some embodiments, recovering butanol from butyl esters includes hydrolyzing esters to carboxylic acids and butanol. In some embodiments, the butyl ester is hydrolyzed in the presence of a hydrolysis catalyst. In some embodiments, the butyl ester is hydrolyzed in the presence of water and the hydrolysis catalyst comprises an acid catalyst, organic acid, inorganic acid, water soluble acid, or water insoluble acid. In some embodiments, the hydrolysis catalyst includes enzymes that can hydrolyze butyl esters to produce carboxylic acids and butanol. In some embodiments, the enzyme is an esterase, lipase, phospholipase, or lysophospholipase. In some embodiments, enzymatic reaction conditions support enzymatic hydrolysis rather than esterification. In some embodiments, enzyme reaction conditions include a cosolvent. In some embodiments, the fatty acid butyl esters, fatty acids, isobutanol, and water are dissolved in the cosolvent, and the free fatty acids do not react with the cosolvent. In some embodiments, the cosolvent is selected from acetone, tert-butanol, 2-Me-2-butanol, 2-Me-2-pentanol, and 3-Me-3-pentanol. In some embodiments, enzymatic reaction conditions include final product removal. In some embodiments, the final product is isobutanol or fatty acid. In some embodiments, isobutanol is removed by vacuum distillation, pervaporartion, permselective filtration, or gas sparging. In some embodiments, fatty acids are removed by precipitation, selective permeation filtration, or electrophoresis. In some embodiments, the hydrolysis reaction occurs in a reaction vessel. In some embodiments, recovering butanol from butyl esters includes transesterifying butyl esters with butanol and fatty acid alkyl esters or acyl glycerides. In some embodiments, the fatty acid alkyl esters comprise fatty acid methyl esters, fatty acid ethyl esters, or fatty acid propyl esters. In some embodiments, the method further comprises providing a natural oil to contact the natural oil with one or more enzymes to convert at least some natural oils to carboxylic acids. In some embodiments, the enzyme is an enzyme that can hydrolyze or transesterify butyl esters to produce butanol. In some embodiments, the enzyme is an esterase, lipase, phospholipase, or lysophospholipase. In some embodiments, the carboxylic acid comprises a fatty acid. In some embodiments, the carboxylic acid has a carbon chain length of 12 to 22 carbons. In some embodiments, at least about 10% butanol is recovered from the butyl ester. In some embodiments, at least about 50% butanol is recovered from the butyl ester. In some embodiments, at least about 90% of butanol is recovered from the butyl esters. In some embodiments, the carboxylic acid is recovered from the butyl ester. In some embodiments, the method includes removing butanol from the fermentation vessel as an extractant stream; And adding the extractant stream to at least two distillation columns. In some embodiments, the distillation column is a superatmospheric distillation column having a steam heated reboiler. In some embodiments, the method includes recovering water and solvent from the distillation column; And recycling the water and the solvent. In some embodiments, the method further comprises recovering heat from the distillation process; And recycling the heat to evaporate the water.

The invention also provides a method of preparing a feedstock comprising the steps of: (a) providing a feedstock; (b) liquefying the feedstock to produce a feedstock slurry; (c) separating the feedstock slurry to produce a product comprising an aqueous stream, an oil stream, and a solid; (d) adding the aqueous stream to a fermentation vessel comprising fermentation broth; (e) saccharifying the aqueous stream; (f) fermenting the glycated aqueous stream to produce butanol, while simultaneously contacting butanol with at least one carboxylic acid and at least one catalyst capable of esterifying the carboxylic acid with butanol, thereby butyl ester of carboxylic acid Producing, the carboxylic acid is present at a concentration sufficient to produce a biphasic mixture; (g) separating the butyl ester containing organic phase from the aqueous phase; And (h) recovering butanol from the butyl ester; A process for producing butanol from a feedstock, optionally in which steps (e) and (f) occur simultaneously. In some embodiments, the method further comprises obtaining an oil from the oil stream and converting at least some oil into carboxylic acid. In some embodiments, the feedstock slurry is separated by centrifugation, filtration, or decantation. In some embodiments, the carboxylic acid comprises a fatty acid. In some embodiments, the carboxylic acid has a carbon chain length of 12 to 22 carbons. In some embodiments, the method further comprises adding the oil to the fermentation vessel before converting at least some oil into the carboxylic acid. In some embodiments, the method further comprises adding additional carboxylic acid to the fermentation vessel. In some embodiments, the oil is converted to carboxylic acid after the step of adding additional carboxylic acid. In some embodiments, the carboxylic acid is corn oil fatty acid, soybean oil fatty acid, or a mixture of corn oil fatty acid and soybean oil fatty acid. In some embodiments, the oil obtained from the oil stream comprises glycerides and the one or more catalysts hydrolyze the glycerides into fatty acids. In some embodiments, the butyl ester of carboxylic acid is butyl ester of fatty acid. In some embodiments, the catalyst is an enzyme capable of esterifying a carboxylic acid with butanol to produce a butyl ester of carboxylic acid. In some embodiments, the enzyme is an esterase, lipase, phospholipase, or lysophospholipase. In some embodiments, the solids are processed to produce animal feed products. In some embodiments, recovering butanol from butyl esters includes hydrolyzing esters to carboxylic acids and butanol. In some embodiments, the butyl ester is hydrolyzed in the presence of a hydrolysis catalyst. In some embodiments, the butyl ester is hydrolyzed in the presence of water and the hydrolysis catalyst comprises an acid catalyst, organic acid, inorganic acid, water soluble acid, or water insoluble acid. In some embodiments, the hydrolysis catalyst includes enzymes that can hydrolyze butyl esters to produce carboxylic acids and butanol. In some embodiments, the enzyme is an esterase, lipase, phospholipase, or lysophospholipase. In some embodiments, the hydrolysis reaction occurs in a reaction vessel. In some embodiments, recovering butanol from butyl esters includes transesterifying butyl esters with butanol and fatty acid alkyl esters or acyl glycerides. In some embodiments, the fatty acid alkyl esters comprise fatty acid methyl esters, fatty acid ethyl esters, or fatty acid propyl esters. In some embodiments, the method further comprises providing a natural oil to contact the natural oil with one or more enzymes to convert at least some natural oils to carboxylic acids. In some embodiments, the enzyme is an enzyme that can hydrolyze or transesterify butyl esters to produce butanol. In some embodiments, the enzyme is an esterase, lipase, phospholipase, or lysophospholipase.

In another embodiment, the fermentation method comprises: providing an aqueous feed stream obtained from biomass, the aqueous feed stream comprising water, a fermentable carbon source derived from biomass, and an oil; Contacting the aqueous feed stream with a catalyst such that at least some of the oil is hydrolyzed to the free fatty acid to produce a catalyzed feed stream comprising the free fatty acid and the catalyst; Contacting the catalyzed feed stream with fermentation broth in a fermentation vessel; Fermenting the fermentable carbon source in a fermentation vessel to produce product alcohol; And contacting the product alcohol with the free fatty acid and the catalyst during fermentation to catalyze the esterification of the free fatty acid with the product alcohol in the fermentation vessel to produce an alcohol ester of the fatty acid. In some embodiments, contacting the feed stream with the catalyst and the fermentation broth and contacting the fermentation and product alcohol with the free fatty acid and the catalyst can occur simultaneously. In some embodiments, the product alcohol is butanol and the alcohol ester of fatty acids is butyl ester of fatty acids.

The present invention provides a fermentation medium comprising a microorganism producing alcohol in the fermentation medium; And contacting the fermentation medium with a catalyst that can esterify the fermentation medium with a carboxylic acid and the alcohol with the carboxylic acid upon fermentation to produce an alcohol ester. do. In some embodiments, the alcohol produced by the microorganism is butanol and the alcohol ester is butyl ester. In some embodiments, the fermentation medium is contacted with a carboxylic acid that is substantially insoluble in the fermentation medium, and a catalyst that can esterify the alcohol with the carboxylic acid to produce an alcohol ester.

The invention also provides a fermentation medium comprising an alcohol, a fermentable carbon source, and a free fatty acid; And contacting the fermentation medium with one or more enzymes capable of esterifying the free fatty acids with alcohols to esterify the free fatty acids with alcohols to produce alcohol esters of the fatty acids. Provides a way to create In some embodiments, the fermentable carbon source is derived from biomass. In some embodiments, the microorganisms in the fermentation medium are recombinant microorganisms. In some embodiments, the alcohol is butanol and the alcohol ester of fatty acids is butyl ester of fatty acids.

In another embodiment, a method of producing a product alcohol includes providing a biomass feedstock comprising water, a fermentable carbon source, and an oil, wherein the oil comprises acyl glycerides; Liquefying the biomass feedstock to produce a liquefied biomass comprising oligosaccharides; Converting at least some of the acyl glycerides into free fatty acids to contact the biomass feedstock or liquefied biomass with a composition comprising one or more enzymes from which the free fatty acids can produce an extractant, the one or more enzymes also Free fatty acids can be esterified with product alcohols with alcohol esters of fatty acids; Contacting the liquefied biomass with a saccharifying enzyme capable of converting the oligosaccharide into a fermentable sugar; Contacting the liquefied biomass with a recombinant microorganism capable of converting the fermentable sugar into a product alcohol, thereby producing a fermentation product comprising the product alcohol; Contacting the product alcohol with the free fatty acid and one or more enzymes to catalyze the esterification of the free fatty acid with the product alcohol to produce an alcohol ester of the fatty acid; And contacting the fermentation product with an extractant. In an embodiment, contact with an extractant produces a two-phase mixture comprising an aqueous phase and an extractant phase, and an ester containing extractant phase is formed by the distribution of the alcohol esters of fatty acids onto the extractant phase. In some embodiments, the product alcohol is butanol and the alcohol ester of fatty acids is butyl ester of fatty acids.

In another embodiment, a method of producing a product alcohol includes providing a biomass feedstock comprising water, a fermentable carbon source, and an oil, wherein the oil comprises acyl glycerides; Liquefying the biomass feedstock to produce a liquefied biomass comprising oligosaccharides; Contacting the liquefied biomass with a composition comprising one or more enzymes capable of converting at least some acyl glycerides into free fatty acids, wherein the one or more enzymes can also esterify the free fatty acids with the product alcohols with alcohol esters of fatty acids. has exist - ; Contacting the liquefied biomass with a saccharifying enzyme capable of converting the oligosaccharide into a fermentable sugar; Contacting recombinant microorganisms capable of converting fermentable sugars to product alcohols upon fermentation with saccharified biomass to produce a fermentation medium comprising product alcohols; And contacting the fermentation medium with a carboxylic acid extractant during fermentation, the fermentation medium comprising one or more enzymes capable of esterifying the free fatty acid with the product alcohol to produce an alcohol ester of the fatty acid. . In a further embodiment of this method, the fermentation medium is contacted with a carboxylic acid that is substantially insoluble in the fermentation medium, and a catalyst that can esterify the alcohol with the carboxylic acid to produce an alcohol ester. In another embodiment of this method, the alcohol produced by the microorganism is butanol and the alcohol ester is butyl ester.

The invention also comprises liquefying a starch or fermentable carbon source in a feedstock to produce a slurry with oligosaccharides; Centrifuging the feedstock slurry to produce a centrifugation product comprising (i) an aqueous layer comprising oligosaccharides, (ii) an oil layer, and (iii) a solid; Supplying the aqueous layer to a fermentation vessel comprising fermentation broth; And fermenting the aqueous layer to produce product alcohol. The product alcohol is then contacted with the carboxylic acid and the catalyst so that the carboxylic acid esters with the product alcohol to produce an alcohol ester. In some embodiments, the oil is a vegetable oil. In another embodiment, the product alcohol is butanol and the alcohol ester of carboxylic acid is butyl ester of fatty acid.

In some embodiments, the method of producing the product alcohol comprises providing a fractionated biomass feedstock comprising water, starch, and / or fermentable carbon sources, and only residual amounts of oil remaining after fractionation of the biomass. Oils include acyl glycerides; Liquefying the fractionated biomass feedstock to produce a liquefied fractionated biomass comprising oligosaccharides; Contacting the liquefied fractionated biomass with a composition comprising one or more enzymes capable of converting at least some residual acyl glycerides into free fatty acids, wherein the one or more enzymes also esterify the free fatty acids with the product alcohols to May produce an alcohol ester; Contacting the liquefied fractionated biomass with a saccharifying enzyme capable of converting the oligosaccharide into a fermentable sugar; Contacting recombinant microorganisms capable of converting fermentable sugars to product alcohols upon fermentation with saccharified biomass to produce a fermentation medium comprising product alcohols; And contacting the fermentation medium with a carboxylic acid extractant during fermentation, the fermentation medium comprising one or more enzymes capable of esterifying the free fatty acid with the product alcohol to produce an alcohol ester of the fatty acid. . In a further embodiment of this invention, the fermentation medium is contacted with a catalyst which can esterify the carboxylic acid and the alcohol with the carboxylic acid in the fermentation medium to produce an alcohol ester. In further embodiments, the carboxylic acid is substantially insoluble in the fermentation medium. In another embodiment of this method, the alcohol produced by the microorganism is butanol and the alcohol ester is butyl ester.

The invention also provides a mash formed from biomass and comprising water and fermentable sugars; Catalysts capable of esterifying free fatty acids with fatty acid alkyl esters and optionally hydrolyzing acyl glycerides to free fatty acids; Alcohol; Free fatty acids; And fatty acid alcohol esters produced in situ from esterification of free fatty acids with alcohols by a catalyst. In some embodiments, the alcohol is butanol and the fatty acid alcohol ester is a fatty acid butyl ester.

The invention also relates to recombinant microorganisms capable of producing alcohols; Fermentable carbon source; And fatty alcohol esters, wherein the fatty alcohol esters are produced upon fermentation. In some embodiments, the recombinant microorganism can produce butanol. In some embodiments, the fatty acid alcohol ester is a fatty acid butyl ester. In some embodiments, the fermentable carbon source comprises a sugar. In some embodiments, the fermentable carbon source comprises methane, the recombinant microorganism can produce methanol and the fatty acid alcohol ester is a fatty acid methyl ester.

Also provided herein are recombinant yeast cells useful for the production of product alcohols. In an embodiment, a recombinant host cell disclosed herein can be any bacterial, yeast or fungal host useful for genetic modification and recombinant gene expression. In another embodiment, the recombinant host cell is Clostridium (C lostridium) inside, Xi thigh eggplant (Zymomonas) in Escherichia coli (Escherichia), A live Monera (Salmonella) genus, Serratia marcescens (Serratia), An El beanie O (Erwinia), A keulrep when Ella (Klebsiella) genus Shigella (Shigella) genus Rhodococcus (Rhodococcus) genus Pseudomonas (Pseudomonas) genus Bacillus (Bacillus) genus Lactobacillus bacteria (Lactobacillus), A enterococci (Enterococcus ) Genus, Alcaligenes genus, genus Klebsiella, genus Paenibacillus , genus Arthrobacter , genus Corynebacterium , genus Brevibacterium , ski irradiation Caro My process (Schizosaccharomyces), A Cluj Vero My process (Kluyveromyces) in, Yarrow subtotal (Yarrowia) in blood teeth (Pichia) genus Candida (Candida), A century nyulra (Hansenula), A director Chen Escherichia (Issatchenkia ), Or Saccharomyces genus. In another embodiment, the host cell is Saccharomyces cerevisiae , Schizosaccharomyces pombe ), Kluyveromyces lactis), Cluj Vero Mai Seth thermostat Toledo Lance (Kluyveromyces thermotolerans), Cluj Vero My Marcia Seth Augustine (Kluyveromyces marxianus), Candida glabrata (Candida glabrata ), Candida albicans ( Candida albicans ), Pichia stiphytis stipitis ), Yarrowia repolitica lipolytica ), E. coli , or L. plantarum . In another embodiment, the host cell is a yeast host cell. In some embodiments, the host cell is a member of the genus Saccharomyces. In some embodiments, the host cell is Kluyveromyces lactis, Candida glabrata or Schizocaromyces pombe. In some embodiments, the host cell is Saccharomyces cerevisiae. Saccharomyces cerevisiae yeasts are known in the art and include the American Type Culture Collection (Rockville, MD) and the Central Bureau voor Schimmelcultures (CBS) Fungal Biodiversity Centre, LeSaffre, Gert Strand AB, Ferm Solutions, North American Bioproducts, Martrex, and Lallemand Available from a variety of suppliers, including but not limited to Saccharomyces cerevisiae BY4741, CEN.PK 113-7D, Ethanol Red® Yeast, Gert Strand Prestige Turbo Yeast , Pro ™ Yeast, Bio-Perm® XR Yeast, Gert Strand Distillers Yeast, FerMax ™ Green Yeast, Permax Gold (Gold) yeast, Thermo Sacred (Thermosacc)? Yeast, BG-1, PE-2, CAT-1, CBS7959, including but not limited to CBS7960, CBS7961 and.

At least one of the enzymes catalyzing the conversion of α-ketoisovalerate to the product of isobutyraldehyde to the substrate, or at least one enzyme catalyzing the conversion of isobutyraldehyde to the product of the substrate from isobutanol to the chromosome Providing a recombinant host cell comprising an isobutanol biosynthetic pathway encoded by an integrated heterologous polynucleotide; And contacting the recombinant host cell with a fermentable carbon source to produce a fermentation broth under conditions in which isobutanol is produced. In some embodiments, the method further comprises adding an extractant to produce a biphasic mixture. In another embodiment, the extractant comprises a carboxylic acid. In some embodiments, the extractant comprises a fatty acid. In another embodiment, the method further comprises adding an esterification enzyme capable of catalyzing the esterification of isobutanol with carboxylic acid.

Provided herein are fermentation media comprising product alcohol, water, fermentable carbon source, and microorganisms producing product alcohol; Contacting the fermentation medium with an extractant during fermentation to produce a biphasic mixture comprising an aqueous phase and an organic phase; And contacting the fermentation medium with a carboxylic acid and an enzyme capable of esterifying the carboxylic acid with a product alcohol. In some embodiments, the extractant comprises a carboxylic acid. In some embodiments, the carboxylic acid is produced by hydrolysis of oil from the biomass feedstock. In some embodiments, the fermentable carbon and carboxylic acid are from the same biomass feedstock source. In some embodiments, the carboxylic acid comprises saturated, monounsaturated, polyunsaturated carboxylic acids having 12 to 22 carbons, and mixtures thereof. In some embodiments, the contact of the fermentation medium with the extractant and the carboxylic acid and enzyme occurs simultaneously. In some embodiments, the microorganism is a genetically modified microorganism (eg, recombinant microorganism or host cell such as recombinant yeast cell).

As used herein, PNY1504, PNY2205, or recombinant host cells; Extractant; And optionally an esterification enzyme. Also provided herein is a composition comprising PNY1504, PNY2205, or recombinant host cells and butyl esters.

Further provided herein is the use of a composition comprising PNY1504, PNY2205, or other recombinant yeast cells, and recombinant yeast cells for producing isobutanol.

The accompanying drawings, which are incorporated in and form a part of this specification, are intended to illustrate the invention, and together with the description serve to explain the principles of the invention and to practice and use the invention by those skilled in the art. To be provided.
≪ 1 >
1 schematically illustrates an exemplary method and system of the present invention wherein a catalyst for alcohol esterification is fed to a fermentation vessel together with carboxylic acid and / or natural oil.
2,
2 schematically illustrates an exemplary method and system of the present invention wherein natural oil is converted to carboxylic acid using a catalyst and the carboxylic acid and catalyst are fed to a fermentation vessel.
3,
3 schematically illustrates an exemplary method and system of the present invention wherein liquefied biomass is contacted with a catalyst for lipid hydrolysis prior to fermentation.
<Fig. 4>
4 schematically illustrates an exemplary method and system of the present invention wherein liquefied and glycated biomass is contacted with a catalyst for lipid hydrolysis prior to fermentation.
5,
FIG. 5 shows the present invention in which significant amounts of lipids and insoluble solids are removed from the liquefied biomass prior to fermentation, the removed lipids are converted to carboxylic acids using a catalyst, and the carboxylic acid and catalyst are fed to the fermentation vessel. An exemplary method and system of FIG.
6,
6 shows the aqueous and solvent phase concentrations of isobutanol produced by fermentation using sucrose as carbon source. The aqueous phase titer (Panel A) is expressed in g / L and the solvent phase type (isobutanol, panel B and isobutanol as FABE, panel C. panel D is total isobutanol in the solvent phase) is expressed in weight percent. .
7,
7 shows the effective titers of isobutanol, g / L over time. The effective titer of this example was calculated based on the initial volume of aqueous fermentation broth after inoculation, as described herein.
8,
8 shows the consumption of sugars expressed in glucose equivalents over time.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification, including definitions, will control. Also, unless otherwise required by context, a singular term will include the plural, and the plural terms will include the singular. All publications, patents, and other references mentioned herein are incorporated by reference in their entirety for all purposes.

Unless otherwise specified, where the following abbreviations are used herein, the abbreviations have the following meanings:

ADH Alcohol Dehydrogenase

ALS acetolactate synthase

AQ aqueous fraction

Butyl ester (s) of BuO-COFA corn oil fatty acid (s)

CALB Candida antarctica lipase B

COFA Corn Oil Fatty Acid (s)

DDGS dry berth

DG diglyceride (s)

DHAD Dihydroxy Acid Dehydratase

End of EOR run

EtOH Ethanol

Ethyl Ester (s) of EtO-COFA Corn Oil Fatty Acid (s)

FABE fatty acid butyl ester (s)

FAEE fatty acid ethyl ester (s)

FAME fatty acid methyl ester (s)

FFA free fatty acid (s)

FOA Fluoroorthoic Acid

HADH horse liver alcohol dehydrogenase

IBA Isobutanol

i-BuOH isobutanol

isobutyl ester (s) of i-BuO-COFA corn oil fatty acid (s)

i-BuO-oleate isobutyl oleate

i-PrOH isopropanol

isopropyl ester (s) of i-PrO-COFA corn oil fatty acid (s)

Product removal in place of ISPR

KARI Ketolic Acid Reductoisomerase

KivD Ketoisovalerate Decarboxylase

MAG monoacylglyceride (s)

MeBOH 2-methyl-1-butanol

2-methyl-1-butyl ester (s) of MeBO-COFA corn oil fatty acid (s)

MeOH Methanol

Methyl ester (s) of MeO-COFA corn oil fatty acid (s)

MG monoglyceride (s)

n-BuOH n-butanol

OA Ole Alcohol

ORG Organic Fraction

PenOH 1-pentanol

1-pentyl ester (s) of PenO-COFA corn oil fatty acid (s)

PrOH 1-propanol

1-propyl ester (s) of PrO-COFA corn oil fatty acid (s)

SOFA soybean oil fatty acid

SSF Simultaneous Saccharification Fermentation

t- BuOH tert-butyl alcohol

TG Triglyceride (s)

3M3P 3-Me-3-pentanol

To further define the present invention, the following terms and definitions are provided herein.

As used herein, the terms “comprise,” “comprising,” “comprise,” “comprising,” “having,” “having,” “containing,” or “comprising,” or any other variant thereof. Will be understood to include the integers or groups of integers mentioned, but not to exclude any other integers or groups of integers. For example, a composition, mixture, process, method, article, or device comprising a list of elements is not necessarily limited to such elements and is not specifically listed or such composition, mixture, process, method, article, or device It may also contain other elements inherent in the. Moreover, unless expressly stated to the contrary, "or" does not mean " comprehensive " or " exclusive " For example, condition A or B is satisfied by any of the following: A is true (or present) and B is false (or not present), A is false (or not present) and B is true (Or present), both A and B are true (or present).

In addition, the indefinite articles "a" and "an" before an element or component of the present invention are intended to be non-limiting in the case of the element or component, ie, the number of beings. Thus, the indefinite article "a" or "an" should be read to include one or at least one, and the singular word of the element or component also includes the plural unless the number obviously means the singular.

The term "invention" or "invention" as used herein is a non-limiting term and is not intended to refer to any single embodiment of a particular invention, and includes all possible embodiments as described in the application. .

As used herein, the term "about," which modifies the amount of a component or reactant of the invention employed, includes, for example, conventional measurement and liquid handling procedures used to prepare concentrates or solutions in practice; Accidental errors in these processes; A change in quantity that can occur through the manufacture of ingredients, differences in source or purity, etc. employed to prepare a composition or perform a method. The term “about” also includes amounts that vary due to different equilibrium conditions for the composition resulting from the particular initial mixture. The claims, whether or not modified by the term "about", include equivalents of the quantity. In one embodiment, the term "about" means within 10% of the indicated value, or within 5% of the indicated value.

As used herein, "biomass" includes natural resources such as corn, sugar cane, wheat, cellulose or lignocellulosic materials, and cellulose, hemicellulose, lignin, starch, oligosaccharides, disaccharides and / or monosaccharides. It refers to a natural product containing hydrolyzable polysaccharides that provide a fermentable sugar comprising the substance and any sugars and starches derived from mixtures thereof. The biomass may also include additional components such as proteins and / or lipids. The biomass may be derived from a single source or the biomass may comprise a mixture derived from more than one source. For example, the biomass may comprise a mixture of corncobs and corncobs, or a mixture of grass and leaves. Biomass includes bioenergy crops, agricultural residues, municipal solid waste, industrial solid waste, sludge from papermaking, yard waste, waste sugar, wood and forest waste One is not limited thereto. Examples of biomass are corn residues, corn cobs, crop residues such as corn husks, corn stalks, grass, wheat, rye, straw, barley, barley straw, hay, rice straw, waxweed, waste paper, sugar cane waste, sorghum, candy Ingredients obtained from milling of sorghum, soybeans, grains, trees, branches, roots, leaves, wood chips, sawdust, shrubs and bushes, vegetables, fruits, flowers, animal compost, and mixtures thereof. For example, the mash, juice, molasses, or hydrolyzate can be produced from the biomass by any processing known in the art for processing the biomass for fermentation purposes, such as milling, processing, and / or liquefaction. And fermentable sugars, and may include water. For example, cellulosic and / or lignocellulosic biomass may be processed by any method known to those skilled in the art to obtain hydrolysates containing fermentable sugars. Particularly useful are the low ammonia pretreatments disclosed in US Patent Application Publication No. 2007 / 0031918A1, which is incorporated herein by reference. Enzymatic glycosylation of cellulosic and / or lignocellulosic biomass typically results in an enzyme consortium to degrade cellulose and hemicellulose to produce hydrolysates containing sugars including glucose, xylose, and arabinose. I use it. (Saccharification enzymes suitable for cellulose and / or lignocellulosic biomass are reviewed in Lynd, et al., Microbiol. Mol. Biol. Rev. 66: 506-577, 2002).

The mash, juice, molasses, or hydrolyzate may comprise feedstock 12 and feedstock slurry 16, as described herein. The aqueous feed stream may be produced or derived from the biomass by any processing known in the art for processing the biomass for fermentation purposes, such as milling, processing, and / or liquefaction, Sugar, for example, and may include water. The aqueous feed stream may comprise feedstock 12 and feedstock slurry 16, as described herein.

As used herein, "biomass yield" refers to the number of grams of biomass produced per gram of carbon substrate produced (ie, cell biomass production).

As used herein, "feedstock" means a feed of a fermentation process, the feed containing a fermentable carbon source, with or without insoluble solids, and optionally, before and after the fermentable carbon source, The containing feed is liberated from starch or obtained from the decomposition of complex sugars by further processing such as liquefaction, saccharification, or other processes. The feedstock includes or is derived from biomass. Suitable feedstocks include, but are not limited to, rye, wheat, corn, corn mash, sugar cane, sugar cane mash, barley, cellulosic material, lignocellulosic material, or mixtures thereof. When referring to "feedstock oil," this term will be understood to include oils produced from a given feedstock.

As used herein, “fermentation medium” means a mixture of water, sugars, dissolved solids, optionally microorganisms that produce alcohol, product alcohols, and all other components of materials contained in fermentation vessels, where product alcohols are present Is prepared by the reaction of sugars with alcohol, water and carbon dioxide (CO ₂ ) by microorganisms. Sometimes, the terms "fermentation broth" and "fermentation mixture" as used herein may be used synonymously with "fermentation medium".

As used herein, "fermentable carbon source" or "fermentable carbon substrate" means a carbon source that can be metabolized by the microorganisms disclosed herein for the production of fermentation alcohol. Suitable fermentable carbon sources include monosaccharides such as glucose or fructose; Disaccharides such as lactose or sucrose; Oligosaccharides; Polysaccharides such as starch or cellulose; C5 sugars such as xylose and arabinose; One carbon substrate comprising methane; And mixtures thereof.

As used herein, "fermentable sugar" refers to one or more sugars that can be metabolized by the microorganisms disclosed herein for the production of fermentation alcohol.

As used herein, “fermentation vessel” means a vessel through which a fermentation reaction is conducted in which product alcohols such as butanol are produced from sugars.

As used herein, "liquefied container" means a container in which liquefaction is performed. Liquefaction is the process by which oligosaccharides are liberated from the feedstock. In some embodiments where the feedstock is corn, the oligosaccharides are liberated from the corn starch content upon liquefaction.

As used herein, "glycosylation vessel" means a vessel in which glycosylation (ie, degradation of oligosaccharides into monosaccharides) is performed. When fermentation and saccharification occur simultaneously, the saccharification vessel and the fermentation vessel may be one of the same vessels.

As used herein, "sugar" refers to oligosaccharides, disaccharides, monosaccharides, and / or mixtures thereof. The term “saccharides” also includes carbohydrates including starch, dextran, glycogen, cellulose, pentosan, and sugars.

As used herein, "glycosylation enzyme" refers to one or more enzymes capable of hydrolyzing the alpha-1, 4-glucoside linkages of polysaccharides and / or oligosaccharides such as glycogen or starch. Glycosylating enzymes may also include enzymes capable of hydrolyzing cellulose or lignocellulosic materials.

As used herein, "insoluble solids" means the non-fermentable portions of the feedstock, such as embryos, fibers, and gluten. For example, the non-fermentable portion of the feedstock includes the feedstock portion that remains as a solid and can absorb liquid from the fermentation broth.

Dry liquor (DDGS) as used herein refers to by-products or by-products from fermentation of feedstock or biomass (eg, fermentation of particles or particle mixtures that produce product alcohols). In some embodiments, DDGS may also refer to an animal feed product resulting from a process of producing product alcohols (eg, butanol, isobutanol, and the like).

As used herein, "product alcohol" refers to any alcohol that can be produced by a microorganism in a fermentation process using biomass as a source of fermentable carbon substrate. Product alcohols include, but are not limited to, C ₁ to C ₈ alkyl alcohols. In some embodiments, the product alcohol is C ₂ to C ₈ alkyl alcohol. In another embodiment, the product alcohol is C ₂ to C ₅ alkyl alcohol. It will be appreciated that C ₁ to C ₈ alkyl alcohols include, but are not limited to, methanol, ethanol, propanol, butanol, and pentanol. Likewise, C ₂ to C ₈ alkyl alcohols include, but are not limited to, ethanol, propanol, butanol, and pentanol. "Alcohol" is also used herein in connection with a product alcohol.

As used herein, “butanol” is used alone or as a mixture thereof, butanol isomer 1-butanol (1-BuOH), 2-butanol (2-BuOH), tert-butanol ( t -BuOH), and / or Isobutanol (also known as iBuOH, i-BuOH or I-BUOH, 2-methyl-1-propanol). Often when referring to esters of butanol, the terms "butyl ester" and "butanol ester" may be used interchangeably.

As used herein, "propanol" refers to the propanol isomeric isopropanol or 1-propanol.

"Pentanol" as used herein refers to pentanol isomer 1-pentanol, 3-methyl-1-butanol, 2-methyl-1-butanol, 2,2-dimethyl-1-propanol, 3-pentanol, 2-pentanol, 3-methyl-2-butanol, or 2-methyl-2-butanol.

The term "alcohol equivalent" as used herein refers to the weight of alcohol obtained by complete hydrolysis of the alcohol ester and subsequent recovery of the alcohol from the alcohol ester amount.

The term "aqueous phase titer" as used herein refers to the concentration of a particular alcohol (eg butanol) in fermentation broth.

As used herein, the term "effective titer" refers to the total amount of alcohol equivalent of a particular alcohol (eg, butanol) produced by fermentation, or alcohol esterification, per liter of fermentation medium. For example, the effective titer of butanol in the unit volume of fermentation is (i) the amount of butanol in the fermentation medium; (ii) the amount of butanol recovered from the organic extractant; (iii) the amount of butanol recovered from the gas phase when gas stripping is used; And (iv) alcohol equivalents of butyl ester in organic or aqueous phase.

As used herein, the term "efficiency" is the effective titer divided by the fermentation time.

As used herein, the term "effective yield" is the total number of grams of product alcohol produced per gram of glucose consumed.

As used herein, "in situ product removal (ISPR)" means the selective removal of certain fermentation products from a biological process, such as fermentation, to adjust the product concentration in a biological process as the product is produced.

As used herein, an "extractant" or "ISPR extractant" is used to extract any product alcohol, such as butanol, or extracts any product alcohol ester produced by the catalyst from the product alcohol and carboxylic acid or lipid. It means an organic solvent used to. Often, the term "solvent" as used herein may be used synonymously with "extractant". In the process described herein, the extractant is water-immiscible.

The term "water immiscible" or "water insoluble" refers to a chemical component such as extractant or solvent that cannot be mixed with an aqueous solution, such as fermentation broth, in such a way as to form one liquid phase.

The term "aqueous phase" as used herein refers to the aqueous phase of a biphasic mixture obtained by contacting fermentation broth with a water immiscible organic extractant. In an embodiment of the process described herein, including fermentation extraction, the term “fermentation broth” then specifically refers to the aqueous phase in biphasic fermentation extraction.

As used herein, the term "organic phase" refers to the non-aqueous phase of the biphasic mixture obtained by contacting the fermentation broth with a water immiscible organic extractant.

As used herein, the term "carboxylic acid" refers to the general formula -COOH, wherein a carbon atom is bonded to an oxygen atom by a double bond to form a carbonyl group (-C = O), and to a hydroxyl group (-OH) by a single bond Any organic compound having The carboxylic acid may be in the form of a protonated carboxylic acid, in the form of a carboxylic acid salt (eg, ammonium, sodium, or potassium salt), or a mixture of protonated carboxylic acid and carboxylic acid salt. The term "carboxylic acid" refers to a mixture or single of a carboxylic acid, such as can be produced, for example, by hydrolysis of fatty acid esters or triglycerides, diglycerides, monoglycerides, and phospholipids derived from biomass. Chemical species (eg, oleic acid).

The term "fatty acid" as used herein refers to carboxylic acids (eg, aliphatic monocarboxylic acids) having saturated or unsaturated C ₄ to C ₂₈ carbon atoms (most often C ₁₂ to C ₂₄ carbon atoms). Say. Fatty acids may also be branched or unbranched. Fatty acids may be derived from or included in animal or vegetable fats, oils or waxes. Fatty acids may occur naturally in the form of glycerides in fats or oils, or may be obtained by hydrolysis or synthesis of fats. The term "fatty acid" may refer to a single species or mixture of fatty acids. In addition, the term "fatty acid" also includes free fatty acids.

The term "fatty alcohol" as used herein refers to an alcohol having an aliphatic chain of C ₄ to C ₂₂ carbon atoms, saturated or unsaturated.

As used herein, the term "fatty aldehyde" refers to an aldehyde having an aliphatic chain of C ₄ to C ₂₂ carbon atoms, saturated or unsaturated.

The term "fatty amide" as used herein refers to an amide having a long aliphatic chain of C ₄ to C ₂₂ carbon atoms, saturated or unsaturated.

The term "fatty ester" as used herein refers to an ester having a long aliphatic chain of C ₄ to C ₂₂ carbon atoms, saturated or unsaturated.

As used herein, "natural oil" refers to a lipid obtained from a plant (eg, biomass) or an animal. As used herein, “vegetable oil” refers to lipids obtained in particular from plants. Often, "lipid" can be used synonymously with "oil" and "acyl glyceride". Natural oils include resins, corn oil, canola oil, capric / caprylic triglyceride oil, castor oil, coconut oil, cottonseed oil, fish oil, jojoba oil, lard oil, linseed oil, neatfoot oil, Ooty Cica oil, palm oil, peanut oil, rapeseed oil, rice bran oil, safflower oil, soybean oil, sunflower oil, tung oil, jatropha oil, and vegetable oil blends.

As used herein, the term "separation" is synonymous with "recovery" and refers to the removal of the compound from the initial mixture to obtain a higher purity or higher concentration of the compound than the purity or concentration of the compound in the initial mixture.

The term “butanol biosynthetic pathway” as used herein refers to an enzyme pathway that produces 1-butanol, 2-butanol, or isobutanol.

The term "1-butanol biosynthetic pathway" as used herein refers to an enzyme pathway that produces 1-butanol from acetyl coenzyme A (acetyl-CoA).

The term "2-butanol biosynthetic pathway" as used herein refers to an enzyme pathway that produces 2-butanol from pyruvate.

The term “isobutanol biosynthetic pathway” as used herein refers to an enzyme pathway that produces isobutanol from pyruvate.

The term “gene” refers to a nucleic acid fragment that can be expressed with a particular protein, optionally including a regulatory sequence (5 ′ noncoding sequence) followed by a coding sequence (3 ′ noncoding sequence). "Native gene" refers to a gene found in nature with its own regulatory sequence. A “chimeric gene” refers to any gene that is not a native gene (ie, modified from its natural state or other sources), including regulatory and coding sequences that are not found together in nature. Thus, chimeric genes may comprise regulatory sequences and coding sequences derived from different sources or regulatory sequences and coding sequences derived from the same source but arranged in a different manner than those found in nature. "Endogenous gene" refers to a natural gene at its natural location in the genome of an organism. A "foreign gene" or "heterologous gene" refers to a gene that is not normally found as a native gene in a host organism, but has been introduced into the host organism by gene transfer. Foreign genes may include native or chimeric genes inserted into non-natural organisms.

As used herein, the term "coding region" refers to a DNA sequence encoding a particular amino acid sequence. An “appropriate regulatory sequence” is located upstream of a coding sequence (5 ′ noncoding sequence), inside or downstream thereof (3 ′ noncoding sequence) and affects transcription, RNA processing or stability, or translation of related coding sequences. Refers to a nucleotide sequence. Regulatory sequences may include promoters, translation leader sequences, introns, polyadenylation recognition sequences, RNA processing sites, effector binding sites, and stem-loop structures.

The term "codon optimization" associated with a coding region or gene of a nucleic acid molecule for transformation of various hosts refers to the coding region of a nucleic acid molecule to reflect the usual codon usage of the host organism without altering the polypeptide encoded by the DNA. Or altering codons in a gene. Codon optimization is within the ordinary skill in the art.

The term "polynucleotide" is intended to include a singular nucleic acid as well as a plurality of nucleic acids, and refers to a nucleic acid molecule or construct, such as messenger RNA (mRNA) or plasmid DNA (pDNA). As used herein, a "gene" is a polynucleotide. The polynucleotide may comprise the nucleotide sequence of a full length cDNA sequence or fragment thereof, including untranslated 5 'and 3' sequences and coding sequences. The polynucleotide may consist of any polyribonucleotide or polydeoxyribonucleotide that may be unmodified RNA or DNA, or modified RNA or DNA (eg, heterologous DNA). For example, polynucleotides may be single- and double stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA, single-stranded or more typical, which is a mixture of single- and double-stranded regions. And may comprise a hybrid molecule comprising DNA and RNA, which may be double stranded or a mixture of single- and double stranded regions. "Polynucleotide" includes chemically, enzymatically or metabolically modified forms.

A polynucleotide sequence may be referred to as "isolated" where it is removed from its natural environment. For example, heterologous polynucleotides encoding polypeptides or polypeptide fragments having dihydroxy acid dehydratase activity contained in a vector are considered isolated for the purposes of the present invention. Further examples of isolated polynucleotides include recombinant polynucleotides maintained in heterologous host cells or purified (partially or substantially) polynucleotides in solution. Isolated polynucleotides or nucleic acids according to the present invention further comprise such molecules produced synthetically. Isolated polynucleotide fragments in polymer form of DNA may consist of one or more segments of cDNA, genomic DNA or synthetic DNA.

As used herein, the term "polypeptide" is intended to include not only a single "polypeptide" but also a plurality of "polypeptides" and molecules consisting of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). Refers to. The term "polypeptide" refers to any chain (s) of two or more amino acids, not a specific length of product. Thus, peptides, dipeptides, tripeptides, oligopeptides, "proteins", "amino acid chains" or any other term used to refer to the chain (s) of two or more amino acids are included within the definition of "polypeptide", The term “polypeptide” may be used instead of or interchangeably with any such term. Polypeptides may be derived from natural biological sources or may be produced by recombinant techniques, but are not necessarily translated from designated nucleic acid sequences. It can be produced in any way, including by chemical synthesis.

A “isolated” polypeptide, or fragment, variant or derivative thereof, is intended a polypeptide that does not exist in its natural environment. No specific level of purification is necessary. For example, an isolated polypeptide can be removed from its native or natural environment. Recombinantly produced polypeptides and proteins expressed in a host cell are considered to be isolated for the purposes of the present invention, such as natural or recombinant polypeptides that are isolated, fractionated or partially or significantly purified by any suitable technique.

As used herein, a "recombinant microorganism" refers to a bacterium or yeast that is modified using recombinant DNA technology, for example, by genetically engineering the host cell to include a biosynthetic pathway, such as a biosynthetic pathway that produces an alcohol such as butanol. Refers to microorganisms.

The present invention satisfies the need for alternative extractive fermentation methods that do not require the distribution of product alcohol between fermentation medium and ISPR extractant as a means of reducing the toxic effects of product alcohols (eg butanol) on microorganisms. In addition, the product alcohol is converted to alcohol esters which are less toxic to microorganisms, while at the same time the lowering of the partition coefficient of the fermentation product extractor is reduced, so that alcohol (alcohol esters and free alcohols which can be converted back to alcohol after separation from the fermentation medium) By providing a process for the production of alcohols such as butanol, in which the yield of the product) is improved, the demand for reducing the partition coefficient of the fermentation product ISPR extractant is met. In addition, the present invention provides a solution to the shortcomings of alternative alcohol product removal processes that can be combined with existing processes (eg solids removal) to provide increased product removal at economical and environmental advantages. do. Thus, the present invention further provides related advantages, as will be apparent from the description of the embodiments described below.

The present invention provides a method of esterifying an alcohol with a carboxylic acid, thereby extracting the resulting alcohol ester from the fermentation medium, after which the alcohol can be recovered from the alcohol ester. The acid can be added directly to the fermentation medium as free fatty acid or can be derived from an oil. The present invention also provides an alcohol fermentation process by hydrolyzing an oil derived from a feedstock into a carboxylic acid that can be used for esterification of the alcohol and / or can serve as a component of an ISPR extractor or an ISPR extractant for extracting an alcohol ester. It provides a method for removing or reducing oil from oil.

Reducing the amount of water present in the reaction system, or using a reaction system using only one or more non-aqueous solvents, is usually required for esterification of alcohols with carboxylic acids when catalyzed by enzymes such as lipases. . Described herein is a surprising discovery that lipase enzymes can effectively promote esterification of product alcohols and carboxylic acids upon fermentation of fermentable carbon sources, resulting in product alcohols. Also described herein is the surprising discovery that esterification of product alcohols with carboxylic acids during fermentation can provide fermentation performance improvements. For example, by obtaining product alcohols (eg butanol) produced in ester form, effectively reducing the concentration of product alcohols in the aqueous phase, thus alleviating the toxic effects of product alcohols on glucose consumption and product production. .

The invention will be explained with reference to the drawings. 1 illustrates an exemplary process flow diagram for the production of fermentation alcohols such as ethanol or butanol in accordance with an embodiment of the present invention. As shown, feedstock 12 may be introduced into the inlet of liquefaction vessel 10 and liquefied to produce feedstock slurry 16. Feedstock 12 contains hydrolyzable polysaccharides that supply fermentable carbon substrates (eg, fermentable sugars such as glucose) and include, but are not limited to, rye, wheat, sugar cane, or corn. Or else may be derived from biomass. In some embodiments, feedstock 12 may be one or more components of fractional biomass, and in other embodiments, feedstock 12 may be milled micromass. In some embodiments, feedstock 12 may be corn, such as dry milled finely grained corn grains, and insoluble particles may include embryos, fibers, and gluten. Insoluble solids are the non-fermentable part of the feedstock 12. In order to proceed with the review herein in connection with the embodiment shown in the figures, the feedstock 12 will often be described as constituting milled finely divided corn in which insoluble solids are not separated therefrom. However, it should be appreciated that the exemplary methods and systems described herein may be modified with different feedstocks, whether or not fractionated, as will be apparent to those skilled in the art. In addition, as will be appreciated by those skilled in the art, maximizing feedstock content (eg, corn content) may maximize sugar content and product titer. In some embodiments, the feedstock 12 may be high oleic corn, so the corn oil derived therefrom is high oleic corn oil having an oleic acid content of at least about 55 wt% oleic acid. In some embodiments, the oleic acid content in the high oleic corn oil can be about 65 wt% or less, compared to the oleic acid content in conventional corn oil. High oleic oil may provide some advantages for use in the process of the present invention, since the hydrolysis of the oil provides free fatty acids with a high oleic acid content to contact the fermentation broth.

The process of liquefying feedstock 12 involves hydrolyzing the polysaccharides in feedstock 12 into sugars, including, for example, dextrins and oligosaccharides, which is a conventional process. Any known liquefaction process, including but not limited to acid processes, acid-enzyme processes, or enzyme processes, and corresponding liquefaction vessels commonly used in the art may be used. These processes can be used alone or in combination. In some embodiments, an enzymatic process can be used, and an appropriate enzyme 14, for example alpha-amylase, is introduced into the inlet of the liquefaction vessel 10. Water may also be introduced into the liquefaction vessel 10. In some embodiments, glycosylating enzymes, such as glucoamylases, may also be introduced into the liquefaction vessel 10. In a further embodiment, lipase may be introduced into the liquefaction vessel 10 to catalyze the conversion of one or more components of the oil into free fatty acids.

Feedstock slurry 16 resulting from liquefaction of feedstock 12 includes a fermentable carbon substrate (eg, sugar), oil, and insoluble solids derived from the feedstock. Feedstock slurry 16 may be withdrawn from the outlet of liquefaction vessel 10. In some embodiments, feedstock 12 is corn or corn kernels, so feedstock slurry 16 is a corn mash slurry. In some embodiments, feedstock 12 is a lignocellulosic feedstock, so feedstock slurry 16 may be lignocellulosic hydrolyzate. In some embodiments, feedstock 12 is sugar cane.

Feedstock slurry 16 is introduced into fermentation vessel 30 together with microorganism 32 Fermentation vessel 30 is configured to ferment slurry 16 to produce alcohol. In particular, the microorganism 32 metabolizes the fermentable sugars in the slurry 16 to release the product alcohol. The microorganism 32 is selected from the group of bacteria, cyanobacteria, filamentous fungi, and yeasts. In some embodiments, microorganism 32 may be a bacterium, such as E. coli. In some embodiments, microorganism 32 may be a fermentable recombinant microorganism. The slurry can include, for example, sugars and water in the form of oligosaccharides, and in some embodiments, less than about 20 g / L of monomeric glucose, less than about 10 g / L, or less than about 5 g / L of monomeric glucose. can do. Suitable methods for measuring the amount of monomeric glucose are well known in the art. Such suitable methods known in the art include HPLC.

In some embodiments, slurry 16 is subjected to a saccharification process to break down complex sugars (eg, oligosaccharides) in slurry 16 into monosaccharides that can be readily metabolized by microorganism 32. Any known glycosylation process commonly used in the art may be used, including but not limited to acid processes, acid-enzyme processes, or enzyme processes. In some embodiments, simultaneous saccharification fermentation (SSF) may occur within fermentation vessel 30 shown in FIG. 1. In some embodiments, enzyme 38, such as glucoamylase, may be introduced into the inlet of fermentation vessel 30 to break down starch or oligosaccharides into glucose that may be metabolized by microorganism 32.

Carboxylic acid 28 and / or natural oil 26 is introduced into fermentation vessel 30 together with catalyst 42. The catalyst 42 may be introduced before, after or simultaneously with the introduction of the enzyme 38. Thus, in some embodiments, the addition of enzyme 38 and catalyst 42 may be staged (eg, catalyst 42, then enzyme 38, or vice versa), or substantially May be concurrent (i.e. immediately after one enzyme / catalyst, such as at the exact time it takes a human or machine to do the addition in one stroke, or as a time it takes the human or machine to do the addition in two strokes) Other enzymes / catalysts). Catalyst 42 may esterify the product alcohol with carboxylic acid 28 to produce an alcohol ester. For example, in the case of butanol production, the catalyst 42 can esterify butanol with carboxylic acid 28 to produce butyl esters.

When natural oil 26 is fed to fermentation vessel 30, at least some acyl glycerides in oil 26 are hydrolyzed to carboxylic acid 28 by contacting oil 26 with catalyst 42. Can be. The acid / oil composition resulting from hydrolysis of oil 26 is typically at least about 17 wt% carboxylic acid 28 (as free fatty acid). In some embodiments, the acid / oil composition resulting from hydrolysis of oil 26 is at least about 20 wt% carboxylic acid, at least about 25 wt% carboxylic acid, at least about 30 wt% carboxylic acid, at least about 35 wt% carboxylic acid, at least about 40 wt% carboxylic acid, at least about 45 wt% carboxylic acid, at least about 50 wt% carboxylic acid, at least about 55 wt% carboxylic acid, at least about 60 wt% carboxylic acid At least about 65 wt% carboxylic acid, at least about 70 wt% carboxylic acid, at least about 75 wt% carboxylic acid, at least about 80 wt% carboxylic acid, at least about 85 wt% carboxylic acid, at least about 90 wt % Carboxylic acid, at least about 95 wt% carboxylic acid, or at least about 99 wt% carboxylic acid. In some embodiments, the resulting acid / oil composition comprises monoglycerides and / or diglycerides from partial hydrolysis of acyl glycerides in oil. In some embodiments, the resulting acid / oil composition comprises a byproduct of glycerol, acyl glyceride hydrolysis. In some further embodiments, the resulting acid / oil composition comprises lysophospholipids from partial hydrolysis of phospholipids in oil.

In some embodiments, after hydrolysis of the acyl glycerides in oil 26, the residual acyl glycerides from oil 26 are from about 0 wt% to at least about 2 wt% fermentation broth composition. In some further embodiments, after hydrolysis of the acyl glycerides in oil 26, the residual acyl glycerides from oil 26 is at least about 0.5 wt% fermentation broth composition. Thus, in some embodiments, the acyl glycerides from oil 26 may be catalytically hydrolyzed to carboxylic acid 28 using catalyst 42, which catalyst 42 may also be carboxylic acid 28. ) Can be esterified with the product alcohol. In some embodiments, a second catalyst (not shown) may be introduced into the fermentation vessel for hydrolysis of acyl glycerides. In addition, acyl glycerides in oils derived from feedstock 12 and present in slurry 16 may also be hydrolyzed to carboxylic acid 28 '(see, eg, the embodiment of FIG. 3). In some embodiments, the concentration of carboxylic acid (eg, fatty acid) in the fermentation vessel exceeds the solubility limit in the aqueous phase and forms a two-phase fermentation mixture comprising the organic phase and the aqueous phase. In some embodiments, the concentration of carboxylic acid in the fermentation broth is typically about 0.8 g / L or less and limited by the solubility of the carboxylic acid in the broth.

In some embodiments, catalyst 42 and the second catalyst, if used, can be one or more enzymes, such as lipase enzymes. In some embodiments, catalyst 42 may be one or more enzymes, such as hydrolase enzymes such as lipase enzymes. Lipase enzymes used are, for example, Absidia , Achromobacter , Aeromonas , Alcaligenes , Alternaria , Aspergillus , arc our bakteo, Aureobasidium (Aureobasidium), Bacillus, Rove Beria (Beauveria), bromo choseu Riggs (Brochothrix), Candida, chromotherapy bakteo (Chromobacter), Corp. Linus (Coprinus), Fusarium (Fusarium), geo-tree glutamicum (Geotricum), Hanse nyulra, trailing coke (Humicola), high spore forehead (Hyphozyma), Lactobacillus, Metairie Clear (Metarhizium), non-cor (Mucor), neck triazole (Nectria), Neuro spokes La (N eurospora) , L indeed My process (Paecilomyces), Penny silryum (Penicillium), Pseudomonas, Li Estonian (Rhizoctonia), Li jomu cor (Rhizomucor), Rhizopus crispus (Rhizopus), Rhodes Pori Stadium (Rhodosporidium), also torulra (Rhodotorula), Saccharomyces in A ball in the recess, suspension (Sus), sports My process (Sporobolomyces), Thermo My process (Thermomyces), thiazol a sports Pasteurella (Thiarosporella), tricot der e (Trichoderma), tying yarn Solarium (Verticillium), and / or Yarrow May be derived from any source, including a weir strain. In a preferred embodiment, the source of lipase is Absidia blakesleena ), Absidia corimbifera corymbifera ), Achromobacter iophagus , Alcaligenes sp . ), Alternaria brassiciola), Aspergillus Playa booth (A spergillus flavus), Aspergillus niger (A spergillus niger ), Aspergillus tubingensis ), Aureobasidium pullulans , Bacillus pumilus), Bacillus stearoyl Terre a brush loose (Bacillus strearothermophilus ), Bacillus subtilis ( Bacillus subtilis ), Brochothrix thermosohata ), Candida cylindracea ( Candida lugosa ( Candida rugosa)), Candida para Li poly urticae (Candida paralipolytica), Candida hits arcs urticae (Candida antarctica) lipase A, Candida hits arcs urticae lipase B, Candida El nobiyi (Candida ernobii ), Candida deformans ( Candida deformans ), Chromobacter viscosum ), Coprinus cinerius), Fusarium oxy Spokane Room (Fusarium oxysporum), Fusarium solani (Fusarium solani), Fusarium solani Fish (Fusarium solani pisi), erecting a cool morum Fusarium (Fusarium roseum culmorum ), Geotricum penicillatum ), H ansenula anomala ), Humicola brebispora brevispora ), Fumi-Cola Breviss Variant Themoidea ( Humicola brevis there is . thermoidea ), Humicola insolens , Lactobacillus curvatus), Rhizopus crispus duck material (Rhizopus oryzae), Penny silryum cycloalkyl europium (Penicillium cyclopium), Penny silryum Crewe Stoke breath (Penicillium crustosum), Penny silryum X pansum (Penicillium expansum), Penny silryum species (Penicillium sp . ) I, Penny silryum species (Penicillium sp . ) II, Pseudomonas Pseudomonas aeruginosa), Pseudomonas Alcaligenes (Pseudomonas alcaligenes), Pseudomonas Sepharose cyano (Pseudomonas cepacia ) (synonym: Burkholderia cepacia )), Pseudomonas fluorescens , Pseudomonas pragis fragi ), Pseudomonas maltophilia), Pseudomonas Men City or (Pseudomonas mendocina), Pseudomonas mepi urticae Li poly urticae (Pseudomonas mephitica lipolytica), Alcaligenes Pseudomonas (Pseudomonas alcaligenes ), Pseudomonas plantari ), Pseudomonas pseudoalcaligenes , Pseudomonas putida ), Pseudomonas stutzeri ), and Pseudomonas wisconsinensis , Rhizoctonia solani ), Rhizomucor miehei , Rhizopus japonicus ), Rhizopus microsporus ), Rhizopus nodosus ), Rhodosporidium toruloides ), Rhodotorula glutinis , Saccharomyces cerevisiae (S accharomyces cerevisiae ), Sporobolomyces shibatanus ), Sus scrofa , Thermomyces ranuginosus ( Thermomyces) lanuginosus ) (formerly Humicola ranuginose) lanuginose)), thiazol a sports Pasteurella Pace olrina (Thiarosporella phaseolina), tricot der Haar town Jia num (Trichoderma harzianum ), Trichoderma reesei ), and Yarrowia lipolytica . In a further preferred embodiment, the lipase is thermomyces lanuginosus ) lipase, Aspergillus sp . ), Lipase, Aspergillus niger (Aspergillus niger) lipase, Aspergillus Tuvinian Genesis (Aspergillus tubingensis ) lipase, Candida antarctica lipase B, Pseudomonas sp . lipase, penicillium roquefort ( Penicillium) roqueforti) lipase, Penny silryum kamem Berti (Penicillium camembertii) lipases, non-cor jabani kusu (Mucor javanicus) lipase, Burke holde Ria Sepharose cyano (Burkholderia cepacia ) Lipase, alkali genesis ( Alcaligenes) sp . ) Lipase, Candida rugosa Lipase, Candida parapsissis ( Candida parapsilosis ) lipase, Candida deformans ( Candida the deformans) lipase, candida Geo tree glutamicum Doom (Geotrichum candidum) Lipase A and B, neuro spokes la Klein Inc. (Neurospora crassa ) lipase, Nectria haemacocaca ( Nectria haematococca) lipase, Fusarium hetero sports rooms (Fusarium heterosporum) lipase, Rhizopus crispus del Sliema (Rhizopus delemar) lipase, Li jomu cor Mie Hay (Rhizomucor miehei ) lipase, Rhizopus arrhizus lipase, and Rhizopus oryzae ) is selected from the group consisting of lipases. Suitable commercial lipase formulations suitable as catalysts (42) include Lipolase® available from Novozymes. 100 L, Lipex? 100L, Lipoclean? 2000T, Lipozyme? CALB L, Novozyme? CALA L, and the palrata (Palatase) 20000L, or Sigma-Aldrich possible Pseudomonas fluorescein sense (Pseudomonas fluorescens) obtained from (SigmaAldrich), Pseudomonas Sepharose cyano (Pseudomonas cepacia ), Mucor Miehei ( Mucor miehei , pig pancreas ( hog pancreas), Candida syringe driver Seah (Candida cylindracea), Candida Lu Test (Candida rugosa), Rhizopus crispus nibe mouse (Rhizopus niveus), Candida hits arcs urticae (Candida antarctica ), Rhizopus arrhizus or Aspergillus .

Phospholipases are enzymes that hydrolyze ester bonds of phospholipids, but many phospholipases can also hydrolyze triglycerides, diglycerides, and monoglycerides (lipid acyl hydrolase (LAH) activity). . As used herein, the term “phospholipase” refers to any phospho that catalyzes, for example, hydrolysis of glycerol phosphate ester bonds, in oils such as crude or vegetable oils, for example. Enzymes having lipase activity. The phospholipase activity of the present invention can produce water extractable phosphorylated bases and diglycerides. Phospholipase activity includes phospholipase C (PLC) activity; PI-PLC activity, phospholipase A (PLA) activity such as phospholipase Al or phospholipase A2 activity; Phospholipase B (PLB) activity, such as phospholipase B1 or phospholipase B2 activity, including lysophospholipase (LPL) activity and / or lysophospholipase-transacylase (LPT A) activity; Phospholipase D (PLD) activity, such as phospholipase Dl or phospholipase D2 activity; And / or patatin activity or any combination thereof.

The term “phospholipase” also includes enzymes having lysophospholipase activity, two substrates of which are 2-lysophosphatidylcholine and H ₂ O, the two products of which are glycerophosphocholine and carbohydrates. Is a carboxylate. The phospholipase Al (PLA1) enzyme removes the 1 position fatty acid, producing free fatty acids and 1-lyso-2-acylphospholipids. Phospholipase A2 (PLA2) enzymes remove the 2-position fatty acids, producing free fatty acids and 1-acyl-2-lysophospholipids. PLA1 and PLA2 enzymes may exhibit intra- or extracellular, membrane binding or solubility. Phospholipase C (PLC) enzymes remove the phosphate moiety to produce 1,2-diacylglycerol and phosphate esters. Phospholipase D (PLD) enzymes produce 1,2-diacylglycerophosphate and base groups. Phospholipases useful in the present invention include filamentous fungal species in various biological sources such as Fusarium, such as F. culmorum , F. heterosporum , Fusarium solanie ( F. solani ), or strains of F. oxysporum ; Or filamentous fungal species in the genus Aspergillus , such as Aspergillus awamori awamori ), Aspergillus poetidus foetidus ), Aspergillus japonicus , Aspergillus niger niger ) or Aspergillus duckbill ( Aspergillus) oryzae ) may be obtained from, but is not limited to. Thermomyces Lanuginus lanuginosus ) phospholipase variants, such as the commercial product Lecitase? Ultra (Novozymes A'S, Denmark) is also useful in the present invention. One or more phospholipases may be immobilized or applied as lyophilized powder in aqueous solution.

Alcohols (eg, butanol) produced by the fermentation of one or more fermentable sugars can be converted to carboxylic esters by enzymatic catalysis in which the carboxylic acid is esterified with alcohol. Enzymes such as lipase, phospholipase, and lysophospholipase can catalyze this reaction; Such enzymes may be inactivated due to one or more factors, including but not limited to hydrodynamic shear or inactivation at the gas-liquid and liquid-liquid interface. In fermentation where the oligosaccharides are additionally converted to one or more fermentable sugars, enzymes (eg, glucoamylases) that convert the oligosaccharides to fermentable sugars may also be inactivated by one or more such same factors.

Inactivation of enzymes at the gas-liquid interface (e.g., with bubbles and) that occurs due to aeration of the fermentation broth and / or is generated by the generation of gaseous carbon dioxide in the broth upon fermentation of one or more fermentable sugars. Which can occur at the interface of fermentation broth) is known in the art. Hen egg white lysozyme and aspergillus duckbill ( Aspergillus oryzae ) ( Thermomyces ranuginosas produced by Novozymes repolarase?) lanuginosus ) Inactivation of lipases was observed at the gas-liquid interface in three different reactor configurations: bubble column, stirred vessel with baffles (no aeration by gas sparging), and falling film (Ghadge, et. al., Chem. Eng. Sci. 58: 5125-5134, 2003). Thermomyces at the gas-liquid interface in a baffled stirred-tank reactor (no aeration by gas sparging) Thermomyces lanuginosus ) lipase ( Aspergillus duck) oryzae ); Inactivation mechanism of Novozymes repolarase 100L?) Has been reported (Patil, et al., AIChE J. 46: 1280-1283, 2000).

Stahmann et al. (Eur. J. Biochem. 244: 220-225, 1997) reported that the Ashbya gossypii lipase was agitated due to the interfacial deactivation of the gas / liquid or liquid / liquid interface. It was reported to be inactivated in water, trioleoylglycerol / water or oleic acid / water mixture within minutes. Elias et al. (Adv. Biochem. Engineering / Biotechnology 59: 47-71, 1998) reported that: (i) some enzymes are inactivated by hydrodynamic shear in the absence of a gas-liquid interface; (ii) for enzymes inactivated by hydrodynamic shear, the inactivation rate is increased in the presence of a gas-liquid interface; (iii) some enzymes are not inactivated in the absence of a gas-liquid interface regardless of the applied hydrodynamic shear; (iv) For enzymes that require a gas-liquid interface for inactivation, the rate of inactivation increases with increasing hydrodynamic shear. Ross et al. (J. Mol. Catal. B: Enzymatic 8: 183-192, 2000) reported that α-Kamo in various aqueous / organic solvent mixtures by passing solvent droplets upward through an aqueous solution of enzyme in a bubble column apparatus. Interfacial inactivation of trypsin, β-chymotrypsin, papain, and porcine liver esterase has been described. Kinetics and mechanisms of shear inactivation of Candida cylindracea lipase in stirred tank reactors have also been reported, and the inactivation mechanism has been found to be due to the shear induced gas-liquid interfacial effect (Lee, et al., Biotechnol. Bioeng. 33: 183-190, 1989).

Under fermentation conditions used in some of the methods described herein, hydrodynamic shear and gas-liquid and liquid-liquid interfaces are present during fermentation, respectively, and can cause enzyme inactivation. At least one enzyme (eg, glucoamylase, lipase, phospholipase, and lysophospholipase) present in the biphasic mixture (eg, fermentation broth and carboxylic acid) under the conditions described herein upon fermentation. The potential effect of each of these factors on the stability and activity of P would not have been predictable based on the prior art. Each of these factors may result in inactivation of one or more enzymes in the fermentation mixture, but sufficient enzymatic activity was maintained during the fermentation to catalyze the esterification of the product alcohol with carboxylic acid to produce carboxylic acid esters. In the reaction where glucoamylase is also present in the biphasic fermentation mixture of fermentation broth and carboxylic acid, sufficient enzymatic activity (ie for converting oligosaccharides to fermentable sugars) was also maintained.

Carboxylic acid (28) can be any carboxylic acid that can be esterified with a product alcohol such as butanol or ethanol to produce an alcohol ester of carboxylic acid. For example, in some embodiments, carboxylic acid 28 may be a free fatty acid, and in some embodiments, carboxylic acid or free fatty acid may have 4 to 28 carbons, and in other embodiments 4 to 22 carbons. Carbon, 8 to 22 carbons in other embodiments, 10 to 28 carbons in other embodiments, 7 to 22 carbons in other embodiments, 12 to 22 carbons in other embodiments, in other embodiments 4 to 18 carbons, 12 to 22 carbons in another embodiment, and 12 18 carbons in another embodiment. In some embodiments, carboxylic acid 28 is one or more of the following fatty acids: azelaic acid, capric acid, caprylic acid, castor oil, coconut oil (ie, lauric acid, myristic acid, pal) As a combination of naturally occurring fatty acids, including butyric acid, caprylic acid, capric acid, stearic acid, caproic acid, arachidic acid, oleic acid, and linoleic acid), isostearic acid, lauric acid, linseed oil, myristic Acids, oleic acid, palm oil, palmitic acid, palm kernel oil, pelagonic acid, ricinoleic acid, sebacic acid, soybean oil, stearic acid, tall oil, resins, and # 12 hydroxystearic acid. In some embodiments, carboxylic acid 28 is one or more dibasic acids.

In some embodiments, carboxylic acid 28 may be a mixture of two or more different fatty acids. In some embodiments, carboxylic acid 28 comprises free fatty acids derived from hydrolysis of acyl glycerides by any method known in the art, including chemical hydrolysis or enzymatic hydrolysis. As described above in some embodiments, carboxylic acid 28 may be derived from natural oil 26 by enzymatic hydrolysis of oil glycerides with enzyme 42 as catalyst 42. In some embodiments, the fatty acids or mixtures thereof include unsaturated fatty acids. The presence of unsaturated fatty acids reduces the melting point, providing an advantage for handling. Among unsaturated fatty acids, ie monounsaturated fatty acids with single carbon-carbon double bonds, can provide an advantage with regard to melting point without compromising adequate thermal and oxidative stability for process considerations.

In some embodiments, natural oil 26 is resin, corn oil, canola oil, capric / caprylic triglyceride oil, castor oil, coconut oil, cottonseed oil, fish oil, jojoba oil, lard oil, linseed oil, right angle Oils, Otissica Oil, Palm Oil, Peanut Oil, Rapeseed Oil, Rice Bran Oil, Safflower Oil, Soybean Oil, Sunflower Oil, Copper Oil, Jatropha Oil, Pumpkin Oil, Grapeseed Oil, and Vegetable Oil Blends (or High Concentrations of Different Chain Length and Unsaturation) Oil, which can be purified to degrees (ie, 18: 1). In some embodiments, natural oil 26 is a mixture of two or more natural oils, such as, for example, a mixture of palm oil and soybean oil. In some embodiments, natural oil 26 is a vegetable oil. In some embodiments, the vegetable oil may be derived from biomass that may, but not necessarily, be used in the fermentation process. The biomass can be the same or different source from which the feedstock 12 is obtained. Thus, for example, in some embodiments, oil 26 may be derived from corn while feedstock 12 may be sugar cane. For example, in some embodiments, oil 26 may be derived from corn, and the biomass source of feedstock 12 is also corn. As will be apparent to those skilled in the art, any possible combination of different biomass sources for oil 26 versus feedstock 12 may be used. In some embodiments, oil 26 is derived from biomass used in the fermentation process. Thus, in some embodiments, as discussed below with respect to FIG. 3, oil 26 is derived directly from feedstock 12 as oil 26 ′. For example, if feedstock 12 is corn, oil 26 'is corn oil, which is a component of the feedstock.

Optionally, ethanol 33 may be supplied to fermentation vessel 30 and included in the fermentation broth. In some embodiments, when recombinant microorganisms with reduced or eliminated expression of butanol biosynthetic pathways and / or pyruvate decarboxylase are used as microorganism 32, microorganism 32 may be a 2-carbon substrate, for survival and growth, For example, it may be necessary to supplement ethanol. Thus, in some embodiments, ethanol 33 may be supplied to fermentation vessel 30.

Surprisingly, however, the method of the present invention in which carboxylic acids, such as fatty acids, are present in a fermentation vessel, can reduce the amount of ethanol 33 that is typically supplied for a given recombinant microorganism without sacrificing the vitality of the recombinant microorganism. I found out. In addition, in some embodiments of the method of the present invention, the alcohol (eg, butanol) production rate without ethanol supplementation may be comparable to the production rate that can be realized when ethanol 33 is supplemented. As further demonstrated by the comparative examples shown in Examples 1 to 14 below, the butanol production rate when fatty acid but not ethanol is in the fermentation vessel is the butanol production rate when neither fatty acid nor ethanol is in fermentation vessel. It may be comparable or larger than that. Thus, in some embodiments, the replenishment amount of ethanol 33 is reduced compared to conventional processes. For example, a typical amount of ethanol added to a fermentation vessel for a microorganism requiring supplementation of a 2-carbon substrate is about 5 g / L anhydrous ethanol (ie, 5 g anhydrous ethanol per liter of fermentation medium). In some embodiments, the fermentation is not supplemented by any ethanol 33. In the latter case, the stream of ethanol 33 is completely removed from the fermentation vessel. Thus, in some embodiments of the present invention, in butanol fermentation or other alcohol fermentation using the costs associated with ethanol 33 of supplementation, and microorganisms that may require supplementation of a 2-carbon substrate for survival and growth. Discomfort associated with the storage bin of ethanol 33 and the supply of ethanol to the fermentation vessel can be reduced or eliminated.

In addition, regardless of ethanol replenishment, in some embodiments, the methods of the present invention may provide high glucose uptake by microorganisms 32 by the presence of fatty acids at fermentation. Fatty acids may be introduced into fermentation vessel 30 as carboxylic acid 28 derived from the hydrolysis of the constituent biomass oil of slurry 16 and / or hydrolyzed from the supplied oil 26. A method for producing product alcohol from a fermentation process in which free fatty acids are produced in one step of the process and contacted with microbial cultures in a fermentation vessel to improve microbial growth rate and glucose consumption is incorporated herein by reference in its entirety. Co-owned US Provisional Application No. 61 / 368,451, filed July 28, 2010, which is hereby incorporated and incorporated herein by reference.

In the fermentation vessel 30, the alcohol produced by the microorganism 32 is esterified with the carboxylic acid 28 using the catalyst 42 to produce an alcohol ester. For example, in the case of butanol production, butanol produced by microorganism 32 is esterified with carboxylic acid 28 using catalyst 42 to produce butyl ester. In situ product removal (ISPR) can remove alcohol esters from fermentation broth. As demonstrated herein, by using a catalyst to form esters with ISPR, fermentation performance can be improved. In some embodiments, by using a catalyst to form an ester with ISPR (eg, liquid-liquid extraction), the effective titer is at least compared to the effective titer of similar fermentation with ISPR without a catalyst producing the ester. About 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100% increase You can. Similarly, in some embodiments, by using a catalyst to form esters with ISPR (eg, liquid-liquid extraction), analogous fermentation (eg, Examples) with ISPR without catalyst to produce esters 9 and 11 to 14, see Table 3), at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100%. In some embodiments, the effective yield is increased by at least about 10%, at least about 20%, at least about 30%, at least about 40%, or at least about 50%. In some embodiments, the resulting fermentation broth after alcohol esterification may comprise free (ie, non-esterified) alcohol, and in some embodiments, the concentration of free alcohol in the fermentation broth after alcohol esterification is determined by the product alcohol being butanol. Fermentation broth after alcohol esterification when 1, 3, 6, 10, 15, 20, 25, 30 25, 40, 45, 50, 55, or 60 g / L or when the product alcohol is ethanol The concentration of free alcohol in is 15, 20, 25, 30 25, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 g / L or less. In some embodiments, the ratio of alcohol esters to alcohol in the fermentation vessel can be about 1: 1. In some embodiments, the alcohol has an effective titer of at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90%. Is converted to an alcohol ester.

In some embodiments, the grain load for water at a sufficient concentration to achieve a final effective titer of at least about 50 g / L, at least about 75 g / L, or at least about 100 g / L is butanol and It can be used for grain mash fermentation, including microorganisms that can produce the same alcohol. In another embodiment, the grain mash fermentation can utilize simultaneous saccharification fermentation (SSF) and the concentration of glucose can be kept relatively low, eg, at least about 75 g / L glucose, on the fermentation broth during fermentation.

In some embodiments, the fatty acid may be added to the fermentation vessel in an amount that is less than about 70% of the fermentation vessel volume, less than about 50% of the fermentation vessel volume, or less than about 30% of the fermentation vessel volume. The amount of fatty acid added to the fermentation vessel may be a means for maintaining the aqueous phase titer of butanol upon fermentation. In another embodiment, the aqueous phase titer of butanol may be maintained at a level of less than about 35 g / L (fermentation broth), less than about 25 g / L (fermentation broth), or less than about 20 g / L (fermentation broth). have. In other embodiments, the amount of active esterase in the fermentation broth may be less than about 100 ppm active enzyme, less than about 50 ppm active enzyme, or less than about 10 ppm active enzyme. In some embodiments, the cell mass used in the fermentation broth may be less than about 50 g dcw / L, less than about 20 g dcw / L, or less than about 10 g dcw / L. In another embodiment, the fermentation process may be performed for at least about 30 hours to at least about 100 hours, at least about 40 hours to at least about 80 hours, or at least about 50 hours to at least about 70 hours.

In some embodiments, sufficient to achieve a final effective titer of at least about 30 g butanol per liter of fermentation broth, at least about 45 g butanol per liter of fermentation broth, or at least about 60 g butanol per liter of fermentation broth. Briggs against water at concentrations can be used for sugar cane fermentation, including microorganisms that can produce butanol. In some embodiments, the fatty acid may be added to the fermentation vessel in an amount that is less than about 70% of the fermentation vessel volume, less than about 50% of the fermentation vessel volume, or less than about 30% of the fermentation vessel volume. The amount of fatty acid added to the fermentation vessel may be a means for maintaining the aqueous phase titer of butanol upon fermentation. In another embodiment, the aqueous phase titer of butanol may be maintained at a level of less than about 35 g / L (fermentation broth), less than about 25 g / L (fermentation broth), or less than about 15 g / L (fermentation broth). have. In another embodiment, the amount of active esterase in the fermentation broth may be less than about 200 ppm active enzyme, less than about 100 ppm active enzyme, or less than about 20 ppm active enzyme. In some embodiments, the cell volume used for the fermentation broth may be at least about 100 g of cells per liter of broth in the initial infusion amount that initially accounts for at least about 30% of the fermentation vessel volume. After 3-7 hours of fermentation, the cell mass can be diluted to at least about 25 g of cells per liter of fermentation broth by addition of sugarcane feed. The cells may continue to grow to at least about 30 g of cells per liter of fermentation broth over a total fermentation time of 8 to 15 hours.

In some embodiments, the fermentation broth is contacted with the extractant during fermentation to produce a biphasic mixture comprising an aqueous phase and an organic phase. In such embodiments, ISPR, including liquid-liquid extraction, can be done conveniently. Liquid-liquid extraction can be performed according to the process described in US Patent Application Publication No. 2009/0305370, which is incorporated herein in its entirety. US Patent Application Publication 2009/0305370 discloses a butanol from fermentation broth using liquid-liquid extraction, comprising contacting the fermentation broth with a water immiscible extractant to produce a biphasic mixture comprising an aqueous phase and an organic phase. It describes how to generate and recover. Typically, the extractant is saturated, monounsaturated, polyunsaturated (and mixtures thereof) C ₁₂ to C ₂₂ fatty alcohols, C ₁₂ to C ₂₂ fatty acid, C acid ester, of ₁₂ to C ₂₂ fatty acids, C ₁₂ to C ₂₂ fat aldehyde , C ₁₂ to C ₂₂ fatty amides, and mixtures thereof. Extractants are also saturated, monounsaturated, polyunsaturated (and mixtures thereof) C ₄ to C ₂₂ fatty alcohols, C ₄ to C ₂₈ fatty acids, esters of C ₄ to C ₂₈ fatty acids, C ₄ to C ₂₂ fatty aldehydes, and Organic extractant selected from the group consisting of mixtures thereof. Extractor (s) for ISPR for use in the processes described herein avoids the consumption of carboxylic acid 28 in fermentation vessel 30 by catalytic esterification of carboxylic acid 28 with an alcohol extractant. It is typically an alcohol-free extractant so that less carboxylic acid is used for esterification with the product alcohol. For example, if oleyl alcohol is used as the ISPR extractant, the oleyl alcohol ester of carboxylic acid may be produced in the fermentation vessel due to the presence of the active catalyst 42, as further demonstrated in Example 24 below. Can be.

With regard to the embodiment of FIG. 1, carboxylic acid 28 may also function as ISPR extractant 28 or a component thereof. As mentioned above, the carboxylic acid 28 may be fed, and / or may be produced in situ when the natural oil 26 is fed to the fermentation vessel 30, and / or the feedstock 16 is It may be produced in situ if it contains oils that can be hydrolyzed. In some embodiments, ISPR extractant 28 includes free fatty acids. In some embodiments, ISPR extractant 28 comprises corn oil fatty acid (COFA). In some embodiments, oil 26 is corn oil, so ISPR extractant 28 is COFA. ISPR extractant (carboxylic acid) 28 is contacted with fermentation broth to produce a biphasic mixture comprising an aqueous phase 34 and an organic phase. The product alcohol esters produced in the fermentation vessel are preferentially distributed into the organic phase to produce the ester containing organic phase 36. In other words, the product alcohol ester is produced at a concentration above the equilibrium concentration of the alcohol ester present in the aqueous phase 34 and, therefore, is preferentially distributed in the organic phase. Any free product alcohol in the fermentation broth is also preferentially partitioned into the ester containing organic phase. The biphasic mixture can be removed from fermentation vessel 30 as stream 39 and introduced into vessel 35 where ester-containing organic phase 36 is separated from aqueous phase 34. Separation of the biphasic mixture 39 into the ester containing organic phase 36 and the aqueous phase 34 is carried out by siphoning, aspiration, decantation, centrifugation using gravity settling tanks, membrane-assisted phase splitting), hydrocyclones, and the like, can be accomplished using any method known in the art. All or part of aqueous phase 34 may be recycled to fermentation vessel 30 as fermentation medium (shown), or discarded and replaced with fresh medium, or treated for removal of any residual product alcohol. 30).

With regard to FIG. 1, ester containing organic phase 36 is introduced into vessel 50, where the alcohol ester is reacted with one or more substances 52 to recover product alcohol 54. Product alcohol 54 may be recovered using any method known in the art to obtain alcohol from alcohol esters. For example, in some embodiments, the product alcohol may be recovered by hydrolysis with a base from an alcohol ester followed by acidification. In another embodiment, the product alcohol ester can be hydrolyzed by water in the presence of a hydrolysis catalyst as material 52. For example, in some embodiments, hydrolysis of the product alcohol ester to alcohol and carboxylic acid 28 (eg, fatty acid when carboxylic acid 28 is a fatty acid) is a lipase as substance 52. , Water soluble acids, inorganic acids, organic acids, or solid acid catalysts. For example, sulfuric acid can be used as an inorganic acid catalyst for alcohol ester hydrolysis. Some suitable hydrolysis catalysts include lipase enzymes; Esterase enzymes; Inorganic strong acids such as sulfuric acid, hydrochloric acid, or phosphoric acid; Organic strong acids such as toluenesulfonic acid or naphthalenesulfonic acid; Or solid acid catalysts such as Amberlyst? Sulfonated polystyrene resins, or zeolites. In some embodiments, hydrolysis of the alcohol ester can be accomplished by using steam as the material 52 to increase the temperature and / or pressurize. In some embodiments, hydrolysis of the alcohol esters can be done in a column, such as a reaction distillation column. Examples 45-54 and 56-58 demonstrate several methods of recovering product alcohol from alcohol esters. In some embodiments, byproduct 56 is obtained from recovery of product alcohol 54. Byproduct 56 does not include carboxylic acid 28 which may be recovered from hydrolysis of the alcohol ester.

In some embodiments, the hydrolysis of the fatty acid present in the ester containing organic phase 36 to the alcohol and free fatty acids of the fatty acid occurs in a ratio of fatty acids to water of about 10: 1 to about 1:10, or in other embodiments And from about 100: 1 to about 1: 100 fatty acid to water ratio. In some embodiments, the alcohol esters of fatty acids are hydrolyzed to water at temperatures below about 100 ° C. In some embodiments, the hydrolysis occurs at temperatures above 100 ° C, above 150 ° C, above 200 ° C, or above 250 ° C.

For example, in some embodiments, the alcohol ester may be transesterified to produce the product alcohol 54, and in some embodiments, the second alcohol ester 56, eg, fatty acid alkyl ester, is also a byproduct And as (56). To achieve this transesterification, the alcohol ester can be contacted with a catalyst capable of transesterifying the alcohol ester to release butanol. In some embodiments, the alcohol ester may be transesterified using glycerol to produce acyl glycerides as product alcohol 54 and byproduct 56. The resulting acyl glycerides may include mono- and diacylglycerides. Some suitable catalysts for the transesterification reaction are, for example, lipase enzymes, in particular the alkoxide salts of the second alcohols, alkyl titanates, soluble inorganic acids such as sulfuric acid and phosphoric acid, soluble organic acids such as toluenesulfonic acid and naphthalenesulfonic acid, and Solid acids, such as Amberlyst? Sulfonated polystyrene resins, or zeolites. Suitable lipases for transesterification or hydrolysis are Burkholderia cepacia ), Thermomyces ranuginosa lanuginosa ), or lipases derived from Candida antarctica . In some embodiments, the lipase is immobilized to a soluble or insoluble support using methods well known to those skilled in the art (eg Immobilization of Enzymes and Cells; Gordon F. Bickerstaff, Editor; Humana Press, Totowa, NJ, USA, 1997 ). Enzyme immobilization may include 1) binding of the enzyme to a porous or nonporous carrier support via covalent support, physical adsorption, electrostatic bonding, or affinity bonding; 2) crosslinking with bifunctional or multifunctional reagents; 3) entrapment in a gel matrix, polymer, emulsion, or any type of membrane; And 4) various techniques, including any combination of these methods. In another embodiment, the lipase cannot be immobilized. In some embodiments, the lipase is soluble. Fatty acid alkyl esters 56 may include, for example, fatty acid methyl esters. Other fatty acid alkyl esters 56 may include, for example, C ₂ to C ₁₂ straight, branched, and cyclic alcohol esters. The product alcohol 54 can then be separated from the reaction mixture comprising the byproduct 56 using any separation means known in the art, such as, for example, distillation. Other suitable separation mechanisms may include, for example, extraction and membrane separation.

In some embodiments, at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, At least about 90%, or at least about 99% of product alcohol is recovered from the alcohol ester.

ISPR extractant (carboxylic acid) 28 may be separated from the alcohol ester prior to reaction of the alcohol ester for the recovery of product alcohol 54. Alternatively, ISPR extractant 28 may be separated from the product alcohol and any byproducts after the reaction of the alcohol ester. The resulting recovered lean extractant 27 is typically a fresh make-up extractor 28 (if supplied, oil (26) for further production and / or extraction of alcohol esters. May be recycled back to the fermentation vessel 30. Alternatively, fresh extractant 28 (or oil 26) may be added continuously to the fermentation vessel to replace the extractant removed in the biphasic mixture stream 39.

In some embodiments, the catalyst 42 may be recovered from the biphasic mixture 39 and reused in one step of the fermentation process, such as in the fermentation itself or in the recovery of the product alcohol.

In some embodiments, one or more additional ISPR extractors 29 (see FIG. 2) may be introduced into fermentation vessel 30 to produce a biphasic mixture comprising an aqueous phase and an organic phase. In this embodiment, the ISPR extractant 29 is an exogenous organic extractant such as oleyl alcohol, behenyl alcohol, cetyl alcohol, lauryl alcohol, myristyl alcohol, stearyl alcohol, 1-undecanol, oleic acid, lauric Acids, myristic acid, stearic acid, methyl myristate, methyl oleate, undecanal, lauric aldehyde, 20-methylundecanal, and mixtures thereof. However, for the reasons mentioned above, ISPR extractant 29 is preferably not alcohol. Instead, the ISPR extractant 29 is preferably a carboxylic acid (eg free fatty acid). In some embodiments, ISPR extractant 29 is COFA. In some embodiments, ISPR extractant 29 is a linseed oil fatty acid, soybean oil fatty acid, jatropha oil fatty acid, or a fatty acid derived from palm oil, castor oil, olive oil, coconut oil, peanut oil, or any seed oil. In some embodiments, ISPR extractant 29 is a bioavailable agent described in co-owned US Provisional Application No. 61 / 368,436, filed July 28, 2010, co-pending, for example, incorporated herein by reference. Fatty acids, fatty alcohols, fatty amides, fatty esters (especially those containing 1 to 8 carbon atoms in the alcohol moiety, for example fatty acid methyl esters and lower levels of fatty acids, obtained from chemical conversion of natural oils such as mass lipids) Alcohol esters), fatty acid glycol esters, hydroxylated triglycerides, and mixtures thereof. In some embodiments, ISPR extractant 29 is a free fatty acid obtained by chemical hydrolysis of biomass lipids. In some embodiments, ISPR extractant 29 is a bioavailable agent described in co-owned US Provisional Application No. 61 / 368,444, filed on July 28, 2010, for example, which is incorporated herein by reference. Free fatty acids resulting from enzymatic hydrolysis of natural oils such as mass lipids.

Product removal in situ may be done in batch mode or continuous mode in fermentation vessel 30. In a continuous mode of product removal in situ, the product is continuously removed from the vessel (or reactor). In batch mode of product removal in situ, a large amount of organic extractant is added to the fermentation vessel and no extractant is removed during the process. For in situ product removal, the organic extractant may be contacted with the fermentation medium at the start of the fermentation to form a biphasic fermentation medium. Alternatively, the organic extractant may be contacted with the fermentation medium after the microorganism has achieved the desired amount of growth, which may be determined by measuring the optical density of the culture. The organic extractant may also contact the fermentation medium when the product alcohol level in the fermentation medium reaches a preselected level. In the case of butanol production according to some embodiments of the present invention, at a point before the butanol concentration reaches a toxic level, the carboxylic acid extractant is contacted with the fermentation medium to esterify butanol with carboxylic acid, thereby obtaining butyl ester Can be produced, and in some embodiments, a two-phase mixture can be produced comprising an aqueous phase and an organic phase comprising a butyl ester. Thus, the concentration of butanol is reduced in the fermentation vessel, as a result minimizing the toxic effect of butanol on the microorganisms. The ester containing organic phase can then be removed from the fermentation vessel after the desired effective titer of the butyl ester is achieved (and can be separated from the fermentation broth making up the aqueous phase). For example, in some embodiments, the ester containing organic phase can be separated from the fermentation broth after the effective titer of the butyl ester is greater than about 10 g / kg fermentation broth. In another embodiment, the ester containing organic phase has an effective titer of butyl ester greater than about 230 g / kg (fermentation broth), greater than about 300 g / kg (fermentation broth), greater than about 400 g / kg (fermentation broth), about 500 After greater than g / kg (fermentation broth), or greater than about 600 g / kg (fermentation broth), it may be separated from the fermentation medium. In another embodiment, the ester containing organic phase may be separated from the fermentation medium after the percent conversion of COFA is at least about 10%, at least about 25%, at least about 50%, at least about 75%, or at least about 100%. have. In some embodiments, the ester containing organic phase is separated from the aqueous phase after fermentation of the available fermentable sugars in the fermentation vessel is substantially complete.

In the embodiment shown in FIG. 1, the alcohol ester is extracted from the fermentation broth in situ, with the separation of the biphasic mixture 39 taking place in a separate vessel 35. In some embodiments, separation of the biphasic mixture may occur in the fermentation vessel, as shown in the embodiments of FIGS. 3 and 4 described below, in which the ester containing organic phase stream 36 exits directly from the fermentation vessel 30. Aqueous phase stream 34 may also exit directly from fermentation vessel 30 and may be treated and recycled for removal of any residual alcohol ester or product alcohol, or discarded and replaced with fresh fermentation medium. The extraction of alcohol esters and product alcohols with organic extractants can be done with or without the removal of microorganisms 32 from the fermentation broth. The microorganism 32 can be removed from the fermentation broth by means known in the art, including but not limited to filtration or centrifugation. For example, aqueous phase stream 34 may comprise microorganisms 32 such as yeast. The microorganism 32 can be easily separated from the aqueous phase stream, for example in a centrifuge (not shown). The microorganism 32 may then be recycled to the fermentation vessel 30 and may increase the production rate of alcohol production over time, thereby increasing the alcohol production efficiency.

In some embodiments, the system and process of FIG. 1 can be modified such that simultaneous saccharification fermentation in fermentation vessel 30 is replaced with a separate saccharification vessel 60 prior to fermentation vessel 30, as will be apparent to those skilled in the art. (See, eg, embodiment of FIG. 4).

In another embodiment, for example in the embodiment of FIG. 2, natural oil 26 (instead of being fed directly into fermentation vessel 30) is fed into vessel 40 to which catalyst 42 is also fed. At least some of the acyl glycerides in oil 26 are hydrolyzed to yield carboxylic acid 28. Then, the product stream from vessel 40 comprising carboxylic acid 28 and catalyst 42 is introduced into fermentation vessel 30. The carboxylic acid 28 and the catalyst 42 are contacted with the product alcohol produced in the fermentation medium in the same manner as described above with respect to FIG. 1 so that the alcohol ester of the product alcohol is a catalyzed ester of the carboxylic acid and the product alcohol. The furnace is created in its original position. Carboxylic acid 28 may also serve as an ISPR extractant, and in some embodiments, sufficient carboxylic acid 28 and / or one or more additional ISPR extractant 29 is introduced into fermentation vessel 30. It is possible to produce a biphasic mixture comprising an aqueous phase and an organic phase and the alcohol esters are partitioned into the organic phase. The remaining process tasks of the embodiment of FIG. 2 are the same as in FIG. 1 and will not be described again in detail.

For example, in some embodiments of the present invention as shown in the embodiment of FIG. 3, the catalyst 42 includes a feedstock comprising an oil 26 'derived from a biomass forming the feedstock 12. May be added to the slurry 16. In the embodiment shown, catalyst 42 may hydrolyze glycerides in oil 26 'to free fatty acid 28'. Thus, after introducing the catalyst 42 into the feedstock slurry 16, at least some of the glycerides in the oil 26 'are hydrolyzed to feedstock slurry with the free fatty acid 28' and the catalyst 42. (18) is generated. For example, if feedstock 12 is corn, oil 26 'is corn oil which is a component of the feedstock and free fatty acid 28' is corn oil fatty acid (COFA).

The feedstock slurry 18 is introduced into the fermentation vessel 30 together with the alcohol producing microorganism 32 and included in the fermentation medium. In some embodiments, enzymes 38, such as glucoamylase, may also be introduced into the fermentation vessel for simultaneous glycosylation of sugars in the slurry 18 and fermentation of alcohols within the fermentation vessel 30. The presence of catalyst 42 (introduced through slurry 18) in the fermentation vessel is the same as described above with respect to FIG. 1, of alcohol and free fatty acid 28 ′ (introduced through slurry 18). The esterification is catalyzed to produce fatty alcohol esters in situ. In some embodiments, butanol producing microorganism 32 is introduced into fermentation vessel 30 along with feedstock slurry 18 for butanol production. Catalyst 42 (introduced through slurry 18) in the fermentation vessel catalyzes the esterification of butanol and free fatty acid 28 '(introduced through slurry 18), thereby in situ fatty acid butyl ester (FABE ). Free fatty acids 28 'may also act as ISPR extractants. For example, if the free fatty acid 28 'is COFA, an alcohol ester of COFA is produced in situ, and the COFA acts as an ISPR extractant or part thereof.

In some embodiments, one or more additional ISPR extractants 29 may be introduced into fermentation vessel 30 to preferentially distribute the alcohol ester (and any free alcohol) from the aqueous phase. In some embodiments, ISPR extractant 29 may be carboxylic acid 28 described with respect to the embodiments of FIGS. 1 and 2. In some embodiments, ISPR extractant 29 is introduced into fermentation vessel 30 as oil 26, and then the oil is hydrolyzed to fatty acids by catalyst 42 to be ISPR extractant 29. In some embodiments, oil 26 is corn oil, so ISPR extractant 29 is COFA. In some embodiments, ISPR extractant 29 includes fatty acids, fatty alcohols, fatty amides, fatty esters (especially from 1 to 8 carbon atoms in the alcohol moiety, as described above with respect to the embodiments of FIGS. 1 and 2). Fatty acid extractant selected from the group consisting of fatty acid methyl esters and lower alcohol esters of fatty acids), fatty acid glycol esters, hydroxylated triglycerides, and mixtures thereof. In another embodiment, ISPR extractant 29 may be a free fatty acid obtained by chemical or enzymatic hydrolysis of biomass lipids. In such embodiments, the biomass lipids for producing the extractant 29 may be from the same or different biomass source from which the feedstock 12 is obtained. For example, in some embodiments, the biomass lipids for producing extractant 29 may be derived from soybean, while the biomass source of feedstock 12 is corn. As will be apparent to those skilled in the art, any possible combination of different biomass sources for extractant 29 versus feedstock 12 may be used. The remaining process tasks of the embodiment of FIG. 2 are the same as in FIG. 1 and will not be described again in detail.

As a non-limiting prophetic example, with respect to the embodiment of FIG. 3, the aqueous suspension of whole cornmeal flour (as feedstock 12), which may nominally contain about 4 wt% of corn oil, is from about 85 ° C. to Treated with amylase (as liquefied enzyme 14) at 120 ° C. for 30 minutes to 2 hours, the resulting liquefied mash 16 was cooled to 65 ° C. to 30 ° C., and the available fatty acid content of lipids into free fatty acids 0.1 ppm to 10 ppm (in some embodiments) lipase (as catalyst 42) at pH 4.5 to 7.5 (in some embodiments, pH 5.5 to 6.5) for a time sufficient to bring the conversion to at least 30% to at least 99%. 0.5 ppm to 1.0 ppm). Liquefied and lipase treated mash 18 may be cooled to about 30 ° C. (eg, using a heat exchanger) and loaded into fermentation vessel 30 with about 25 wt% to 30 wt% dry corn solids. Glucose can be produced by saccharification of the liquefied mash 18 during fermentation by addition of glucoamylase (as glycosylase 38). The resulting fermentation broth may contain much less than the amount of corn oil (eg, about 1.2 wt% corn oil) that may be present in the broth using liquefied mash that has not been treated with lipase 42. In particular, by lipase treatment 42, the COFA 28 '(and some diglycerides (DG) or monoglycerides (MG)) of corn oil lipid 26' (triglyceride (TG)) It can lead to conversion and reduce the rate of accumulation of lipid 26 'in the COFA ISPR extraction solvent 28' or 29 '. The lipase treatment 42 can also lead to the conversion of butanol produced during fermentation to the butyl ester of COFA, wherein the butyl ester is high for dissolution into the COFA phase 36 during liquid-liquid extraction ISPR. Has a partition coefficient. At the end of fermentation, the COFA phase 36 containing the butyl ester of COFA can be separated from the fermentation broth (in vessels 30/35), butanol 54 is butanol 54 and COFA 27 ), Such as by using one of several methods, including but not limited to hydrolysis of esters using lipase 52, solid acid catalyst 52, or steam 52, for example. May be recovered from the organic mixture 36 (in vessel 50).

For example, in another embodiment as shown in the embodiment of FIG. 4, the system and process of FIG. 3 allows simultaneous glycosylation fermentation (SSF) in the fermentation vessel 30 to take precedence over the fermentation vessel 30. It can be changed to be replaced with the saccharification container 60. 4 is substantially the same as FIG. 3, except that it includes a separate saccharification vessel 60 containing enzyme 38, and catalyst 42 is applied to the liquefied, glycated feedstock stream 62. Is introduced. The feedstock slurry 16 is introduced into the saccharification vessel 60 together with an enzyme 38 such as glucoamylase so that the oligosaccharide form sugars in the slurry 16 can be broken down into monosaccharides. Liquefied, saccharified feedstock stream 62 exits saccharification vessel 60 into which catalyst 42 is introduced. Feedstock stream 62 comprises monosaccharides and oils 26 'derived from the feedstock and insoluble solids. Oil 26 'is hydrolyzed by introduction of catalyst 42 to produce a liquefied, saccharified feedstock slurry 64 having free fatty acids 28' and catalyst 42.

Alternatively, in some embodiments, the catalyst 42 is a glycosylated enzyme ( Can be added together with 38) to simultaneously produce glucose and hydrolyze the oil lipid 26 'to the free fatty acid 28'. The addition of enzyme 38 and catalyst 42 may be stepwise (eg, catalyst 42, then enzyme 38, or vice versa) or simultaneously. However, in contrast to the embodiment of FIG. 1 where addition of catalyst 42 to fermentation vessel 30 during SSF also substantially simultaneously converts product alcohol to alcohol ester, the alcohol ester is a slurry containing catalyst 42. It is not produced until 64 is introduced into fermentation vessel 30. Alternatively, in some embodiments, slurry 62 may be introduced into fermentation vessel 30 and catalyst 42 is added directly to fermentation vessel 30.

In the embodiment of FIG. 4, slurry 64 is introduced into fermentation vessel 30 along with alcohol producing microorganisms 32 which metabolize monosaccharides to produce product alcohols. The presence of catalyst 42 (introduced through slurry 64) in the fermentation vessel is the same as described above with respect to FIG. The esterification is catalyzed to produce fatty alcohol esters in situ. The free fatty acid 28 'may also act as an ISPR extractant to preferentially distribute the alcohol ester (and any free alcohol) from the aqueous phase. In some embodiments, one or more additional ISPR extractants 29 may also be introduced into fermentation vessel 30 as described above with respect to FIG. 3. The remaining process tasks of the embodiment of FIG. 4 are the same as in FIG. 3 and will not be described again in detail.

In some embodiments, including any of the above described embodiments with respect to FIGS. 1-4, insoluble solids may be removed from feedstock slurry 16 prior to introduction into fermentation vessel 30. For example, as shown in the embodiment of FIG. 5, feedstock slurry 16 is introduced into an inlet of separator 20 configured to discharge insoluble solids as a solid phase or wet cake 24. For example, in some embodiments, separator 20 may comprise a filter press, vacuum filtration, mechanical pressure filtration, or centrifuge (eg, decanter centrifuge) to separate insoluble solids from feedstock slurry 16. It may include. In some embodiments, any conventional centrifuge used in the industry, including, for example, decanter ball centrifuges, tricanter centrifuges, disk stack centrifuges, filtration centrifuges, or decanter centrifuges to separate insoluble solids. Can be used. In some embodiments, the removal of insoluble solids from the feedstock slurry 16 may include filtration, vacuum filtration, belt filters, pressure filtration, filtration with screens, screen separation, great or grating, porous grating, floatation, hydroclone, It can be accomplished by filter press, screw press, gravity settling tank, vortex separator, or any method that can be used to separate the solids from the liquid. Optionally, in some embodiments, separator 20 may also be configured to remove some or substantially all of oil 26 'present in feedstock slurry 16. In such embodiments, separator 20 is known in the art for removing oil from an aqueous feed stream, including but not limited to siphoning, decantation, centrifugation with gravity settling tanks, phase separation with membranes, and the like. It may be any suitable separator. Residual feedstock comprising sugar and water is discharged to fermentation vessel 30 as an aqueous stream 22.

For example, in some embodiments, separator 20 comprises an aqueous layer containing sugar and water (ie, stream 22), a solid layer containing insoluble solids (ie, wet cake 24), and an oil layer A tricanter centrifuge 20 that agitates or rotates the feedstock slurry 16 to produce a centrifugal product comprising (i.e., oil stream 26 '). In this case, the catalyst 42 may be contacted with the removed oil 26 'to produce a stream of free fatty acids 28' and catalyst 42. The free fatty acid 28 'and the stream of catalyst 42 are then introduced into the fermentation vessel 30 and contacted with the fermentation broth in the same manner as described above with respect to FIG. 1, whereby the product alcohol in the fermentation broth Catalytic esterification of the fatty acid to the alcoholic esters can be achieved in situ.

The free fatty acid 28 'may also serve as the ISPR extractor 28' and one or more additional ISPR extractants 29 may also be introduced into the fermentation vessel 30. Thus, the feedstock oil 26 'can be catalytically hydrolyzed with carboxylic acids to reduce the amount of lipids present in the ISPR extractant while producing an ISPR extractant as well. The ester containing organic phase 36 may be separated from the aqueous phase 34 of the biphasic mixture 39 in vessel 35, and the product alcohol may be recovered from the alcohol ester in vessel 50 (see FIG. 1). . The remaining process tasks of the embodiment of FIG. 5 are the same as in FIG. 3 and will not be described again in detail.

When the wet cake 24 is removed through the centrifuge 20, in some embodiments, some oil from the feedstock 12, such as corn oil, is applied to the wet cake 24 when the feedstock is corn. Remaining. The wet cake 24 may be washed with additional water in the centrifuge once the aqueous solution 22 is discharged from the centrifuge 20. Cleaning of the wet cake 24 will recover the sugars (eg, oligosaccharides) present in the wet cake, and the recovered sugars and water can be recycled to the liquefaction vessel 10. After washing, the wet cake 24 may be combined with the solubles and then dried to produce dry liquor (DDGS) via any suitable known process. The production of DDGS from the wet cake 24 produced in the centrifuge 20 has several advantages. Since insoluble solids do not reach the fermentation vessel, the DDGS does not have an enclosed extractant and / or product alcohol such as butanol, and is not subject to the conditions of the fermentation vessel and does not come into contact with the microorganisms present in the fermentation vessel. All these advantages make it easier to process and sell DDGS as, for example, animal feed. In some embodiments, the oil 26 'is not drained separately from the wet cake 24, but rather the oil 26' is included as part of the wet cake 24 and is eventually present in DDGS. In this case, the oil may be separated from the DDGS and converted to ISPR extractant 29 for subsequent use in the same or different alcohol fermentation process. Methods and systems for removing insoluble solids from feedstock slurry (16) by centrifugation are co-owned US, filed June 18, 2010, co-pending, the entire contents of which are incorporated herein by reference. It is described in detail in provisional application 61 / 356,290.

As mentioned above, the oil 26 'may be separated from the DDGS using any suitable known process, including, for example, a solvent extraction process. In one embodiment of the present invention, DDGS is loaded into an extraction vessel and washed with a solvent such as hexane to remove oil 26 '. Other solvents that may be used include, for example, petroleum distillates such as isobutanol, isohexane, ethanol, petroleum ether, or mixtures thereof. After oil 26 'extraction, DDGS can be treated to remove any residual solvent. For example, DDGS can be heated using any method known in the art to evaporate any residual solvent. After solvent removal, DDGS can be subjected to a drying process to remove any residual water. The processed DDGS can be used as a feed supplement for animals such as poultry, livestock, and domestic pets.

After extraction from DDGS, a mixture of the resulting oil 26 'and the solvent can be collected for separation of the oil 26' from the solvent. In one embodiment, the oil 26 '/ solvent mixture may be treated by evaporation so that the solvent is evaporated and the oil may be collected and recycled. The recovered oil may be converted to ISPR extractant 29 for subsequent use in the same or different alcohol fermentation process.

In addition to recovering solids, it may be desirable to recover other byproducts of the fermentation process. In one embodiment, fatty acid esters (eg, fatty acid isobutyl esters) may be recovered to increase the yield of carbohydrates, for example, to product alcohols (eg, butanol). This can be achieved using a solvent that extracts fatty acid isobutyl esters from the byproducts, for example, by combining and mixing several byproduct streams and drying the product of the blending and mixing steps. Such solvent type extraction systems for recovering corn oil triglycerides from DDGS are described in US Patent Application Publication No. 2010/0092603, the teachings of which are incorporated herein by reference.

In one embodiment of solvent extraction of fatty acid esters, the solids can be separated from the whole stillage (“separated solids”), where the stream is by far the largest portion of the fatty acids in the unmixed byproduct stream. It is because it contains ester. This separated solid may then be placed in an extractor and washed with a solvent. In one embodiment, the separated solids are turned at least once to ensure that all sides of the separated solids are washed with solvent. After washing, the resulting mixture of lipids and solvent, known as micelles, is collected for the separation of extracted lipids from the solvent. For example, the resulting mixture of lipids and solvent can be deposited in the separator for further processing. During the extraction process, because the solvent wets the separated solids, the solvent not only transports the lipids into solution but also collects the solid particulates. Such “particulates” are typically undesirable impurities in micelles, and in one embodiment, micelles can be discharged from the extractor or separator through a device that separates or scrubs the particulates from the micelles.

To separate the lipids and solvents contained in the micellar, the micellar can be transferred to a distillation step. In this step, the micellar can be treated, for example, via an evaporator that heats the micellar to a temperature that is high enough to cause evaporation of the solvent, but not high enough to adversely affect or evaporate the extracted lipids. As the solvent evaporates, it can be collected and recycled to a condenser, for example for later use. Separation of the solvent from the micelles yields a crude stock and can be further processed to separate water, fatty acid esters (eg fatty acid isobutyl esters), fatty acids, and triglycerides.

After extraction of the lipid, the solids are conveyed out of the extractor and a stripping process can be performed to remove residual solvent. Recovery of residual solvents is important for process economics. In one embodiment, the wet solids may be transported to a desolventizer in an environment where no vapor leaks to preserve and collect solvents that temporarily evaporate from the wet solids. As the solid is injected into the desolventizer, the solid can be heated to remove the residual solvent by evaporation. In order to heat the solids, the desolventizer may include a mechanism for dispensing the solids in one or more trays, which solids may be eaten directly or, for example, by direct contact with heated air or steam. It may be indirectly heated by heating the conveying tray. To facilitate the transfer of solids from one tray to another, the tray that carries the solids may include openings through which the solids can pass from one tray to the next. From the desolventizer, the solids can optionally be delivered to a mixer, where the solids are mixed with other byproducts before being delivered to the dryer. An example of solids extraction is described in Example 63. In this embodiment, the solids are fed to the desolventizer, where the solids are in contact with steam. In one embodiment, steams and solids in the desolventizer may be countercurrent. The solids can then exit the desolventizer and be fed to a dryer or a mixer into which the various byproducts can optionally be mixed. The vapor exiting the desolventizer can be condensed and optionally mixed with micelles and then fed to the decanter. The water rich phase exiting the decanter can be fed to a distillation column where hexane is removed from the water rich stream. In one embodiment, the hexane depleted water rich stream exits the bottom of the distillation column and can be recycled back to the fermentation process, for example, can be used to slurry the ground corn solids. In other embodiments, the overhead and bottom products can be recycled to the fermentation process. For example, the lipid rich bottoms product can be fed to a feed of a hydrolysis device. The overhead product can be condensed and fed to the decanter, for example. The hexane rich stream exiting this decanter can optionally be used as part of the solvent feed of the extractor. The water rich phase exiting this decanter can be fed to a column stripping hexane from water. As will be appreciated by those skilled in the art, the process of the present invention can be modified in various ways to optimize the fermentation process for the production of product alcohols such as butanol.

In another embodiment of solvent extraction of fatty acid esters, the solids may be separated from the solvent and beer drained from the fermentation prior to being introduced into the preflash column as a heterogeneous mixture. Wet cakes of these solids may be formed using separation devices such as screen filters or centrifuges. The screened cake of solids can be substituted and washed with hydrous isobutanol to remove fatty acid esters retained in the wet solids. Alternatively, the centrifuged cake of solids may be re-pulped in brine isobutanol and separated again to remove fatty acid esters retained in the wet solids. An example of an embodiment of such solids extraction is described in Example 63.

In further embodiments, byproducts (or byproducts) may be derived from the mash used in the fermentation process. For example, corn oil may be separated from the mash and such corn oil may contain triglycerides, free fatty acids, diglycerides, monoglycerides, and phospholipids (see, eg, Example 66). . Corn oil may optionally be added to other byproducts (or byproducts) at different rates, thus exhibiting, for example, the ability to vary the amount of triglycerides in the resulting byproducts. In this way, the fat content of the resulting byproduct can be adjusted to obtain a low fat, high protein animal feed, for example better suited to the needs of the cow compared to the high fat product.

In one embodiment, crude corn oil isolated from the mash can be further processed into cooking oil for consumer use, or it can also be used as an ingredient in animal feed because its high triglyceride content makes it a good source of metabolic energy. have. In other embodiments, it may also be used as feedstock for biodiesel or renewable diesel.

In one embodiment, the extractant byproduct may be used, in whole or in part, as a component of the animal feed byproduct, or as a feedstock for biodiesel or renewable diesel.

In further embodiments, the solids can be separated from the mash and can include triglycerides and free fatty acids. Such solids (or streams) can be used as animal feed, which is recovered as centrifuge from the discharge or after drying. Solids (or wet cakes) may be particularly suitable as feed for ruminants (eg, dairy cows) because of their high content of available lysine and bypass or lumen non-degradable proteins. For example, such solids can be particularly valuable in high protein, low fat feeds. In other embodiments, such solids can be used as the base, that is, other byproducts such as syrup can be added to the solids to produce a product that can be used as animal feed. In other embodiments, different amounts of other byproducts can be added to the solids to suit the properties of the resulting product, to meet the needs of a particular animal species.

The composition of the solid separated from the total distillation waste liquid as described in Example 62 may comprise, for example, crude protein, fatty acid, and fatty acid isobutyl ester. In one embodiment, such compositions (or byproducts) can be used in wet or dry conditions, for example as animal feed requiring high protein (eg high lysine), low fat, and high fiber content. In other embodiments, fat may be added to such compositions, for example, from other byproduct streams, if a high fat, low fiber animal feed is desired. In one embodiment, such high fat, low fiber animal feed can be used for swine or poultry. In further embodiments, the non-aqueous composition of Condensed Distillers Solubles (CDS) (see, eg, Example 66) can be, for example, proteins, fatty acids, and fatty acid isobutyl esters, and other dissolved and Suspended solids such as salts and carbohydrates. Such CDS compositions can be used, for example, in wet or dry conditions as animal feed requiring high protein, low fat, high mineral salt feed ingredients. In one embodiment, such a composition can be used as a component of a dairy ration.

In another embodiment, oil from the fermentation process may be recovered by evaporation. Such non-aqueous compositions may include fatty acid isobutyl esters and fatty acids (see, eg, Example 66), and such compositions (or streams) may be fed to a hydrolysis device to recover isobutanol and fatty acids. . In a further embodiment, this stream can be used as feedstock for biodiesel production.

The various streams generated by the production of alcohols (eg butanol) through the fermentation process can be combined in various ways to produce many byproducts. For example, chalcedony from mash is used to produce fatty acids for use as extractant, and when the lipids are extracted by an evaporator for other uses, the residual stream is blended and processed to yield crude protein, crude fat, triglycerides, fatty acids, And by-product compositions comprising fatty acid isobutyl esters. In one embodiment, such compositions comprise at least about 20 to 35 wt% crude protein, at least about 1 to 20 wt% crude fat, at least about 0 to 5 wt% triglycerides, at least about 4 to 10 wt% fatty acids, and fatty acid isobutyl Esters at least about 2 to 6 wt%. In one particular embodiment, the byproduct composition may comprise about 25 wt% crude protein, about 10 wt% crude fat, about 0.5 wt% triglycerides, about 6 wt% fatty acids, and about 4 wt% fatty acid isobutyl esters. .

In another embodiment, the lipid is extracted by an evaporator, the fatty acids are used for other purposes, about 50 wt% of crude corn from the mash and the residual stream is blended and processed, and the resulting byproduct composition is crude protein, crude fat, triglycerides. Rides, fatty acids, and fatty acid isobutyl esters. In one embodiment, such compositions comprise at least about 25 to 31 wt% crude protein, at least about 6 to 10 wt% crude fat, at least about 4 to 8 wt% triglycerides, at least about 0 to 2 wt% fatty acids, and fatty acid isobutyl Esters at least about 1 to 3 wt%. In one particular embodiment, the byproduct composition may comprise about 28 wt% crude protein, about 8 wt% crude fat, about 6 wt% triglycerides, about 7 wt% fatty acids, and about 1 wt% fatty acid isobutyl ester. .

In another embodiment, 50 wt% of the solids separated from the total distillation waste and the corn oil extracted from the mash are combined and the resulting byproduct composition comprises crude protein, crude fat, triglycerides, fatty acids, fatty acid isobutyl esters, lysine, neutral detergents. Insoluble fibers (NDF), and acid resistant fibers (ADF). In one embodiment, such a composition` comprises at least about 26 to 34 wt% crude protein, at least about 15 to 25 wt% crude fat, at least about 12 to 20 wt% triglycerides, at least about 1 to 2 wt% fatty acids, isobutyl fatty acid Ester at least about 2-4 wt%, lysine at least about 1-2 wt%, NDF at least about 11-23 wt%, and ADF at least about 5-11 wt%. In one specific embodiment, the byproduct composition comprises about 29 wt% crude protein, about 21 wt% crude fat, about 16 wt% triglycerides, about 1 wt% fatty acid, about 3 wt% fatty acid isobutyl ester, about 1 wt% lysine. , About 17 wt% NDF, and about 8 wt% ADF. The high fat, triglyceride, and lysine content and low fiber content of such by-product compositions may be desirable as feed for pigs and poultry.

As mentioned above, the various streams generated by the production of alcohols (eg, butanol) through the fermentation process can be combined in various ways, by-product compositions comprising crude protein, crude fat, triglycerides, fatty acids, and fatty acid isobutyl esters. Can be generated. For example, a composition comprising at least about 6% crude fat and at least about 28% crude protein can be used as an animal feed product for milking animals. Compositions comprising at least about 6% crude fat and at least about 26% crude protein may be used as animal feed products for kennel livestock, while compositions comprising at least about 1% crude fat and at least about 27% crude protein may be used for wintering cattle. ) As an animal feed product. Compositions comprising at least about 13% crude fat and at least about 27% crude protein can be used as poultry animal feed products. Compositions comprising at least about 18% crude fat and at least about 22% crude protein can be used as animal feed products for unit animals. Thus, the various streams can be formulated to be customized as feed products for specific animal species.

In one embodiment, the one or more streams generated by the production of alcohols (eg, butanol) through the fermentation process are in various combinations comprising at least about 90% COFA that can be used as a fuel source, such as biodiesel. Can be generated.

As an example of one embodiment of the method of the invention, milled grain (eg, corn processed by hammer mill) and one or more enzymes are combined to produce a slurried grain. This slurried grain is cooked, liquefied and optionally flashed with flash vapor to obtain a cooked mash. The cooked mash is then filtered to remove the suspended solids to form a wet cake and filtrate. Filtration can be accomplished by various methods, such as centrifugation, screening, or vacuum filtration, and this filtration step can remove at least about 80% to at least about 99% of the suspended solids from the mash.

The wet cake is reslurried with water and refiltered to remove additional starch, resulting in a cleaned filter cake. The slurry process may be repeated several times, for example, one to five times. The water used to reslurry the wet cake can be recycled to the water generated during the fermentation process. The filtrate produced by the slurry and refiltration process can be returned to the initial mixing stage to produce a slurry with the milled grain. The filtrate can be heated or cooled before the mixing step.

The cleaned filter cake may be reslurried into vias in a number of steps in the production process. For example, the cleaned filter cake may be reslurried into a beer after the fermentation vessel, before the preflash column, or at the feed point of a distiller's grain dryer. The cleaned filter cake can be dried separately from other byproducts to produce DDGS or animal feed products or can be used directly as a wet cake.

The filtrate produced as a result of the initial mixing step can be further processed as described herein. For example, the filtrate can be heated by steam or process to process heat exchange. Saccharification enzymes can be added to the filtrate, and the dissolved starch of the filtrate can be partially or fully saccharified. The saccharified filtrate can be cooled by a number of means, such as interprocess exchange, exchange with cooling water, or exchange with chilled water.

The cooled filtrate can then be added to the fermentation vessel and recombinant yeast capable of producing microorganisms suitable for alcohol production, such as butanol. In addition, ammonia and recycle stream may also be added to the fermentation vessel. Such a process may comprise at least one fermentation vessel, at least two fermentation vessels, at least three fermentation vessels, or at least four fermentation vessels. The carbon dioxide generated during fermentation can be vented to a scrubber to reduce air emissions (eg butanol air emissions) and increase product yield.

The solvent may be added to the fermentation vessel through a regeneration loop or may be added directly to the fermentation vessel. The solvent may be one or more organic compounds capable of dissolving or reacting with an alcohol (eg butanol) and solubility in water may be limited. The solvent may be taken from the fermentation vessel in succession as a single liquid phase or two liquid phase materials, or the solvent may be recovered batchwise as a single or two liquid phase materials.

The vias may be degassed. The vias may be heated prior to degassing, for example, by inter-process exchange using hot mash or inter-process exchange using preflash column overhead product. The vapor may then be vented to a condenser and then to a scrubber. The degassed vias may further be heated by, for example, inter-process heat exchange with other streams in the distillation zone.

Preheated vias and solvents can be injected into a preflash column that can be retrofitted from the beer column of a conventional dry grinding fuel ethanol plant. This column can be operated with sub-atmospheric pressure driven by water vapor taken from an evaporator train or a mash cooking step. The overhead product of the preflash column may be condensed by interprocess heat exchange, including heat exchange with some combination of cooling water, and heat exchange with preflash column feed. The liquid condensate can be run with an alcohol / water decanter (eg, butanol / water decanter).

The preflash column bottoms product can be advanced to a solvent decanter. The preflash column bottoms product can substantially strip free alcohol (eg, butanol). The decanter may be a still well, centrifuge, or hydroclonal. Water is substantially separated from the solvent phase in this decanter to form an aqueous phase. The aqueous phase, including suspended and dissolved solids, can be centrifuged to produce the wet cake and Dean stills. The wet cake may be combined with other streams and dried to produce DDGS, or may be sold separately from other streams which are dried to produce DDGS, or may be sold as a wet cake. The aqueous phase can be partially partitioned to provide a backset that is used to reslurry the filter cake described above. Dividing also provides Dean Stilage, which can be pumped to the evaporator for further processing.

The organic phase produced in the solvent decanter may be an ester of an alcohol (eg butanol). The solvent can be hydrolyzed to regenerate the reactive solvent and recover additional alcohol (eg butanol). Alternatively, the organic phase can be filtered and sold as product. Hydrolysis can be heat accelerated, homogeneously catalyzed, or heterogeneously catalyzed. Hydrolysis can also occur by enzymatic reactions. The heat injection into this process can be a heating furnace, hot oil, electric heat injection, or high pressure steam. The water added to promote hydrolysis may be recycled water stream, fresh water, or steam.

The cooled hydrolysis solvent may be pumped into a negative pressure solvent column where substantially alcohol (eg, butanol) may be stripped with steam. Such steam may be water vapor from an evaporator, steam from the flash stage of the mash process, or steam from a boiler (eg, US Patent Application Publication No. 2009/0171129, incorporated herein by reference). Reference). The rectification column of a conventional dry grinding fuel ethanol plant may be suitable as a solvent column. The tower can be modified to act as a solvent column. The bottom product of the solvent column may be cooled by, for example, cooling water or interprocess heat exchange. The cooled bottom product may be decanted to remove residual water, which may be recycled to another step using the process or to a mash step.

The solvent column overhead product may be cooled by exchange with chilled water or by inter-process heat exchange, and the condensate may be shared with the preflash column overhead product (eg, butanol / water decanter) May be performed. Other mixed water and alcohol (eg butanol) streams may be added to this decanter including scrubber bottoms product and condensate from the degassing step. The vent containing carbon dioxide may be directed to the water scrubber. The aqueous layer of this decanter may be fed to a solvent column or may strip alcohol (eg butanol) in a small dedicated distillation column. The aqueous layer can be preheated by interprocess exchange using a preflash column overhead product, a solvent column overhead product, or a solvent column bottom product. This dedicated column can be modified from the side stripper of a conventional dry grinding fuel ethanol process.

The organic layer of alcohol / water decanter (eg butanol / water decanter) may be pumped to an alcohol (eg butanol) column. This column may be a superatmospheric column and may be driven by steam condensation in the reboiler. Feed to the column can be heated by inter-process heat exchange to reduce the energy demand to operate the column. Such interprocess heat exchangers may comprise a preflash column splitter, a solvent column splitter, a product of a hydrolysis device, water vapor from an evaporator, or a butanol column bottoms product. The condensate of the alcohol (eg butanol) column vapor can be cooled and returned to an alcohol / water decanter (eg butanol / water decanter). The alcohol (eg butanol) column bottoms product may be cooled by inter-process heat exchange, including exchange with an alcohol (eg butanol) column feed, further cooled with cooling water, filtered, It is sold as product alcohol (eg butanol).

Dean stilges generated from the preflash column bottoms described above can be advanced to multiple effect evaporators. Such evaporators may have two, three or more stages. The evaporator may have a form of four bodies by two effects, similar to the conventional design of a fuel ethanol plant, or may have three bodies by three effects, or may have other forms. Dean Stilage can be imported in any effect. At least one of the first effect bodies may be heated with steam from a superatmospheric alcohol (eg butanol) column. The vapor can be taken from the lowest pressure effect, providing heat in the form of water vapor to the negative pressure preflash column and the solvent column. Syrup from the evaporator can be added to the hair dryer.

Carbon dioxide emissions from fermentation vessels, degassers, alcohol / water decanters (eg butanol / water decanters) and other sources can be run with water scrubbers. The water supplied to the top of this scrubber may be fresh make-up water or recycle water. Recycled water can be treated to remove volatile organic compounds (eg, biological digestion) and cooled. The scrubber bottoms product can be released to an alcohol / water decanter (eg, butanol / water decanter), a solvent column, or used with other recycled water to reslurminate the wet cake described above. The condensate from the evaporator can be subjected to anaerobic biological digestion or other processes to purify the water before it is recycled to reslurry the filter cake.

When corn is used as a source of milled grain, corn oil may be separated from the process stream at any of several points. For example, the centrifuge may be operated to produce a corn oil stream after filtration of the cooked mash, or the preflash column water centrifuge may be operated to produce a corn oil stream. Medium concentration syrup for the final syrup may be centrifuged to produce a corn oil stream.

In another example of embodiment of the method of the present invention, the material discharged from the fermentation vessel is processed in a separation system comprising devices such as centrifuges, sedimentation baths, hydroclones, and the like, and combinations thereof, to directly or slight regeneration. High concentrations of live yeast can be recovered which can later be recycled for reuse in subsequent fermentation batches. This separation system also produces an organic stream comprising fatty esters (eg isobutyl fatty esters) and alcohols (eg isobutanol) resulting from fermentation and an aqueous stream containing only traces of immiscible organics. can do. This aqueous stream can be used to repulp and pump the low starch solids separated and rinsed from the liquefied mash before and after stripping of alcohol (eg isobutanol) content. This has the advantage that it may be possible to avoid transferring these solids from the liquefaction zone to the grain drying and syrup blend areas using a long belt driven conveying system. In addition, this pre-distillation wastewater produced after the alcohol (eg isobutanol) has been stripped will need to be separated into Dean Stilridge and wet cake fractions using existing or new separation equipment, which Dean Stilridge Partially backed bags will be formed to blend with cook water to produce a new batch of fermentable mash. Another advantage of this embodiment is that any residual dissolved starch retained in the water of the solid separated from the liquefied mash may be partially captured and recovered through this bagset. Alternatively, the yeast contained in the solids stream may be considered non-viable and may be redispersed in the aqueous stream, and this distilled combined stream of any alcohol (eg butanol) content remaining from the fermentation. have. Non-viable organisms can be further isolated for use as nutrients in the proliferation process.

In other embodiments, the multiphase material may be left at the bottom of the preflash column and treated in a separation system as described above. The concentrated solids can be redispersed in the aqueous stream and this combined stream can be used to repulp and pump the washed low starch solids separated from the liquefied mash.

The process described above and other processes described herein can be demonstrated using numerical modeling, such as Aspen modeling (see, eg, US Pat. No. 7,666,282). For example, commercial modeling software Aspen Plus? (Aspen Technology, Inc., Burlington, Mass.) Was developed in New York, NY, to develop an aspen model for integrated butanol fermentation, purification, and water management processes. It can be used with physical property databases, such as DIPPR, available from the American Institute of Chemical Engineers, Inc. Such process modeling can perform a number of basic engineering calculations, such as mass and energy balance, vapor / liquid equilibrium, and reaction rate calculations. In order to generate an aspen model, the input of information may include, for example, experimental data, water content and composition of the feedstock, temperature for mash cooking and flashing, glycosylation conditions (eg, enzyme feed, starch conversion, temperature, pressure), Fermentation conditions (eg, microbial feed, glucose conversion, temperature, pressure), degassing conditions, solvent columns, preflash columns, condensers, evaporators, centrifuges, and the like.

The present invention also provides systems and methods for producing fermentation products such as product alcohols through fermentation, and systems and methods for increasing biomass processing productivity and cost efficiency. In some embodiments, the product alcohol is butanol. The feedstock may be liquefied to produce a feedstock slurry, the feedstock slurry comprising soluble sugars and insoluble solids. If the feedstock slurry is fed directly into the fermentation vessel, insoluble solids can interfere with the efficient removal and recovery of product alcohols such as butanol from the fermentation vessel. In particular, when liquid-liquid extraction is used to extract butanol from the fermentation broth, the presence of insoluble particulates interferes with the contact between the extractant and the fermentation broth to reduce the rate of mass transfer from butanol to the extractant; Creating an emulsion in the fermentation vessel, hindering good phase separation of the extractant and fermentation broth; Reducing the efficiency of recovery and recycling of the extractant since at least some extractant and butanol are “enclosed” in solids that are eventually removed as DDGS; Low fermentation vessel volume efficiency because the solids occupy the volume of the fermentation vessel and the extractant is released more slowly from the fermentation broth; And contamination with corn oil may cause system inefficiencies, including but not limited to shortening the life cycle of the extractant. All these effects result in high capital and operating costs. In addition, extractants "enclosed" in DDGS can impair DDGS value and eligibility for sale as animal feed. Thus, to avoid and / or minimize these problems, at least some insoluble particles (or solids) are removed from the feedstock slurry prior to addition of sugars present in the feedstock slurry to the fermentation vessel. The extraction activity and butanol production efficiency are increased when the extraction is done in a fermentation broth containing an aqueous solution from which insoluble particles have been removed, compared to the extraction carried out in a fermentation broth containing an aqueous solution from which insoluble particles have not been removed.

Extractive fermentation in the absence of insoluble solids results in a high mass transfer rate of product alcohol from the fermentation broth to the extractant, good phase separation of the extractant from fermentation in or out of the fermentation vessel, and high extractant droplet ascent rate. This can result in a low hold up. Also, for example, the extractant droplets held up in the fermentation broth during fermentation are released more quickly from the fermentation broth more quickly, resulting in less free extractant in the fermentation broth, thus reducing the amount of extractant lost in the process. You can. Also, for example, the microorganisms can be recycled and additional equipment in downstream processing, such as, for example, some or all of the beer column and / or pre-distillation waste liquor centrifuges can be removed. In addition, for example, the possibility that the extractant is lost in the DDGS is eliminated. In addition, for example, the recycling capacity of microorganisms can increase the overall production rate of product alcohol, lower the overall titer requirement, and / or lower the aqueous titer requirement, resulting in healthier microorganisms and higher production rates. Can bring Furthermore, for example, the capital cost can be reduced; Increase fermentation vessel productivity as the volume is used more efficiently because extractant holdup is minimized and insoluble solids are not present; The stirrer can be removed from the fermentation vessel for use in continuous fermentation or small fermentation vessels in plants in undeveloped areas.

Examples of increased extraction efficiency include, for example, stabilization partition coefficients, increased (eg, faster or more complete) phase separation, increased liquid-liquid mass transfer coefficients, working at low titers, increased process stream recirculation, fermentation Increased volumetric efficiency, increased feedstock (e.g. corn) load supply, increased butanol titer tolerance of microorganisms (e.g. recombinant microorganisms), recycling water, saving energy, increasing recycling of extractant, and And / or recycling of microorganisms.

For example, the volume of the fermentation vessel occupied by the solids will be reduced. Thus, the effective volume of fermentation vessels available for fermentation can be increased. In some embodiments, the volume of fermentation vessel available for fermentation is increased to at least about 10%.

For example, stabilization of the distribution coefficients can be achieved. Since the corn oil in the fermentation vessel can be reduced by removing solids from the feedstock slurry prior to fermentation, the extractant is less in contact with the corn oil combined with the extractant and, if present in sufficient quantity, can lower the partition coefficient. Thus, the reduction of corn oil introduced into the fermentation vessel leads to a more stable partition coefficient of the extractant in the fermentation vessel. In some embodiments, the partition coefficient is reduced to less than about 10% for 10 fermentation cycles.

Extraction of butanol with an extractant, for example, because the high mass transfer rate of the product alcohol from the fermentation broth to the extractant (eg, in the form of a high mass transfer coefficient) will result in an increased production efficiency of the product alcohol. Efficiency can be increased. In some embodiments, the mass transfer coefficient is increased at least twice (see Examples 4 and 5).

In addition, phase separation between the fermentation broth and the extractant may be increased, which reduces the likelihood of emulsion formation, resulting in an increased production efficiency of the product alcohol. For example, phase separation may occur faster or may be more complete. In some embodiments, phase separation may occur within 24 hours in which no previously noticeable phase separation has been observed. In some embodiments, phase separation occurs at a rate of at least about 2 times, at least about 5 times, or at least about 10 times compared to phase separation where solids have not been removed (see Examples 6 and 7).

In addition, recovery and recycling of the extractant may be increased. The extractant will not be "enclosed" in the solids, and can eventually be removed as DDGS, resulting in increased production efficiency of the product alcohol (see Examples 8 and 9). In addition, less dilution of the extractant with corn oil may result in less degradation of the extractant (see Example 10).

In addition, the flow rate of the extractant can be reduced, which lowers the operating cost, resulting in an increase in the production efficiency of the product alcohol.

In addition, the holdup of the extractant will be reduced as a result of the extractant droplets rising at a high rate, resulting in an increase in the production efficiency of the product alcohol. Reducing the amount of insoluble solids in the fermentation vessel will also result in increased production efficiency of the product alcohol.

In addition, the stirrer can be removed from the fermentation vessel because it no longer needs to suspend insoluble solids, thus saving capital cost and energy, and increasing the production efficiency of the product alcohol.

1-5 provide various non-limiting embodiments of methods and systems that include a fermentation process in which alcohol esters are produced in situ, extracted from fermentation medium, and reacted to recover product alcohol. 1-5 also provide various non-limiting embodiments of methods and systems using carboxylic acids that can be esterified with product alcohols and can simultaneously act as ISPR extractant. 1-5 also provide various non-limiting embodiments of methods and systems for converting lipids in a feedstock into carboxylic acids that can be esterified with product alcohols and at the same time can act as an ISPR extractant.

In some embodiments, including any of the above described embodiments described with respect to FIGS. 1-5, the fermentation broth in fermentation vessel 30 is genetically modified to produce butanol via a biosynthetic pathway from at least one fermentable carbon source ( That is, genetically engineered) at least one recombinant microorganism 32. In particular, recombinant microorganisms can be grown in fermentation broth containing the appropriate carbon substrate. Additional carbon substrates include monosaccharides such as fructose and galactose; Oligosaccharides such as lactose, maltose, or sucrose; Polysaccharides such as starch or cellulose; Or mixtures thereof and crude mixtures from renewable feedstocks, such as, but not limited to, cheese whey permeate, corn steep liquor, beet molasses, and barley malt. Other carbon substrates can include ethanol, lactate, succinate or glycerol.

In addition, the carbon substrate may be a single carbon substrate, for example carbon dioxide, or methanol, which has demonstrated metabolic conversion to important biochemical intermediates. In addition to mono and bi carbon substrates, methylotrophic organisms are also known to utilize a number of other carbon containing compounds such as methylamine, glucosamine and various amino acids for metabolic activity. For example, methyltropic yeast is known to form trehalose or glycerol using carbon derived from methylamine (Bellion, et al., Microb. Growth C1 Compd., [Int. Symp.], 7th ( 1993), 415-32, Editor (s): Murrell, J. Collin; Kelly, Don P. Publisher: Intercept, Andover, UK). Likewise, Candida of various species will metabolize alanine or oleic acid (Sulter et al., Arch. Microbiol. 153: 485-489, 1990). Thus, the carbon source used in the present invention may include a wide variety of carbon containing substrates, and is believed to be limited only by the choice of organism.

Although all of the aforementioned carbon substrates and mixtures thereof are considered suitable, in some embodiments, the carbon substrate is xylose and / or arabinose for yeast cells modified to use glucose, fructose and sucrose, or C5 sugars. And mixtures thereof with C5 sugars. Sucrose can be derived from renewable sugar sources such as sugar cane, sugar beet, cassava, sweet sorghum and mixtures thereof. Glucose and dextrose may be derived from renewable grain sources through saccharification of starch-based feedstocks, including grains such as corn, wheat, rye, barley, oats and mixtures thereof. Fermentable sugars are also cellulosic or lignocellulosic biomass that can be regenerated through pretreatment and saccharification processes, as described, for example, in US Patent Application Publication No. 2007/0031918 A1, which is incorporated herein by reference. Can be derived from. In addition to a suitable carbon source (from aqueous stream 22), the fermentation broth should contain other ingredients known to those skilled in the art, suitable for the growth of cultures and for the promotion of enzymatic pathways for the production of suitable minerals, salts, cofactors, buffers and product alcohols. do.

From the above discussion and examples, those skilled in the art can identify the basic features of the present invention, and various changes and modifications of the present invention can be made without departing from the present invention to adapt the present invention to various uses and conditions. For example, in some embodiments, alcohol esterification and extraction according to the present invention may be used for pre-fermentation during the cultivation of microorganisms 32, ie prior to fermentation in fermentation vessel 30. Can be. Typically, microorganisms 32, such as yeast, can be grown from species culture at desired cell concentrations before harvesting and inoculating into fermentation vessel 30, as known in the art.

Carbon source feedstock is an important cost factor in the production of microorganisms such as yeast production, so biomass yield for sugar is an important optimization criterion. Since ATP yield from alcohol fermentation is much lower than that from catabolism of respirable sugars, the occurrence of alcohol fermentation adversely affects biomass yield and is attempted to be avoided during yeast production (ie, cultivation). Nevertheless, the cultivation of microorganisms in a cultivation medium can produce a predetermined amount of fermentation products, including alcohol. For example, in Saccharomyces cerevisiae yeast, alcohol fermentation and respiration occur simultaneously whenever the specific growth rate (μ) and / or the sugar concentration in aerobic culture exceeds a threshold (eg See, for example, van Hoek, et al., Biotechnol. Bioeng. 68: 517-523, 2000). To achieve high biomass yields, yeast growth is typically controlled by respiratory conditions using, for example, fed-batch fermentation techniques for species culture. For example, sugars are fed at low rates, resulting in low sugar concentrations and low rates of sugar absorption in culture, so that sugar metabolism can occur substantially in the respiratory tract. Under these conditions, high biomass yields can be obtained and accumulation of toxic products can be minimized. Indeed, in large scale fed-batch industrial processes, cells may encounter concentration gradients due to inefficient mixing (see, eg, Enfors, et al., J. Biotechnol. 85: 175-185, 2001). The production and reassimilation of fermentation byproducts may be one of the reasons for the reduction of biomass yield per glucose in large scale bioreactors compared to laboratory scale.

However, under these conditions, fermentation products, including butanol, for example, in culture of butanol producing yeast may not be resynchronized and may accumulate in the medium and thus be toxic to microorganisms at high concentrations. If product accumulation exceeds a critical cell growth inhibitory concentration (eg, cell growth is lower than growth that can be limited by feeding), fed-batch control loss can occur. According to the present invention, by using alcohol esterification and extraction to remove butanol from the medium, fed-batch fermentation can be advanced despite the problems of inefficient mixing and butanol toxicity.

Thus, according to some embodiments, the culturing medium is contacted with catalyst 42 and carboxylic acid 28 to produce alcohol esters by esterification of the product alcohols, ultimately in glucose biomass in large scale bioreactors. Yield can be improved. In addition, the concentration of product alcohol in the medium is controlled by alcohol esterification, thus minimizing or avoiding the deleterious effects of product alcohol on microorganisms. In some embodiments, the alcohol ester may be extracted from the alcohol and cultivation medium recovered from the alcohol ester in the same manner as described above with respect to extraction of the alcohol ester from the fermentation vessel 30 and recovery of the product alcohol 54. . In some embodiments, alcohol esterification according to the present invention may be used to esterify the product alcohol in the cultivation medium and the fermentation medium. In this embodiment, the high yield of product alcohol as a whole not only recovers the alcohol esters (and free product alcohols) from the fermentation medium, but also recovers the alcohol esters produced at the time of the cultivation (eg, alcohol esters from the propagation tank). And / or recover product alcohol). In some embodiments, alcohol esterification according to the present invention can be used as a pre-fermentation for the removal of alcohol from the cultivation medium, although conventional ISPR of product alcohol is used to remove product alcohol upon fermentation in fermentation vessel 30. Can be used for

Thus, alcohol esterification and extraction according to the present invention can be used at various stages in the alcohol fermentation process without departing from the present invention.

Alcohol products produced by the process of the invention have many uses, for example as reagents, solvents, and fuels. Butanol produced by the process of the claims is used for the production of fuels (eg biofuels), fuel additives, esters that can be used as diesel or biodiesel fuels, alcohols, feedstock chemicals in the plastics industry, It can be used directly as a component of formulated products such as cosmetics, and as chemical intermediates. Butanol can also be used as solvent for paints, coatings, varnishes, resins, gums, dyes, fats, waxes, resins, shellacs, rubbers, and alkaloids. Thus, the present invention provides an alternative method for producing alcohols including butanol that can support the high demand of such industrial chemicals.

Recombinant Microorganisms and Butanol Biosynthetic Pathways

Without wishing to be bound by theory, the processes described herein are believed to be useful with any alcohol producing microorganism, in particular recombinant microorganisms, which produce alcohol with titers exceeding their acceptable range.

Alcohol producing microorganisms are known in the art. For example, methanol is produced by fermentative oxidation of methane by methanetrophic bacteria (e.g., Methylosinus trichosporium) and methanol (C ₁ alkyl alcohol) The acid and the carboxylic acid are contacted with a catalyst capable of esterifying with methanol to produce a methanol ester of the carboxylic acid. Yeast strain CEN.PK113-7D (CBS 8340, the Centraal Buro voor Schimmelculture; van Dijken, et al., Enzyme Microb. Techno. 26: 706-714, 2000) can produce ethanol and ethanol to carboxylic acid And contacting the carboxylic acid with a catalyst capable of esterifying with ethanol to produce an ethyl ester (see, eg, Example 36).

Recombinant microorganisms producing alcohols are also known in the art (eg, Ohta, et al., Appl. Environ. Microbiol. 57: 893-900, 1991; Underwood, et al. , Appl. Environ.Microbiol. 68: 1071-1081, 2002; Shen and Liao, Metab. Eng. 10: 312-320, 2008; Hahnai, et al., Appl. Environ.Microbiol. 73: 7814-7818, 2007; US Pat. No. 5,514,583; US Pat. No. 5,712,133; WO 1995/028476; Feldmann, et al., Appl. Microbiol. Biotechnol. 38: 354-361, 1992; Zhang, et al. , Science 267: 240-243, 1995; US Patent Application Publication No. 2007/0031918 A1; US Patent 7,223,575; US Patent 7,741,119; US Patent Application Publication 2009/0203099 A1; US Patent Application Publication 2009 / 0246846 A1 and International Patent Application Publication No. WO 2010/075241).

Suitable recombinant microorganisms capable of producing butanol are known in the art and certain suitable microorganisms capable of producing butanol are described herein. Recombinant microorganisms that produce butanol through the biosynthetic pathway include Clostridium, Zymonas, E. coli, Salmonera, Serratia, Erbinia, Klebsiella, Shigella, Rhodococcus and Pseudomonas. , Genus Bacillus, Lactobacillus, Enterococci, Alkali genus, Klebsiella, Fanibacilli, Articular spores, Corynebacterium, Brevibacterium, Ski investigation caromyces, Klui Members of the genus Veromaises, Jarowia, Pichia, Candida, Hansenula, Ischenchenia, or Saccharomyces. In one embodiment, the recombinant microorganism is Escherichia coli , Lactobacillus plantarum ), Kluyveromyces lactis ), Kluyveromyces marxianus , and Saccharomyces cerevisiae ) can be selected from the group consisting of. In one embodiment, the recombinant microorganism is yeast. In one embodiment, the recombinant microorganism is my access to my process, Xi Kosaka a saccharide (Zygosaccharomyces), ski irradiation Caro My process, deck Mosquera (Dekkera), sat rulrop sheath (Torulopsis), Breda Gaetano My process (Brettanomyces), and Crabtree-positive yeast selected from a number of Candida species. Crabtree positive yeast species are Saccharomyces kluyveri , Saccharomyces kluyveri , Schicharomyces pombe , Saccharomyces bayanus , Saccharomyces bayanus and Saccharomyces mikitae . (Saccharomyces mikitae), my process to the saccharide's para doksu (Saccharomyces paradoxus), Zygosaccharomyces my process to look Shi Xi Kosaka (rouxii), and Candida glabrata (Candida glabrata ), but is not limited thereto.

In some embodiments, the host cell is Saccharomyces cerevisiae . Saccharomyces cerevisiae yeasts are known in the art and include the American Type Culture Collection (Rockville, MD), the Centraalbureau voor Schimmelcultures (CBS) Fungal Biodiversity Centre, Lesaf. , Gert Strand Abs, Firm Solutions, North American Bioproducts, Matrex, and Lall Mans are available from a variety of suppliers. Saccharomyces cerevisiae BY4741, CEN.PK 113-7D, Ethanol Red? Yeast, Firm Pro ™ Yeast, Bio-Perm? XR Yeast, Gert Strand Prestige Batch Turbo Alcohol Yeast, Gert Strand Pot Distillers Yeast, Gert Strand Distillers Turbo Yeast, Permax ™ Green Yeast, Permax ™ Gold Yeast, Thermosac? Yeast, BG-1, PE-2, CAT-1, CBS7959, CBS7960, and CBS7961.

Butanol production using fermentation using microorganisms, and microorganisms that produce butanol, are disclosed, for example, in US Patent Application Publication No. 2009/0305370, which is incorporated herein by reference. In some embodiments, the microorganism comprises a butanol biosynthetic pathway. In some embodiments, at least one, at least two, at least three, or at least four polypeptides that catalyze the conversion of substrates to substrates of the pathway are encoded by heterologous polynucleotides of the microorganism. In some embodiments, all polypeptides that catalyze the conversion of products in substrates of the pathway are encoded by heterologous polynucleotides of the microorganism. In some embodiments, the microorganism comprises lowering or elimination of pyruvate decarboxylase activity. Microorganisms substantially free of pyruvate decarboxylase activity are described in US Patent Application Publication No. 2009/0305363, which is incorporated herein by reference. Microorganisms that are substantially free of enzymes with NAD dependent glycerol-3-phosphate dehydrogenase activity, such as GPD2, are also described herein.

Suitable biosynthetic pathways for the production of butanol are known in the art and certain suitable routes are described herein. In some embodiments, the butanol biosynthetic pathway comprises at least one gene that is heterologous to the host cell. In some embodiments, the butanol biosynthetic pathway comprises more than one gene that is heterologous to the host cell. In some embodiments, the butanol biosynthetic pathway comprises a heterologous gene encoding a polypeptide corresponding to every step of the biosynthetic pathway.

Certain suitable proteins with the ability to catalyze the conversion of the indicated substrates to the products are described herein, and other suitable proteins are provided in the art. For example, US Patent Application Publication Nos. 2008/0261230, 2009/0163376, and 2010/0197519, which are incorporated herein by reference, describe acetohydroxy acid isomeroreductases; US Patent Application Publication No. 2010/0081154, which is incorporated herein by reference, describes dihydroxy acid dehydratase; Alcohol dehydrogenases are described in US Patent Application Publication No. 2009/0269823, which is incorporated herein by reference.

It is well known to those skilled in the art that multiple levels of sequence identity are useful in identifying polypeptides from other species that have the same or similar functions or activities and are suitable for use in the recombinant microorganisms described herein. Useful examples of percent identity include, but are not limited to, 75%, 80%, 85%, 90% or 95%, or any integer ratio of 75% to 100%, such as 75%, 76%, 77% , 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94 %, 95%, 96%, 97%, 98% or 99% may be useful in describing the present invention.

1-butanol biosynthetic pathway

Biosynthetic pathways for the production of 1-butanol, and suitable polypeptides that can be used and polynucleotides encoding such polypeptides are described in US Patent Application Publication No. 2008/0182308 A1 by Donaldson et al., Which is incorporated herein by reference. It is described. Such biosynthetic pathways include the conversion of the following substrates to products:

a) conversion of acetyl-CoA to acetoacetyl-CoA, which may be catalyzed by, for example, acetyl-CoA acetyltransferase;

b) acetoacetyl-CoA to 3-hydroxybutyryl-CoA, which can be catalyzed, for example, by 3-hydroxybutyryl-CoA dehydrogenase;

c) conversion of 3-hydroxybutyryl-CoA to crotonyl-CoA, which may be catalyzed by, for example, crotonase;

d) conversion of crotonyl-CoA to butyryl-CoA, which may be catalyzed, for example, by butyryl-CoA dehydrogenase;

e) conversion of butyraldehyde to butyryl-CoA, which may be catalyzed, for example, by butyraldehyde dehydrogenase; And

f) conversion of butyraldehyde to 1-butanol, which may be catalyzed by, for example, 1-butanol dehydrogenase.

In some embodiments, the 1-butanol biosynthetic pathway comprises at least one gene, at least two genes, at least three genes, at least four genes, or at least five genes that are heterologous to yeast cells. In some embodiments, the recombinant host cell comprises a heterologous gene for conversion of each substrate of the 1-butanol biosynthetic pathway to a product.

2-butanol biosynthetic pathway

Biosynthetic pathways for the production of 2-butanol, and suitable polypeptides that can be used and polynucleotides encoding such polypeptides are all incorporated herein by reference in US Patent Application Publication No. 2007/0259410 A1 by Donaldson et al. And 2007 / 0292927A1, and WO 2007/130521. One 2-butanol biosynthetic pathway involves the conversion of the following substrates to products:

a) conversion of pyruvate to alpha-acetolactate, which may be catalyzed by, for example, acetolactate synthase;

b) conversion of alpha-acetolactate to acetoin, which may be catalyzed by, for example, acetolactate decarboxylase;

c) acetoin to 2,3-butanediol, which may be catalyzed, for example, by butanediol dehydrogenase;

d) conversion of 2,3-butanediol to 2-butanone, which may be catalyzed, for example, by butanediol dehydratase; And

e) Conversion of 2-butanone to 2-butanol, which may be catalyzed by, for example, 2-butanol dehydrogenase.

In some embodiments, the 2-butanol biosynthetic pathway comprises at least one gene, at least two genes, at least three genes, or at least four genes that are heterologous to yeast cells. In some embodiments, the recombinant host cell comprises a heterologous gene for conversion of each substrate of the 2-butanol biosynthetic pathway to a product.

Isobutanol Biosynthesis Pathway

Biosynthetic pathways for the production of isobutanol, and appropriate polypeptides that can be used and polynucleotides encoding such polypeptides are described in US Patent Application Publication No. 2007/0092957 A1 and International Patent Application Publication No. WO 2007 / 050671. One isobutanol biosynthetic pathway involves the conversion of the following substrates to products:

a) conversion of pyruvate to acetolactate, which may be catalyzed by, for example, acetolactate synthase;

b) acetolactate to 2,3-dihydroxyisovalerate, which can be catalyzed, for example, by acetohydroxy acid reductoisomerase;

c) conversion of 2,3-dihydroxyisovalerate to α-ketoisovalerate, which may be catalyzed by, for example, acetohydroxy acid dehydratase;

d) conversion of isobutyraldehyde to a-ketoisovalerate, which may be catalyzed, for example, by branched keto acid decarboxylase; And

e) Conversion of isobutyraldehyde to isobutanol, which can be catalyzed, for example, by branched chain alcohol dehydrogenase.

E.C. corresponding to the appropriate polypeptide sequence encoding the enzyme catalyzing the conversion of the isobutanol biosynthetic pathway to the product and the appropriate enzyme for the indicated pathway step. Numbers include, but are not limited to, those of Tables AA and BB. Prescribed E.C. Suitable enzymes associated with numbers will be readily available to those skilled in the art, for example, via the BRENDA database (http://www.brenda-enzymes.org/).

TABLE AA

TABLE BB

At least one of an enzyme catalyzing step c) and an enzyme selected from the group of enzymes catalyzing step e) is encoded by a heterologous polynucleotide integrated into a chromosome of a microorganism, comprising steps a) to e) (above) Provided herein are recombinant microorganisms comprising isobutanol biosynthetic pathways. In some embodiments, the enzyme catalyzing step c) is encoded by a heterologous polynucleotide integrated into the chromosome of the microorganism and the enzyme catalyzing step e) is encoded by a heterologous polynucleotide integrated into the chromosome of the microorganism.

Provided herein are polynucleotides suitable for recombinant microorganisms that include butanol biosynthetic pathways such as isobutanol biosynthetic pathways. Such polynucleotides include the coding region of the alsS gene from Bacillus subtilis (nucleotide positions 457-2172 of SEQ ID NO: 1) and the ilvC gene from Lactococcus lactis (nucleotides 3634-4656 of SEQ ID NO: 1), and either or Plasmids comprising both. In addition, the coding region of the alsS gene from Bacillus subtilis expressed from the yeast CUP1 promoter (nucleotides 2-449 of SEQ ID NO: 1), followed by the CYC1 terminator (nucleotides 2181-2430 of SEQ ID NO: 1) for expression of ALS (SEQ ID NO: Lactococcus expressed from the yeast ILV5 promoter (2433-3626 of SEQ ID NO: 1), followed by the ILV5 terminator (nucleotide 4682-5304 of SEQ ID NO: 1) for expression of the chimeric gene having the nucleotide positions 457-2172) and KARI Suitable are chimeric genes having the coding region of the ilvC gene from Lactis (nucleotides 3634-4656 of SEQ ID NO: 1), and plasmids comprising either or both of the chimeric genes.

Suitable polynucleotides include the coding region of the ilvD gene from Streptococcus mutans (nucleotide positions 3313-4849 of SEQ ID NO: 2), the coding region of codon optimized horse liver alcohol dehydrogenase (nucleotides 6286-7413 of SEQ ID NO: 2), lactose Plasmids comprising the coding region of the codon optimized kivD gene from Caucus lactis (nucleotides 9249-10895 of SEQ ID NO: 2), and any or all or any combination thereof. In addition, ilvD from Streptococcus mutans expressed from the Saccharomyces cerevisiae FBA1 promoter (nucleotides 2109-3105 of SEQ ID NO: 2), followed by the FBA1 terminator (nucleotides 4858-5857 of SEQ ID NO: 2) for expression of DHAD Chimeric genes having the coding region of the gene (nucleotide positions 3313-4849 of SEQ ID NO: 2); Of codon-optimized equine hepatic alcohol dehydrogenase expressed from Saccharomyces cerevisiae GPM1 promoter (nucleotides 7425-8181 of SEQ ID NO: 2) followed by ADH1 terminator (nucleotides 5962-6277 of SEQ ID NO: 2) for expression of ADH. Chimeric genes having a coding region (nucleotides nucleotide 6286-7413 of SEQ ID NO: 2); And the coding region of the codon optimized kivD gene from Lactococcus lactis expressed from the TDH3 promoter (nucleotides 10896-11918 of SEQ ID NO: 2) followed by the TDH3 terminator (nucleotides 8237-9235 of SEQ ID NO: 2) for expression of KivD ( Chimeric genes having nucleotides 9249-10895 of SEQ ID NO: 2, and plasmids comprising any, all, or any combination of these chimeric genes are suitable. Suitable polynucleotides also include polynucleotides having at least about 75% identity with defined coding regions and chimeric genes, and plasmids comprising such polynucleotides.

In some embodiments, the isobutanol biosynthetic pathway comprises at least one gene, at least two genes, at least three genes, or at least four genes that are heterologous to yeast cells. In some embodiments, the recombinant host cell comprises a heterologous gene for the conversion of each substrate of the isobutanol biosynthetic pathway to a product.

Suitable strains include the specific applications cited and incorporated herein by reference and the strains described in US Provisional Application No. 61 / 380,563, filed September 7, 2010. Construction of certain suitable strains, including the strains used in the examples, is provided herein.

Construction of Saccharomyces cerevisiae strain BP1083 ("NGCI-070"; PNY1504)

Strain BP1064 is derived from CEN.PK 113-7D (CBS 8340; Central Bureau voor Schimmelcultures (CBS) Fungal Biodiversity Center) and includes deletions of the following genes: URA3, HIS3, PDC1, PDC5 BP1064 was transformed with plasmids pYZ090 (SEQ ID NO: 1, described in US Provisional Application No. 61 / 246,844) and pLH468 (SEQ ID NO: 2) to generate strain NGCI-070 (BP1083, PNY1504).

Deletion in which the entire coding sequence is completely removed by homologous recombination with a PCR fragment comprising either the G418 resistance marker or the URA3 gene for selection of the transformant and regions of homology upstream and downstream of the target gene. Generated. G418 resistance markers flanking the loxP site were removed using Cre recombinase. The URA3 gene was removed by homologous recombination to produce scarless deletions or, if the loxP site was flanked, using Cre recombinase.

Scarris deletion procedures were employed from Akada et al., Yeast, 23: 399-405 (2006). In general, PCR cassettes for each scarris deletion were made by combining four fragments, namely A-B-U-C, by overlapping PCR. The PCR cassette consisting of the native CEN.PK 113-7D URA3 gene, along with the promoter (250 bp upstream of the URA3 gene) and terminator (150 bp downstream of the URA3 gene), is a selectable / counter-selectable marker, URA3 (fragment U) It contained. Fragments A and C, each 500 bp in length, correspond to 500 bp upstream of the target gene (fragment A) and 3 '500 bp of the target gene (fragment C). Fragments A and C were used for integration of the cassette into the chromosome by homologous recombination. Fragment B (500 bp in length) corresponds to 500 bp adjacent downstream of the target gene, and since direct repetition of the sequence corresponding to fragment B was generated upon incorporation of the cassette into the chromosome, from homologous recombination chromosome Used for excision of the URA3 marker and fragment C. Using the PCR product ABUC cassette, the URA3 marker was first integrated into the chromosome by homologous recombination and then excised from the chromosome. Early integration resulted in the deletion of genes other than 3 '500 bp. Upon ablation, the 3 '500 bp region of the gene was also deleted. For integration of genes using this method, the gene to be integrated was included in a PCR cassette between fragments A and B.

URA3 fruition

To delete the endogenous URA3 coding region, the ura3 :: loxP-kanMX-loxP cassette was PCR amplified from pLA54 template DNA (SEQ ID NO: 3). pLA54 contains the Kluyveromyces lactis TEF1 promoter and kanMX marker, and the loxP site is flanked to allow recombination using Cre recombinase and removal of the marker. Fusion? PCR was performed using DNA polymerase (New England BioLabs Inc., Ipswich, Mass., USA) and primers BK505 and BK506 (SEQ ID NOs: 4 and 5). The URA3 portion of each primer was derived from the 5 'region upstream of the promoter and 3' region downstream of the coding region to allow replacement of the URA3 coding region due to the integration of the loxP-kanMX-loxP markers. The technique (Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp. 201-202) was used to transform into CEN.PK 113-7D and transformants were 30 ° C. Were selected from YPD containing G418 (100 μg / mL) in. Transformants were screened to demonstrate correct integration by PCR using primers LA468 and LA492 (SEQ ID NOs: 6 and 7, respectively), and CEN.PK 113-7D It was named ura3 :: kanMX.

HIS3 deletion

Fusion of four fragments to a PCR cassette for Scaris HIS3 deletion? High Fidelity PCR Master Mix (New England Biolabs, Inc., Ipswich, Mass.) And Gentra? Puregene? Amplification was done using CEN.PK 113-7D genomic DNA as a template made from Yeast / Bact, kit (Qiagen, Valencia, Calif.). HIS3 fragment A was amplified using primer oBP452 (SEQ ID NO: 14) and primer oBP453 (SEQ ID NO: 15) containing a 5 'tail homologous to the 5' end of HIS3 fragment B. HIS3 fragment B comprises primer oBP454 (SEQ ID NO: 16) comprising a 5 'tail homologous to the 3' end of HIS3 fragment A and a 5 'tail homologous to the 5' end of HIS3 fragment U Was amplified using primers oBP455 (SEQ ID NO: 17). HIS3 fragment U comprises primer oBP456 (SEQ ID NO: 18) comprising a 5 'tail homologous to the 3' end of HIS3 fragment B and a 5 'tail homologous to the 5' end of HIS3 fragment C Was amplified using primers oBP457 (SEQ ID NO: 19). HIS3 fragment C was amplified using primers oBP458 (SEQ ID NO: 20) and primer oBP459 (SEQ ID NO: 21) comprising a 5 'tail homologous to the 3' end of HIS3 fragment U. PCR products were purified with a PCR Purification Kit (Qiagen, Valencia, CA). HIS3 fragment AB was generated by overlapping PCR by mixing HIS3 fragment A and HIS3 fragment B and amplifying with primers oBP452 (SEQ ID NO: 14) and oBP455 (SEQ ID NO: 17). HIS3 fragment UC was generated by overlapping PCR by mixing HIS3 fragment U and HIS3 fragment C and amplifying using primers oBP456 (SEQ ID NO: 18) and oBP459 (SEQ ID NO: 21). The resulting PCR product was purified on agarose gel and then purified by gel extraction kit (Qiagen, Valencia, CA). HIS3 ABUC cassettes were generated by overlapping PCR by mixing HIS3 fragments AB and HIS3 fragments UC and amplifying using primers oBP452 (SEQ ID NO: 14) and oBP459 (SEQ ID NO: 21). PCR products were purified with a PCR Purification Kit (Qiagen, Valencia, CA).

CEN.PK 113-7D Create competent cells of Δura3 :: kanMX and use the Frozen-EZ Yeast Transformation II ™ Kit (Zymo, Irvine, CA, USA) Research Corporation) was transformed with the HIS3 ABUC PCR cassette. Transformation mixtures were plated at 30 ° C. on synthetic complete medium lacking uracil supplemented with 2% glucose. Gentra for transformants with his3 knockout? Fuzzy? Genomic DNA prepared with the East / Bat Kit (Qiagen, Valencia, CA) was screened by PCR using primers oBP460 (SEQ ID NO: 22) and oBP461 (SEQ ID NO: 23). The correct transformants were selected with strain CEN.PK 113-7D Δura3 :: kanMX Δhis3 :: URA3.

KanMX marker removal from Δura3 site and URA3 marker removal from Δhis3 site

The CEN.PK 113-7D Δura3 :: kanMX Δhis3 :: URA3 was replaced using the Frozen-Easy East Transformation II ™ Kit (Zymo Research Corporation, Irvine, CA) with pRS423 :: PGAL1-cre (SEQ ID NO: 66 KanMX markers were removed by plating on synthetic complete media lacking histidine and uracil supplemented with 2% glucose at 30 ° C., US Provisional Application No. 61 / 290,639. Transformants were grown in YP supplemented with 1% galactose for about 6 hours at 30 ° C. to induce Cre recombinase and KanMX marker ablation, and plated on YPD (2% glucose) plates at 30 ° C. for recovery. It was. The isolates were grown overnight in YPD and plated on synthetic complete medium containing 5-fluoroorroic acid (5-FOA, 0.1%) at 30 ° C. to select isolates that lacked the URA3 marker. 5-FOA resistant isolates were grown and plated on YPD for removal of the pRS423 :: PGAL1-cre plasmid. Growth loss on KanMX markers, URA3 markers, and pRS423 :: PGAL1-cre plasmids was checked for isolates by analyzing growth on YPD + G418 plates, synthetic complete media plates lacking uracil, and synthetic complete media plates lacking histidine. It was. The exact isolate, susceptible to G418 and nutritional to uracil and histidine, was selected as strain CEN.PK 113-7D Δura3 :: loxP Δhis3 and named BP857. Gentra for deletion and marker removal? Fuzzy? PCR using genomic DNA prepared with the East / Bat Kit (Qiagen, Valencia, Calif.), Primers oBP450 (SEQ ID NO: 24) and oBP451 (SEQ ID NO: 25) for Δura3 and primer oBP460 for Δhis3 (SEQ ID NO: 22) and oBP461 (SEQ ID NO: 23).

PDC6  fruition

Fusion of four fragments to a PCR cassette for Scarris PDC6 deletion? High Fidelity PCR Master Mix (New England Biolabs, Inc., Ipswich, Mass.) And Gentra? Fuzzy? Amplification was done using CEN.PK 113-7D genomic DNA as a template made with the Yeast / Back kit (Qiagen, Valencia, CA). PDC6 fragment A was amplified using primer oBP440 (SEQ ID NO: 26) and primer oBP441 (SEQ ID NO: 27) comprising a 5 'tail homologous to the 5' end of PDC6 fragment B. PDC6 fragment B comprises primer oBP442 (SEQ ID NO: 28) comprising a 5 'tail homologous to the 3' end of PDC6 fragment A, and a 5 'tail homologous to the 5' end of PDC6 fragment U Was amplified using primers oBP443 (SEQ ID NO: 29). PDC6 fragment U comprises primer oBP444 (SEQ ID NO: 30) comprising a 5 'tail homologous to the 3' end of PDC6 fragment B, and a 5 'tail homologous to the 5' end of PDC6 fragment C Was amplified using primers oBP445 (SEQ ID NO: 31). PDC6 fragment C was amplified using primer oBP446 (SEQ ID NO: 32), and primer oBP447 (SEQ ID NO: 33) comprising a 5 'tail homologous to the 3' end of PDC6 fragment U. PCR products were purified with a PCR Purification Kit (Qiagen, Valencia, CA). PDC6 fragment AB was generated by overlapping PCR by mixing PDC6 fragment A and PDC6 fragment B and amplifying with primers oBP440 (SEQ ID NO: 26) and oBP443 (SEQ ID NO: 29). PDC6 fragment UC was generated by overlapping PCR by mixing PDC6 fragment U and PDC6 fragment C and amplifying using primers oBP444 (SEQ ID NO: 30) and oBP447 (SEQ ID NO: 33). The resulting PCR product was purified on agarose gel and then purified by gel extraction kit (Qiagen, Valencia, CA). PDC6 ABUC cassettes were generated by overlapping PCR by mixing PDC6 fragment AB and PDC6 fragment UC and amplifying using primers oBP440 (SEQ ID NO: 26) and oBP447 (SEQ ID NO: 33). PCR products were purified with a PCR Purification Kit (Qiagen, Valencia, CA).

Competent cells of CEN.PK 113-7D Δura3 :: loxP Δhis3 were made and transformed with the PDC6 ABUC PCR cassette using the Frozen-Easy East Transformation II ™ Kit (Zymo Research Corporation, Irvine, CA). I was. Transformation mixtures were plated at 30 ° C. on synthetic complete medium lacking uracil supplemented with 2% glucose. Gentra for transformants with pdc6 knockout? Fuzzy? Genomic DNA prepared with the East / Bat Kit (Qiagen, Valencia, CA) was screened by PCR using primers oBP448 (SEQ ID NO: 34) and oBP449 (SEQ ID NO: 35). The correct transformants were selected with strain CEN.PK 113-7D Δura3 :: loxP Δhis3 Δpdc6 :: URA3.

CEN.PK 113-7D Δura3 :: loxP Δhis3 Δpdc6 :: URA3 was grown overnight in YPD and plated on synthetic complete medium containing 5-fluoro-orotic acid (0.1%) at 30 ° C. Isolates with missing markers were selected. Gentra for deletion and marker removal? Fuzzy? It was confirmed by sequencing with primers oBP448 (SEQ ID NO: 34) and oBP449 (SEQ ID NO: 35) by PCR using genomic DNA prepared with the East / Bact kit (Qiagen, Valencia, CA). The absence of the PDC6 gene from the isolate was demonstrated by negative PCR results using primers specific for the coding sequence of PDC6, oBP554 (SEQ ID NO: 36) and oBP555 (SEQ ID NO: 37). The correct isolate was selected as strain CEN.PK 113-7D Δura3 :: loxP Δhis3 Δpdc6 and named BP891.

PDC1 deletion ilvDSm integration

The PDC1 gene was deleted and replaced with the ilvD coding region from Streptococcus mutans ATCC No. 700610. A fragment followed by fusion of the ilvD coding region from Streptococcus mutans for the PCR cassette for PDC1 deletion-ilvDSm integration? High Fidelity PCR Master Mix (New England Biolabs, Inc., Ipswich, Mass.) And Gentra? Fuzzy? Amplification was performed using NYLA83 genomic DNA as a template made with the East / Bat Kit (Qiagen, Valencia, CA). NYLA83 is a strain comprising the PDC1 deletion-ilvDSm integration described in US Patent Application Publication No. 2009/0305363, the entire contents of which are incorporated herein by reference (US Patent Application Publication No. 5, which is incorporated herein by reference in its entirety). Construct described in 2011/0124060). PDC1 fragment A-ilvDSm (SEQ ID NO: 141) was amplified using primer oBP513 (SEQ ID NO: 38) and primer oBP515 (SEQ ID NO: 39) comprising a 5 'tail homologous to the 5' end of PDC1 fragment B . Fusion of B, U, and C Fragments for PCR Cassettes for PDC1 Deletion-ilvDSm Integration? High Fidelity PCR Master Mix (New England Biolabs, Inc., Ipswich, Mass.) And Gentra? Fuzzy? Amplification was done using CEN.PK 113-7D genomic DNA as a template made with the Yeast / Back kit (Qiagen, Valencia, CA). Primer OBP516 (SEQ ID NO: 40) comprising a 5 'tail homologous to the 3' end of PDC1 fragment A-ilvDSm, and a 5 'tail homologous to the 5' end of PDC1 fragment U Amplified using a primer containing oBP517 (SEQ ID NO: 41). PDC1 fragment U comprises primer oBP518 (SEQ ID NO: 42) comprising a 5 'tail homologous to the 3' end of PDC1 fragment B, and a 5 'tail homologous to the 5' end of PDC1 fragment C Amplification using primers oBP519 (SEQ ID NO: 43). PDC1 fragment C was amplified using primer oBP520 (SEQ ID NO: 44), and primer oBP521 (SEQ ID NO: 45) comprising a 5 'tail homologous to the 3' end of PDC1 fragment U. PCR products were purified with a PCR Purification Kit (Qiagen, Valencia, CA). PDC1 fragment A-ilvDSm-B was generated by overlapping PCR by mixing PDC1 fragment A-ilvDSm and PDC1 fragment B and amplifying using primers oBP513 (SEQ ID NO: 38) and oBP517 (SEQ ID NO: 41). PDC1 fragment UC was generated by overlapping PCR by mixing PDC1 fragment U and PDC1 fragment C and amplifying using primers oBP518 (SEQ ID NO: 42) and oBP521 (SEQ ID NO: 45). The resulting PCR product was purified on agarose gel and then purified by gel extraction kit (Qiagen, Valencia, CA). Nested PCR by mixing the PDC1 A-ilvDSm-BUC cassette (SEQ ID NO: 142) with the PDC1 fragments A-ilvDSm-B and PDC1 fragment UC and amplifying using primers oBP513 (SEQ ID NO: 38) and oBP521 (SEQ ID NO: 45) Generated by PCR products were purified with a PCR Purification Kit (Qiagen, Valencia, CA).

Competent cells of CEN.PK 113-7D Δura3 :: loxP Δhis3 Δpdc6 were made and PDC1 A-ilvDSm-BUC using the Frozen-Easy East Transformation II ™ Kit (Zymo Research Corporation, Irvine, CA). Transformed with a PCR cassette. Transformation mixtures were plated at 30 ° C. on synthetic complete medium lacking uracil supplemented with 2% glucose. Gentra transformants with pdc1 knockout ilvDSm integration? Fuzzy? Genomic DNA prepared with the East / Bat Kit (Qiagen, Valencia, Calif.) Was screened by PCR using primers oBP511 (SEQ ID NO: 46) and oBP512 (SEQ ID NO: 47). The absence of the PDC1 gene from the isolate was demonstrated by negative PCR results using primers specific for the coding sequence of PDC1, oBP550 (SEQ ID NO: 48) and oBP551 (SEQ ID NO: 49). The correct transformants were selected with strain CEN.PK 113-7D Δura3 :: loxP Δhis3 Δpdc6 Δpdc1 :: ilvDSm-URA3.

CEN.PK 113-7D Δura3 :: loxP Δhis3 Δpdc6 Δpdc1 :: ilvDSm-URA3 were grown overnight in YPD and played on synthetic complete medium containing 5-fluoro-orotic acid (0.1%) at 30 ° C. To isolate the isolates with missing URA3 markers. Gentric deletion of PDC1, integration of ilvDSm, and marker removal? Fuzzy? It was confirmed by sequencing with primers oBP511 (SEQ ID NO: 46) and oBP512 (SEQ ID NO: 47) by PCR using genomic DNA prepared with the East / Bact kit (Qiagen, Valencia, CA). The correct isolate was selected as strain CEN.PK 113-7D Δura3 :: loxP Δhis3 Δpdc6 Δpdc1 :: ilvDSm and named BP907.

PDC5 deletion sadB integration

The PDC5 gene was deleted and replaced with the sadB coding region from Acromobacter xyloxidans. First, a segment of the PCR cassette for PDC5 deletion-sadB integration was cloned into the plasmid pUC19-URA3MCS.

pUC19-URA3MCS is based on pUC19 and contains the sequence of the URA3 gene from Saccharomyces cerevisiae located within the multiple cloning site (MCS). pUC19 contains pMB1 replicon and a gene encoding beta-lactamase for replication and selection in E. coli. In addition to the coding sequences for URA3, sequences from upstream and downstream of these genes were included for expression of the URA3 gene in yeast. Vectors can be used for cloning purposes and can be used as yeast integration vectors.

Fusion of DNA comprising a URA3 coding region with 250 bp upstream and 150 bp downstream of the URA3 coding region from Saccharomyces cerevisiae CEN.PK 113-7D genomic DNA? Primer oBP438 (SEQ ID NO: 12), and XbaI, comprising the BamHI, AscI, PmeI, and FseI restriction sites, using a high fidelity PCR master mix (New England Biolabs, Ipswich, Mass.) Amplification was carried out using primers oBP439 (SEQ ID NO: 13) comprising PacI, and NotI restriction sites. Genome DNA Gentra? Fuzzy? Prepared using the Yeast / Back Kit (Qiagen, Valencia, CA). PCR product and pUC19 (SEQ ID NO: 150) were ligated with T4 DNA ligase after digestion with BamHI and XbaI to generate vector pUC19-URA3MCS. The vector was confirmed by sequencing by primers oBP264 (SEQ ID NO: 10) and oBP265 (SEQ ID NO: 11).

 The coding sequence of sadB and PDC5 fragment B were cloned into pUC19-URA3MCS to generate the sadB-BU portion of the PDC5 A-sadB-BUC PCR cassette. Using the coding sequence of sadB as a template, pLH468-sadB (SEQ ID NO: 67) was used to generate a primer oBP530 (SEQ ID NO: 50) containing an AscI restriction site, and a 5 'tail with homology to the 5' end of PDC5 fragment B. Amplification was carried out using the containing primer oBP531 (SEQ ID NO: 51). PDC5 fragment B was amplified using primer oBP532 (SEQ ID NO: 52) comprising a 5 'tail with homology to the 3' end of sadB, and primer oBP533 (SEQ ID NO: 53) comprising a PmeI restriction site. PCR products were purified with a PCR Purification Kit (Qiagen, Valencia, CA). sadB-PDC5 fragment B was generated by overlap PCR by mixing sadB and PDC5 fragment B PCR products and amplifying with primers oBP530 (SEQ ID NO: 50) and oBP533 (SEQ ID NO: 53). The resulting PCR product was digested with AscI and PmeI and ligated into the corresponding sites of pUC19-URA3MCS after digestion with appropriate enzymes using T4 DNA ligase. The resulting plasmid was amplified by sadB-fragment B-fragment U using primer oBP536 (SEQ ID NO: 54) and primer oBP546 (SEQ ID NO: 55) comprising a 5 'tail homologous to the 5' end of PDC5 fragment C. Used as a template for PDC5 fragment C was amplified using primer oBP547 (SEQ ID NO: 56), and primer oBP539 (SEQ ID NO: 57) comprising a 5 'tail homologous to the 3' end of PDC5 sadB-fragment B-fragment U. PCR products were purified with a PCR Purification Kit (Qiagen, Valencia, CA). Nested PCR by mixing PDC5 sadB-fragment B-fragment U-fragment C, PDC5 sadB-fragment B-fragment U and PDC5 fragment C and amplifying with primers oBP536 (SEQ ID NO: 54) and oBP539 (SEQ ID NO: 57) Produced by The resulting PCR product was purified on agarose gel and then purified by gel extraction kit (Qiagen, Valencia, CA). A primer oBP542 containing a 5 'tail with a PDC5 A-sadB-BUC cassette (SEQ ID NO: 143) and a PDC5 sadB-fragment B-fragment U-fragment C with homology to 50 nucleotide contiguous upstream of the native PDC5 coding sequence (SEQ ID NO: 58), and primers amplified using oBP539 (SEQ ID NO: 57). PCR products were purified with a PCR Purification Kit (Qiagen, Valencia, CA).

Create competent cells of CEN.PK 113-7D Δura3 :: loxP Δhis3 Δpdc6 Δpdc1 :: ilvDSm and use the Frozen-Easy East Transformation II ™ Kit (Zymo Research Corporation, Irvine, CA) Transformed with -sadB-BUC PCR cassette. Transformation mixtures were plated on synthetic complete medium lacking uracil (without glucose) supplemented with 1% ethanol at 30 ° C. Gentra transformants with pdc5 knockout sadB integration? Fuzzy? Genomic DNA prepared with the East / Bat Kit (Qiagen, Valencia, Calif.) Was screened by PCR using primers oBP540 (SEQ ID NO: 59) and oBP541 (SEQ ID NO: 60). The absence of the PDC5 gene from the isolate was demonstrated by negative PCR results using primers specific for the coding sequence of PDC5, oBP552 (SEQ ID NO: 61) and oBP553 (SEQ ID NO: 62). The correct transformants were selected as strain CEN.PK 113-7D Δura3 :: loxP Δhis3 Δpdc6 Δpdc1 :: ilvDSm Δpdc5 :: sadB-URA3.

CEN.PK 113-7D Δura3 :: loxP Δhis3 Δpdc6 Δpdc1 :: ilvDSm Δpdc5 :: sadB-URA3 was grown overnight in YPE (1% ethanol), supplemented with ethanol at 30 ° C. (without glucose) and 5-fluoro- Plated on synthetic complete medium containing orotic acid (0.1%) to select isolates that lacked the URA3 marker. Gentric deletion of PDC5, integration of sadB, and marker removal? Fuzzy? Genomic DNA prepared with the East / Bat Kit (Qiagen, Valencia, Calif.) Was confirmed by PCR using primers oBP540 (SEQ ID NO: 59) and oBP541 (SEQ ID NO: 60). The correct isolate was selected as strain CEN.PK 113-7D Δura3 :: loxP Δhis3 Δpdc6 Δpdc1 :: ilvDSm Δpdc5 :: sadB and named BP913.

GPD2 Fruit

To delete the endogenous GPD2 coding region, the gpd2 :: loxP-URA3-loxP cassette (SEQ ID NO: 151) was PCR amplified using loxP-URA3-loxP (SEQ ID NO: 68) as template DNA. loxP-URA3-loxP contains URA3 markers from flanked loxP recombinase sites (ATCC No 77107). fusion? PCR was performed using DNA polymerase (New England Biolabs, Ipswich, Mass.) And primers LA512 and LA513 (SEQ ID NOs: 8 and 9). The GPD2 portion of each primer was derived from the 5 'region upstream of the GPD2 coding region and the 3' region downstream of the coding region so that the GPD2 coding region was replaced by the integration of the loxP-URA3-loxP marker. PCR products were transformed into BP913 and transformants were selected on synthetic complete medium supplemented with 1% ethanol (no glucose) and lacking uracil. Transformants were screened to demonstrate correct integration by PCR using primers oBP582 and AA270 (SEQ ID NOs 63 and 64).

URA3 markers were transformed with pRS423 :: PGAL1-cre (SEQ ID NO: 66) and recycled by plating on synthetic complete medium supplemented with 1% ethanol and deficient in histidine at 30 ° C. Transformants were streaked on synthetic complete medium supplemented with 1% ethanol, containing 5-fluoro-orotic acid (0.1%) and incubated at 30 ° C. to select isolates that lacked the URA3 marker. 5-FOA resistant isolates were grown in YPE (1% ethanol) for removal of the pRS423 :: PGAL1-cre plasmid. Deletion and marker removal was confirmed by PCR using primers oBP582 (SEQ ID NO: 63) and oBP591 (SEQ ID NO: 65). The correct isolate was selected as strain CEN.PK 113-7D Δura3 :: loxP Δhis3 Δpdc6 Δpdc1 :: ilvDSm Δpdc5 :: sadB Δgpd2 :: loxP and named PNY1503 (BP1064).

BP1064 was transformed with plasmids pYZ090 (SEQ ID NO: 1) and pLH468 (SEQ ID NO: 2) to generate strain NGCI-070 (BP1083; PNY1504).

Construction of Saccharomyces cerevisiae strain PNY2205

The strain, PNY2205 was derived from PNY1503 (BP1064) described above.

Typically, deletions from which the entire coding sequence was removed were formed by homologous recombination with PCR fragments comprising regions of homology upstream and downstream of the URA3 gene and the target gene for selection of the transformants. The URA3 gene was removed by homologous recombination to form a scarris deletion. Gene integration was formed in a similar way.

Scarris deletion procedures were employed from Akada et al., Yeast, 23: 399, 2006. In general, PCR cassettes for each scarris deletion were made by combining four fragments, namely A-B-U-C, by overlapping PCR. In some cases, each fragment was first cloned into a plasmid before the entire cassette was amplified by PCR for the deletion / integration procedure. Along with the promoter (250 bp upstream of the URA3 gene) and terminator (150 bp downstream of the URA3 gene) regions, the PCR cassette consisting of the native CEN.PK 113-7D URA3 gene provides a selectable / counter-selectable marker, URA3 (fragment). U). Fragments A and C, each generally 500 bp in length, corresponded to 500 bp contiguous upstream of the target gene (fragment A) and 3 '500 bp (fragment C) of the target gene. Fragments A and C were used for integration of the cassette into the chromosome by homologous recombination. Fragment B (500 bp in length) corresponds to 500 bp adjacent downstream of the target gene, and since direct repetition of the sequence corresponding to fragment B was generated upon incorporation of the cassette into the chromosome, homologous recombination from the chromosome Used for ablation of the URA3 marker and fragment C.

Using the PCR product ABUC cassette, the URA3 marker was first integrated into the chromosome by homologous recombination and then excised from the chromosome. Early integration resulted in the deletion of genes other than 3 '500 bp. Upon ablation, the 3 '500 bp region of the gene was also deleted. For integration of genes using this method, the gene to be integrated was included in a PCR cassette between fragments A and B.

FRA2 deletion

The FRA2 deletion was designed to delete 250 nucleotides from the 3 'end of the coding sequence, leaving the first 113 nucleotides of the intact FRA2 coding sequence. In-frame termination codons appeared 7 nucleotides downstream of the deletion. Fusion of four fragments to a PCR cassette for Scarris FRA2 deletion? High Fidelity PCR Master Mix (New England Biolabs, Inc., Ipswich, Mass.) And Gentra? Fuzzy? Amplification was done using CEN.PK 113-7D genomic DNA as a template made with the Yeast / Back kit (Qiagen, Valencia, CA). FRA2 fragment A was amplified using primers oBP594 (SEQ ID NO: 152) and primer oBP595 (SEQ ID NO: 153) comprising a 5 'tail homologous to the 5' end of FRA2 fragment B. FRA2 fragment B comprises primer oBP596 (SEQ ID NO: 154) comprising a 5 'tail homologous to the 3' end of FRA2 fragment A, and a 5 'tail homologous to the 5' end of FRA2 fragment U Was amplified using primers oBP597 (SEQ ID NO: 155). FRA2 fragment U comprises a primer oBP598 (SEQ ID NO: 156) comprising a 5 'tail homologous to the 3' end of FRA2 fragment B, and a 5 'tail homologous to the 5' end of FRA2 fragment C Was amplified using primers oBP599 (SEQ ID NO: 157). FRA2 fragment C was amplified using primers oBP600 (SEQ ID NO: 158) and oBP601 (SEQ ID NO: 159) comprising a 5 'tail homologous to the 3' end of FRA2 fragment U. PCR products were purified with a PCR Purification Kit (Qiagen, Valencia, CA). FRA2 fragment AB was generated by overlapping PCR by mixing FRA2 fragment A and FRA2 fragment B and amplifying with primers oBP594 (SEQ ID NO: 152) and oBP597 (SEQ ID NO: 155). FRA2 fragment UC was generated by overlapping PCR by mixing FRA2 fragment U and FRA2 fragment C and amplifying using primers oBP598 (SEQ ID NO: 156) and oBP601 (SEQ ID NO: 159). The resulting PCR product was purified on agarose gel and then purified by gel extraction kit (Qiagen, Valencia, CA). FRA2 ABUC cassettes were generated by overlapping PCR by mixing FRA2 fragment AB and FRA2 fragment UC and amplifying with primers oBP594 (SEQ ID NO: 152) and oBP601 (SEQ ID NO: 159). PCR products were purified with a PCR Purification Kit (Qiagen, Valencia, CA).

Competent cells of PNY1503 were made and transformed with the FRA2 ABUC PCR cassette using the Frozen-Easy East Transformation II ™ Kit (Zymo Research Corporation, Irvine, CA). Transformation mixtures were plated on synthetic complete medium lacking uracil supplemented with 1% ethanol at 30 ° C. Gentra for transformants with fra2 knockouts? Fuzzy? Genomic DNA prepared with the East / Bat Kit (Qiagen, Valencia, Calif.) Was screened by PCR using primers oBP602 (SEQ ID NO: 160) and oBP603 (SEQ ID NO: 161). The correct transformants were grown in YPE (yeast extract, peptone, 1% ethanol) and plated on synthetic complete medium containing 5-fluoro-orotic acid (0.1%) at 30 ° C. Lost isolates were selected. Gentra for deletion and marker removal? Fuzzy? Genomic DNA prepared using the East / Bact kit (Qiagen, Valencia, Calif.) Was identified by PCR using primers oBP602 (SEQ ID NO: 160) and oBP603 (SEQ ID NO: 161). The absence of the FRA2 gene from the isolate was demonstrated by negative PCR results using primers specific for the deleted coding sequence of FRA2, oBP605 (SEQ ID NO: 162) and oBP606 (SEQ ID NO: 163). Select the correct isolate with strain CEN.PK 113-7D MATa ura3Δ :: loxP his3Δ pdc6Δ pdc1Δ :: P [PDC1] -DHAD | ilvD_Sm-PDC1t pdc5Δ :: P [PDC5] -ADH | sadB_Ax-PDC5t gpd2Δ :: loxP fra2 It was named PNY1505 (BP1135).

ADH1 deletion and kivD Ll (y) integration

The ADH1 gene was deleted and replaced with a kivD coding region from codon optimized Lactococcus lactis for expression in Saccharomyces cerevisiae. As described in U.S. Provisional Application No. 61 / 356,379, filed Jun. 18, 2010, which is incorporated herein by reference, a cloris cassette for ADH1 deletion-kivD_Ll (y) integration is first cloned into plasmid pUC19-URA3MCS. It was. The vector is based on pUC19 and comprises the sequence of the URA3 gene from Saccharomyces cerevisiae CEN.PK 113-7D located within the multiple cloning site (MCS). pUC19 includes pMB1 replicon and a gene encoding beta-lactamase for replication and selection in E. coli. In addition to the coding sequences for URA3, sequences from upstream (250 bp) and downstream (150 bp) of these genes are present for expression of the URA3 gene in yeast.

KivD coding region from codon optimized Lactococcus lactis for expression in Saccharomyces cerevisiae using pLH468 (US Provisional Application No. 61 / 246,709, filed Sep. 29, 2009) as a template Amplified with primer oBP562 (SEQ ID NO: 164) comprising the PmeI restriction site, and primer oBP563 (SEQ ID NO: 165) comprising a 5 'tail homologous to the 5' end of ADH1 fragment B. From genomic DNA prepared as above, ADH1 fragment B, primer oBP564 (SEQ ID NO: 166) comprising a 5 'tail with homology to the 3' end of kivD_Ll (y), and primer oBP565 comprising a FseI restriction site (SEQ ID NO: 167). PCR products were purified with a PCR Purification Kit (Qiagen, Valencia, CA). kivD_Ll (y) -ADH1 fragment B was generated by overlapping PCR by mixing kivD_Ll (y) and ADH1 fragment B PCR products and amplifying with primers oBP562 (SEQ ID NO: 164) and oBP565 (SEQ ID NO: 167). The resulting PCR product was digested with PmeI and FseI and ligated into the corresponding sites of pUC19-URA3MCS after digestion with appropriate enzymes using T4 DNA ligase. ADH1 fragment A was amplified from genomic DNA with primer oBP505 (SEQ ID NO: 168) comprising a SacI restriction site, and primer oBP506 (SEQ ID NO: 169) comprising an AscI restriction site. The ADH1 fragment A PCR product was digested with SacI and AscI and ligated into the corresponding site of the plasmid containing kivD_Ll (y) -ADH1 fragment B using T4 DNA ligase. ADH1 fragment C was amplified from genomic DNA using primer oBP507 (SEQ ID NO: 170) comprising a PacI restriction site, and primer oBP508 (SEQ ID NO: 171) comprising a SalI restriction site. The ADH1 Fragment C PCR product was digested with PacI and SalI and ligated into the corresponding site of the plasmid containing ADH1 Fragment A-kivD_Ll (y) -ADH1 Fragment B using T4 DNA ligase. Primers oBP674 (SEQ ID NO: 173) comprising the AscI restriction site from the hybrid promoter UAS (PGK1) -P _FBA1 from the vector pRS316-UAS (PGK1) -P _FBA1- GUS (SEQ ID NO: 172), and a primer comprising the PmeI restriction site; Amplified using oBP675 (SEQ ID NO: 174). The UAS (PGK1) -P _FBA1 PCR product was digested with AscI and PmeI and ligated into the corresponding site of the plasmid containing the kivD_Ll (y) -ADH1 fragment ABC using T4 DNA ligase. The whole integration cassette was amplified from the resulting plasmid using primers oBP505 (SEQ ID NO: 168) and oBP508 (SEQ ID NO: 171) and purified with a PCR purification kit (Qiagen, Valencia, CA).

Competent cells of PNY1505 were made and transformed with the ADH1-kivD_Ll (y) PCR cassette constructed above using the Frozen-Easy East Transformation II ™ kit (Zymo Research Corporation, Irvine, CA). Transformation mixtures were plated on synthetic complete medium lacking uracil supplemented with 1% ethanol at 30 ° C. Transformants were grown in YPE (1% ethanol) and plated on synthetic complete medium containing 5-fluoro-orthoic acid (0.1%) at 30 ° C. to select isolates lacking the URA3 marker. . Deletion of ADH1 and integration of kivD_Ll (y), Gentra? Fuzzy? External primers oBP495 (SEQ ID NO: 175) and oBP496 (SEQ ID NO: 176), and kivD_Ll (y) specific primers oBP562 using genomic DNA prepared using the East / Back kit (Qiagen, Valencia, CA) (SEQ ID NO: 164) and external primer oBP496 (SEQ ID NO: 176) by PCR. The exact isolate was strain CEN.PK 113-7D MATa ura3Δ :: loxP his3Δ pdc6Δ pdc1Δ :: P [PDC1] -DHAD | ilvD_Sm-PDC1tpdc5Δ :: P [PDC5] -ADH | sadB_Ax-PDC5t gpd2Δ: Δ alodhP fra :: UAS (PGK1) P [FBA1] -kivD_Ll (y) -ADH1t was selected and named PNY1507 (BP1201). PNY1507 was transformed with isobutanol pathway plasmids pYZ090 (SEQ ID NO: 1) and pBP915 (described below).

Construction of the pRS316-UAS (PGK1) -FBA1p-GUS Vector

To clone the cassette UAS (PGK1) -FBA1p (SEQ ID NO: 177), first 602 bp FBA1 promoter (FBA1p) was extracted from the genomic DNA of CEN.PK primers T-FBA1 (SalI) (SEQ ID NO: 178) and B-FBA1 (SpeI). (SEQ ID NO: 179), and the PGK1p promoter was removed by SalI / SpeI digestion of the plasmid and then cloned into the SalI and SpeI sites on plasmid pWS358-PGK1p-GUS (SEQ ID NO: 180), pWS358-FBA1p- GUS was generated. The pWS358-PGK1p-GUS plasmid was generated by inserting the PGK1p and beta-glucuronidase gene (GUS) DNA fragments into multiple cloning sites of pWS358 derived from the pRS423 vector (Christianson, et al., Gene 110: 119 -122, 1992). Secondly, the resulting pWS358-FBA1p-GUS plasmid was digested with a DNA fragment comprising SalI and SacI-FBA1p promoter, GUS gene, and gel purified FBAt terminator-cloned into the SalI / SacI site on pRS316 to pRS316- FBA1p-GUS was generated. Third, CEN 118 bp DNA fragment comprising an upstream activating sequence (UAS) located between the 3-phosphoglycerate kinase (PGK1) open reading frame, ie, position -519 and position-402 upstream of UAS (PGK1). PCR amplification from the genomic DNA of .PK using primers TU / PGK1 (KpnI) (SEQ ID NO: 181) and BU / PGK1 (SalI) (SEQ ID NO: 182). The PCR product was digested with KpnI and SalI and cloned into the KpnI / SalI site on pRS316-FBA1p-GUS to generate pRS316-UAS (PGK1) -FBA1p-GUS.

Construction of the unified vector pUC19-kan :: pdc1 :: FBA-alsS :: TRX1

1.7 kb BbvCI / PacI fragments were converted from pRS426 :: GPD :: alsS :: CYC (US Patent Application Publication No. 2007/0092957) to pRS426 :: FBA :: ILV5 :: CYC (US Patent Application Publication No. 2007/0092957, FBA-alsS-CYCt cassette was constructed, which was then digested with BbvCI / PacI to release the ILV5 gene. Ligation reactions were transformed with E. coli TOP10 cells and transformants were screened by PCR using primers N98SeqF1 (SEQ ID NO: 183) and N99SeqR2 (SEQ ID NO: 184). The FBA-alsS-CYCt cassette was filed on June 18, 2010 at pUC19-URA3 :: ilvD-TRX1 (incorporated herein by reference) at the AflII site (Klenow fragment was used to form suitable ends for ligation). US Pat. No. 61 / 356,379, which was isolated from the vector using BglII and NotI for cloning into clone “B”). Obtain a transformant comprising an alsS cassette in both orientations of the vector, primers N98SeqF4 (SEQ ID NO: 185) and N1111 (SEQ ID NO: 185) for array "A" and N98SeqF4 (SEQ ID NO: 185) for array "B" and Confirmed by PCR using N1110 (SEQ ID NO: 187). The geneticin selective version of the "A" array vector was then removed from the URA3 gene (1.2 kb NotI / NaeI fragment) and the Geneticin cassette described above (June 2010, incorporated herein by reference). (SEQ ID NO: 655 of US Provisional Application No. 61 / 356,379, filed on May 18). The Klenow fragment is used to form all the ends suitable for ligation, and the transformants are screened by PCR using primers BK468 (SEQ ID NO: 188) and N160SeqF5 (SEQ ID NO: 189) to give the same orientation as the previous URA3 marker. Clones with nettysin resistance genes were selected. The resulting clones were designated as pUC19-kan :: pdc1 :: FBA-alsS :: TRX1 (clone A) (SEQ ID NO: 190).

The pUC19-kan :: pdc1 :: FBA-alsS integration vector described above was linearized with PmeI and transformed with PNY1507 (described above). PmeI cleaves the vector in the cloned pdc1-TRX1 intergenic region, inducing targeted integration at that location (Rothstein, Methods Enzymol. 194: 281-301, 1991). Transformants were selected from YPE plus 50 μg / ml G418. Patched transformants were screened by PCR for integration events using primers N160SeqF5 (SEQ ID NO: 189) and oBP512 (SEQ ID NO: 47). Two transformants were indirectly tested for acetolactate synthase function by assessing the ability of the strain to produce isobutanol. To do this, additional isobutanol pathway genes were fed onto an E. coli-yeast shuttle vector (pYZ090ΔalsS and pBP915, described below). One clone, strain MATa ura3Δ :: loxP his3Δ pdc6Δ pdc1Δ :: P [PDC1] -DHAD | ilvD_Sm-PDC1t-pUC19-loxP-kanMX-loxP-P [FBA1] -ALS | alsS_Bs-CYC1t pdcP5Δ :: P ] -ADH | sadB_Ax-PDC5t gpd2Δ :: loxP fra2Δ adh1Δ :: UAS (PGK1) P [FBA1] -kivD_Ll (y) -ADH1t was designated PNY2204. PNY2205 is PNY2204 transformed with pYZ090ΔalsS and pBP915 plasmid.

Isobutanol  Path Plasmid ( pYZ090 Δ alsS  And pBP915 )

pYZ090 (SEQ ID NO: 1) was digested with SpeI and NotI to remove most of the CUP1 promoter and all alsS coding sequences and CYC terminators. Vectors were then self-ligated after treatment with Klenow fragments and transformed into E. coli Stbl3 cells selected for ampicillin resistance. Removal of the DNA region was confirmed for two independent clones by DNA sequencing via ligation junction by PCR using primers N191 (SEQ ID NO: 191). The resulting plasmid was named pYZ090ΔalsS (SEQ ID NO: 192).

pBP915 was constructed by deleting 957 base pairs of the kivD gene and the TDH3 promoter upstream of kivD from pLH468 (SEQ ID NO: 2, US Provisional Application No. 61 / 246,709, filed Sep. 29, 2009). pLH468 was digested with SwaI and large fragments (12896 bp) were purified on agarose gels and then purified with a gel extraction kit (Qiagen, Valencia, CA). Isolated DNA fragments were self-ligated with T4 DNA ligase and used to transform the electrocompetent TOP10 Escherichia coli (Invitrogen, Carlsbad, Calif.). Plasmids from the transformants were isolated and checked for appropriate deletion by restriction analysis with SwaI restriction enzymes. Isolates were also sequenced with primers oBP556 (SEQ ID NO: 193) and oBP561 (SEQ ID NO: 194) over the deletion site. Clones with appropriate deletions were named pBP915 (pLH468ΔkivD) (SEQ ID NO: 195).

Construction of Strains NYLA74, NYLA83, and NYLA84

Insertion inactivation of endogenous PDC1 and PDC6 genes of Saccharomyces cerevisiae, PDC1, PDC5, and PDC6 genes encode three major isozymes of pyruvate decarboxylase, are described as follows:

Building the pRS425 :: GPM-sadB

DNA fragments (disclosed in US Patent Application Publication No. 2009/0269823) encoding butanol dehydrogenase (SEQ ID NO: 70) from Acromobacter xyloxyxidans were cloned. The coding region (SEQ ID NO: 69) of these genes, named sadB for secondary alcohol dehydrogenase, was used in accordance with the recommended protocol for Gram-negative bacteria, using standard conditions? Fuzzy? From acromobacter xyloxyoxysidans genomic DNA prepared using the kit (Qiagen, Valencia, Calif.), Forward and reverse primers N473 and N469 (SEQ ID NOs: 74 and 75), respectively Amplified using. The PCR product was cloned into pCR? 4 BLUNT (Invitrogen ™, Carlsbad, Calif.) To generate TOPR? -Blunt to generate pCR4Blunt :: sadB, which was isolated from E. coli Mark-1 cells (Mach). -1 cells). The plasmid was then isolated from four clones and the sequence was verified.

The sadB coding region was PCR amplified from pCR4Blunt :: sadB. PCR primers included an additional 5 'sequence that overlaps the yeast GPM1 promoter and ADH1 terminator (N583 and N584 provided as SEQ ID NOs: 76 and 77). PCR products were then cloned in Saccharomyces cerevisiae (Ma, et al., Gene 58: 201-216, 1987) using the "gap repair" method as follows. GPM1 promoter (SEQ ID NO: 72), kivD coding region from Lactococcus lactis (SEQ ID NO: 71), and ADH1 terminator (SEQ ID NO: 73) (described in US Patent Application Publication No. 2007/0092957 A1, Example 17) The yeast-E. Coli shuttle vector pRS425 :: GPM :: kivD :: ADH was digested with BbvCI and PacI restriction enzymes to free the kivD coding region. About 1 μg of the remaining vector fragment was transfected into Saccharomyces cerevisiae strain BY4741 with 1 μg sadB PCR product. Transformants were selected in synthetic complete medium lacking leucine. Appropriate recombination events producing pRS425 :: GPM-sadB were confirmed by PCR using primers N142 and N459 (SEQ ID NOs: 108 and 109).

pdc6 :: Construction of PGPM1-sadB Integrated Cassette and PDC6 Deletion:

The GPM-sadB-ADHt segment (SEQ ID NO: 79) from pRS425 :: GPM-sadB (SEQ ID NO: 78) was linked to the URA3r gene from pUC19-URA3r to create a pdc6 :: PGPM1-sadB-ADH1t-URA3r integration cassette. . pUC19-URA3r (SEQ ID NO: 80) includes a URA3 marker from pRS426 (ATCC No. 77107) flanked by a 75 bp homologous repeat sequence, allowing removal of the URA3 marker and homologous recombination in vivo. . fusion? DNA polymerase (New England Biolabs, Ipswich, Mass.), And primers 114117-11A through 114117A-11D (SEQ ID NOs: 81, 82, 83, and 84), and 114117-13A and SOE PCR (Horton, et al., Gene 77: 61-68, 1989) using pRS425 :: GPM-sadB and pUC19-URA3r plasmid DNA as templates, together with 114117-13B (SEQ ID NOs 85 and 86). The two DNA segments were linked to each other.

External primers for SOE PCR (114117-13A and 114117-13B) included a region of about 50 bp of 5 'and 3' homologous to the upstream and downstream regions of the PDC6 promoter and terminator, respectively. The completed cassette PCR fragment was transformed with BY4700 (ATCC No. 200866) and the transformants were maintained on synthetic complete medium supplemented with uracil and supplemented with 2% glucose at 30 ° C. using standard genetic techniques ( Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp. 201-202. Transformants were screened by PCR using primers 112590-34G and 112590-34H (SEQ ID NOs 87 and 88), and 112590-34F and 112590-49E (SEQ ID NOs 89 and 90) to delete the PDC6 coding region. The integration of the PDC6 locus was demonstrated. The URA3r markers were recycled by plating on synthetic complete medium supplemented with 2% glucose and 5-FOA at 30 ° C. according to standard protocols. Marker removal was confirmed by patching colonies from 5-FOA plates with SD-URA medium demonstrating no growth. The resulting identified strains have the following genotypes: BY4700 pdc6 :: PGPM1-sadB-ADH1t.

pdc1 :: Construction of PPDC1-ilvD Integration Cassette and PDC1 Deletion:

fusion? PLH468 and as a template, with DNA polymerase (New England Biolabs Inc., Ipswich, Mass.) And primers 114117-27A to 114117-27D (SEQ ID NOs: 111, 112, 113, and 114). ilvD-FBA1t segment from pLH468 (SEQ ID NO: 2) by SOE PCR (described in Horton, et al., Gene 77: 61-68, 1989) using pUC19-URA3r plasmid DNA (SEQ ID NO: 2) No. 91) was linked to the URA3r gene from pUC19-URA3r to make a pdc1 :: PPDC1-ilvD-FBA1t-URA3r integration cassette.

External primers for SOE PCR (114117-27A and 114117-27D) included about 50 bp regions of 5 'and 3' homologous to the downstream region of the PDC1 promoter and the downstream region of the PDC1 coding sequence. The completed cassette PCR fragments were transformed with BY4700 pdc6 :: PGPM1-sadB-ADH1t and the transformants were maintained on synthetic complete medium at 30 ° C. deficient in uracil and supplemented with 2% glucose at 30 ° C. using standard genetic techniques. (Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp. 201-202). Transformants were screened by PCR using primers 114117-36D and 135 (SEQ ID NOs: 92 and 93), and primers 112590-49E and 112590-30F (SEQ ID NOs: 90 and 94) to identify PDC1 that lacked the PDC1 coding sequence. The integration of the locus was demonstrated. The URA3r markers were recycled by plating on synthetic complete medium supplemented with 2% glucose and 5-FOA at 30 ° C. according to standard protocols. Marker removal was confirmed by patching colonies from 5-FOA plates with SD-URA medium demonstrating no growth. The resulting identified strain “NYLA67” has the following genotype: BY4700 pdc6 :: PGPM1-sadB-ADH1t pdc1 :: PPDC1-ilvD-FBA1t.

HIS3 deletion

To delete the endogenous HIS3 coding region, the his3 :: URA3r2 cassette was PCR amplified from the URA3r2 template DNA (SEQ ID NO: 95). URA3r2 includes URA3 markers from pRS426 (ATCC No. 77107) flanked by 500 bp homologous repeat sequences, allowing removal of URA3 markers and homologous recombination in vivo. fusion? PCR was performed using DNA polymerase (New England Biolabs, Inc., Ipswich, Mass.) And primers 114117-45A and 114117-45B (SEQ ID NOs: 96 and 97) to yield approximately 2.3 kb PCR products. Produced. The HIS3 portion of each primer was derived upstream of the 5 'region of the HIS3 promoter and downstream of the 3' region of the coding region to allow replacement of the HIS3 coding region due to the integration of the URA3r2 marker. PCR products were transformed into NYLA67 using standard genetic techniques (Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp. 201-202) and transformants were 30 Selection was made on synthetic complete media supplemented with 2% glucose at 캜 and lacking uracil. Transformants were screened by replica plating on synthetic complete medium supplemented with 2% glucose at 30 ° C. and histidine deficient, demonstrating correct integration. The URA3r markers were recycled by plating on synthetic complete medium supplemented with 2% glucose and 5-FOA at 30 ° C. according to standard protocols. Marker removal was confirmed by patching colonies from 5-FOA plates with SD-URA medium demonstrating no growth. The resulting identified strain named NYLA73 has the following genotype: BY4700 pdc6 :: PGPM1-sadB-ADH1t pdc1 :: PPDC1-ilvD-FBA1t Δhis3.

Construction of the pdc5 :: kanMX Integrated Cassette and PDC5 Deletion:

Fusion pdc5 :: kanMX4 cassette? Strain YLR134W chromosomal DNA (ATCC No) using DNA polymerase (New England Biolabs Inc., Ipswich, Mass.) And primers PDC5 :: KanMXF and PDC5 :: KanMXR (SEQ ID NOs: 98 and 99) 4034091) yielded approximately 2.2 kb PCR product. The PDC5 portion of each primer was derived upstream of the 5 'region of the PDC5 promoter and downstream of the 3' region of the coding region to allow replacement of the PDC5 coding region due to integration of the kanMX4 marker. PCR products were transformed into NYLA73 using standard genetic techniques (Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp. 201-202) and transformants were 30 Selection was made on YP medium supplemented with 1% ethanol and geneicin (200 μg / mL) at &lt; RTI ID = 0.0 &gt; Transformants were screened by PCR to demonstrate correct integration at the PDC locus where the PDC5 coding region was replaced using primers PDC5kofor and N175 (SEQ ID NOs: 100 and 101). The exact transformants identified have the following genotypes: BY4700 pdc6 :: PGPM1-sadB-ADH1t pdc1 :: PPDC1-ilvD-FBA1t Δhis3 pdc5 :: kanMX4. The strain was named NYLA74.

Plasmid vectors pRS423 :: CUP1-alsS + FBA-budA and pRS426 :: FBA-budC + GPM-sadB were transformed into NYLA74 to produce butanediol producing strain (NGCI-047).

Plasmid vectors pLH475-Z4B8 (SEQ ID NO: 140) and pLH468 were transformed into NYLA74 to produce isobutanol producing strain (NGCI-049).

Deletion of HXK2 (hexokinase II):

fusion hxk2 :: URA3r cassette? PCR amplification from the URA3r2 template (described above) using DNA polymerase (New England Biolabs Incorporated, Ipswich, Mass.) And primers 384 and 385 (SEQ ID NOs: 102 and 103), approximately 2.3 kb PCR product was generated. The HXK2 portion of each primer was derived upstream of the 5 'region of the HXK2 promoter and downstream of the 3' region of the coding region, allowing the integration of the URA3r2 marker to replace the HXK2 coding region. PCR products were transformed into NYLA73 using standard genetic techniques (Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp. 201-202) and transformants were 30 Selection was made on synthetic complete media lacking uracil supplemented with 2% glucose at ° C. Transformants were screened by PCR, using primers N869 and N871 (SEQ ID NOs: 104 and 105) to demonstrate correct integration at the HXK2 locus where the HXK2 coding region was replaced. URA3r2 markers were recycled by plating onto synthetic complete medium supplemented with 2% glucose and 5-FOA at 30 ° C. according to standard protocols. Marker removal was confirmed by patching colonies from 5-FOA plates with SD-URA medium, demonstrating no growth and correct marker removal by PCR using primers N946 and N947 (SEQ ID NOs: 106 and 107). The resulting identified strain named NYLA83 has the following genotype: BY4700 pdc6 :: PGPM1-sadB-ADH1t pdc1 :: PPDC1-ilvD-FBA1t Δhis3 Δhxk2.

Construction of the pdc5 :: kanMX Integrated Cassette and PDC5 Deletion:

The pdc5 :: kanMX4 cassette was PCR amplified as described above. PCR fragments were transformed into NYLA83 and transformants were selected and screened as described above. The correct transformant identified as NYLA84 has the following genotype: BY4700 pdc6 :: PGPM1-sadB-ADH1t pdc1 :: PPDC1-ilvD-FBA1t Δhis3 Δhxk2 pdc5 :: kanMX4.

Plasmid vectors pLH468 and pLH532 were used as standard genetic techniques (Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY) to strain NYLA84 (BY4700 pdc6 :: PGPM1-sadB-ADH1t pdc1: (PPDC1-ilvD-FBA1t Δhis3 Δhxk2 pdc5 :: kanMX4) and simultaneously transformed the resulting “butanologen NYLA84” at 30 ° C. with synthetic histidine and uracil deficient and supplemented with 1% ethanol Kept in phase.

Expression vector pLH468

pLH468 plasmid (SEQ ID NO: 2) was constructed for the expression of DHAD, KivD, and HADH in yeast and is described in US Patent Application Publication No. 2009/0305363, which is incorporated herein by reference. pLH486 was expressed from the Saccharomyces cerevisiae FBA1 promoter (nucleotides 2109-3105), followed by the ilvD gene from Streptococcus mutans (nucleotide positions 3313-4849) expressed from the FBA1 terminator (nucleotides 4858-5857) for expression of DHAD. A chimeric gene having a coding region of; Coding region of codon-optimized hepatic alcohol dehydrogenase (nucleotide 6286-7413) expressed from Saccharomyces cerevisiae GPM1 promoter (nucleotides 7425-8181) followed by ADH1 terminator (nucleotides 5962-6277) for expression of ADH. Chimeric genes having; And the coding region of the codon-optimized kivD gene from Lactococcus lactis (nucleotides 9249-10895) expressed from the TDH3 promoter (nucleotides 10896-11918) followed by the TDH3 terminator (nucleotides 8237-9235) for expression of KivD. It was constructed to contain chimeric genes.

Coding regions for Lactococcus lactis ketoisovalate decarboxylase (KivD) and hepatic alcohol dehydrogenase (HADH) are optimized for expression in Saccharomyces cerevisiae (SEQ ID NOs: 71 and 118, respectively) And DNA2.0, Inc., Menlo Park, Calif., Based on the codons provided for the plasmids pKivDy-DNA2.0 and pHadhy-DNA2.0. The encoded proteins are SEQ ID NOs: 117 and 119, respectively. Individual expression vectors for KivD and HADH were constructed. To assemble pLH467 (pRS426 :: PTDH3-kivDy-TDH3t), the vector pNY8 (SEQ ID NO: 121; also designated pRS426.GPD-ald-GPDt, US Patent Application Publication No. 2008/0182308, incorporated herein by reference No., described in Example 17), was digested with AscI and SfiI enzymes to remove the GPD promoter and ald coding region. TDH3 promoter fragment (SEQ ID NO: 122) from pNY8 was PCR amplified using 5 'primer OT1068 and 3' primer OT1067 (SEQ ID NOs 123 and 124) to add an AscI site at the 5 'end and a SpeI site at the 3' end. It was. The pNY8 vector fragment digested with AscI / SfiI was ligated with the SpeI-SfiI fragment containing the TDH3 promoter PCR product digested with AscI and SpeI and the codon optimized kivD coding region isolated from the vector pKivD-DNA2.0. Triple ligation produced the vector pLH467 (pRS426 :: PTDH3-kivDy-TDH3t). pLH467 was verified by restriction mapping and sequencing.

pLH435 (pRS425 :: PGPM1-Hadhy-ADH1t) was derived from the vector pRS425 :: GPM-sadB (SEQ ID NO: 78) described in US Provisional Application No. 61 / 058,970, Example 3, which is incorporated herein by reference. pRS425 :: GPM-sadB is the coding region from the GPM1 promoter (SEQ ID NO: 72), butanol dehydrogenase of Acromobacter xyloxidans (sadB; DNA SEQ ID NO: 69; Protein SEQ ID NO: 70: US Patent Application Publication No. 2009 / 0269823), and a pRS425 vector (ATCC No. 77106) with a chimeric gene containing the ADH1 terminator (SEQ ID NO: 73). pRS425 :: GPMp-sadB contains BbvI and PacI sites at the 5 'and 3' ends of the sadB coding region, respectively. The NheI site was added to the 5 'end of the sadB coding region by site-directed mutagenesis using primers OT1074 and OT1075 (SEQ ID NOs 126 and 127) to generate vector pRS425-GPMp-sadB-NheI, Proven by sequencing. decomposing pRS425 :: PGPM1-sadB-NheI into NheI and PacI to drop out the sadB coding region, and to the NheI-PacI fragment containing the codon optimized HADH coding region from vector pHadhy-DNA2.0 Ligation resulted in pLH435.

To combine KivD and HADH expression cassettes in a single vector, the yeast vector pRS411 (ATCC No. 87474) was digested with SacI and NotI, and in a triple ligation reaction, SalI from pLH435 containing the PGPM1-Hadhy-ADH1t cassette. Along with the -NotI fragment, the SacI-SalI fragment from pLH467 containing the PTDH3-kivDy-TDH3t cassette was ligated. This resulted in the vector pRS411 :: PTDH3-kivDy-PGPM1-Hadhy (pLH441) and was validated by restriction mapping.

To generate co-expression vectors for all three genes in the lower isobutanol pathway, ilvD, kivDy, and Hadhy, pRS423 FBA ilvD (Strep) (SEQ ID NO: 128) was used, which was used as a source of IlvD genes in the United States. Patent Application Publication No. 2010/0081154. This shuttle vector contains an F1 origin of replication (nucleotides 1423-1879) for maintenance in E. coli and a 2 micron origin (nucleotides 8082-9426) for replication in yeast. The vector has an FBA1 promoter (nucleotides 2111 to 3108; SEQ ID NO: 120) and an FBA terminator (nucleotides 4861 to 5860; SEQ ID NO: 129). It also has His markers for selection in yeast (nucleotides 504-1163), and ampicillin resistance markers for selection in E. coli (nucleotides 7092-7949). The ilvD coding region (nucleotides 3116-4828; SEQ ID NO: 115; Protein SEQ ID NO: 116) from Streptococcus mutans UA159 (ATCC No. 700610) is present between the FBA promoter and the FBA terminator that form a chimeric gene for expression. There is also a lumio tag fused to the ilvD coding region (nucleotides 4829-4849).

The first step consists of pac423 FBA ilvD (Strep) (also called pRS423-FBA (SpeI) -IlvD (Streptococcus mutans) -Rumio) blunt ended SacII sites using SacI and SacII (T4 DNA polymerase). To give a vector with a total length of 9,482 bp. The second step was to isolate the kivDy-hADHy cassette from pLH441 using SacI and KpnI (KpnI site blunt terminated using T4 DNA polymerase) to provide a 6,063 bp fragment. This fragment was ligated with a 9,482 bp vector fragment from pRS423-FBA (SpeI) -IlvD (Streptococcus mutans) -Lumio. This produced vector pLH468 (pRS423 :: PFBA1-ilvD (Strep) Lumio-FBA1t-PTDH3-kivDy-TDH3t-PGPM1-hadhy-ADH1t), which was confirmed by restriction mapping and sequencing.

build pLH532

pLH532 plasmid (SEQ ID NO: 130) was constructed for expression of ALS and KARI in yeast. pLH532 is a pHR81 vector (ATCC No. 87541) containing the following chimeric genes: 1) CUP1 promoter (SEQ ID NO: 139), acetolactate synthase coding region from Bacillus subtilis (AlsS; SEQ ID NO: 137; protein sequence Number 138) and CYC1 terminator 2 (SEQ ID NO: 133); 2) ILV5 promoter (SEQ ID NO: 134), Pf5.IlvC coding region (SEQ ID NO: 132) and ILV5 terminator (SEQ ID NO: 135); And 3) FBA1 promoter (SEQ ID NO: 136), Saccharomyces cerevisiae KARI coding region (ILV5; SEQ ID NO: 131); And CYC1 terminator.

The Pf5.IlvC coding region is a sequence encoding KARI from Pseudomonas fluorescens, which was described in US Patent Application Publication No. 2009/0163376, which is incorporated herein by reference.

The Pf5.IlvC coding region was synthesized by DNA2.0, Inc. (Menlo Park, CA; SEQ ID NO: 132) based on codons optimized for expression in Saccharomyces cerevisiae. .

pYZ090 build

pYZ090 (SEQ ID NO: 1) is based on the pHR81 (ATCC No. 87541) backbone, Bacillus subtilis expressed from the yeast CUP1 promoter (nucleotides 2-449) followed by the CYC1 terminator (nucleotides 2181-2430) for expression of ALS Lactococcus lock expressed from the yeast ILV5 promoter (2433-3626), followed by the ILV5 terminator (nucleotides 4682-5304) for the expression of the chimeric gene having the coding region of the alsS gene from the nucleotide (nucleotide positions 457-2172), and KARI It was constructed to contain a chimeric gene with the coding region (nucleotides 3634-4656) of the ilvC gene derived from TIS.

pYZ067 build

pYZ067 was constructed to contain the following chimeric genes: 1) Streptococcus expressed from yeast FBA1 promoter (nucleotides 1161-2250) followed by FBA terminator (nucleotides 4005-4317) for expression of dihydroxy acid dehydratase (DHAD) Coating region of ilvD gene from mutans UA159 (nucleotide positions 2260-3971), 2) horse expressed from yeast GPM promoter (nucleotides 5819-6575) followed by ADH1 terminator (nucleotides 4356-4671) for the expression of alcohol dehydrogenase Coding region for hepatic ADH (nucleotides 4680-5807), and 3) lactose expressed from the yeast TDH3 promoter (nucleotides 8830-9493) followed by the TDH3 terminator (nucleotides 5682-7161) for expression of ketoisovalerate decarboxylase Coding region of the KivD gene from Caucus lactis (nucleotides 7175-8821).

Constructing pRS423 :: CUP1-alsS + FBA-budA and pRS426 :: FBA-budC + GPM-sadB and pLH475-Z4B8

The construction of pRS423 :: CUP1-alsS + FBA-budA and pRS426 :: FBA-budC + GPM-sadB and pLH475-Z4B8 is described in US Patent Application Publication No. 2009/0305363, which is incorporated herein by reference.

Construction of Saccharomyces cerevisiae strain PNY2242

Strain PNY2242 was constructed at various stages from PNY1507 (described above). First, the chimeric gene was composed of the FBA1 promoter, the alsS coding region, and the CYC1 terminator was integrated upstream of the chromosome XII and TRX1 genes. The sequence of the modified locus is provided as SEQ ID NO: 196. Next, two copies of the gene encoding the hepatic alcohol dehydrogenase were incorporated into chromosomes VII and XVI. On chromosome VII, the chimeric gene was composed of the PDC1 promoter, hADH coding region, and the ADH1 terminator was placed at the fra2Δ locus (the initial deletion of FRA2 is described above). The sequence of the modified locus is provided as SEQ ID NO: 197. On chromosome XVI, the chimeric genes consisted of the PDC5 promoter, hADH coding region, and the ADH1 terminator was integrated into the region previously occupied by the long term repeat element YPRCdelta15. The sequence of the modified locus is provided as SEQ ID NO: 198. The native genes YMR226c and ALD6 were then deleted. Removal of YMR226c was just a scarris deletion of the coding region. The sequence of the modified locus is provided as SEQ ID NO: 199. The ALD6 coding region plus 700 bp upstream sequence was deleted using CRE-lox mediated marker removal (method described above), and the resulting locus contained one loxP site. The sequence of the modified locus is provided as SEQ ID NO: 200. Finally, plasmids were introduced into strains for expression of KARI (pLH702, SEQ ID NO: 201) and DHAD (pYZ067DkivDDhADH, SEQ ID NO: 202), resulting in strain PNY2242.

Where recombinant microorganisms produce isobutanol, under certain embodiments, the microorganisms exhibit high specific productivity. In addition, the volume ratio was improved to about 50%.

Without wishing to be bound by theory, it is believed that the methods described herein provide extractive fermentation methods with improved product alcohol production efficiency. As mentioned above, alcohol production using fermentation by microorganisms may be inefficient due to the alcohol toxicity threshold of the microorganisms. In some embodiments, the methods herein provide means for converting product alcohols into substances that are less toxic to microorganisms. For example, the product alcohol can be contacted with the carboxylic acid in the presence of a catalyst that esterifies the alcohol to the carboxylic acid, thereby producing an alcohol ester that is less toxic to microorganisms. In addition, the production of alcohol esters from product alcohols leads to low concentrations of product alcohols in the fermentation medium. Reducing the concentration of the product alcohol minimizes the toxic effect of the product alcohol on the microorganism, thus leading to an improvement in the production efficiency of the product alcohol.

The carboxylic acid can act as an extractant and the alcohol ester can be partitioned into the extractant. However, the partition coefficient of the extractant may be lowered by lipid contamination. To reduce the lowering of the partition coefficient of the extractant, the lipid present in the fermentation medium can be converted to the extractant, thus minimizing lipid contamination. In some embodiments, the methods herein provide a means to convert lipids present in the feedstock or biomass into extractants by catalytic hydrolysis of the lipids to carboxylic acids. Carboxylic acids produced by such hydrolysis can act as extractants or can be esterified with product alcohols to produce alcohol esters. Thus, the methods described herein provide means for maintaining the partition coefficient of the extractant (eg, lipid hydrolysis) and minimizing the toxic effects of the product alcohol (eg, esterification of the product alcohol).

The carboxylic acid can be supplied to the fermentation vessel or can be derived from lipids (eg biomass) supplied to the fermentation vessel by hydrolysis. The amount of carboxylic acid should be sufficient to form a biphasic mixture comprising an organic phase and an aqueous phase. That is, an appropriate concentration of carboxylic acid (ie, extractant) is contacted with the fermentation broth to form a biphasic mixture. Alcohol esters produced in the fermentation broth will preferentially be distributed to the organic phase since these esters are produced at concentrations above the equilibrium concentration of the aqueous phase. The alcohol ester containing organic phase can be separated from the fermentation broth, the product alcohol can be recovered from the organic phase and the extractant can be recycled to the fermentation vessel.

In addition, although various embodiments of the present invention have been described above, it should be understood that these are given by way of example only and not limitation. It will be apparent to those skilled in the art that various changes in form and details may be made without departing from the spirit and scope of the invention. Accordingly, the breadth and scope of the present invention should not be limited by any of the above-described exemplary embodiments, but should be defined by the claims and their equivalents.

All publications, patents, and patent applications mentioned in the specification are indicative of proficiency in the art to which the invention pertains, and to the same extent as if each individual publication, patent, or patent application was specifically indicated to be incorporated by reference. It is incorporated herein by reference.

Example

The following non-limiting examples will further illustrate the present invention. Although the following examples include corn as feedstock and COFA as carboxylic acid, it should be understood that other biomass sources may be used as feedstock, and acids other than COFA may be provided as carboxylic acids without departing from the present invention. do. In addition, although the following examples include butanol and butyl ester production, other alcohols, including ethanol, and alcohol esters can be produced without departing from the present invention.

As used herein, the meaning of the abbreviations used was as follows: "g" means gram (s), "kg" means kilogram (s), and "L" means liter (s) "ML" means milliliter (s), "μl" means microliter (s), "mL / L" means milliliter (s) per liter, and "mL / min" means Milliliter (s) per minute, "DI" means deionization, "uM" means micrometer (s), "nm" means nanometer (s), "w / v" Means weight / volume, "OD" means optical density, "OD ₆₀₀ " means optical density at a wavelength of 600 nM, "dcw" means dry cell weight, and "rpm" Revolutions per minute, "° C" means degrees Celsius, "° C / min" means degrees Celsius per minute, "slpm" means standard liters (s) per minute, "ppm" Means one millionth and "pdc" is followed by the enzyme number Attached to pyruvate means bait Cartesian decarboxylase enzyme.

General way

Seed flask growth

Saccharomyces cerevisiae strains genetically engineered to produce isobutanol from a carbohydrate source, with pdc1 deletion, pdc5 deletion, and pdc6 deletion, were 0.55 to 1.1 g / L dcw (OD ₆₀₀ 1.3) in seed flasks from the freeze culture. To 2.6-Thermo Helios α Thermo Fisher Scientific Inc., Waltham, Mass., USA. Cultures were grown at 23-26 ° C. in an incubator rotating at 300 rpm. Frozen cultures were previously stored at -80 ° C. The composition of the first seed flask medium was as follows:

3.0 to 5.0 g / L dextrose

3.0 to 3.5 g / L ethanol, anhydrous

3.7 g / L ForMedium ™ Synthetic Complete Amino Acids (Kaiser) Drop Out:

Without HIS, without URA (Reference number DSCK162CK)

Difco yeast nitrogen base free of 6.7 g / L amino acid (No. 291920).

8-12 milliliters of the first seed flask culture was transferred to a 2 L flask and grown at 30 ° C. in an incubator rotating at 300 rpm. The second seed flask has 220 mL of the following medium:

30.0 g / L dextrose

5.0 g / L ethanol, anhydrous

3.7 g / L Fordium ™ Synthetic Complete Amino Acid Dropout:

Without HIS, without URA (Reference number DSCK162CK)

6.7 g / L Amino Acid Free Difco Yeast Nitrogen Base (No. 291920)

0.2 M MES buffer titrated to pH 5.5-6.0.

Cultures were grown to 0.55 to 1.1 g / L dcw (OD ₆₀₀ 1.3 to 2.6). An addition of 30 mL of a solution containing 200 g / L peptone and 100 g / L yeast extract was added at this cell concentration. Then, 0.2 uM filter sterilized HD Osenol (OCENOL)? An addition of 250-300 mL of 90/95 oleyl alcohol (Cognis, Monheim, Germany) was added to the flask. The culture is continued to grow to> 4 g / L dcw (OD ₆₀₀ > 10) before harvesting and adding to fermentation.

Fermentation manufacturer

Initial Fermentation Vessel Manufacturing

House water was injected into a glass jacked 2 L fermentation vessel (Sartorius AG, Göttingen, Germany) to a liquefied weight of 66%. The pH probe (Hamilton Easyferm Plus K8, Part No. 238627, Hamilton Bonaduz AG, Bonaduz, Switzerland) was used with the Sartorius DCU-3 Control Tower Calibration. Corrected through the menu. Zero calibration was performed at pH = 7. Span correction was performed at pH = 4. The probe was then injected into the fermentation vessel through a stainless steel head plate. Dissolved oxygen probe (pO ₂ probe) was also injected into the fermentation vessel through the head plate. The tubing used to carry the nutrients, the cultivation solution, the extraction solvent, and the base was attached to the head plate and the ends were foiled. The entire fermentation vessel was injected into a Steris (Steris Corporation, Mentor, Ohio) autoclave and sterilized for 30 minutes in a liquid cycle.

The fermentation vessel was removed from the autoclave and placed in a load cell. The jacket water supply and return lines were connected to household water and clean drain, respectively. The condenser coolant inline and the coolant outline were connected to a 6-L recycle temperature bath operating at 7 ° C. The vent line, which transfers the gas from the fermentation vessel, was connected to a transfer line connected to a Thermo mass spectrometer (Prima dB, Thermo Fisher Scientific Inc., Waltham, Mass.). The sparger line was connected to the gas supply line. Tubing for addition of nutrients, cultivation fluid, extraction solvent, and base was plumbed or clamped through the pump.

Fermentation vessel temperature was adjusted to 55 ° C. with a thermocouple and domestic water circulation loop. Wet corn kernels (# 2 yellow maize maize) were ground using a hammer mill with a 1.0 mm screen, and then the obtained whole cornmeal was fed at an injection amount of 29 to 30% (dry corn solids weight) of the liquefaction reactant. Added to fermentation vessel.

Before liquefaction ( pre - liquefaction ) Lipase  process

Lipase enzyme stock was added to the fermentation vessel to a final lipase concentration of 10 ppm. The fermentation vessel was maintained at 55 ° C., 300 rpm, and 0.3 slpm N ₂ overlay for> 6 hours. After the lipase treatment was completed, liquefaction was performed as described later (liquefaction).

liquefaction

Alpha-amylase was added to the fermentation vessel according to its specification while the fermentation vessel was mixed at 300 to 1200 rpm with sterile house N ₂ added at 0.3 slpm through a sparger. The temperature setpoint was changed from 55 ° C to 85 ° C. When the temperature reached> 80 ° C, the liquefaction cooking time was started and the liquefaction cycle was maintained at> 80 ° C for 90 to 120 minutes. After completion of the liquefaction cycle, the fermentation vessel temperature set point was set to a fermentation temperature of 30 ° C. N ₂ was directed back from the sparger to the headspace to prevent bubble generation without the addition of a chemical antifoam.

After liquefaction ( post - liquefaction ) Lipase  process

After completion of the liquefaction cycle (liquefaction), the fermentation vessel temperature was set to 55 ° C instead of 30 ° C. If necessary, a bolus addition of acid or base was performed to adjust the pH manually to pH = 5.8. Lipase enzyme stock was added to the fermentation vessel to a final lipase concentration of 10 ppm. The fermentation vessel was maintained at 55 ° C., 300 rpm, and 0.3 slpm N ₂ overlay for> 6 hours. After the lipase treatment was completed, the fermentation vessel temperature was set to 30 ° C.

Lipase heat inactivation treatment (heat kill treatment method)

To inactivate the lipase, the fermentation vessel temperature was maintained at> 80 ° C for> 15 minutes. After the heat deactivation treatment was completed, the fermentation vessel temperature was set to 30 ° C.

Addition of nutrients before inoculation

Ethanol (7 mL / L, volume after inoculation, 200 proof, anhydrous) was added to the fermentation vessel immediately before inoculation. Thiamine was added at a final concentration of 20 mg / L immediately before inoculation, and 100 mg / L nicotinic acid was also added.

Before vaccination Orail  Alcohol or Corn Oil Fatty Acids Added

Immediately after inoculation, 1 L / L of oleyl alcohol or corn oil fatty acid (volume after inoculation) was added.

Fermentation operation

Fermentation vessel inoculation

The fermentation vessel pO ₂ probe was calibrated to zero with N ₂ added to the fermentation vessel. While sterile air sparging at 300 rpm, the fermentation vessel pO ₂ probe was calibrated to its span. The fermentation vessel was inoculated at> 4 g / L dcw after the second seed flask. The shake flask was removed from the incubator / shaker for 5 minutes to separate the oleyl alcohol phase and the aqueous phase. The aqueous phase (110 mL) was transferred to a sterile inoculation vessel. The inoculum was injected into the fermentation vessel through a peristaltic pump.

Fermentation vessel operating conditions

Fermentation vessels were operated at 30 ° C. during the entire growth and production phase. It was possible to bring the pH of 5.7 to 5.9 down to a control setpoint of 5.2 without adding pH to any acid. The pH was controlled to pH = 5.2 with ammonium hydroxide during the remaining growth and production stages. Aseptic air was added to the fermentation vessel at 0.3 slpm through a sparger during the remaining growth and production stages. pO ₂ was set to be controlled at 3.0% using the Statorius DCU-3 control box PID control loop, with only agitation control, the stirrer minimum set at 300 rpm and the maximum set at 2000 rpm. α-amylase (glucoamylase) was added to feed glucose through co-glycosylation fermentation of liquefied corn mash. Glucose was maintained in excess (1-50 g / L) as long as starch could be used for saccharification.

analysis

Gas analysis

Process air was analyzed on a Thermo Prima (Thermo Fisher Scientific Incorporated, Waltham, Mass.) Mass spectrometer. This was the same process air that was sterilized and then added to each fermentation vessel. The offgas of each fermentation vessel was analyzed on the same mass spectrometer. These thermo prima dBs are calibrated at 6:00 AM every Monday morning. For calibration checks, a schedule was made through Gas Works v1.0 (Thermo Fisher Scientific, Inc., Waltham, Mass.) Software associated with the mass spectrometer. The calibrated gas was as follows:

Carbon dioxide was checked at 5% and 15% during the calibration cycle along with the gas in other known containers. Oxygen was checked at 15% with gas in other known containers. Based on the off-gas analysis of each fermentation vessel, the amount of stripped isobutanol, consumed oxygen, and carbon dioxide breathed into the off-gas was determined using a mass spectrometry analysis of the mass spectrometer and gas flow to the fermentation vessel (mass flow controller). It was measured by. The gas treatment rate per hour was calculated and then added up during fermentation.

Cell mass measurement

A 0.08% Trypan Blue solution was prepared by diluting 0.4% Trypan Blue in NaCl (VWR BDH8721-0) 1: 5 with 1 × PBS. A 1.0 mL sample was taken from the fermentation vessel and injected into a 1.5 mL Eppendorf centrifuge tube and centrifuged for 5 min at 14,000 rpm at Eppendorf, 5415C. After centrifugation, the upper solvent layer was removed using an m200 Variable Channel BioHit pipette with 20-200 μl Bioheat Pipette Tips. Care was taken not to remove the layer between the solvent layer and the aqueous layer. Once the solvent layer was removed, the samples were resuspended using Vortex-Genie® set at 2700 rpm.

A series of dilutions was needed to prepare the ideal concentration for the hemocytometer count. When the OD is 10, a 1:20 dilution was performed to achieve 0.5 OD, which would give an ideal amount of cells, 20-30, counted per square. In order to reduce inaccuracies in dilution due to corn solids, multiple dilutions with cut 100-1000 μl bioheat pipettes were required. The tip was cut about 1 cm to increase the aperture, preventing the tip from clogging. For a 1:20 final dilution, an initial 1: 1 dilution of the fermentation sample and 0.9% NaCl solution was prepared. Next, a 1: 1 dilution of the previous solution (ie, an initial 1: 1 dilution) and a 0.9% NaCl solution (second dilution) were generated, followed by 1: 1 of the second dilution and trypan blue solution. Five dilutions were generated. Samples were swirled between each dilution and the cutting tips rinsed with 0.9% NaCl and trypan blue solution.

The cover slip was carefully placed on top of the hemocytometer (Hausser Scientific Bright-Line 1492). Aliquots (10 μl) were taken from the final trypan blue dilution and injected into the hemocytometer using an m20 barrierable channel bioheat pipette with 2-20 μl bioheat pipette tips. The hemocytometer was placed on a Zeis Axioskop 40 microscope at 40 × magnification. The center quadrant was divided into 25 squares and then the four corners and center squares of the two chambers were counted and recorded. After counting the two chambers, the average is taken and multiplied by the dilution factor (20), then multiplied by the number of squares of the hemocytometer's quadrant, 25, divided by 0.0001 mL, which is the counted quadrant volume. The sum of these calculations is the cell count per mL.

Aqueous phase  Of fermentation products LC  analysis

Samples were refrigerated until ready for processing. Samples were removed from cold storage and brought to room temperature (about 1 hour). About 300 μl of sample was transferred to a 0.2 um centrifuge filter (Nanosep® MF modified nylon centrifuge filter) using an m1000 barrierable channel bioheat pipette with a 100 to 1000 μl bioheat pipette tip, followed by eppen Dorf, 5415C, was centrifuged for 5 minutes at 14,000 rpm. About 200 μl filtered sample was transferred to a 1.8 auto sampler vial with 250 μl glass vial insert with polymer pits. Prior to vortexing the sample using a Vortex-Genie® set at 2700 rpm, the vial was capped using a screw cap with a PTFE septum.

The Agilent 1200 Series LC with a two-part sample, isocratic pump, vacuum degasser, heated column compartment, sampler cooling system, UV DAD detector and RI detector. It was flowed in. The column used was Bio-Rad Cation H refill, Aminex HPX-87H with a 30 × 4.6 guard column, 300 × 7.7. The column temperature was 40 ° C. and the mobile phase was 0.01 N sulfuric acid at a flow rate of 0.6 mL / min for 40 minutes. The results are shown in Table 1.

[Table 1]

GC Analysis of Fermentation Products in Solvent Phase

Samples were refrigerated until ready for processing. Samples were removed from cold storage and brought to room temperature (about 1 hour). About 150 μl of sample was transferred to a 1.8 auto sampler vial with a 250 μl glass vial insert with polymer pits using an m1000 barrier channel bioheat pipette with 100 to 1000 μl bioheat pipette tips. The vial was capped using a screw cap with a PTFE septum.

Samples were then flowed in an Agilent 7890A GC with 7683B injector and G2614A auto sampler. The column was an HP-InnoWax column (30 m × 0.32 mm ID, 0.25 μm film). The carrier gas was helium at a flow rate of 1.5 mL / min measured at 45 ° C. with a constant head pressure; The injector split was 1:50 at 225 ° C .; The oven temperature was 1.5 minutes at 45 ° C. for 1.5 minutes, 45 ° C. to 160 ° C. at 10 ° C./min, followed by 14 minutes at 230 ° C. at 35 ° C./min, during a 29 minute flow time. Flame ionization detection was used with 40 mL / min helium makeup gas at 260 ° C. The results are shown in Table 2.

[Table 2]

Samples for analyzing fatty acid butyl esters were flowed on an Agilent 6890 GC equipped with a 7683B injector and a G2614A auto sampler. The column was an HP-DB-FFAP column (15 mx 0.53 mm ID (Megabore), 1-micron film thickness column (30 mx 0.32 mm ID, 0.25 μm film). Carrier gas measured at 45 ° C. with constant head pressure. Helium at a flow rate of 3.7 mL / min; injector split 1:50 at 225 ° C .; oven temperature from 100 ° C. to 250 ° C. at 100 ° C. and 2.0 min at 100 ° C. during a 26 minute flow time. It was then 9 minutes at 250 ° C. Flame ionization detection was used with 40 mL / min helium constituent gas at 300 ° C. The following GC standards (Nu-Chek Prep; Elsyon, MN) , Isobutyl palmitate, isobutyl stearate, isobutyl oleate, isobutyl linoleate, isobutyl linoleate, isobutyl arachidate .

Examples 1 to 14 describe various fermentation conditions that can be used in the required method. As an example, some of the fermentations were subjected to lipase treatment before liquefaction, and others were lipase treatment after liquefaction. In another example, the thermal inactivation treatment was performed on the fermentation. After fermentation, the effective isobutanol titer (Eff Iso Titer), i.e. the total number of grams of isobutanol produced per liter of the aqueous phase, was determined. The results are shown in Table 3.

Example 1-(control)

Experimental identifier 2010Y014 included: seed flask growth method, initial fermentation vessel preparation method, liquefaction method, nutrient addition method before inoculation, fermentation vessel inoculation method, fermentation vessel operating condition method, and all analytical methods. Oleyl alcohol was added in a single batch 0.1-1.0 hours after inoculation. Butanologen was NGCI-070.

Example 2

Experiment identifier 2010Y015 included: seed flask growth method, initial fermentation vessel preparation method, liquefaction method, liquefaction method after liquefaction, nutrient addition method before inoculation, fermentation vessel inoculation method, fermentation vessel operating condition method, and all analytical methods. . Oleyl alcohol was added in a single batch 0.1-1.0 hours after inoculation. Butanologen was NGCI-070.

Example 3

Experiment identifier 2010Y016 included: seed flask growth method, initial fermentation vessel preparation method, liquefaction method, liquefaction method after liquefaction, nutrient addition method before inoculation except ethanol, fermentation vessel inoculation method, fermentation vessel operating conditions Methods, and all analysis methods. Oleyl alcohol was added in a single batch 0.1-1.0 hours after inoculation. Butanologen was NGCI-070.

Example 4

Experiment identifier 2010Y017 included: seed flask growth method, initial fermentation vessel preparation method, liquefaction method, post-liquefaction heat sterilization method, nutrient addition method before inoculation except ethanol, fermentation vessel inoculation method, fermentation vessel operation Conditional methods, and all analytical methods. Oleyl alcohol was added in a single batch 0.1-1.0 hours after inoculation. Butanologen was NGCI-070.

Example 5

Experimental identifier 2010Y018 included: seed flask growth method, initial fermentation vessel preparation method, liquefaction method, liquefaction method after liquefaction except for adding only 7.2 ppm of lipase after liquefaction, post-liquefaction heat sterilization method, Nutrient addition method before inoculation, fermentation vessel inoculation method, fermentation vessel operating condition method, and all analytical methods. Oleyl alcohol was added in a single batch 0.1-1.0 hours after inoculation. Butanologen was NGCI-070.

Example 6-(control)

Experiment identifier 2010Y019 included: seed flask growth method, initial fermentation vessel preparation method, liquefaction method, post-liquefaction heat sterilization method, nutrient addition method before inoculation, fermentation vessel inoculation method, fermentation vessel operating condition method, and all assays Way. Oleyl alcohol was added in a single batch 0.1-1.0 hours after inoculation. Butanologen was NGCI-070.

Example 7-(control)

Experiment identifier 2010Y021 included: seed flask growth method, initial fermentation vessel preparation method, lipase treatment method before liquefaction, liquefaction method, heat sterilization treatment during liquefaction, nutrient addition method before inoculation, fermentation vessel inoculation method, fermentation vessel operation Conditional methods, and all analytical methods. Oleyl alcohol was added in a single batch 0.1-1.0 hours after inoculation. Butanologen was NGCI-070.

Example 8

Experiment identifier 2010Y022 included: seed flask growth method, initial fermentation vessel preparation method, liquefaction method, nutrient addition method before inoculation, fermentation vessel inoculation method, fermentation vessel operating condition method, and all analytical methods. Oleyl alcohol was added in a single batch 0.1-1.0 hours after inoculation. Butanologen was NGCI-070.

Example 9

Experiment identifier 2010Y023 included: seed flask growth method, initial fermentation vessel preparation method, liquefaction method, liquefaction method after liquefaction, non-heat sterilization treatment, nutrient addition method before inoculation, fermentation vessel inoculation method, fermentation vessel operating condition method , And all analytical methods. Corn oil fatty acid produced as crude corncob was added in a single batch 0.1-1.0 hours after inoculation. Butanologen was NGCI-070.

Example 10

Experiment identifier 2010Y024 included: seed flask growth method, initial fermentation vessel preparation method, lipase treatment method before liquefaction, liquefaction method, heat sterilization during liquefaction, nutrient addition method before inoculation except no addition of ethanol Fermentation vessel inoculation method, fermentation vessel operating condition method, and all analytical methods. Oleyl alcohol was added in a single batch 0.1-1.0 hours after inoculation. Butanologen was NGCI-070.

Example 11

Experiment identifier 2010Y029 included: seed flask growth method, initial fermentation vessel preparation method, lipase treatment method before liquefaction, liquefaction method, heat sterilization treatment during liquefaction, nutrient addition method before inoculation, fermentation vessel inoculation method, fermentation vessel operation Conditional methods, and all analytical methods. Corn oil fatty acid produced as crude corncob was added in a single batch 0.1-1.0 hours after inoculation. Butanologen was NGCI-070.

Example 12

Experiment identifier 2010Y030 included: seed flask growth method, initial fermentation vessel preparation method, liquefaction method before liquefaction, liquefaction method, heat sterilization during liquefaction, nutrient addition method before inoculation except no addition of ethanol Fermentation vessel inoculation method, fermentation vessel operating condition method, and all analytical methods. Corn oil fatty acid produced as crude corncob was added in a single batch 0.1-1.0 hours after inoculation. Butanologen was NGCI-070.

Example 13-(Control)

Experiment identifier 2010Y031 included: seed flask growth method, initial fermentation vessel preparation method, liquefaction method, liquefaction method after liquefaction, non-heat sterilization method, nutrient addition method before inoculation, except no addition of ethanol, fermentation Vessel inoculation method, fermentation vessel operating condition method, and all assay methods. Corn oil fatty acid produced as crude corncob was added in a single batch 0.1-1.0 hours after inoculation. Butanologen was NGCI-070.

Example 14

Experiment identifier 2010Y032 included: seed flask growth method, initial fermentation vessel preparation method, liquefaction method, liquefaction method after liquefaction, non-heat sterilization treatment, nutrient addition method before inoculation, fermentation vessel inoculation method, fermentation vessel operating condition method , And all analytical methods. Corn oil fatty acid produced as crude corncob was added in a single batch 0.1-1.0 hours after inoculation. Butanologen was NGCI-070.

[Table 3]

Examples 15 and 16 show a comparison of fermentation and isobutanol production in the presence and absence of lipase treatment after liquefaction. The results are shown in Tables 4 and 5.

Example  15

Experimental identifier 2010Y026 included: seed flask growth method, initial fermentation vessel preparation method, liquefaction method, liquefaction method after liquefaction, nutrient addition method before inoculation, fermentation vessel inoculation method, fermentation vessel operating condition method, and all analytical methods . Corn oil fatty acid produced as crude corncob was added in a single batch 0.1-1.0 hours after inoculation. Corn oil fatty acid extraction solvent was added in the same volume as the broth volume. Butanologen was PNY2205. In the fermentation time of 46 hours to 61 hours, 274 g of 50% w / w sterile glucose solution was added because the glucose produced by the corn mash was almost consumed.

Example 16

Experiment identifier 2010Y027 included: seed flask growth method, initial fermentation vessel preparation method, liquefaction method, nutrient addition method before inoculation, fermentation vessel inoculation method, fermentation vessel operating condition method, and all analytical methods. HD Ossenol? 90/95 (oleyl alcohol, CAS No. 143-28-2, Cognis, Monheim, Germany) was added in a single batch 0.1-1.0 hours after inoculation. Oleyl alcohol extraction solvent was added in the same volume as the broth volume. Butanologen was PNY2205.

[Table 4]

[Table 5]

Examples 17-22 show a comparison of the effects of fresh extract versus recycled extract on fermentation and isobutanol production. The results are shown in Table 6. For these examples, 2 L and 10 L fermentations were prepared as described below.

10 L preseed flask growth

Saccharomyces cerevisiae strains (strain PNY2242 described above) genetically engineered to produce isobutanol from a carbohydrate source with pdc1 deletion, pdc5 deletion, and pdc6 deletion (see above strain PNY2242) from seed cultures (125 mL vented) vented) in a 10 mL synthetic medium in a flask) to 0.6-0.7 g / L dcw (OD ₆₀₀ 1.5-2.5-Thermo Helios α Thermo Fisher Scientific Inc., Waltham, Mass.). Cultures were grown at 29-31 ° C. in an incubator rotating at 260 rpm. Frozen cultures were previously stored at -80 ° C. The composition of the synthetic seed flask medium was as follows:

10.0 g / L dextrose

3.5 mL / L ethanol, anhydrous

3.7 g / L Fordium ™ Synthetic Complete Amino Acid Dropout:

Without HIS, without URA (Reference number DSCK162CK)

6.7 g / L Amino Acid Free Difco Yeast Nitrogen Base (No. 291920)

1: 1 :: Tween 80: 1% ergosterol in ethanol

Two milliliters of the first seed flask culture was transferred to 25 mL in a 250 mL vented flask and grown at 29-31 ° C. in an incubator rotating at 260 rpm. The second seed flask uses the same synthetic medium as used above.

Cultures were grown to 0.6-0.7 g / L dcw (OD ₆₀₀ 1.0-3.0). 8 mL of this second flask culture was then added to three flasks (2 L, vented, baffle flasks) with 200 mL of synthetic medium. Cultures were grown in incubator at 29-31 ° C. for 18-24 hours. Three seed flasks use the same synthetic medium as used in the two first seed flasks. These three flasks (flask broth 600 mL) were used to inoculate the growth tank with a final aqueous phase volume of 6 L.

10 L propagation tank liquefaction

10 L, prepared for use with B. Braun BioStat C fermentation vessels. Inline pH probes were injected into the fermentation vessel. Zero calibration was performed at pH = 7. Span correction was performed at pH = 4. The probe was then injected into the fermentation vessel through the side port. Dissolved oxygen probe (pO ₂ probe) was also injected into the fermentation vessel through the side port. The tubing used to carry the nutrients, the cultivation solution, the extraction solvent, and the base was attached to the head plate and the ends were foiled. Valves for harvesting and sampling were sterilized with low pressure steam and steam traps at> 121 ° C for> 20 minutes.

Fermentation vessel temperature was adjusted to 30 ° C. with a thermocouple and domestic water circulation loop. Wet corn kernels (# 2 yellow maize maize) were ground using a hammer mill with a 1.0 mm screen, and then the obtained whole cornmeal was fed at an injection amount of 10-20% (dry corn solids weight) of the liquefaction reactant. Added to fermentation vessel. Difco yeast extract was added to the fermentation vessel at 0.5% w / w of the total batch weight.

Alpha-amylase was added to the fermentation vessel according to its specification while the fermentation vessel was mixed at 300-1500 rpm with sterile house N ₂ added at 1-2 slpm via a sparger. To ensure good mixing, the temperature setpoint was changed from 55 ° C. to 95 ° C. in a 5 ° C. step change while maintaining 5-15 minutes in each step. When the temperature reached> 90 ° C, the liquefaction cooking time was initiated and the liquefaction cycle was maintained at> 90 ° C for 60 minutes. After completion of the liquefaction cycle, the fermentation vessel temperature set point was set to a fermentation temperature of 30 ° C. N ₂ was directed back from the sparger to the headspace to prevent bubble generation without the addition of a chemical antifoam.

10L breeder tank operation

The fermentation vessel pO ₂ probe was calibrated to zero with N ₂ added to the fermentation vessel. While sterile air sparging at 400 rpm, the fermentation vessel pO ₂ probe was calibrated to its span. Fermentation vessels were inoculated at the end of the preseed flask growth stage. Three shake flasks were removed from the incubator / shaker and added to sterile containers. The contents of the sterilization vessel were added to 5.3-5.5 L of liquefaction mash prepared during the propagation tank liquefaction process.

Fermentation temperature was controlled to 29-31 ° C. Stirring speed was fixed at 400 rpm. Air was sparged to 2.0 slpm during the entire fermentation. The pH was controlled between 5.4 and 5.5 using NH ₄ OH and PID control loops in the fermentation vessel. The back pressure set in the fermentation vessel controlled by the PID loop controlling the back pressure control valve was 30 to 50 kPa (0.3 to 0.5 bar).

16-20 hours after inoculation, glucoamylase (1.8 mL of Distillase® L-400, Genencor, Palo Alto, Calif.) Was added to initiate simultaneous saccharification fermentation and dissolved. Glucose was released from starch. Also, the 5.5 L HD Osenol? 90/95 (oleyl alcohol, Cognis, Monheim, Germany) was added to the fermentation vessel. After 34 to 36 hours, the stirrer speed was reduced to 100 rpm. After 10 minutes, the stirrer was turned off and the air flow for the fermentation vessel was changed from sparge mode to overlay mode.

10L production tank liquefied

10 L production tank liquefaction was performed as described above. The fermentation vessel temperature was controlled to 30 ° C. with thermocouples and domestic water circulation loops. Wet corn kernels (# 2 yellow maize maize) are ground using a hammer mill with a 1.0 mm screen and the resulting corncob flour is fermented with an injection amount of 25 to 35% (dry corn solids weight) of the liquefaction reactant. Was added. 75 mL of 100 × vitamin solution (2 g / L thiamine and 10 g / L nicotinic acid) was added to the fermentation vessel. Alpha-amylase was added to the fermentation vessel as described above. In addition, after returning the fermentation vessel to 30 ° C., 6-7 mL / L of absolute ethanol was added to the fermentation vessel.

10L production tank operation

The fermentation vessel pO ₂ probe was calibrated to zero with N ₂ added to the fermentation vessel. While sterile air sparging at 400 rpm, the fermentation vessel pO ₂ probe was calibrated to its span. Fermentation vessels were inoculated into growth tanks. Aseptic transfer was performed from the growth tank after 36 hours of growth time in the growth tank and fermentation agitation was stopped for> 10 minutes. This resulted in a significant separation of the oleyl alcohol and the aqueous phase. Aseptic conveyance was done from the harvesting valve of the propagation tank, located at the bottom of this fermentation vessel. About 10% v / v was added to the production tank based on the final nonsolvent volume of the tank after transfer.

Fermentation temperature was controlled to 29-31 ° C. Stirring speed was fixed at 400 rpm. Air was sparged to 2.0 slpm during the entire fermentation. The pH was controlled between 5.2 and 5.3 using NH ₄ OH and PID control loops in the fermentation vessel. The back pressure set in the fermentation vessel controlled by the PID loop controlling the back pressure control valve was 30 to 50 kPa (0.3 to 0.5 bar).

Immediately before inoculation, 25 to 35% v / v Cognis Emery? 610 soybean fatty acid was aseptically added to the fermentation vessel. Fermentation vessels were inoculated with 10% v / v fermentation broth after completion of the 10 L propagation tank operating method. Immediately after inoculation, glucoamylase (Distylase® L-400) was added to release glucose from starch. Additional glucoamylase additions were made when needed to maintain glucose excess. Immediately after inoculation, lipase (Novozymes repolarase-100L) was added to the fermentation vessel at 4-15 ppm.

2 L preseed flask growth

2 L preseed flask growth was prepared using Saccharomyces cerevisiae strain (strain PNY2242 described above) and 0.6-0.7 g in seed flask (10 mL synthetic medium in 125 mL vented flask) from freeze culture. / L dcw (OD ₆₀₀ 1.5-2.5-Thermo Helios α Thermo Fisher Scientific Inc., Waltham, Mass.). Cultures were grown at 29-31 ° C. in an incubator rotating at 260 rpm. Frozen cultures were previously stored at -80 ° C. The composition of the synthetic seed flask medium was as follows:

10.0 g / L dextrose

3.5 mL / L ethanol, anhydrous

3.7g / L Fordium Synthetic Fully Amino Acid (Kaiser) Drop Out:

Without HIS, without URA (Reference number DSCK162CK)

6.7 g / L Amino Acid Free Difco Yeast Nitrogen Base (No. 291920)

1: 1 :: Twin 80: 1% ergosterol in ethanol

Two milliliters of the first seed flask culture was transferred to 25 mL in a 250 mL vented flask and grown at 29-31 ° C. in an incubator rotating at 260 rpm. The second seed flask uses the same synthetic medium as used above.

Cultures were grown to 0.6-0.7 g / L dcw (OD ₆₀₀ 1.0-3.0). 4 mL of this second flask culture was then added to 100 mL of corn mash centrate in a 2 L flask. Cultures were grown in incubator at 29-31 ° C. for 18-24 hours. Then 500 mL of HD Osenol? 90/95 (oleyl alcohol, Cognis, Monheim, Germany) was added to the flask. The flask was grown for 6-8 hours. Then 1.2 g distilase in 80 mL of deionized water? 2 mL of L-400 (Genenco, Palo Alto, Calif.) Was added to the centrate to release glucose from the dissolved starch in the centrate. Cultures were continued to grow for 18 to 24 hours. Final biomass concentrations were 6-12 g / L dcw.

Corn was liquefied with the following recipe to prepare corn mash centrate:

1150 g tap water

340.5 g 1 mm screened ground maize

13.5 g yeast extract (Difco No. 9102333, low dusting)

27 g peptone

4.1 g urea

40.5 mg nicotinic acid

40.5 mg thiamine.

The material was then centrifuged for 30 minutes in a Sorval RC5C centrifuge. Supernatants were separated from solid pellets. The supernatant was heated in a steric autoclave for 5 minutes of liquid cycle, which is defined as the centrate.

2 L Fermentation Manufacture

2 L initial fermentation vessel manufacturing

A 2 L initial fermentation vessel preparation was prepared as described above. The fermentation vessel temperature was controlled to 55 ° C. with thermocouple and domestic water circulation loops. Wet corn kernels (# 2 yellow maize maize) are ground using a hammer mill with a 1.0 mm screen and the resulting corncob meal is fermented in an amount of 25-30% (dry corn solids weight) of the liquefaction reactant. Was added. In addition, liquefaction was continued as mentioned above. Alpha-amylase was added to the fermentation vessel according to its specification while the fermentation vessel was mixed at 300 to 1200 rpm with sterile house N ₂ added at 0.3 slpm through a sparger.

Add before 2 L inoculation

The following nutrients were added to the fermentation vessel after inoculation and on the basis of volume after inoculation:

30 mg / L nicotinic acid

30 mg / L Thiamine

1% ergosterol w / v solution in 1 mL / L 1: 1: Twin 80: ethanol

6.3 mL / L ethanol

2 g / L urea.

2 L fermentation vessel inoculation

The fermentation vessel pO ₂ probe was calibrated to zero with N ₂ added to the fermentation vessel. While sterile air sparging at 300 rpm, the fermentation vessel pO ₂ probe was calibrated to its span. Fermentation vessels were inoculated at the end of the preseed flask growth stage. The shake flask was removed from the incubator / shaker and centrifuged for 30 minutes. The liquid (oleyl alcohol and aqueous supernatant) was discarded and the cell pellet was resuspended in preseed flask growth medium (synthetic medium). 100 mL of the aqueous phase was transferred to a sterile inoculation vessel. The inoculum was injected into the fermentation vessel through a peristaltic pump.

Lipase addition after 2 L inoculation

Repolarase? A solution (100 L stock) was prepared at an enzyme concentration of 1.2 to 1.4 mg / mL. The solution was inoculated into the fermentation vessel and then added to the fermentation at the desired ppm concentration based on the nonsolvent volume. The addition time was <1 hour after inoculation of the fermentation vessel.

Add 2 L soybean oil fatty acid

In a fermentation vessel, Virgin Cognis Emery? 610 recycled cognis emery containing soy fatty acids or 0-30% by weight fatty acid butyl ester? 0.1-0.5 L / L (volume after inoculation) of 610 soy fatty acids were added.

2L fermentation vessel operating conditions

Fermentation vessels were operated at 30 ° C. during the entire growth and production phase. It was possible to bring the pH of 5.7 to 5.9 down to a control setpoint of 5.25 without adding pH to any acid. The pH was controlled to pH = 5.2 with ammonium hydroxide during the remaining growth and production stages. Sterile air was added to the fermentation vessel through a sparger at 0.2-0.3 slpm during the remaining growth and production stages. pO ₂ was not controlled. The stirrer was set to fixed rpm at 300 rpm. The stirring shaft has two Rushton impellers below the aqueous level and one pitched blade impeller above the aqueous level. Glucoamylase was added to feed glucose via co-glycosylation fermentation of liquefied corn mash. Starch was kept above glucose (1-50 g / L) as long as available for saccharification.

For cell volume measurement using the procedure described above, 5-20 mL samples were taken from the fermentation vessel, injected into a centrifuge tube and centrifuged. In addition, analytical methods such as gas analysis and LC analysis of the fermentation product in the aqueous phase and GC analysis of the fermentation product in the solvent phase were performed as described above.

Fermentation conditions for Examples 17-22 are provided below and a summary of the results (regenerated soybean fatty acids with virgin soybean oil fatty acids and fatty acid butyl esters) are shown in Table 6.

Example  17

Experimental identifier GLNOR1050 included: 10 L preseed flask growth, 10 L propagation tank liquefaction, 10 L propagation tank operation, 10 L production tank liquefaction, 10 ppm repolarase added to the fermentation vessel? 10L production tank operation with 100L (Genenco), extractant: Virgin Cognis Emery? 610 Soy Fatty Acids (Virgin Soybean Oil Fatty Acids). Liquid solvent and nonsolvent material were separated by Sorbal RC-12 centrifuge, and all analytical methods.

Example 18

Experiment Identifier Glono 1051 included: 10 L presided flask growth, 10 L growth tank liquefaction, 10 L growth tank operation, 10 L production tank liquefaction, 4 ppm repolarase added to the fermentation vessel? 10L production tank operation with 100L (Genenco), extractant: Virgin Cognis Emery? 610 Soy Fatty Acids (Virgin Soybean Oil Fatty Acids). Liquid solvent and nonsolvent material were separated by Sorbal RC-12 centrifuge, and all analytical methods.

Example 19

Identifier 2011Y029 included: 2 L presided flask growth, 2 L fermentation preparation, 2 L liquefaction, addition before 2 L inoculation, 2 L fermentation vessel inoculation, lipase addition after 2 L inoculation to a final concentration of 10 ppm, 2 L Regenerated Soybean Oil Fatty Acid Addition (Regenerated Cognis Emery of Example 56A—610 Soybean Fatty Acid and Fatty Acid Butyl Ester—50% v / v Solvent Load), 2 L Fermentation Vessel Operating Conditions, and All Analytical Methods.

Example 20

Identifier 2011Y030 included: 2 L presided flask growth, 2 L fermentation preparation, 2 L liquefaction, addition before 2 L inoculation, 2 L fermentation vessel inoculation, lipase addition after 2 L inoculation to a final concentration of 10 ppm, 20 0.4 L / L (volume after inoculation) virgin cognis emery with 30% fatty acid butyl ester? 610 soybean fatty acid addition, 2 L fermentation vessel operating conditions, and all analytical methods.

Example 21

Identifier 2011Y031 included: 2 L presided flask growth, 2 L fermentation preparation, 2 L liquefaction, addition before 2 L inoculation, 2 L fermentation vessel inoculation, lipase addition after 2 L inoculation to a final concentration of 10 ppm, 2 L Regenerated Soybean Oil Fatty Acid Addition (Regenerated Cognis Emery of Example 56B—610 Soybean Fatty Acid and Fatty Acid Butyl Ester-10% v / v Solvent Load), 2 L Fermentation Vessel Operating Conditions, and All Analytical Methods.

Example  22

Identifier 2011Y032 included: 2 L presided flask growth, 2 L fermentation preparation, 2 L liquefaction, addition before 2 L inoculation, 2 L fermentation vessel inoculation, lipase addition after 2 L inoculation to a final concentration of 10 ppm, 0.4 L / L (volume after inoculation) Virgin Cognis Emery? 610 soybean fatty acid addition, 2 L fermentation vessel operating conditions, and all analytical methods.

TABLE 6

Example 23

The following examples illustrate the production of isobutanol by fermentation using sucrose as fermentable carbon source.

Generation of biomass

Inoculum: Seed medium was prepared to initiate growth of isobutanogen. The composition of the seed medium was as follows: ammonium sulfate, 5 g / L; Potassium dihydrogen phosphate, 3 g / L; Magnesium sulfate heptahydrate, 0.5 g / L; Ethanol, 3.2 g / L; Yeast extract (BBL), 5 g / L; Glucose, 10 g / L; MES buffer, 150 mmol / L; Biotin, 50 μg / L; And 1 L water, 15 g EDTA, 4.5 g zinc sulfate heptahydrate, 0.8 g manganese chloride dihydrate, 0.3 g cobalt chloride hexahydrate, 0.3 g copper sulfate pentahydrate, 0.4 g disodium molybdenum dihydrate, 4.5 g calcium chloride dihydrate , Microelement solution containing 3 g iron sulfate heptahydrate, 1 g boric acid, 0.1 g potassium iodide, 1 mL / L. After adjusting the pH to 5.5, the medium filter was sterilized through a 0.22 μ sterile filter device.

Preparation of 10 L Fermentation Vessel for Biomass Production

A single vial of isobutanologen PNY2205 was aseptically transferred to a 15 mL seed medium in a 125 mL vented flask for overnight growth at 30 ° C. and 260 rpm shaking. The culture was aseptically transferred to 500 mL of the same medium in a 2 L baffle, vented flask to grow overnight with shaking at 30 ° C. and 260 rpm and 10 L Sarto prepared when the culture reached OD ₆₀₀ 7. Transfer to Rius C fermentation vessel (Sartorius AG, Göttingen, Germany).

A 10 L Sartorius C fermentation vessel was prepared with 6 L initial volume of growth medium. Growth medium composition and preparation were as follows: prior to sterilization, ammonium sulfate, 1 g / L; Potassium dihydrogen phosphate, 5 g / L; Magnesium sulfate heptahydrate, 2 g / L; Yeast extract (Amberex ™ 695), 2 g / L; Antifoam Sigma 204, 0.5 mL / L; Biotin, 100 μg / L; And 1 mL / L trace element solution (prepared in 1 L water: 15 g EDTA, 4.5 g zinc sulfate heptahydrate, 0.8 g manganese chloride dihydrate, 0.3 g cobalt chloride hexahydrate, 0.3 g copper sulfate pentahydrate, 0.4 g disodium) Molybdenum dihydrate, 4.5 g calcium chloride dihydrate, 3 g iron sulfate heptahydrate, 1 g boric acid, 0.1 g potassium iodide). After steam sterilization at 121 ° C. at the appropriate location, the vessel was cooled and 60 g of feed medium was added. Feed medium was prepared as follows: sucrose, 50% solution, 2.97 L; Biotin, 1.4 mg; 34 mL of trace mineral solution; Titrated and steam sterilized to pH 7.5 with 5N sodium hydroxide; After sterilization and cooling, 320 mL of a 20% (w / v) filter sterile solution of 130 mL ethanol and yeast extract (Ambrex ™ 695) was added. Thus, the initial sugar concentrations of the 10 L fermentation vessel were 3.7 g / L sucrose, 0.8 g / L glucose, and 0.8 g / L fructose.

Fermentation was controlled at pH 5.5 (with ammonium hydroxide addition), 30 ° C., air flow at 2.0 standard liters per minute, 30% dissolved oxygen by stirring control, and 0.5 barg back pressure. After inoculation, the sugar was consumed until the residual measurement of glucose was less than 0.1 g / L, then the feeding program was started; This occurred at the fermentation time that elapsed 11 hours. The program was set to maintain the sucrose limit at a programmed growth rate of 0.1 / hr until an OD ₆₀₀ of 20 (about 8 g / L dry cell weight) was achieved. The growth rate actually measured in this experiment was 0.18 / hr. The target OD ₆₀₀ was reached after 20 hours of fermentation time.

Once the goal was achieved, the culture was harvested aseptically and centrifuged in a Sorbal RC12BP centrifuge. The resulting pellet was resuspended to a final volume of 300 mL using isobutanol production medium, as described below. This culture was used as inoculum for isobutanol producing fermentation vessels.

Isobutanol production

Preparation of Production Fermentation Vessel: Two 1 liter glass apples (Applicorn Inn, Netherlands) combined with Sartorius Biostat B Plus Twin Control Unit (Sartorius AG, Göttingen, Germany) for isobutanol production Corp. (Applikon, Inc.) fermentation vessel was used. Fermentation vessels were prepared with 1 L deionized water and sterilized by autoclaving at 121 ° C. for 30 minutes. Once the fermentation vessel was cooled down, the water was aseptically removed and the amount of filter sterilized product medium added, as shown in Table 7. This production medium composition was as follows: Amino acid free yeast nitrogen base (Difco), 6.7 g / L; Yeast synthetic drop out medium supplement, 2.70 g / L, containing no histidine, leucine, tryptophan, and uracil (Sigma); Tryptophan, 1.6 mg / L; Leucine, 8 mg / L; Ethanol, 2.8 g / L; Antifoam sigma 204, 0.2 mL / L; Sucrose, 25 g / L. Immediately before inoculation, the filter sterilized lipase solution was as shown in Table 7. Repolarase? Lipase solution was prepared by diluting L100 (Sigma) to a final concentration of 1.25 mg protein / mL in 10 mM potassium phosphate buffer, pH 7. Prior to addition to the fermentation vessel, the solution was prepared and stored at 5 ° C. for 1 day.

[Table 7]

The fermentation vessel was controlled with pH 5.2 (with 20% potassium hydroxide addition), 30 ° C., air flow of 0.2 standard liters per minute, and 3% dissolved oxygen by stirring control.

Fermentation vessels were each inoculated with 40 mL of concentrated biomass at an initial OD _{600 of} 20-25 (about 8-10 g / L dry cell weight). In the volume of filter sterilized soybean oil fatty acid (SOFA) shown in Table 7, a 4 mL addition of filter sterilized vitamin solution (thiamine-HCl, 1 mg / mL; nicotinic acid in water, 1 mg / mL) was added at the time of inoculation. . Samples (5-10 mL) were taken out every 2 to 3 hours and placed in a YSI Select Biochemistry Analyzer (YSI, Inc., Yellow Springs, Ohio). Glucose and sucrose were analyzed by As sucrose was consumed, a feed of 50% sucrose (w / w) was added to maintain a concentration of 5 to 30 g / L. The aqueous and organic phases of the samples were separated and analyzed by the HPLC method described above via Agilent 1100 HPLC. For the analysis of organic acids and alcohols, Shodex? A Sugar SH1011 column was used with a 0.01 N sulfuric acid mobile phase. For analysis of sucrose, glucose, and fructose, BioRad Aminex with 0.01 M Na ₂ HPO ₄ (pH 8) mobile phase? HPX-87N column was used.

Each fermentation vessel to which lipase was added had isobutanol in the low concentration aqueous phase and free isobutanol in the solvent phase. The aqueous and solvent phase concentrations of isobutanol are shown in FIG. 6. Adding more lipase at the same solvent loading also led to lower aqueous phase titers of isobutanol and free isobutanol in lower solvents, and more isobutanol as FABE.

Cultures containing lipase led to higher effective titers of isobutanol than control fermentation vessels containing no lipase. 7 shows the effective titers of isobutanol. In this example, effective titers were calculated based on the initial measured weight of the broth in the fermentation vessel and the initial measured weight of the solvent injected into the fermentation vessel after inoculation. Fermentation assumed a solvent density of 0.88 g / mL and an aqueous broth density of 1.00 g / mL. Addition of more lipase at lower solvent loading led to higher effective titers of isobutanol (D vs C), but not to the relative volume increase of solvent (C vs B).

The spent sugar, calculated as glucose equivalent, was higher in the fermentation vessel to which lipase was added, as shown in FIG. 8. The glucose equivalent consumed is calculated with each mole of sucrose counted to 2 moles of glucose and each mole of fructose counted to 1 mole of glucose from the remaining measured sugar supplied to the fermentation vessel, and then Converted to grams by molecular weight. The spent glucose equivalent concentration is also calculated based on the initial volume of fermentation broth after inoculation.

Example 24

Orail  Simultaneous with in situ product removal with alcohol Glycation  Liquefied Corn for Fermentation Hourly Lipase  process

For samples of broth and oleyl alcohols taken from the fermentation carried out as described above in Examples 1, 2 and 3, E. Wt% lipid (fatty acid methyl ester, derivatized as FAME) according to the method described by G. Bligh and WJ Dyer (Canadian Journal of Biochemistry and Physiology, 37: 911-17, 1959, hereinafter referred to as Ref. 1), and wt% free fatty acids (FFA, fatty acid methyl ester, derivatized as FAME) were analyzed. Also, for liquefied corn mash prepared for each of the three fermentations, repolarase? Wt% lipid and wt% after treatment with 100 L (Novozymes) (10 ppm of lipolase-total soluble protein (BCA protein assay, Sigma Aldrich) per kg liquefied reaction containing 30 wt% ground corn kernels) FFA was analyzed. The fermentation described in Examples 2 and 3 containing no lipase added to the liquefied corn mash of Example 1 (control), and containing the liquefied corn mash treated with lipase (lipase not thermally inactivated) did not add ethanol. Except that except for the fermentation described in Example 3.

The% FFA in the lipase treated liquefied corn mash prepared for the fermentation run as described in Examples 2 and 3 was 88% and 89%, respectively, compared to 31% of lipase untreated (Example 1). At 70 hours (end of run (EOR)), the concentrations of FFA in the OA phase of the fermentation run as described in Examples 2 and 3 (containing active lipase) were 14% and 20%, respectively, and the corresponding lipid increase ( Corn oil fatty acid methyl ester derivative) as measured by GC / MS due to lipase catalytic esterification of COFA by OA, which was initially produced by lipase catalytic hydrolysis of corn oil in liquefied corn mash; The production of oleyl palmitate, oleyl stearate, and oleyl oleate was confirmed by GC / MS, and the fourth ester was tentatively identified as oleyl linoleate. Results for FFA and lipid analysis are shown in Table 8.

[Table 8]

Example 25

Thermal inactivation of lipase in lipase treated liquefied corn mash to limit the production of oleyl alcohol esters of corn oil free fatty acids

Tap water (918.4 g) was added to the jacketed 2-L resin kettle, followed by 474.6 g wet weight (417.6 g dry weight) of whole corn flour (1.0 mm screen of hammer mill) with stirring. The mixture was heated to 55 ° C. with stirring at 300 rpm and the pH was adjusted to 5.8 with 2 N sulfuric acid. To the mixture was added 14.0 g of an aqueous solution containing 0.672 g of Spezyme®-FRED L (Genenco, Palo Alto, Calif.) And the temperature of the mixture was stirred under stirring at 600 rpm and pH 5.8. Increased to 85 ° C. After 120 minutes at 85 ° C., the mixture was cooled to 50 ° C. and a 45.0 mL aliquot of the resulting liquefied corn mash was transferred to a 50-mL polypropylene centrifuge tube and stored frozen at −80 ° C.

In the first reaction, 50 g of liquefied corn mash prepared as described above was prepared with 10 ppm lipolase? Without inactivation of lipase for 1 hour at 85 ° C. 100 L (Novozymes) was mixed at 55 ° C. for 6 hours, and the mixture was cooled to 30 ° C. In the second reaction, 50 g of liquefied corn mash was mixed with 10 ppm lipolase for 6 hours at 55 ° C, then heated to 85 ° C for 1 hour (lipase inactivation) and then cooled to 30 ° C. In the third reaction, 50 g of liquefied corn mash are mixed at 55 ° C. for 6 hours without lipase addition, heated at 85 ° C. for 1 hour, and the mixture is cooled to 30 ° C., adding 38 g of oleyl alcohol, The resulting mixture was stirred at 30 ° C. for 73 h. In the fourth reaction, 50 g of liquefied corn mash was mixed at 55 ° C. for 6 hours without lipase addition, then heated to 85 ° C. for 1 hour and then cooled to 30 ° C. Each of the four reaction mixtures was sampled at 6 hours, then 38 g of oleyl alcohol were added and the resulting mixture was stirred at 30 ° C. and sampled at 25 and 73 hours. According to the method described in Reference 1, as a wt% lipid (fatty acid methyl ester, derivatized as FAME) and wt% free fatty acid (FFA, fatty acid methyl ester, FAME) on a sample (liquefied mash and oleyl alcohol (OA)) Derivatized).

The% FFA in the OA phase of the second reaction run with thermal inactivation of the lipase prior to OA addition was 40% FFA at 25 and 73 hours when the lipase of the lipase treated liquefied corn mash was not thermally inactivated (first reaction). And 99% at 25 hours and 95% at 73 hours, respectively, compared to only 21% FFA. No significant change in% FFA was observed in the two control reactions without lipase addition. The results are shown in Table 9.

TABLE 9

Example 26

Thermal Inactivation of Lipase in Lipase Treated Liquefied Corn Mash for Simultaneous Saccharification Fermentation with In Situ Product Removal with Oleyl Alcohol

Three fermentations were performed as described above in Examples 4, 5, and 6. No lipase was added to the liquefied corn mash in Examples 4 and 6 prior to fermentation, and heat-inactivated immediately after the lipase treatment (using 7.2 ppm of repolarase? Total soluble protein) of the liquefied corn mash in the fermentation described in Example 5. (To completely inactivate the lipase), followed by nutrient addition and fermentation before inoculation. The% FFA in liquefied corn mash prepared without lipase treatment for the fermentation performed as described in Examples 4 and 6 was 31% and 34%, respectively, compared to 89% of the lipase treatment (Example 5). During the fermentation described in Table 10, the concentration of FFA in the OA phase was not reduced in any of the three fermentations, including those containing thermally inactivated lipases. The% FFA in the OA phase of the fermentation performed according to Example 5 (heat inactivation of lipase before fermentation) was equivalent to 33% FFA in the remaining two fermentations (Examples 4 and 6) where the liquefied corn mash was not treated with lipase. By comparison, it was 95% at 70 hours (end of run (EOR)). The results are shown in Table 10.

[Table 10]

Example 27

Lipase treatment of whole cornmeal before liquefaction

Tap water (1377.6 g) was added to each of the two jacketed 2-L resin kettles, then 711.9 g wet weight (625.8 g dry weight) of whole corn flour (1.0 mm screen of hammer mill) was added to each kettle with stirring. . Each mixture was heated to 55 ° C. with stirring at 300 rpm and the pH was adjusted to 5.8 with 2 N sulfuric acid. To each mixture was added 21.0 g of an aqueous solution containing 1.008 g of Spezimme-FRED L (Genenco, Palo Alto, Calif.). Then in one mixture, repolarase? Add 10.5 mL (21 mg total soluble protein, 10 ppm lipase final concentration) in an aqueous 100L solution, and add lipolase to another mixture? 1.05 mL (2.1 mg total soluble protein, 1.0 ppm lipase final concentration) of 100 L solution solution was added. Samples were recovered from each reaction mixture at 55 ° C. at 1 h, 2 h, 4 h and 6 h, then the temperature of the mixture was increased to 85 ° C. under stirring at 600 rpm and pH 5.8, and the mixture was first 85 ° C. Samples were taken when they reached. After 120 minutes at 85 ° C., samples are taken, the mixture is cooled to 50 ° C. and the final sample of the resulting liquefied corn mash is transferred to a 50-mL polypropylene centrifuge tube; All samples were stored frozen at -80 ° C.

In two separation reactions, 50 g samples of 10 ppm lipase treated liquefied corn mash prepared as described above or 55 g samples of 1.0 ppm lipase treated liquefied corn mash were oleyl alcohol (OA) at 30 ° C. for 20 h. (38 g), followed by centrifugation of the liquefied mash and OA in each reaction mixture, wt% lipid (fatty acid methyl ester, derivatized as FAME) for each phase according to the method described in Reference 1 And wt% free fatty acid (FFA, fatty acid methyl ester, derivatized as FAME). The% FFA in the OA phase of the liquefied mash / OA mixture prepared by thermal inactivation of 10 ppm lipase at liquefaction was 62 in the OA phase of the liquefied mash / OA mixture prepared by thermal deactivation of 1.0 ppm lipase at liquefaction. Compared to only% FFA, it was 98% at 20 h. The results are shown in Table 11.

TABLE 11

Example  28

Lipase screening for treatment of whole cornmeal before liquefaction

Seven reaction mixtures containing tap water (67.9 g) and whole corn flour (35.1 g wet weight, ground to 1.0 mm screen using a hammer mill) at pH 5.8 were stirred at 55 ° C. in a flask with a stopper. 3-mL samples (t = 0 h) were removed from each flask, and the samples were immediately frozen in dry ice, followed by 1 mg total soluble protein (10 ppm final concentration in the reaction mixture) of one of the following lipases (Novozymes) Approximately 0.5 mL of 10 mM sodium phosphate buffer, pH 7.0, was added to the sample in each flask: Lipolase? 100 L, Repex? 100L, lipoclin? 2000T, Lipozyme? CALB L, novozyme? CALA L, and palatase 20000L; Lipase was not added to the seventh flask. The resulting mixture was stirred at 55 ° C. in a stoppered flask, and 3-mL samples were recovered from each reaction mixture at 1 h, 2 h, 4 h and 6 h, wt% according to the method described in Reference 1 Lipids (fatty acid methyl esters, derivatized as FAME) and wt% free fatty acids (FFA, fatty acid methyl esters, derivatized as FAME) are frozen directly in dry ice until analysis, and the percentage of free fatty acid content is summed to the total total concentration of lipids. And free fatty acids were measured for each sample. The results are shown in Table 12.

[Table 12]

Example 29

Lipase Treatment of Whole Corn Flour Prior to Simultaneous Saccharification Fermentation Using In Situ Removal with Oleyl Alcohol

Three fermentations were performed as described above in Examples 7, 8, and 10. For the fermentation performed as described in Examples 7 and 10, lipase (10 ppm of lipolase—total soluble protein) was added to the suspension of ground corn and heated to 55 ° C. for 6 h before liquefaction, thereby making it thermally inactive lipase A liquefied corn mash containing was produced. No lipase was added to the suspension of ground corn used to prepare the liquefied corn mash for the fermentation described in Example 8, but the suspension was subjected to the same heating step at 55 ° C. before liquefaction. The% FFA in the lipase treated liquefied corn mash prepared for the fermentation run as described in Examples 7 and 10 was 83% and 86%, respectively, compared to 41% of lipase untreated (Example 8). During the fermentation, the concentration of FFA was not reduced in any fermentation, including containing thermally inactivated lipases. The% FFA in the OA phase of the fermentation carried out according to Examples 7 and 10 (heat inactivation of lipase before fermentation) was only 49% FFA of the fermentation performed according to Example 8 in which the cornmeal flour was not treated with lipase before liquefaction. As compared to 97% at 70 h (end of run (EOR)), respectively. The results are shown in Table 13.

[Table 13]

Example 30

For Simultaneous Saccharification Fermentation Using In Situ Product Removal Using Corn Oil Fatty Acids (COFA) Whole corn  Flour or liquefied corn Hourly Lipase  process

Five fermentations were performed as described above in Examples 9, 11, 12, 13, and 14. For the fermentation performed as described in Examples 9, 13, and 14, lipase (10 ppm of lipolase-total soluble protein) was added after liquefaction and the lipase was not thermally inactivated. Fermentations performed as described in Examples 9 and 14 resulted in 5 g / L of ethanol added prior to inoculation, while fermentations performed as described in Example 13 had no ethanol added. The fermentation carried out as described in Examples 11 and 12 was carried out in a suspension of ground maize before the liquefaction of 10 ppm lipolase? Total soluble protein was added to induce heat inactivation of the lipase upon liquefaction. Fermentations performed as described in Example 11 resulted in 5 g / L of ethanol added prior to inoculation, while fermentations performed as described in Example 12 had no ethanol added. The final total grams of isobutanol (i-BuOH) present on the COFA of fermentation containing active lipase is i-BuOH present on the COFA of fermentation containing inactive lipase (including i-BuOH present as FABE) It was significantly larger than the final total grams of. The final total grams of isobutanol (i-BuOH) present in the fermentation broth containing the active lipase (aqueous phase) were only slightly less than the final total grams of i-BuOH present in the fermentation broth containing the inactive lipase. Therefore, the total production of i-BuOH (as a combination of free i-BuOH and isobutyl ester of FAFA) was significantly higher in the presence of active lipase compared to that obtained in the presence of thermally inactivated lipase. The results are shown in Tables 14 and 15.

[Table 14]

 [Table 15]

Example 31

Production of Isobutyl COFA Ester by Phospholipase Catalytic Reaction of Isobutanol with Corn Oil Fatty Acid (COFA)

2- (N-morpholino) ethanesulfonic acid aqueous buffer (0.20 M, pH 5.3), isobutanol (2-methyl-1-propanol), phospholipase (phospholipase A; sigma aldrich, L3295-250) And a reaction mixture containing corn oil fatty acid prepared from corn oil at 30 ° C. (Table 16), and the sample was recovered from each reaction mixture at a predetermined time, immediately centrifuged, the aqueous layer and the organic layer were separated, Butanol (i-BuOH) and isobutyl esters of corn oil fatty acids (i-BuO-COFA) were analyzed (Table 17).

[Table 16]

TABLE 17

Example 32

Dependence of Isobutyl-COFA Ester Concentration on Aqueous / COFA Ratio in Lipase Catalysis

2- (N-morpholino) ethanesulfonic acid aqueous buffer (0.20 M, pH 5.2), isobutanol (2-methyl-1-propanol), lipase (lipolase-100 L; Novozymes) and corn oil The reaction mixture containing corn oil fatty acid prepared from (Table 18) was stirred at 30 ° C., and the sample was recovered from each reaction mixture at a predetermined time, immediately centrifuged, the aqueous layer and the organic layer were separated, and isobutanol (i -BuOH) and isobutyl esters of corn oil fatty acids (i-BuO-COFA) were analyzed (Table 19).

[Table 18]

TABLE 19

Example 33

Dependence of Butyl-COFA Ester Concentration on Ester Alcohols in Lipase Catalysis

2- (N-morpholino) ethanesulfonic acid aqueous buffer (0.20 M, pH 5.2), isobutanol (2-methyl-1-propanol) or n-butanol, lipase (lipolase? 100 L; Novozymes ) And the reaction mixture containing corn oil fatty acid prepared from corn oil (Table 20) was stirred at 30 ° C., and the sample was recovered from each reaction mixture at a predetermined time, immediately centrifuged, the aqueous layer and the organic layer were separated, Isobutanol (i-BuOH) or n-butanol (n-BuOH) and isobutyl- or butyl esters (BuO-COFA) of corn oil fatty acids were analyzed (Table 21).

TABLE 20

TABLE 21

Example  34

With isobutanol  Oleic Lipase  By catalytic reaction Isobutyl Oleate  produce

2- (N-morpholino) ethanesulfonic acid aqueous buffer (0.20 M, pH 5.2), isobutanol (2-methyl-1-propanol), lipase (0 ppm or 10 ppm lipolase-100 L; novozyme And reaction mixture (Table 22) containing oleic acid (Alfa Aesar) at 30 ° C., the sample was recovered from each reaction mixture at a predetermined time, immediately centrifuged, and the aqueous and organic layers Separately, isobutanol (i-BuOH) and isobutyl oleate (i-BuO-oleate) were analyzed (Table 23).

Table 22

TABLE 23

Example 35

Comparison of the Production of Isobutyl Oleate by Lipase Catalysis of Isobutanol and Oleic Acid

2- (N-morpholino) ethanesulfonic acid aqueous buffer (MES, 0.20 M, pH 5.2), isobutanol (2-methyl-1-propanol), oleic acid (alpha Aisa), and lipase (10 ppm) ( Lipolase from Novozymes 100 L, Repex 100 L, Lipozyme CALB L, Novozyme CALA L, Palatase, or Lipase (10 ppm), Pseudomonas Fluoresence from Sigma Aldrich, The reaction mixture (Table 24) containing Pseudomonas Sephacia, Mucor Miehei, Porcine Pancreas, Candida Silindracea, Rizopus Niveus, Candida Antartica, Rizopus Arhijus or Aspergillus) After stirring, the sample was recovered from each reaction mixture at a predetermined time, immediately centrifuged, and the aqueous layer and the organic layer were separated to analyze isobutanol (i-BuOH) and isobutyl oleate (i-BuO-oleate). (Table 25).

TABLE 24

TABLE 25

Example  36

Production of Ethyl-COFA Ester by Lipase Catalysis of Ethanol and Corn Oil Fatty Acid (COFA)

Corn prepared from 2- (N-morpholino) ethanesulfonic acid aqueous buffer (0.20 M, pH 5.5), ethanol, lipase (lipolase? 100 L or lipozyme? CALB L; Novozymes) and corn oil The reaction mixture containing milk fatty acid (Table 26) was stirred at 30 ° C., the sample was recovered from each reaction mixture with stirring at a predetermined time, centrifuged immediately, the aqueous layer and the organic layer were separated, and the ethanol and corn oil fatty acids were separated. Ethyl ester (EtO-COFA) was analyzed (Table 27).

TABLE 26

TABLE 27

Example  37

Production of Ethyl-COFA Ester by Lipase Catalytic Reaction of Ethanol and Corn Oil Fatty Acid (COFA) in Yeast Fermentation

Wild-type yeast strain CEN.PK113-7D was grown overnight in medium containing amino acid free yeast nitrogen base (6.7 g / L), dextrose (25 g / L), and MES buffer (0.1 M at pH 5.5). . The overnight culture was diluted with fresh medium, resulting in an optical density of 0.1 at 600 nm. Diluted cultures were aliquoted into six 250 mL sealed cap shake flasks with 25 mL per flask. Four of the cultures were supplemented with two lipase enzyme stocks (Lipozyme® CALB L or Lipolase® 100L of 2 mg protein / mL 10 mM phosphate buffer (pH 7.0)) to give a final lipase concentration of 10 ppm in the medium. To be made. Corn oil fatty acid (COFA) was added to the three aqueous cultures in the flask (without enzyme, CALB L, or Lipolase® 100 L) in a 1: 1 volume ratio. One flask was not supplemented. Cultures were grown for 23 h at 30 ° C. and 250 rpm in a temperature controlled shake incubator. Samples for cell volume measurements were phase separated in a 15 mL conical bottom tube. The optical density of the sample at 600 nm was measured in a 20-fold dilution in brine. Centrifuge the sample for chromatographic analysis (5 mL aqueous culture or 10 mL culture / COFA emulsion) directly at 4000 rpm for 5 minutes on a TX-400 swinging bucket rotor in a 15 mL conical bottom tube. It was. For aqueous samples, a 0.22 μm spin filter was used prior to analysis. Aqueous samples were analyzed on a Shodex SH1011 column with a SH-G guard column using a 0.01 M sulfuric acid mobile phase at 50 ° C. and a flow rate of 0.5 mL per minute. Sugars and alcohols were detected with refractive index and 210 nm absorption and quantified using a standard curve. Samples were taken from aqueous culture (without COFA) or culture / COFA emulsion and analyzed for ethyl ester of COFA as described in the previous examples. The results are shown in Tables 28 and 29.

[Table 28]

[Table 29]

Example 38

Production of Methyl-COFA Ester by Lipase Catalytic Reaction of Methanol with Corn Oil Fatty Acid (COFA)

2- (N-morpholino) ethanesulfonic acid aqueous buffer (0.20 M, pH 5.5), methanol, lipase (lipolase? 100 L (novovozymes), lipozyme? CALB L (novovozymes), lyso A reaction mixture containing Fus Arhijus lipase (Sigma Aldrich), and Candida silindrasia lipase (Sigma Aldrich) and corn oil fatty acids prepared from corn oil (Table 30) is stirred at 30 ° C. and the sample is stirred at a predetermined time. The mixture was recovered from each reaction mixture with stirring, centrifuged immediately, the aqueous and organic layers were separated, and the ethyl esters of ethanol and corn oil fatty acids (EtO-COFA) were analyzed (Table 31).

[Table 30]

[Table 31]

Example 39

Production of 1-propyl-COFA Ester by Lipase Catalytic Reaction of 1-Propanol with Corn Oil Fatty Acid (COFA)

2- (N-morpholino) ethanesulfonic acid aqueous buffer (0.20 M, pH 5.5), 1-propanol, lipase (lipolase? 100 L (Novozymes), lipozyme? CALB L (Novozymes) , A reaction mixture containing Rizopus arhijus lipase (Sigma Aldrich), and Candida silindrasia lipase (Sigma Aldrich)) and corn oil fatty acid prepared from corn oil (Table 32) was stirred at 30 ° C., and the sample was The mixture was recovered from each reaction mixture with stirring at a predetermined time, centrifuged immediately, the aqueous layer and the organic layer were separated and analyzed for 1-propyl ester (PrO-COFA) of 1-propanol and corn oil fatty acid (Table 33).

Table 32

[Table 33]

Example 40

Production of 1-pentyl-COFA Ester by Lipase Catalytic Reaction of 1-Pentanol with Corn Oil Fatty Acid (COFA)

2- (N-morpholino) ethanesulfonic acid aqueous buffer (0.20 M, pH 5.5), 1-pentanol, lipase (lipolase? 100 L (Novozymes), lipozyme? CALB L (Novozymes) ), Rizopus arhijus lipase (Sigma Aldrich), and a reaction mixture containing Candida silindrasia lipase (Sigma Aldrich) and corn oil fatty acids prepared from corn oil (Table 34) were stirred at 30 ° C. and the samples were The mixture was recovered from each reaction mixture with stirring at a predetermined time, centrifuged immediately, the aqueous layer and the organic layer were separated and analyzed for 1-pentyl ester (PenO-COFA) of 1-pentanol and corn oil fatty acid (Table 35).

[Table 34]

[Table 35]

Example 41

Production of 2-methyl - 1 -butyl-COFA ester by lipase catalyzed reaction of 2-methyl - 1 -butanol with corn oil fatty acid (COFA)

2- (N-morpholino) ethanesulfonic acid aqueous buffer (0.20 M, pH 5.5), 2-methyl-1-butanol, lipase (lipolase? 100 L (Novozymes), lipozyme? CALB L ( Novozymes), lycopus arhijus lipase (Sigma-Aldrich), and reaction mixture containing candida silindrasia lipase (Sigma-Aldrich) and corn oil fatty acid prepared from corn oil (Table 36) were stirred at 30 ° C and The sample was recovered from each reaction mixture with stirring at a predetermined time, immediately centrifuged, the aqueous layer and the organic layer were separated, and 2-methyl-1-butyl ester of 2-methyl-1-butanol and corn oil fatty acid (MeBO- COFA) was analyzed (Table 37).

[Table 36]

[Table 37]

Example 42

Production of Isopropyl-COFA Ester by Lipase Catalytic Reaction of Isopropanol with Corn Oil Fatty Acid (COFA)

2- (N-morpholino) ethanesulfonic acid aqueous buffer (0.20 M, pH 5.5), isopropanol (2-propanol), lipase (lipolase? 100 L (Novozymes), lipozyme? CALB L ( Novozymes), lycopus arhijus lipase (Sigma-Aldrich), and a reaction mixture containing candida silindrasia lipase (Sigma-Aldrich) and corn oil fatty acids prepared from corn oil (Table 38) were stirred at 30 ° C and The sample was recovered from each reaction mixture with stirring at a predetermined time, immediately centrifuged, the aqueous layer and the organic layer were separated and analyzed for isopropyl esters of isopropanol and corn oil fatty acid (i-PrO-COFA) (Table 39). ).

[Table 38]

[Table 39]

Example 43

Comparison of partition coefficients for isobutanol between water and extractant

Aqueous isobutanol solution (30 g / L) was mixed with corn oil fatty acid (COFA), or oleic acid or corn oil triglyceride, and their measured partition coefficient was compared to the measured partition coefficient for oleyl alcohol in the table. Recorded. The results are shown in Table 40.

[Table 40]

Example 44

Production of Corn Oil Fatty Acids

In a 5-liter (5L) round bottom flask with a mechanical stirrer, thermocouple, heating mantle, condenser and nitrogen tee, crude corn (non food grade, recovered from an ethanol fermentation plant) 750 g, water 2112 g and 285 g of 50% sodium hydroxide solution were injected. The mixture was heated to 90 ° C. and maintained for 2 hours, during which time it became one thick emulsion-like single phase. At the end of this period, TLC indicates no corn oil remains in the mixture. The mixture was then cooled to 74 ° C. and the mixture acidified by adding 900 g of 25% sulfuric acid. Then, it cooled to 50 degreeC and discharged the aqueous layer. The oil layer was washed twice with 1500 mL of 40 ° C. water, then once with 1 liter of saturated brine. Dried over magnesium sulfate and filtered through Celite. 610 g of a clear red oil were obtained. AOCS method Titration to free fatty acids via Ca 5a-40 indicates that the fatty acid content, expressed in oleic acid, is 95%. The sample was silanized by reacting 104 mg of the sample with 100 uL of N-methyl-N- (trimethylsilyl) trifluoroacetamide in 1 mL of anhydrous pyridine. Gas chromatographic mass spectrometry (GCMS) of the silanized products reveals the presence of TMS derivatives of 16: 0, 18: 2, 18: 1, 18: 0, and 20: 0 acids.

Example 45

Chemical Synthesis of FABE

The 3L flask was equipped with a mechanical stirrer, thermocouple, nitrogen inlet, heating mantle and condenser. The flask was charged with COFA (595 g) (prepared in Example 44), isobutanol (595 g), and sulfuric acid (12 g). The mixture was refluxed for 1.5 hours at which time the condenser was removed and replaced with a steel head. The distillate was collected over 3 hours with an initial head temperature of 90 ° C. and a final head temperature of 105 ° C. The mixture was then cooled to room temperature and 500 mL of deionized water was added. The layers were separated and the organic layer was washed five times with 500 mL of deionized water. It was then washed once with 500 mL of 10% calcium chloride solution followed by 6 times with 500 mL of deionized water. The oil was then dried over magnesium sulfate and filtered through a celite bed to give 601 g of a clear red oil. GC analysis showed that 0.36 wt% of isobutanol was present. GC / MS analysis shows that isobutyl palmitate, isobutyl stearate, isobutyl oleate, isobutyl linoleate, and isobutyl linoleate are present.

Example 46

Recovery of Butanol Using Inorganic Acid Catalyst

A 1 liter round bottom flask with a 30.5 cm (12 ") column packed with a magnetic stirrer and a Rasching ring with a steel head and nitrogen inlet on top was used. In the flask, synthesized in Example 45 254 g of FABE, 255 g of COFA, 100 mL of water, and 5 g of sulfuric acid were injected and heated to a pot temperature of 93 ° C. The head temperature was equilibrated to 89.7 ° C. The first cut was made to have a head temperature of 89 to 94 ° C. Collected at reflux ratio to maintain.

The reaction was cooled down and left at room temperature for 3 days. GC analysis of the pot reveals a total of 1 g of isobutanol present in the pot. Distillation was started again and three more cuts of 25 mL each were collected. Bag (100) mL of water was added to the pot after collecting cut # 2. Four cuts were collected and analyzed with the results shown in Table 41.

GC analysis was done using Hewlett Packard 6890 GC using a 30m FFAP column. The sample was dissolved in isopropanol and 1-pentanol was added as internal standard. Standard curves are isobutanol, isobutyl palmitate, isobutyl stearate, isobutyl oleate, isobutyl linoleate, isobutyl linoleate, isobutyl arachidate, palmitic acid, stearic acid, oleic acid, linoleic acid and linolenic acid Written for. FABE content is the sum of butyl esters and COFA content is the sum of fatty acids.

[Table 41]

Example 47

Recovery of Butanol Using Organic Acid Catalyst

A 1 liter three neck round bottom flask with magnetic stirrer, thermocouple, addition funnel and steel head was used. To the flask was injected 100 g FABE, 100 g COFA, 5 g p-toluenesulfonic acid, and 25 mL water, synthesized as in Example 45. Isobutanol analysis of the initial pot reveals the presence of 1.1 g of isobutanol (contaminants of FABE). The pot was heated to 125 ° C. When the pot reached 116 ° C., the head temperature was 96 ° C. and 125 mL water was added over 2.5 hours. Six cuts were collected over the time water was added and analyzed by GC as in Example 46. The results are provided in Table 42.

[Table 42]

Butanol analysis of the remaining steel pot reveals that 0.9 g of free isobutanol is present. The initial COFA: FABE mixture analyzed was 45 wt% FABE. The final pot analyzed was 32 wt% FABE.

Example 48

Hydrolysis of FABE with Water at High Temperature

Into a 1 liter autoclave, 300 mL of FABE and 300 mL of water synthesized as in Example 45 were injected. Sealed and purged with nitrogen. Agitation was initiated and then heated to 250 ° C. over 45 minutes and samples were recovered every hour after the attainment temperature. Samples were analyzed by GC as in Example 46. The oil phase sample showed composition as a function of time shown in Table 43.

[Table 43]

Example 49

Hydrolysis of FABE with Dilute Acid at High Temperature

In a 1 liter autoclave, 450 g of a 75/25 mixture of FABE and COFA synthesized as in Example 45 and 150 g of 2% sulfuric acid were injected. Sealed and purged with nitrogen. Agitation was initiated and then heated to 225 ° C. over 45 minutes and the samples were recovered every hour after reaching the temperature. Samples were analyzed by GC as in Example 46. The oily sample showed composition as a function of time shown in Table 44.

[Table 44]

Example 50

Hydrolysis of FABE with Sulfuric Acid in Solvent at 100 ° C

A solution of 5 g FABE synthesized as in Example 45, 5 g 25% sulfuric acid, and 60 g diethylene glycol dimethyl ether were prepared. Ten (10) grams of solution were added to each of the five vials and then sealed. All vials were heated to 100 ° C. and one vial was removed from the heater and analyzed every hour. The resulting composition is measured by GC (as described in Example 46) and shown in Table 45.

TABLE 45

Example  51

Hydrolysis of FABE by Reactive Distillation

The 12-liter flask was attached to an insulated 5.1 cm x 76.2 cm (2 "x 30") column with a feed inlet and steel head on top. 1 liter of a column Pro-Pak ^(Pro-pak)? (316 SS 0.41 ㎝ (0.16 " )) as Amberlist of steel packing and 500 ^g? 36 solid acid catalyst (Dow (Dow)) was packed at random. Injected to 6 liters of water to the flask, and stir. Heat Controlled to bring the water distillation rate to about 1.8 mL / min FAFA synthesized according to the method described in Example 45 was added to the top of the column at a rate of 2 g / min The feed was continued for a total of 60 minutes. The distillation was continued for an additional 30 minutes A total of 194 g of distillate was collected, which contained 2.1 g of isobutanol, indicating a 9% conversion of FABE to butanol based on the amount of FABE fed.

Example 52

Hydrolysis of FABE by Countercurrent Steam

The device as described in Example 50 was modified by the addition of a heat tape wound around a steel column. The temperature of the upper half of the column was adjusted to 115 ° C and the temperature of the lower half of the column to 104 ° C. The pot was boiled and the pot heat was adjusted until the water distilled at a rate of 1.5-2 mL / min. FABE (346 g) synthesized according to the method described in Example 45 was fed to the top of the packed column over 3 hours with distillation continuing. After the feeding period, distillation was continued for an additional 90 minutes. A total of 486 g of distillate was collected, which contained 30.1 g of isobutanol. This represents a 39% conversion of FABE to isobutanol.

Example 53

Hydrolysis Catalyzed by Insoluble Organic Acids

Into a 1 liter three necked round bottom flask equipped with an oil bath, a mechanical stirrer, a nitrogen inlet, a water inlet under water, and a steel head, 150 g of FABE, 50 g of water, and 5 g of dodecylbenzene sulfonic acid were injected. The oil bath was heated to 95-100 ° C. and a slow nitrogen sweep was initiated. Distillate cuts were collected every 30 minutes for a total of 5 hours. After 3 hours, water was fed to the steel pot at a rate of 15 mL / hr. Distillate cuts were analyzed for isobutanol content by the GC method described in Example 46, and the results are shown in Table 46. About 44% of isobutanol contained in FABE was collected over 5 hours.

TABLE 46

Example 54

Hydrolysis Catalyzed by Solid Acid Catalyst

Into a 1 liter three necked round bottom flask with oil bath, mechanical stirrer, nitrogen inlet under water, water inlet under water, and steel head, 150 g of FABE and 50 g of dry Amberlyst 15 solid acid catalyst were injected. The flask was heated to 110 ° C. in an oil bath and water was added at a rate of 15 mL / hr through a syringe pump. Distillation fractions were collected every 30 minutes for a total of 5 hours. The fractions were analyzed for isobutanol content by the GC method described in Example 46 and the results are shown in Table 47. About 44% of the theoretical amount of isobutanol contained in FABE was collected over 5 hours.

TABLE 47

Example 55

Hydrolysis Catalyzed by Water Soluble Organic Acid Catalyst

200 g of FABE and 10 g of p-toluenesulfonic acid were injected into a 1 liter flask with a mechanical stirrer, a nitrogen inlet under water, a water inlet under water, and a steel head. The flask was stirred and heated to 110 ° C. in an oil bath, at which time water was added via a syringe pump at a rate of 20 mL / hr. Still fractions were collected every 30 minutes for a total of 3 hours. The fractions were analyzed for isobutanol content by the GC method described in Example 46 and the results are shown in Table 48. About 30% of the theoretical amount of isobutanol contained in FABE was collected over 5 hours.

TABLE 48

Example 56

Hydrolysis of Solvent Phase from Fermentation

A. Solvent Phase 1

The solvent phase from the fermentation shown in Example 17 was analyzed by the GC method shown in Example 46 and the results are shown in Table 49. The analysis shows fatty acids and FABE with small amounts of material with predominantly residence times consistent with FAEE. But analysis of butyl ester and acid shows the proportion of 62% FABE and 39% fatty acid.

Solvent phase (1.25 liters, 1090 g) and 1.25 liters of water were injected into a 1 gallon autoclave. The autoclave was sealed and heated to 250 ° C. and held at that temperature for 4 hours. The autoclave was then cooled and opened to give an emulsion. The mixture was filtered through a celite bed and the layers separated. The organic layer was washed three times with 1 liter of water. The sample was then heated to 50 ° C. and purged with nitrogen for 6 hours. GC analysis is free of i-BuOH and shows a ratio of 33% FABE and 67% fatty acids. Obtained amber oil (993.9 g). Detailed compositional analysis of the initial solvent phase from the fermentation of Example 17 and the solvent phase after hydrolysis are shown in Table 50.

B. Solvent Phase 2

The solvent phase from the fermentation shown in Example 18 was analyzed by the GC method shown in Example 46 and the results are shown in Table 49. The analysis shows fatty acids and FABE with small amounts of material with predominantly residence times consistent with FAEE. Only the analysis of butyl ester and acid shows the proportion of 45% FABE and 55% fatty acid.

Solvent (1.25 liters, 1100 g) and 1.25 liters of water were injected into a 1 gallon autoclave. The autoclave was sealed and heated to 250 ° C. and held at that temperature for 4 hours. The autoclave was then cooled and opened to give an emulsion. The mixture was filtered through a celite bed and the layers separated. The organic layer was washed three times with 1 liter of water. The sample was then heated to 50 ° C. and purged with nitrogen for 6 hours. GC analysis is free of i-BuOH and shows a ratio of 28% FABE and 72% fatty acids. Obtained amber oil (720.5 g).

[Table 49]

TABLE 50

Example  57

Recovery of Product Alcohols-Hydrolysis Using Lipase Catalysts

FABE was synthesized from corn oil fatty acid according to the method described in Example 44. Novozyme 435 (Novo 435, Candida Antarkica Lipase B, fixed on acrylic resin) was purchased from Sigma Aldrich (St. Louis, MO). Candida Antarctica Lipase B was purchased from Novozymes (Franklinton, NC). t- BuOH, acetone, ethanol, methanol, and glycerol were all purchased from Sigma Aldrich (St. Louis, MO). For gas chromatography (GC) analysis, the gas chromatograph used was Hewlett Packard 5890 Series II GC chromatogram, methyl pentadecanoate was used as internal standard.

A. atmospheric pressure, 40 ℃

To a mixture of 2 mL FABE and 5 mL water, 40 mg Novozyme 435 was added, the reaction mixture was poured into 20 mL vials and incubated at 40 ° C. on a rotary shaker (300 rpm). The reaction mixture was analyzed using GC during the 24 hour reaction to form the following% conversion profiles shown in Table 51:

[Table 51]

B. Atmospheric pressure, 40 ° C., 65 ° C. and 80 ° C., without organic solvent

Together with Part A of this example, these data show how the equilibrium changes with temperature.

To a mixture of 1 mL FABE and 2 mL water, 20 mg Novozyme 435 was added and the reaction mixture was spun at 40 ° C. for 45 h in a septum-capped 6 mL vial. The reaction mixture was analyzed using GC to show 18.2% conversion of FABE at equilibrium.

To a mixture of 1 g FABE and 2 mL water, 20 mg Novozyme 435 was added and the reaction mixture was spun at 65 ° C. for 42 h in a 6 mL vial capped with a septum. The reaction mixture was analyzed using GC to show 19.8% conversion of FABE at equilibrium.

To a mixture of 1 g FABE and 2 mL water, 20 mg Novozyme 435 was added and the reaction mixture was spun at 80 ° C. for 42 h in a 6 mL vial capped with a septum. The reaction mixture was analyzed using GC, showing 21.4% conversion of FABE at equilibrium.

C. Organic Solvents for Equilibrium (t- BuOH Example showing the effect of

To three reaction mixtures containing 0.25 mL FABE, 0.75 mL t-BuOH, and 0.1-0.3 mL water, 20 mg novozyme 435 was added to rotate the mixture in a 6 mL vial capped with a septum at 40 ° C. overnight. The mixture was then equilibrated. The reaction mixture was analyzed using GC after 24 h of reaction, resulting in 77-82% FABE conversion given in Table 52. Substitution of t-BuOH with 3-Me-3-pentanol under similar reaction conditions yielded a FABE hydrolysis yield of 70-80%.

Table 52

D. Acetone as a Solvent

To three reaction mixtures containing 0.25 mL FABE, 0.75 mL acetone and 0.1-0.3 mL water, 20 mg Novozyme 435 was added to leave the mixture in a septum capped 6 mL vial overnight at 40 ° C. At that time, the mixture reached equilibrium. The reaction mixture was analyzed using GC after 24 h of reaction showing 71-78% FABE conversion given in Table 53.

Table 53

E. FABE  I- at hydrolysis to conversion BuOH Showing the effect of elimination of nitrogen-nitrogen under atmospheric pressure Purging

In a 25 mL round bottom flask, 2 mL FABE, 5 mL water, and 40 mg novozyme 435 were injected. The reaction mixture was heated to 95 ° C. and i-BuOH produced during the reaction was removed by bubbling nitrogen through the reaction mixture. Samples were taken from the mixture during the reaction and the organic phase was analyzed using GC. As shown in Table 54, 94% conversion was achieved after 6 h:

[Table 54]

F. Example showing removal effect of i-BuOH during hydrolysis on conversion-vacuum distillation

To a 25 mL round bottom flask was injected 3 mL FABE, 7.5 mL water, and 60 mg Novozyme 435. The flask was attached to a vacuum distillation apparatus and the pressure was set to 12.1 kPa (91 mm Hg). The reaction mixture was then heated to 74 ° C. and i-BuOH produced during the reaction was distilled off. Samples were taken from the mixture during the reaction and the organic phase was analyzed using GC. As shown in Table 55, a conversion of 91% was achieved after 10 h.

TABLE 55

G. Example showing removal effect of i-BuOH during hydrolysis on conversion-vacuum distillation example-FABE / COFA starting ratio change: 23% FABE : 77% COFA  v / v

To a 25 mL round bottom flask was injected 0.69 mL FABE, 2.31 mL COFA, 7.5 mL water, and 60 mg Novozyme 435. The flask was attached to a vacuum distillation apparatus and the pressure was set to 12.1 kPa (91 mm Hg). The reaction mixture was then heated to 74 ° C. and i-BuOH produced during the reaction was distilled off. Samples were taken from the mixture during the reaction and the organic phase was analyzed using GC. As shown in Table 56, a 98% conversion was achieved after 10 h.

TABLE 56

H. Example showing removal effect of i-BuOH during hydrolysis on conversion-vacuum distillation example - FABE / COFA starting ratio change: 70% FABE : 30% COFA v / v

In a 25 mL round bottom flask was injected 2.1 mL FABE, 0.9 mL COFA, 7.5 mL water, and 60 mg Novozyme 435. The flask was attached to a vacuum distillation apparatus and the pressure was set to 12.1 kPa (91 mm Hg). The reaction mixture was then heated to 74 ° C. and i-BuOH produced during the reaction was distilled off. Samples were taken from the mixture during the reaction and the organic phase was analyzed using GC. As shown in Table 57, 96% conversion was achieved after 10 h:

Table 57

I. Example showing free Cal B enzyme during FABE hydrolysis under vacuum distillation conditions

Two round bottom flasks were charged with 3 mL (2.7 g) FABE and 7.5 mL H ₂ O, respectively. 5.9 mg Candida antarctica lipase B was added to one mixture and 0.59 mg enzyme was added to the other. The reaction flask was separately connected to a distillation apparatus and exposed to a pressure of 12.1 kPa (91 mm Hg). The reaction mixture was heated to 65-68 ° C. Over 10 hours samples were taken from the reaction mixture and analyzed using gas chromatography. Final FABE conversion was 96 and 78%, respectively. From experiments, it can be seen that reducing the enzyme concentration by 10-fold reduces the rate and conversion to 3x and 18%, respectively. The results are shown in Table 58.

TABLE 58

Example 58

Recovery of product alcohols-transesterification

FABE was synthesized from corn oil fatty acid according to the method described in Example 44; Novozyme 435 (Candida antarctica lipase B, fixed in acrylic resin) was purchased from Sigma Aldrich (St. Louis, MO). Candida Antarctica Lipase B was purchased from Novozymes (Franklinton, NC). t-BuOH, acetone, ethanol, methanol, and glycerol were all purchased from Sigma Aldrich (St. Louis, MO). For GC analysis, the gas chromatograph used was Hewlett Packard 5890 Series II GC chromatogram, methyl pentadecanoate was used as internal standard.

A. Lipase Test-FABE to FAME

Reagents used were t-BuOH (Aldrich); MeOH (Aldrich); Novozyme 435 (Aldrich); PS30 (Amano Enzymes, Inc., Berkholderia Sephacia, Elgin, Ill.); Repolarase? 100T (thermomyses ranuginosa, fixed in silica, Novozymes, Franklinton, NC); Repolarase? 100L (Thermomysis ranuginosa, Novozymes, Franklinton, NC); Lipozyme? TLIM (Fixed Thermomyses ranuginosa, Novozymes, Franklinton, NC); Lipoclin? 2000T (immobilization mixture of lipase; Novozymes, Franklinton, NC); NZL-103-LYO (lipase from Rizomukor Miehei, Novozymes, Franklinton, NC).

To a 6 mL vial, 500 mg FABE (1.48 mmol), 400 μl t-BuOH, 60 μl MeOH (1.48 mmol), 3 μl water, and 2.5 mg lipase were added (see Table 57). The resulting mixture was injected into an incubator / shaker and left overnight at 40 ° C. GC analysis of the reaction mixture showed 9 to 56% conversion. The results are shown in Table 59.

[Table 59]

B. FABE to FAME Conversion-Optimizing the Amount of Methanol

To a 6 mL vial, 500 mg FABE (1.48 mmol), 400 μl t-BuOH, 60-240 μl MeOH (1.48-5.92 mmol), 3 μl water, and 2.5 mg novozyme 435 were added. The resulting mixture was injected into an incubator / shaker and left overnight at 40 ° C. GC analysis of the reaction mixture showed a conversion of 53-73%, as shown in Table 60.

TABLE 60

C. Optimization of the amount of enzyme-from FABE to FAME

To a 6 mL vial, 500 mg FABE (1.48 mmol), 400 μl t-BuOH, 132 μl MeOH (3.26 mmol), and 5-25 mg novozyme 435 were added. The resulting mixture was injected into an incubator / shaker and left overnight at 40 ° C. GC analysis of the reaction mixture showed 76-79% conversion, as shown in Table 61.

TABLE 61

D. Solvent (t- BuOH Minimize the amount of)- FABE in FAME in

To a 6 mL vial, 500 mg FABE (1.48 mmol), 0-300 μl t-BuOH, 132 μl MeOH (3.26 mmol), and 10 mg novozyme 435 were added. The resulting mixture was injected into an incubator / shaker and left overnight at 40 ° C. GC analysis of the reaction mixture showed a conversion of 30-81%, as shown in Table 62.

[Table 62]

E. Minimization of the amount of solvent (3-Me-3-pentanol)-from FABE to FAME

To a 6 mL vial, 500 mg FABE (1.48 mmol), 0-300 μl 3-Me-3-pentanol, 132 μl MeOH (3.26 mmol), and 10 mg novozyme 435 were added. The resulting mixture was injected into an incubator / shaker and left overnight at 40 ° C. GC analysis of the reaction mixture showed a conversion of 30 to 78%, as shown in Table 63.

Table 63

F. FABE to FAME Conversion without Solvent

To a mixture of FABE (500 mg, 1.48 mmol) and methanol (0.13 μl, 3.25 mmol), 40 mg novozyme 435 was added and the reaction mixture was stirred at 40 ° C. overnight. The mixture was then filtered and analyzed by GC showing 76% conversion.

G. Enzyme Recycling-FABE to FAME

To a 6 mL vial, 500 mg FABE (1.48 mmol), 400 μl t-BuOH, 132 μl MeOH (3.26 mmol), and 10 mg novozyme 435 were added. The resulting mixture was injected into an incubator / shaker and left overnight at 40 ° C. Thereafter, the reaction mixture was filtered, the conversion was analyzed using GC, and a filter cake containing immobilized enzyme was used for another conversion from FABE to FAME. The process was repeated 10 times (Table 64). From the experiment, it can be seen that the enzyme can be recycled up to 10 times without loss of conversion in the overnight reaction.

[Table 64]

H. FABE to FAEE Conversion

To a 6 mL vial capped with a septum, 0.8 mL FABE (2.08 mmol) and 0.2 mL EtOH (3.43 mmol) were added to form a single phase. No enzyme was added to the vial or 20 mg Novozyme 435 was added. The vials were then incubated at 25 ° C. and 40 ° C. for 17 h in an incubator shaker (300 rpm), after which the solution was analyzed by gas chromatography to determine the contents shown in Table 65 and the percent conversion from FABE to FAEE. Provided.

TABLE 65

Example 59

Glycerol Degradation of FABE

To 6 mL vials capped with septum, 0.75 mL t-BuOH, 0.25 mL FABE, 0.1 mL (0.126 g) glycerol + 4 μl H ₂ O, 20 mg enzyme, respectively, were formed to form a single phase. The reaction was incubated with various lipases at 40 ° C. on a rotary shaker at 300 rpm. After 20 h, the sample was analyzed by gas chromatography to obtain the contents shown in Table 66. Comparison of the COFA and FABE content indicates that the product is i-BuOH and a mixture of COFA and acyl glycerol (˜64% acyl glycerol / 36% COFA on a molar basis).

TABLE 66

A. Water and Glycerol / FABE  Dependence

To a 6 mL vial capped with a septum, 0.75 mL t-BuOH, 0.25 mL FABE, 0.1 or 0.2 g glycerol + 20 mg each of Amano PS-30 or Novozyme 435 was added to form a single phase. No water was added. The reaction was incubated at 40 ° C. on a 300 rpm rotary shaker. After 20 h, the sample was analyzed by gas chromatography to yield the contents shown in Table 67. Comparison of the COFA and FABE content indicates that the product is i-BuOH and mainly acyl glycerol (mainly monoglycerides). In relation to the previous examples, the proportion of the product in the form of acyl glycerol increases without water being added and increases with glycerol / FABE (˜91% acyl glycerol / 9% COFA, 1.6 glycerol / FABE, mol on a molar basis). / mol use, and ˜95% acyl glycerol / 5% COFA, 3.2 glycerol / FABE, mol / mol on a molar basis). If no water is added, the enzymatic activity of Amano PS-30 is removed. However, Novozyme 435 with water (~ 3% w / w) in the acrylic resin to be immobilized is still active.

TABLE 67

B. Enzyme Concentration Dependency

To a 6 mL vial capped with a septum, 0.75 mL t-BuOH, 0.25 mL FABE, 0.2 g glycerol (glycerol / FABE 3.2 / 1, mol / mol) + 2 or 20 mg of Novozyme 435 (Novo 435) was added. . No water was added. The reaction was incubated at 40 ° C. on a 300 rpm rotary shaker, and the reaction was analyzed by gas chromatography as a function of time. Yields are shown in Table 68. About 97% of the reacted FABEs were converted to acyl glycerol (mainly monoglycerides) on a molar basis.

TABLE 68

The reaction rate is linearly changed, and 2 and 20 ㎎ of Novozymes 435 to about ₁ t _{/ 2,} respectively, 208 minutes and 20 minutes with respect to enzyme concentration. However, the reaction reached almost the same yield of FABE conversion after 24 h.

This final reaction was repeated using 0.75 mL of 3-methyl-3-pentanol instead of 0.75 mL of t-BuOH. The degree of FABE hydrolysis obtained after 24 h was the same for both solvents. The advantage of 3-methyl-3-pentanol is that with a boiling point of 122 ° C., i-BuOH can first be distilled into pure form (boiling point 108 ° C.). Since 3-methyl-3-pentanol can then be distilled and recycled for the hydrolysis reaction, acyl glycerol, COFA, and glycerol present in the concentrate are recycled to the fermentation tank for reuse in the production of FABE. Can be. Tertiary alcohols act alone as a solvent and have the advantage of not reacting with fatty acids in the presence of CALB to produce fatty acid alkyl esters.

C. Organic cosolvent  In the absence FABE Glycerol breakdown ( FABE in COFA  + Acyl glycerol)-glycerol concentration dependence

1 gram (1 g) of FABE was mixed with 2 mL of 50, 70, 90, and 100% (w / w) glycerol to obtain 20 mg Lipobond (Sprinkler Technologies, Trieste, Italy). Sprin Technologies)) into 6 mL vials capped with a septum. The vial was tumbled by spinning at 62 ° C. for 24 h. As the glycerol concentration in the aqueous phase increases, the proportion of product in the acyl glycerol form increases (Table 69, mainly monoglycerides). However, FABE conversion does not indicate glycerol concentration dependence.

TABLE 69

Example 60

COFA of FAEE  And Monoacyl  Conversion to glycerol

The examples below show that COFA can be esterified with EtOH or glycerol in high yield under mild conditions using immobilized enzymes.

Novozyme 435 (Candida antarctica lipase B, fixed in acrylic resin) was purchased from Sigma Aldrich (St. Louis, MO). Acetone, t-BuOH, ethanol, methanol, and glycerol were all purchased from Sigma Aldrich (St. Louis, MO). For GC analysis, the gas chromatograph used was Hewlett Packard 5890 Series II GC chromatogram, methyl pentadecanoate was used as internal standard.

A. COFA to FAEE + i-BuOH Conversion with Ethanol

Corn oil fatty acid (COFA, 0.25 g) was dissolved in 2.0 mL EtOH to form a single phase. By adding 20 mg of Candida antarctica lipase B (CALB) immobilized on acrylic resin (Novozyme 435) (containing 1.7 mg of enzyme), the suspension was sealed with a diaphragm cap at 40 ° C. on a rotary shaker (300 rpm). Incubate for 24 h in mL glass vials. The reaction was virtually complete with 98% of COFA converted to FAEE (fatty acid ethyl ester). GC analysis after 24 h showed 98% conversion from COFA to fatty acid ethyl esters, as shown in Table 70.

[Table 70]

B. Conversion of COFA to Monoacylglycerides (MAG) + i-BuOH with Glycerol

Corn oil fatty acid (COFA, 0.25 g) and 0.325 g of glycerol were dissolved in 2.0 mL acetone. There was a large top phase and a small residual glycerol containing phase in which most components were dissolved. By adding 20 mg of Candida antarctica lipase B (CALB) immobilized on acrylic resin (Novozyme 435) (containing 1.7 mg of enzyme), the suspension was sealed with a diaphragm cap at 40 ° C. on a rotary shaker (300 rpm). Incubate for 24 h in mL glass vials. GC on the top indicated that 87% of COFA was converted to acyl glycerides (expected primarily to be monoacylglycerides). The results are shown in Table 71.

[Table 71]

Example  61

COFA to FAME Conversion

The following examples show that COFA can be esterified with MeOH, EtOH or glycerol in high yield under mild conditions using immobilized lipase.

A. COFA to FAME Conversion without Solvent

To a 6 mL vial, 500 mg COFA (1.48 mmol), 132 μl MeOH (3.26 mmol), and 10 mg novozyme 435 were added. The resulting mixture was injected into an incubator / shaker and left overnight at 40 ° C. GC analysis of the reaction mixture showed 95% conversion.

B. Determining Changes Over Time of COFA to FAME Reactions

To a 6 mL vial, 500 mg COFA (1.48 mmol), 132 μl MeOH (3.26 mmol), and 10 mg novozyme 435 were added. Samples were incubated at 40 ° C. in an incubator / shaker and time points were taken during the reaction and analyzed using GC. The results are shown in Table 72.

[Table 72]

C. Addition of Higher MeOH to COFA → FAME Reaction

To a 6 mL vial, 500 mg COFA (1.48 mmol), 180, 240, 300, or 1320 μl MeOH (4.44, 5.92, 7.41, and 14.82 mmol), and 10 mg novozyme 435 were added. The resulting mixture was injected into an incubator / shaker and left overnight at 40 ° C. GC analysis of the reaction mixture showed 96-97% conversion. The results are shown in Table 73.

[Table 73]

Example 62

This example illustrates the removal of solids from distillation waste and extraction by desolventizers to recover fatty acids, esters, and triglycerides from solids. Upon fermentation, the solids are separated from the total distillation waste liquor and fed to the desolventizer and contacted with steam 997.9 kg / hr (1.1 tons / hr). The flow rates of the whole distillation waste liquid wet cake (extractor feed), solvent, extractor micellar, and extractor discharge solids are shown in Table 74. The values in the table are simply tons / hr.

[Table 74]

Solids discharged from the desolventizer are fed to the dryer. The steam exiting the desolventizer contains 499.0 kg / hr (0.55 tons / hr) of hexane and 999.7 kg / hr (1.102 tons / hr) of water. This stream is condensed and fed to the decanter. The water rich phase exiting the decanter contains about 360 ppm of hexane. This stream is fed to a distillation column where hexane is removed from the water rich stream. The hexane rich stream exiting the top of the distillation column is condensed and fed to the decanter. The organic rich stream exiting the decanter is fed to the distillation column. Steam (9997.2 kg / hr (11.02 tons / hr)) is fed to the bottom of the distillation column. The overhead and bottom product concentrations for this column are shown in Table 75. The value in the table is tonnes / hr.

[Table 75]

Example 63

Solid Extraction

Preparation of Isobutanol

In a 100 mL volumetric flask, 65 g of anhydrous reagent grade isobutanol (supplied from Aldrich) was combined with 10 g of distilled water and shaken until a clear colorless uniform phase was obtained. An additional 10 g of distilled water was added to the measuring flask and shaken again to obtain two continuous clear colorless liquid layers. The top layer is typically considered to be hydrous isobutanol containing 20 wt% moisture and the bottom layer is mainly water, typically having 8 wt% dissolved alcohol.

Extraction and Substitution Cleaning Using Screen Filtration

The fermentation was completed using the recycled fatty acid (Example 19). The 185 g portion representing the resulting heterogeneous mixture was removed and the Nalgene? Over 5 minutes using a slight vacuum (-5.0 kPa (-20 (H 2 O))) at the bottom. Passed through a sealed 80 mesh screen dish supported in a plastic filter funnel. The filtrate was partitioned into 50.9 g hazy aqueous phase and 90.5 g reddish brown oil phase, which contained dispersed fines but did not contain precipitated particulates. The wet cake remained in the screen dish. A sample of 1.5 g of this microscopic wet cake was removed and air dried. While maintaining a mild vacuum under the screen dish until no more liquid droplets were collected, the hydrous isobutanol (23 g) was withdrawn from the top layer inside the flask and passed through the wet cake over 5 minutes. The total filtrate mass of 18 g consisted of a small amount of immiscible bottom hazy aqueous layer and yellow clear hydrous isobutanol layer. The wet cake was removed from the screen dish and a total mass of 38.4 g was recovered. A sample of 1.5 g of this washed wet cake was removed and air dried. Analysis of the dry sample of the fine solids revealed that it contained 53.35 wt% of total fat on the basis of triglycerides, and 15.9 wt% of triglycerides on the basis of analysis of the dry sample of the washed solids. It was found to contain total fat.

Extraction Using Centrifugation and Riesler Wash

The fermentation was completed using the recycled fatty acid (Example 19). A 225 g portion representing the resulting heterogeneous mixture was recovered and centrifuged using a Beckman Coulter Allegra 64R machine at 10,000 rpm for 10 minutes. 67.2 g of the clear reddish brown oil phase was decanted. The residue was centrifuged again and 95.1 g of hazy aqueous centrate was decanted. A 1.5 g sample of wet solids was removed, air dried, 56.5 g recovered and transferred to a 400 mL beaker. Hydrated isobutanol (20 g) removed from the upper layer of the flask was added to the beaker, the wet solid was repulped and stirring was performed for 5 minutes. An additional 32 g of hydrous isobutanol was added with 32 g of centrate and the solid was stirred for 5 minutes in an aqueous suspension just below the stationary organic layer. The mixture was then centrifuged at 10,000 rpm for 10 minutes to decant the clear yellow hydrous isobutanol layer and again centrifuged to separate and dry a 1.5 g sample of the washed wet solid. Analysis of the dry sample of the microscopic wet solids revealed that it contained 21.6 wt% of total fat on the basis of triglycerides, and 4.04 wt on the basis of triglycerides when the dry sample of the washed wet solids was analyzed. Found to contain% total fat.

Example  64

Removal of Corn Oil by Removal of Insoluble Solids

About 1000 g of liquefied corn mash was prepared in a 1 L glass, jacketed resin kettle. The kettle was set up with mechanical stirring, temperature control, and pH control. The following protocol was used: the ground corn was mixed with tap water (26 wt% corn on a dry basis), the slurry was heated to 55 ° C. with stirring, the pH was adjusted to 5.8 with NaOH or H ₂ SO ₄ , and alpha-amylase (0.02 wt% on dry corn basis) was added, continuous heating to 85 ° C., pH adjusted to 5.8, maintained at 85 ° C. for 2 hours while maintaining pH at 5.8, and cooled to 25 ° C. The corn used was the yellow corncob from Pioneer (3335). Grinding in a hammer mill using a 1 mm screen. The moisture content of the ground corn was measured to be about 11.7 wt%, and the starch content of the ground corn was measured to be about 71.4 wt% on a dry corn basis. The alpha-amylase enzyme is Liquozyme from Novozymes (Franklinton, NC). SC DS. The total amount of ingredients used was 294.5 g of ground corn (11.7% moisture), 705.5 g of tap water, and 0.059 g of liquezyme? SC DS. H ₂ O (4.3 g) was added to dilute the enzyme and a total of 2.3 g of 20% NaOH solution was added to control pH. About 952 g of mash were recovered. Note that there was a loss due to the mash being attached to the walls of the kettle and CF bottle.

The liquefied corn mash was centrifuged at 5000 rpm (7260 g's) for 30 minutes at 40 ° C to remove insoluble solids from the aqueous solution of oligosaccharides. By removing the solids by centrifugation, the free corn oil as the separated organic liquid phase on top of the aqueous phase is removed. About 1.5 g of corn oil was recovered from the organic layer suspended above the aqueous phase. By hexane extraction, it was determined that the ground corn used to produce the liquefied mash contained about 3.5 wt% corn oil on a dry corn basis. This corresponds to about 9 g of corn oil fed to the liquefaction process with ground corn.

About 1 g of corn oil was recovered from the organic layer suspended above the aqueous phase. About 617 g of liquefied starch solution was recovered, leaving about 334 g of wet cake. The wet cake contained most of the insoluble solids present in the liquefied mash. The liquefied starch solution contained about 0.2 wt% insoluble solids. The wet cake contained about 21 wt% insoluble solids. The wet cake was washed with 1000 g of tap water to remove oligosaccharides still in the cake. This was done by mixing the cake with water to form a slurry. The slurry was then centrifuged under the same conditions used to centrifuge the original mash to recover the washed solids. Removal of the washed solids by centrifugation removed additional free corn oil as a separated organic liquid phase on top of the aqueous phase. Corn oil was recovered from the organic layer suspended above the aqueous phase.

To remove substantially all of the liquefied starch, the wet solid was washed two more times with 1000 g of tap water each time. The final washed solid was dried in a vacuum oven at 80 ° C. and about 67.7 kPa (20 inch Hg) vacuum overnight. The amount of corn oil remaining in the dry solids, perhaps embryos, was determined by hexane extraction. A 3.60 g sample of relatively dry solids (about 2 wt% moisture) was determined to contain 0.22 g of corn oil. This result corresponds to 0.0624 g corn oil / g dry solids. This is for the washed solids, meaning that the wet solids are free of residual oligosaccharides. After centrifuging the liquefied corn mash to separate the free corn oil layer and the aqueous oligosaccharide solution from the wet cake, it was determined that about 334 g of wet cake containing about 21 wt% insoluble solids remained. This corresponds to a wet cake containing about 70.1 g of insoluble solids. In 0.0624 g corn oil / g dry solids, the solids in the wet cake should contain about 4.4 g of corn oil.

Example 65

Lipid analysis

Lipid analysis was performed by conversion of various fatty acid containing compounds to fatty acid methyl esters ("FAME") by transesterification. Glycerides and phospholipids were transesterified using sodium methoxide in methanol. Glycerides, phospholipids, and free fatty acids were transesterified using acetyl chloride in methanol. The resulting FAME was subjected to 30-m × 0.25 mm (id) OMEGAWAX ™ (Supelco, Sigma-Aldrich, St. Louis, MO) column after dilution in toluene / hexane (2: 3). It was analyzed by gas chromatography using Agilent 7890 GC assembled. The oven temperature was increased from 160 ° C. to 200 ° C. at 5 ° C./min and then from 200 ° C. to 250 ° C. (10 min hold) at 10 ° C./min. FAME peaks recorded through GC analysis were identified by their retention time when compared to known methyl esters (ME), and the FAME peak area was determined using a known amount of internal standard (C15: 0 triglycerides, samples). And taken through a transesterification procedure). Thus, the approximate amount (mg) of any fatty acid FAME (“mg FAME”) is calculated according to the following formula: (FAME peak area / 15: 0 FAME peak area for a specific fatty acid) * (internal standard C15: 0 FAME (mg)). The FAME result can then be corrected for mg of the corresponding fatty acid by dividing by an appropriate molecular weight conversion factor of 1.052. All internal and reference standards are obtained from Nu-Jeop Prep, Inc.

The fatty acid results obtained for the sample transesterified using sodium methoxide in methanol are converted to the corresponding triglyceride levels by multiplying the molecular weight conversion factor of 1.045. Triglycerides generally comprise about 80-90% of the glycerides in the sample studies for this example, with the remainder being diglycerides. Monoglycerides and phospholipid contents are generally negligible. The total fatty acid results obtained for the sample transesterified using acetyl chloride in methanol are corrected for glyceride content by subtracting the fatty acids measured for the same sample using the sodium methoxide procedure. The result is the free fatty acid content of the sample.

The distribution of glyceride content (monoglycerides, diglycerides, triglycerides, and phospholipids) is measured using thin layer chromatography. The oil solution dissolved in 6: 1 chloroform / methanol is spotted near the bottom of the glass plate precoated with silica gel. The spot is then chromatographed over the plate using a 70: 30: 1 hexane / diethyl ether / acetic acid solvent system. Separate spots corresponding to monoglycerides, diglycerides, triglycerides, and phospholipids are then detected by staining the plates with iodine vapor. The spot is then peeled off from the transesterified plate using acetyl chloride in the methanol procedure and analyzed by gas chromatography. The ratio of the sum of the peak areas for each spot to the sum of the peak areas for all the spots is the distribution of the various glycerides.

Example  66

This example illustrates the recovery of byproducts from the mash. Corn oil was prepared from the mash under the conditions described in Example 64 except that a tricanter centrifuge (Flottweg Z23-4, ball diameter 230 mm, length to diameter ratio 4: 1) was used with the following conditions: Separated:

Ball speed: 5000 rpm

Differential velocity: 10 rpm

Feed rate: 3 gpm

Phase separator disc: 138 mm

Impeller setting 144 mm.

The separated corn oil measured 81% triglycerides, 6% free fatty acids, 4% diglycerides, and 5% of the total of phospholipids and monoglycerides as measured by the method and thin layer chromatography described in Example 65. .

Solids separated from the mash under the conditions described above had a moisture content of 58% as measured by weight loss upon drying and 1.2% triglycerides and 0.27% as measured by the method described in Example 65. Free fatty acids are shown.

The composition of the solids separated from the total distillation waste in Table 78, the oil extracted between the evaporator stages, the byproduct extractant and the condensed liquor (CDS), the composition of the total distillation waste in Table 76 and the assumptions in Table 77 (Tricanter Calculations on the assumption of separation in a centrifuge). Aspen Plus values in Table 75? From the model (Aspen Technologies, Inc., Burlington, Mass., USA). This model assumes that corn oil is not extracted from the mash. On a dry basis, the protein content of the cells, dissolved solids, and suspended solids is estimated to be about 50%, 22%, and 35.5%, respectively. The composition of the byproduct extractant is estimated to be 70.7% fatty acid and 29.3% fatty acid isobutyl ester on a dry basis.

Table 76

Table 77

Table 78

In addition, while various embodiments of the invention have been described above, it is obvious that these are given as examples only and are not limited to them. It will be apparent to those skilled in the art that various changes in form and details may be made without departing from the spirit and scope of the invention. Accordingly, the breadth and scope of the present invention should not be limited by any of the above-described exemplary embodiments, but should be defined by the following claims and their equivalents.

All publications, patents, and patent applications mentioned in the specification are indicative of proficiency in the art to which the invention pertains, and to the same extent as if each individual publication, patent, or patent application was specifically indicated to be incorporated by reference. It is incorporated herein by reference.

                         SEQUENCE LISTING <110> Butamax (TM) Advanced Biofuels   <120> PRODUCTION OF ALCOHOL ESTERS AND IN SITU PRODUCT REMOVAL DURING        ALCOHOL FERMENTATION <130> CL5103 <150> US 61 / 368,429 <151> 2010-07-28 <150> US 61 / 356,290 <151> 2010-06-18 <150> US 61 / 368,451 <151> 2010-07-28 <150> US 61 / 368,436 <151> 2010-07-28 <150> US 61 / 368,444 <151> 2010-07-28 <150> US 13 / 160,766 <151> 2011-06-15 <150> US 61 / 440,034 <151> 2011-02-07 <150> US 61 / 379,546 <151> 2010-09-02 <160> 202 <170> PatentIn version 3.5 <210> 1 <211> 11856 <212> DNA <213> Artificial sequence <220> <223> Plasmid <400> 1 tcccattacc gacatttggg cgctatacgt gcatatgttc atgtatgtat ctgtatttaa 60 aacacttttg tattattttt cctcatatat gtgtataggt ttatacggat gatttaatta 120 ttacttcacc accctttatt tcaggctgat atcttagcct tgttactagt tagaaaaaga 180 catttttgct gtcagtcact gtcaagagat tcttttgctg gcatttcttc tagaagcaaa 240 aagagcgatg cgtcttttcc gctgaaccgt tccagcaaaa aagactacca acgcaatatg 300 gattgtcaga atcatataaa agagaagcaa ataactcctt gtcttgtatc aattgcatta 360 taatatcttc ttgttagtgc aatatcatat agaagtcatc gaaatagata ttaagaaaaa 420 caaactgtac aatcaatcaa tcaatcatcg ctgaggatgt tgacaaaagc aacaaaagaa 480 caaaaatccc ttgtgaaaaa cagaggggcg gagcttgttg ttgattgctt agtggagcaa 540 ggtgtcacac atgtatttgg cattccaggt gcaaaaattg atgcggtatt tgacgcttta 600 caagataaag gacctgaaat tatcgttgcc cggcacgaac aaaacgcagc attcatggcc 660 caagcagtcg gccgtttaac tggaaaaccg ggagtcgtgt tagtcacatc aggaccgggt 720 gcctctaact tggcaacagg cctgctgaca gcgaacactg aaggagaccc tgtcgttgcg 780 cttgctggaa acgtgatccg tgcagatcgt ttaaaacgga cacatcaatc tttggataat 840 gcggcgctat tccagccgat tacaaaatac agtgtagaag ttcaagatgt aaaaaatata 900 ccggaagctg ttacaaatgc atttaggata gcgtcagcag ggcaggctgg ggccgctttt 960 gtgagctttc cgcaagatgt tgtgaatgaa gtcacaaata cgaaaaacgt gcgtgctgtt 1020 gcagcgccaa aactcggtcc tgcagcagat gatgcaatca gtgcggccat agcaaaaatc 1080 caaacagcaa aacttcctgt cgttttggtc ggcatgaaag gcggaagacc ggaagcaatt 1140 aaagcggttc gcaagctttt gaaaaaggtt 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tatacaaaat atcgataagt tattcttgcc 2880 caccaattta aggagcctac atcaggacag tagtaccatt cctcagagaa gaggtataca 2940 taacaagaaa atcgcgtgaa caccttatat aacttagccc gttattgagc taaaaaacct 3000 tgcaaaattt cctatgaata agaatacttc agacgtgata aaaatttact ttctaactct 3060 tctcacgctg cccctatctg ttcttccgct ctaccgtgag aaataaagca tcgagtacgg 3120 cagttcgctg tcactgaact aaaacaataa ggctagttcg aatgatgaac ttgcttgctg 3180 tcaaacttct gagttgccgc tgatgtgaca ctgtgacaat aaattcaaac cggttatagc 3240 ggtctcctcc ggtaccggtt ctgccacctc caatagagct cagtaggagt cagaacctct 3300 gcggtggctg tcagtgactc atccgcgttt cgtaagttgt gcgcgtgcac atttcgcccg 3360 ttcccgctca tcttgcagca ggcggaaatt ttcatcacgc tgtaggacgc aaaaaaaaaa 3420 taattaatcg tacaagaatc ttggaaaaaa aattgaaaaa ttttgtataa aagggatgac 3480 ctaacttgac tcaatggctt ttacacccag tattttccct ttccttgttt gttacaatta 3540 tagaagcaag acaaaaacat atagacaacc tattcctagg agttatattt ttttacccta 3600 ccagcaatat aagtaaaaaa ctgtttaaac agtatggcag ttacaatgta ttatgaagat 3660 gatgtagaag tatcagcact tgctggaaag 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ccaaaattaa tagcctatcg cgaagctgca 4560 aaaaatcttg aaattgaaaa aattggggca gagctacgtc aagcaatgcc attcacacaa 4620 tctggtgatg acgatgcctt taaaatctat cagtaaggcc ctgcaggcca gaggaaaata 4680 atatcaagtg ctggaaactt tttctcttgg aatttttgca acatcaagtc atagtcaatt 4740 gaattgaccc aatttcacat ttaagatttt ttttttttca tccgacatac atctgtacac 4800 taggaagccc tgtttttctg aagcagcttc aaatatatat attttttaca tatttattat 4860 gattcaatga acaatctaat taaatcgaaa acaagaaccg aaacgcgaat aaataattta 4920 tttagatggt gacaagtgta taagtcctca tcgggacagc tacgatttct ctttcggttt 4980 tggctgagct actggttgct gtgacgcagc ggcattagcg cggcgttatg agctaccctc 5040 gtggcctgaa agatggcggg aataaagcgg aactaaaaat tactgactga gccatattga 5100 ggtcaatttg tcaactcgtc aagtcacgtt tggtggacgg cccctttcca acgaatcgta 5160 tatactaaca tgcgcgcgct tcctatatac acatatacat atatatatat atatatatgt 5220 gtgcgtgtat gtgtacacct gtatttaatt tccttactcg cgggtttttc ttttttctca 5280 attcttggct tcctctttct cgagcggacc ggatcctccg cggtgccggc agatctattt 5340 aaatggcgcg ccgacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg 5400 tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat 5460 gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat 5520 tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt 5580 aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag 5640 cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa 5700 agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg 5760 ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct 5820 tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac 5880 tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca 5940 caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat 6000 accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact 6060 attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc 6120 ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga 6180 taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg 6240 taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg 6300 aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca 6360 agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta 6420 ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca 6480 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg 6540 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga 6600 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa 6660 tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc 6720 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg 6780 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac 6840 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct 6900 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc 6960 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg 7020 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg 7080 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct 7140 ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga 7200 taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg 7260 cagcgagtca gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc 7320 gcgttggccg attcattaat gcagctggca cgacaggttt cccgactgga aagcgggcag 7380 tgagcgcaac gcaattaatg tgagttagct cactcattag gcaccccagg ctttacactt 7440 tatgcttccg gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc acacaggaaa 7500 cagctatgac catgattacg ccaagctttt tctttccaat tttttttttt tcgtcattat 7560 aaaaatcatt acgaccgaga ttcccgggta ataactgata taattaaatt gaagctctaa 7620 tttgtgagtt tagtatacat gcatttactt ataatacagt tttttagttt tgctggccgc 7680 atcttctcaa atatgcttcc cagcctgctt ttctgtaacg ttcaccctct accttagcat 7740 cccttccctt tgcaaatagt cctcttccaa caataataat gtcagatcct gtagagacca 7800 catcatccac ggttctatac tgttgaccca atgcgtctcc cttgtcatct aaacccacac 7860 cgggtgtcat aatcaaccaa tcgtaacctt catctcttcc acccatgtct ctttgagcaa 7920 taaagccgat aacaaaatct ttgtcgctct tcgcaatgtc aacagtaccc ttagtatatt 7980 ctccagtaga tagggagccc ttgcatgaca attctgctaa catcaaaagg cctctaggtt 8040 cctttgttac ttcttctgcc gcctgcttca aaccgctaac aatacctggg cccaccacac 8100 cgtgtgcatt cgtaatgtct gcccattctg ctattctgta tacacccgca gagtactgca 8160 atttgactgt attaccaatg tcagcaaatt ttctgtcttc gaagagtaaa aaattgtact 8220 tggcggataa tgcctttagc ggcttaactg tgccctccat ggaaaaatca gtcaagatat 8280 ccacatgtgt ttttagtaaa caaattttgg gacctaatgc ttcaactaac tccagtaatt 8340 ccttggtggt acgaacatcc aatgaagcac acaagtttgt ttgcttttcg tgcatgatat 8400 taaatagctt ggcagcaaca ggactaggat gagtagcagc acgttcctta tatgtagctt 8460 tcgacatgat ttatcttcgt ttcctgcagg tttttgttct gtgcagttgg gttaagaata 8520 ctgggcaatt tcatgtttct tcaacactac atatgcgtat atataccaat ctaagtctgt 8580 gctccttcct tcgttcttcc ttctgttcgg agattaccga atcaaaaaaa tttcaaggaa 8640 accgaaatca aaaaaaagaa taaaaaaaaa atgatgaatt gaaaagcttg catgcctgca 8700 ggtcgactct agtatactcc gtctactgta cgatacactt ccgctcaggt ccttgtcctt 8760 taacgaggcc ttaccactct tttgttactc tattgatcca gctcagcaaa ggcagtgtga 8820 tctaagattc tatcttcgcg atgtagtaaa actagctaga ccgagaaaga gactagaaat 8880 gcaaaaggca cttctacaat ggctgccatc attattatcc gatgtgacgc tgcatttttt 8940 tttttttttt tttttttttt tttttttttt tttttttttt ttttttgtac aaatatcata 9000 aaaaaagaga atctttttaa gcaaggattt tcttaacttc ttcggcgaca gcatcaccga 9060 cttcggtggt actgttggaa ccacctaaat caccagttct gatacctgca tccaaaacct 9120 ttttaactgc atcttcaatg gctttacctt cttcaggcaa gttcaatgac aatttcaaca 9180 tcattgcagc agacaagata gtggcgatag ggttgacctt attctttggc aaatctggag 9240 cggaaccatg gcatggttcg tacaaaccaa atgcggtgtt cttgtctggc aaagaggcca 9300 aggacgcaga tggcaacaaa cccaaggagc ctgggataac ggaggcttca tcggagatga 9360 tatcaccaaa catgttgctg gtgattataa taccatttag gtgggttggg ttcttaacta 9420 ggatcatggc ggcagaatca atcaattgat gttgaacttt caatgtaggg aattcgttct 9480 tgatggtttc ctccacagtt tttctccata atcttgaaga ggccaaaaca ttagctttat 9540 ccaaggacca aataggcaat ggtggctcat gttgtagggc catgaaagcg gccattcttg 9600 tgattctttg cacttctgga acggtgtatt gttcactatc ccaagcgaca ccatcaccat 9660 cgtcttcctt tctcttacca aagtaaatac ctcccactaa ttctctaaca acaacgaagt 9720 cagtaccttt agcaaattgt ggcttgattg gagataagtc taaaagagag tcggatgcaa 9780 agttacatgg tcttaagttg gcgtacaatt gaagttcttt acggattttt agtaaacctt 9840 gttcaggtct aacactaccg gtaccccatt taggaccacc cacagcacct aacaaaacgg 9900 catcagcctt cttggaggct tccagcgcct catctggaag tggaacacct gtagcatcga 9960 tagcagcacc accaattaaa tgattttcga aatcgaactt gacattggaa cgaacatcag 10020 aaatagcttt aagaacctta atggcttcgg ctgtgatttc ttgaccaacg tggtcacctg 10080 gcaaaacgac gatcttctta ggggcagaca ttacaatggt atatccttga aatatatata 10140 aaaaaaaaaa aaaaaaaaaa aaaaaaaaat gcagcttctc aatgatattc gaatacgctt 10200 tgaggagata cagcctaata tccgacaaac tgttttacag atttacgatc gtacttgtta 10260 cccatcattg aattttgaac atccgaacct gggagttttc cctgaaacag atagtatatt 10320 tgaacctgta taataatata tagtctagcg ctttacggaa gacaatgtat gtatttcggt 10380 tcctggagaa actattgcat ctattgcata ggtaatcttg cacgtcgcat ccccggttca 10440 ttttctgcgt ttccatcttg cacttcaata gcatatcttt gttaacgaag catctgtgct 10500 tcattttgta gaacaaaaat gcaacgcgag agcgctaatt tttcaaacaa agaatctgag 10560 ctgcattttt acagaacaga aatgcaacgc gaaagcgcta ttttaccaac gaagaatctg 10620 tgcttcattt ttgtaaaaca aaaatgcaac gcgagagcgc taatttttca aacaaagaat 10680 ctgagctgca tttttacaga acagaaatgc aacgcgagag cgctatttta ccaacaaaga 10740 atctatactt cttttttgtt ctacaaaaat gcatcccgag agcgctattt ttctaacaaa 10800 gcatcttaga ttactttttt tctcctttgt gcgctctata atgcagtctc ttgataactt 10860 tttgcactgt aggtccgtta aggttagaag aaggctactt tggtgtctat tttctcttcc 10920 ataaaaaaag cctgactcca cttcccgcgt ttactgatta ctagcgaagc tgcgggtgca 10980 ttttttcaag ataaaggcat ccccgattat attctatacc gatgtggatt gcgcatactt 11040 tgtgaacaga aagtgatagc gttgatgatt cttcattggt cagaaaatta tgaacggttt 11100 cttctatttt gtctctatat actacgtata ggaaatgttt acattttcgt attgttttcg 11160 attcactcta tgaatagttc ttactacaat ttttttgtct aaagagtaat actagagata 11220 aacataaaaa atgtagaggt cgagtttaga tgcaagttca aggagcgaaa ggtggatggg 11280 taggttatat agggatatag cacagagata tatagcaaag agatactttt gagcaatgtt 11340 tgtggaagcg gtattcgcaa tattttagta gctcgttaca gtccggtgcg tttttggttt 11400 tttgaaagtg cgtcttcaga gcgcttttgg ttttcaaaag cgctctgaag ttcctatact 11460 ttctagagaa taggaacttc ggaataggaa cttcaaagcg tttccgaaaa cgagcgcttc 11520 cgaaaatgca acgcgagctg cgcacataca gctcactgtt cacgtcgcac ctatatctgc 11580 gtgttgcctg tatatatata tacatgagaa gaacggcata gtgcgtgttt atgcttaaat 11640 gcgtacttat atgcgtctat ttatgtagga tgaaaggtag tctagtacct cctgtgatat 11700 tatcccattc catgcggggt atcgtatgct tccttcagca ctacccttta gctgttctat 11760 atgctgccac tcctcaattg gattagtctc atccttcaat gctatcattt cctttgatat 11820 tggatcatat gcatagtacc gagaaactag aggatc 11856 <210> 2 <211> 15539 <212> DNA <213> Artificial sequence <220> <223> Plasmid <400> 2 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180 accataaatt cccgttttaa gagcttggtg agcgctagga gtcactgcca ggtatcgttt 240 gaacacggca ttagtcaggg aagtcataac acagtccttt cccgcaattt tctttttcta 300 ttactcttgg cctcctctag tacactctat atttttttat gcctcggtaa tgattttcat 360 tttttttttt ccacctagcg gatgactctt tttttttctt agcgattggc attatcacat 420 aatgaattat acattatata aagtaatgtg atttcttcga agaatatact aaaaaatgag 480 caggcaagat aaacgaaggc aaagatgaca gagcagaaag ccctagtaaa gcgtattaca 540 aatgaaacca agattcagat tgcgatctct ttaaagggtg gtcccctagc gatagagcac 600 tcgatcttcc cagaaaaaga ggcagaagca gtagcagaac aggccacaca atcgcaagtg 660 attaacgtcc acacaggtat agggtttctg gaccatatga tacatgctct ggccaagcat 720 tccggctggt cgctaatcgt tgagtgcatt ggtgacttac acatagacga ccatcacacc 780 actgaagact gcgggattgc tctcggtcaa gcttttaaag aggccctagg ggccgtgcgt 840 ggagtaaaaa ggtttggatc aggatttgcg cctttggatg aggcactttc cagagcggtg 900 gtagatcttt cgaacaggcc gtacgcagtt gtcgaacttg gtttgcaaag ggagaaagta 960 ggagatctct cttgcgagat gatcccgcat tttcttgaaa gctttgcaga ggctagcaga 1020 attaccctcc acgttgattg tctgcgaggc aagaatgatc atcaccgtag tgagagtgcg 1080 ttcaaggctc ttgcggttgc cataagagaa gccacctcgc ccaatggtac caacgatgtt 1140 ccctccacca aaggtgttct tatgtagtga caccgattat ttaaagctgc agcatacgat 1200 atatatacat gtgtatatat gtatacctat gaatgtcagt aagtatgtat acgaacagta 1260 tgatactgaa gatgacaagg taatgcatca ttctatacgt gtcattctga acgaggcgcg 1320 ctttcctttt ttctttttgc tttttctttt tttttctctt gaactcgacg gatctatgcg 1380 gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggaaat tgtaagcgtt 1440 aatattttgt taaaattcgc gttaaatttt tgttaaatca gctcattttt taaccaatag 1500 gccgaaatcg gcaaaatccc ttataaatca aaagaataga ccgagatagg gttgagtgtt 1560 gttccagttt ggaacaagag tccactatta aagaacgtgg actccaacgt caaagggcga 1620 aaaaccgtct atcagggcga tggcccacta cgtgaaccat caccctaatc aagttttttg 1680 gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag ggagcccccg atttagagct 1740 tgacggggaa agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc 1800 gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt 1860 aatgcgccgc tacagggcgc gtccattcgc cattcaggct gcgcaactgt tgggaagggc 1920 gcggtgcggg cctcttcgct attacgccag ctggcgaaag ggggatgtgc tgcaaggcga 1980 ttaagttggg taacgccagg gttttcccag tcacgacgtt gtaaaacgac ggccagtgag 2040 cgcgcgtaat acgactcact atagggcgaa ttgggtaccg ggccccccct cgaggtcgac 2100 ggcgcgccac tggtagagag cgactttgta tgccccaatt gcgaaacccg cgatatcctt 2160 ctcgattctt tagtacccga ccaggacaag gaaaaggagg tcgaaacgtt tttgaagaaa 2220 caagaggaac tacacggaag ctctaaagat ggcaaccagc cagaaactaa gaaaatgaag 2280 ttgatggatc caactggcac cgctggcttg aacaacaata ccagccttcc aacttctgta 2340 aataacggcg gtacgccagt gccaccagta ccgttacctt tcggtatacc tcctttcccc 2400 atgtttccaa tgcccttcat gcctccaacg gctactatca caaatcctca tcaagctgac 2460 gcaagcccta agaaatgaat aacaatactg acagtactaa ataattgcct acttggcttc 2520 acatacgttg catacgtcga tatagataat aatgataatg acagcaggat tatcgtaata 2580 cgtaatagct gaaaatctca aaaatgtgtg ggtcattacg taaataatga taggaatggg 2640 attcttctat ttttcctttt tccattctag cagccgtcgg gaaaacgtgg catcctctct 2700 ttcgggctca attggagtca cgctgccgtg agcatcctct ctttccatat ctaacaactg 2760 agcacgtaac caatggaaaa gcatgagctt agcgttgctc caaaaaagta ttggatggtt 2820 aataccattt gtctgttctc ttctgacttt gactcctcaa aaaaaaaaat ctacaatcaa 2880 cagatcgctt caattacgcc ctcacaaaaa cttttttcct tcttcttcgc ccacgttaaa 2940 ttttatccct catgttgtct aacggatttc tgcacttgat ttattataaa aagacaaaga 3000 cataatactt ctctatcaat ttcagttatt gttcttcctt gcgttattct tctgttcttc 3060 tttttctttt gtcatatata accataacca agtaatacat attcaaacta gtatgactga 3120 caaaaaaact cttaaagact taagaaatcg tagttctgtt tacgattcaa tggttaaatc 3180 acctaatcgt gctatgttgc gtgcaactgg tatgcaagat gaagactttg aaaaacctat 3240 cgtcggtgtc atttcaactt gggctgaaaa cacaccttgt aatatccact tacatgactt 3300 tggtaaacta gccaaagtcg gtgttaagga agctggtgct tggccagttc agttcggaac 3360 aatcacggtt tctgatggaa tcgccatggg aacccaagga atgcgtttct ccttgacatc 3420 tcgtgatatt attgcagatt ctattgaagc agccatggga ggtcataatg cggatgcttt 3480 tgtagccatt ggcggttgtg ataaaaacat gcccggttct gttatcgcta tggctaacat 3540 ggatatccca gccatttttg cttacggcgg aacaattgca cctggtaatt tagacggcaa 3600 agatatcgat ttagtctctg tctttgaagg tgtcggccat tggaaccacg gcgatatgac 3660 caaagaagaa gttaaagctt tggaatgtaa tgcttgtccc ggtcctggag gctgcggtgg 3720 tatgtatact gctaacacaa tggcgacagc tattgaagtt ttgggactta gccttccggg 3780 ttcatcttct cacccggctg aatccgcaga aaagaaagca gatattgaag aagctggtcg 3840 cgctgttgtc aaaatgctcg aaatgggctt aaaaccttct gacattttaa cgcgtgaagc 3900 ttttgaagat gctattactg taactatggc tctgggaggt tcaaccaact caacccttca 3960 cctcttagct attgcccatg ctgctaatgt ggaattgaca cttgatgatt tcaatacttt 4020 ccaagaaaaa gttcctcatt tggctgattt gaaaccttct ggtcaatatg tattccaaga 4080 cctttacaag gtcggagggg taccagcagt tatgaaatat ctccttaaaa atggcttcct 4140 tcatggtgac cgtatcactt gtactggcaa aacagtcgct gaaaatttga aggcttttga 4200 tgatttaaca cctggtcaaa aggttattat gccgcttgaa aatcctaaac gtgaagatgg 4260 tccgctcatt attctccatg gtaacttggc tccagacggt gccgttgcca aagtttctgg 4320 tgtaaaagtg cgtcgtcatg tcggtcctgc taaggtcttt aattctgaag aagaagccat 4380 tgaagctgtc ttgaatgatg atattgttga tggtgatgtt gttgtcgtac gttttgtagg 4440 accaaagggc ggtcctggta tgcctgaaat gctttccctt tcatcaatga ttgttggtaa 4500 agggcaaggt gaaaaagttg cccttctgac agatggccgc ttctcaggtg gtacttatgg 4560 tcttgtcgtg ggtcatatcg ctcctgaagc acaagatggc ggtccaatcg cctacctgca 4620 aacaggagac atagtcacta ttgaccaaga cactaaggaa ttacactttg atatctccga 4680 tgaagagtta aaacatcgtc aagagaccat tgaattgcca ccgctctatt cacgcggtat 4740 ccttggtaaa tatgctcaca tcgtttcgtc tgcttctagg ggagccgtaa cagacttttg 4800 gaagcctgaa gaaactggca aaaaatgttg tcctggttgc tgtggttaag cggccgcgtt 4860 aattcaaatt aattgatata gttttttaat gagtattgaa tctgtttaga aataatggaa 4920 tattattttt atttatttat ttatattatt ggtcggctct tttcttctga aggtcaatga 4980 caaaatgata tgaaggaaat aatgatttct aaaattttac aacgtaagat atttttacaa 5040 aagcctagct catcttttgt catgcactat tttactcacg cttgaaatta acggccagtc 5100 cactgcggag tcatttcaaa gtcatcctaa tcgatctatc gtttttgata gctcattttg 5160 gagttcgcga ttgtcttctg ttattcacaa ctgttttaat ttttatttca ttctggaact 5220 cttcgagttc tttgtaaagt ctttcatagt agcttacttt atcctccaac atatttaact 5280 tcatgtcaat ttcggctctt aaattttcca catcatcaag ttcaacatca tcttttaact 5340 tgaatttatt ctctagctct tccaaccaag cctcattgct ccttgattta ctggtgaaaa 5400 gtgatacact ttgcgcgcaa tccaggtcaa aactttcctg caaagaattc accaatttct 5460 cgacatcata gtacaatttg ttttgttctc ccatcacaat ttaatatacc tgatggattc 5520 ttatgaagcg ctgggtaatg gacgtgtcac tctacttcgc ctttttccct actcctttta 5580 gtacggaaga caatgctaat aaataagagg gtaataataa tattattaat cggcaaaaaa 5640 gattaaacgc caagcgttta attatcagaa agcaaacgtc gtaccaatcc ttgaatgctt 5700 cccaattgta tattaagagt catcacagca acatattctt gttattaaat taattattat 5760 tgatttttga tattgtataa aaaaaccaaa tatgtataaa aaaagtgaat aaaaaatacc 5820 aagtatggag aaatatatta gaagtctata cgttaaacca cccgggcccc ccctcgaggt 5880 cgacggtatc gataagcttg atatcgaatt cctgcagccc gggggatcca ctagttctag 5940 agcggccgct ctagaactag taccacaggt gttgtcctct gaggacataa aatacacacc 6000 gagattcatc aactcattgc tggagttagc atatctacaa ttgggtgaaa tggggagcga 6060 tttgcaggca tttgctcggc atgccggtag aggtgtggtc aataagagcg acctcatgct 6120 atacctgaga aagcaacctg acctacagga aagagttact caagaataag aattttcgtt 6180 ttaaaaccta agagtcactt taaaatttgt atacacttat tttttttata acttatttaa 6240 taataaaaat cataaatcat aagaaattcg cttactctta attaatcaaa aagttaaaat 6300 tgtacgaata gattcaccac ttcttaacaa atcaaaccct tcattgattt tctcgaatgg 6360 caatacatgt gtaattaaag gatcaagagc aaacttcttc gccataaagt cggcaacaag 6420 ttttggaaca ctatccttgc tcttaaaacc gccaaatata gctcccttcc atgtacgacc 6480 gcttagcaac agcataggat tcatcgacaa attttgtgaa tcaggaggaa cacctacgat 6540 cacactgact ccatatgcct cttgacagca ggacaacgca gttaccatag tatcaagacg 6600 gcctataact tcaaaagaga aatcaactcc accgtttgac atttcagtaa ggacttcttg 6660 tattggtttc ttataatctt gagggttaac acattcagta gccccgacct ccttagcttt 6720 tgcaaatttg tccttattga tgtctacacc tataatcctc gctgcgcctg cagctttaca 6780 ccccataata acgcttagtc ctactcctcc taaaccgaat actgcacaag tcgaaccctg 6840 tgtaaccttt gcaactttaa ctgcggaacc gtaaccggtg gaaaatccgc accctatcaa 6900 gcaaactttt tccagtggtg aagctgcatc gattttagcg acagatatct cgtccaccac 6960 tgtgtattgg gaaaatgtag aagtaccaag gaaatggtgt ataggtttcc ctctgcatgt 7020 aaatctgctt gtaccatcct gcatagtacc tctaggcata gacaaatcat ttttaaggca 7080 gaaattaccc tcaggatgtt tgcagactct acacttacca cattgaggag tgaacagtgg 7140 gatcacttta tcaccaggac gaacagtggt aacaccttca cctatggatt caacgattcc 7200 ggcagcctcg tgtcccgcga ttactggcaa aggagtaact agagtgccac tcaccacatg 7260 gtcgtcggat ctacagattc cggtggcaac catcttgatt ctaacctcgt gtgcttttgg 7320 tggcgctact tctacttctt ctatgctaaa cggctttttc tcttcccaca aaactgccgc 7380 tttacactta ataactttac cggctgttga catcctcagc tagctattgt aatatgtgtg 7440 tttgtttgga ttattaagaa gaataattac aaaaaaaatt acaaaggaag gtaattacaa 7500 cagaattaag aaaggacaag aaggaggaag agaatcagtt cattatttct tctttgttat 7560 ataacaaacc caagtagcga tttggccata cattaaaagt tgagaaccac cctccctggc 7620 aacagccaca actcgttacc attgttcatc acgatcatga aactcgctgt cagctgaaat 7680 ttcacctcag tggatctctc tttttattct tcatcgttcc actaaccttt ttccatcagc 7740 tggcagggaa cggaaagtgg aatcccattt agcgagcttc ctcttttctt caagaaaaga 7800 cgaagcttgt gtgtgggtgc gcgcgctagt atctttccac attaagaaat ataccataaa 7860 ggttacttag acatcactat ggctatatat atatatatat atatatgtaa cttagcacca 7920 tcgcgcgtgc atcactgcat gtgttaaccg aaaagtttgg cgaacacttc accgacacgg 7980 tcatttagat ctgtcgtctg cattgcacgt cccttagcct taaatcctag gcgggagcat 8040 tctcgtgtaa ttgtgcagcc tgcgtagcaa ctcaacatag cgtagtctac ccagtttttc 8100 aagggtttat cgttagaaga ttctcccttt tcttcctgct cacaaatctt aaagtcatac 8160 attgcacgac taaatgcaag catgcggatc ccccgggctg caggaattcg atatcaagct 8220 tatcgatacc gtcgactggc cattaatctt tcccatatta gatttcgcca agccatgaaa 8280 gttcaagaaa ggtctttaga cgaattaccc ttcatttctc aaactggcgt caagggatcc 8340 tggtatggtt ttatcgtttt atttctggtt cttatagcat cgttttggac ttctctgttc 8400 ccattaggcg gttcaggagc cagcgcagaa tcattctttg aaggatactt atcctttcca 8460 attttgattg tctgttacgt tggacataaa ctgtatacta gaaattggac tttgatggtg 8520 aaactagaag atatggatct tgataccggc agaaaacaag tagatttgac tcttcgtagg 8580 gaagaaatga ggattgagcg agaaacatta gcaaaaagat ccttcgtaac aagattttta 8640 catttctggt gttgaaggga aagatatgag ctatacagcg gaatttccat atcactcaga 8700 ttttgttatc taattttttc cttcccacgt ccgcgggaat ctgtgtatat tactgcatct 8760 agatatatgt tatcttatct tggcgcgtac atttaatttt caacgtattc tataagaaat 8820 tgcgggagtt tttttcatgt agatgatact gactgcacgc aaatataggc atgatttata 8880 ggcatgattt gatggctgta ccgataggaa cgctaagagt aacttcagaa tcgttatcct 8940 ggcggaaaaa attcatttgt aaactttaaa aaaaaaagcc aatatcccca aaattattaa 9000 gagcgcctcc attattaact aaaatttcac tcagcatcca caatgtatca ggtatctact 9060 acagatatta catgtggcga aaaagacaag aacaatgcaa tagcgcatca agaaaaaaca 9120 caaagctttc aatcaatgaa tcgaaaatgt cattaaaata gtatataaat tgaaactaag 9180 tcataaagct ataaaaagaa aatttattta aatgcaagat ttaaagtaaa ttcacggccc 9240 tgcaggcctc agctcttgtt ttgttctgca aataacttac ccatcttttt caaaacttta 9300 ggtgcaccct cctttgctag aataagttct atccaataca tcctatttgg atctgcttga 9360 gcttctttca tcacggatac gaattcattt tctgttctca caattttgga cacaactctg 9420 tcttccgttg ccccgaaact ttctggcagt tttgagtaat tccacatagg aatgtcatta 9480 taactctggt tcggaccatg aatttccctc tcaaccgtgt aaccatcgtt attaatgata 9540 aagcagattg ggtttatctt ctctctaatg gctagtccta attcttggac agtcagttgc 9600 aatgatccat ctccgataaa caataaatgt ctagattctt tatctgcaat ttggctgcct 9660 agagctgcgg ggaaagtgta tcctatagat ccccacaagg gttgaccaat aaaatgtgat 9720 ttcgatttca gaaatataga tgaggcaccg aagaaagaag tgccttgttc agccacgatc 9780 gtctcattac tttgggtcaa attttcgaca gcttgccaca gtctatcttg tgacaacagc 9840 gcgttagaag gtacaaaatc ttcttgcttt ttatctatgt acttgccttt atattcaatt 9900 tcggacaagt caagaagaga tgatatcagg gattcgaagt cgaaattttg gattctttcg 9960 ttgaaaattt taccttcatc gatattcaag gaaatcattt tattttcatt aagatggtga 10020 gtaaatgcac ccgtactaga atcggtaagc tttacaccca acataagaat aaaatcagca 10080 gattccacaa attccttcaa gtttggctct gacagagtac cgttgtaaat ccccaaaaat 10140 gagggcaatg cttcatcaac agatgattta ccaaagttca aagtagtaat aggtaactta 10200 gtctttgaaa taaactgagt aacagtcttc tctaggccga acgatataat ttcatggcct 10260 gtgattacaa ttggtttctt ggcattcttc agactttcct gtattttgtt cagaatctct 10320 tgatcagatg tattcgacgt ggaattttcc ttcttaagag gcaaggatgg tttttcagcc 10380 ttagcggcag ctacatctac aggtaaattg atgtaaaccg gctttctttc ctttagtaag 10440 gcagacaaca ctctatcaat ttcaacagtt gcattctcgg ctgtcaataa agtcctggca 10500 gcagtaaccg gttcgtgcat cttcataaag tgcttgaaat caccatcagc caacgtatgg 10560 tgaacaaact taccttcgtt ctgcactttc gaggtaggag atcccacgat ctcaacaaca 10620 ggcaggttct cagcatagga gcccgctaag ccattaactg cggataattc gccaacacca 10680 aatgtagtca agaatgccgc agcctttttc gttcttgcgt acccgtcggc catataggag 10740 gcatttaact cattagcatt tcccacccat ttcatatctt tgtgtgaaat aatttgatct 10800 agaaattgca aattgtagtc acctggtact ccgaatattt cttctatacc taattcgtgt 10860 aatctgtcca acagatagtc acctactgta tacattttgt ttactagttt atgtgtgttt 10920 attcgaaact aagttcttgg tgttttaaaa ctaaaaaaaa gactaactat aaaagtagaa 10980 tttaagaagt ttaagaaata gatttacaga attacaatca atacctaccg tctttatata 11040 cttattagtc aagtagggga ataatttcag ggaactggtt tcaacctttt ttttcagctt 11100 tttccaaatc agagagagca gaaggtaata gaaggtgtaa gaaaatgaga tagatacatg 11160 cgtgggtcaa ttgccttgtg tcatcattta ctccaggcag gttgcatcac tccattgagg 11220 ttgtgcccgt tttttgcctg tttgtgcccc tgttctctgt agttgcgcta agagaatgga 11280 cctatgaact gatggttggt gaagaaaaca atattttggt gctgggattc tttttttttc 11340 tggatgccag cttaaaaagc gggctccatt atatttagtg gatgccagga ataaactgtt 11400 cacccagaca cctacgatgt tatatattct gtgtaacccg ccccctattt tgggcatgta 11460 cgggttacag cagaattaaa aggctaattt tttgactaaa taaagttagg aaaatcacta 11520 ctattaatta tttacgtatt ctttgaaatg gcagtattga taatgataaa ctcgaactga 11580 aaaagcgtgt tttttattca aaatgattct aactccctta cgtaatcaag gaatcttttt 11640 gccttggcct ccgcgtcatt aaacttcttg ttgttgacgc taacattcaa cgctagtata 11700 tattcgtttt tttcaggtaa gttcttttca acgggtctta ctgatgaggc agtcgcgtct 11760 gaacctgtta agaggtcaaa tatgtcttct tgaccgtacg tgtcttgcat gttattagct 11820 ttgggaattt gcatcaagtc ataggaaaat ttaaatcttg gctctcttgg gctcaaggtg 11880 acaaggtcct cgaaaatagg gcgcgcccca ccgcggtgga gctccagctt ttgttccctt 11940 tagtgagggt taattgcgcg cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat 12000 tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg 12060 ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag 12120 tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt 12180 ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg 12240 ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg 12300 gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag 12360 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga 12420 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct 12480 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc 12540 tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg 12600 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc 12660 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca 12720 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag 12780 ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct 12840 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc 12900 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga 12960 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca 13020 cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat 13080 taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac 13140 caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt 13200 gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt 13260 gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag 13320 ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct 13380 attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt 13440 gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc 13500 tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt 13560 agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg 13620 gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg 13680 actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct 13740 tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc 13800 attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt 13860 tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt 13920 tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg 13980 aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta tcagggttat 14040 tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg 14100 cgcacatttc cccgaaaagt gccacctgaa cgaagcatct gtgcttcatt ttgtagaaca 14160 aaaatgcaac gcgagagcgc taatttttca aacaaagaat ctgagctgca tttttacaga 14220 acagaaatgc aacgcgaaag cgctatttta ccaacgaaga atctgtgctt catttttgta 14280 aaacaaaaat gcaacgcgag agcgctaatt tttcaaacaa agaatctgag ctgcattttt 14340 acagaacaga aatgcaacgc gagagcgcta ttttaccaac aaagaatcta tacttctttt 14400 ttgttctaca aaaatgcatc ccgagagcgc tatttttcta acaaagcatc ttagattact 14460 ttttttctcc tttgtgcgct ctataatgca gtctcttgat aactttttgc actgtaggtc 14520 cgttaaggtt agaagaaggc tactttggtg tctattttct cttccataaa aaaagcctga 14580 ctccacttcc cgcgtttact gattactagc gaagctgcgg gtgcattttt tcaagataaa 14640 ggcatccccg attatattct ataccgatgt ggattgcgca tactttgtga acagaaagtg 14700 atagcgttga tgattcttca ttggtcagaa aattatgaac ggtttcttct attttgtctc 14760 tatatactac gtataggaaa tgtttacatt ttcgtattgt tttcgattca ctctatgaat 14820 agttcttact acaatttttt tgtctaaaga gtaatactag agataaacat aaaaaatgta 14880 gaggtcgagt ttagatgcaa gttcaaggag cgaaaggtgg atgggtaggt tatataggga 14940 tatagcacag agatatatag caaagagata cttttgagca atgtttgtgg aagcggtatt 15000 cgcaatattt tagtagctcg ttacagtccg gtgcgttttt ggttttttga aagtgcgtct 15060 tcagagcgct tttggttttc aaaagcgctc tgaagttcct atactttcta gagaatagga 15120 acttcggaat aggaacttca aagcgtttcc gaaaacgagc gcttccgaaa atgcaacgcg 15180 agctgcgcac atacagctca ctgttcacgt cgcacctata tctgcgtgtt gcctgtatat 15240 atatatacat gagaagaacg gcatagtgcg tgtttatgct taaatgcgta cttatatgcg 15300 tctatttatg taggatgaaa ggtagtctag tacctcctgt gatattatcc cattccatgc 15360 ggggtatcgt atgcttcctt cagcactacc ctttagctgt tctatatgct gccactcctc 15420 aattggatta gtctcatcct tcaatgctat catttccttt gatattggat catactaaga 15480 aaccattatt atcatgacat taacctataa aaataggcgt atcacgaggc cctttcgtc 15539 <210> 3 <211> 4586 <212> DNA <213> Artificial sequence <220> <223> Template <400> 3 gggtaccgag ctcgaattca ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg 60 gcgttaccca acttaatcgc cttgcagcac atcccccttt cgccagctgg cgtaatagcg 120 aagaggcccg caccgatcgc ccttcccaac agttgcgcag cctgaatggc gaatggcgcc 180 tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata tggtgcactc 240 tcagtacaat ctgctctgat gccgcatagt taagccagcc ccgacacccg ccaacacccg 300 ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg 360 tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgagacgaa 420 agggcctcgt gatacgccta tttttatagg ttaatgtcat gataataatg gtttcttaga 480 cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa 540 tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt 600 gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg 660 cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag 720 atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg 780 agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg 840 gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt 900 ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga 960 cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac 1020 ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc 1080 atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc 1140 gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac 1200 tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag 1260 gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg 1320 gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta 1380 tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg 1440 ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata 1500 tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt 1560 ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc 1620 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct 1680 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa 1740 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gtccttctag 1800 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc 1860 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg 1920 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca 1980 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat 2040 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg 2100 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc 2160 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc 2220 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc 2280 cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg 2340 cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga 2400 gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc 2460 attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa 2520 ttaatgtgag ttagctcact cattaggcac cccaggcttt acactttatg cttccggctc 2580 gtatgttgtg tggaattgtg agcggataac aatttcacac aggaaacagc tatgaccatg 2640 attacgccaa gcttgcatgc ctgcaggtcg actctagagg atccccgcat tgcggattac 2700 gtattctaat gttcagataa cttcgtatag catacattat acgaagttat ctagggattc 2760 ataaccattt tctcaatcga attacacaga acacaccgta caaacctctc tatcataact 2820 acttaatagt cacacacgta ctcgtctaaa tacacatcat cgtcctacaa gttcatcaaa 2880 gtgttggaca gacaactata ccagcatgga tctcttgtat cggttctttt ctcccgctct 2940 ctcgcaataa caatgaacac tgggtcaatc atagcctaca caggtgaaca gagtagcgtt 3000 tatacagggt ttatacggtg attcctacgg caaaaatttt tcatttctaa aaaaaaaaag 3060 aaaaattttt ctttccaacg ctagaaggaa aagaaaaatc taattaaatt gatttggtga 3120 ttttctgaga gttccctttt tcatatatcg aattttgaat ataaaaggag atcgaaaaaa 3180 tttttctatt caatctgttt tctggtttta tttgatagtt tttttgtgta ttattattat 3240 ggattagtac tggtttatat gggtttttct gtataacttc tttttatttt agtttgttta 3300 atcttatttt gagttacatt atagttccct aactgcaaga gaagtaacat taaaactcga 3360 gatgggtaag gaaaagactc acgtttcgag gccgcgatta aattccaaca tggatgctga 3420 tttatatggg tataaatggg ctcgcgataa tgtcgggcaa tcaggtgcga caatctatcg 3480 attgtatggg aagcccgatg cgccagagtt gtttctgaaa catggcaaag gtagcgttgc 3540 caatgatgtt acagatgaga tggtcagact aaactggctg acggaattta tgcctcttcc 3600 gaccatcaag cattttatcc gtactcctga tgatgcatgg ttactcacca ctgcgatccc 3660 cggcaaaaca gcattccagg tattagaaga atatcctgat tcaggtgaaa atattgttga 3720 tgcgctggca gtgttcctgc gccggttgca ttcgattcct gtttgtaatt gtccttttaa 3780 cagcgatcgc gtatttcgtc tcgctcaggc gcaatcacga atgaataacg gtttggttga 3840 tgcgagtgat tttgatgacg agcgtaatgg ctggcctgtt gaacaagtct ggaaagaaat 3900 gcataagctt ttgccattct caccggattc agtcgtcact catggtgatt tctcacttga 3960 taaccttatt tttgacgagg ggaaattaat aggttgtatt gatgttggac gagtcggaat 4020 cgcagaccga taccaggatc ttgccatcct atggaactgc ctcggtgagt tttctccttc 4080 attacagaaa cggctttttc aaaaatatgg tattgataat cctgatatga ataaattgca 4140 gtttcatttg atgctcgatg agtttttcta agtttaactt gatactacta gattttttct 4200 cttcatttat aaaatttttg gttataattg aagctttaga agtatgaaaa aatccttttt 4260 tttcattctt tgcaaccaaa ataagaagct tcttttattc attgaaatga tgaatataaa 4320 cctaacaaaa gaaaaagact cgaatatcaa acattaaaaa aaaataaaag aggttatctg 4380 ttttcccatt tagttggagt ttgcattttc taatagatag aactctcaat taatgtggat 4440 ttagtttctc tgttcgtttt tttttgtttt gttctcactg tatttacatt tctatttagt 4500 atttagttat tcatataatc tataacttcg tatagcatac attatacgaa gttatccagt 4560 gatgatacaa cgagttagcc aaggtg 4586 <210> 4 <211> 80 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 4 ttccggtttc tttgaaattt ttttgattcg gtaatctccg agcagaagga gcattgcgga 60 ttacgtattc taatgttcag 80 <210> 5 <211> 81 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 5 gggtaataac tgatataatt aaattgaagc tctaatttgt gagtttagta caccttggct 60 aactcgttgt atcatcactg g 81 <210> 6 <211> 38 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 6 gcctcgagtt ttaatgttac ttctcttgca gttaggga 38 <210> 7 <211> 31 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 7 gctaaattcg agtgaaacac aggaagacca g 31 <210> 8 <211> 90 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 8 gtattttggt agattcaatt ctctttccct ttccttttcc ttcgctcccc ttccttatca 60 gcattgcgga ttacgtattc taatgttcag 90 <210> 9 <211> 90 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 9 ttggttgggg gaaaaagagg caacaggaaa gatcagaggg ggaggggggg ggagagtgtc 60 accttggcta actcgttgta tcatcactgg 90 <210> 10 <211> 21 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 10 tcggtgcggg cctcttcgct a 21 <210> 11 <211> 21 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 11 aatgtgagtt agctcactca t 21 <210> 12 <211> 57 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 12 aattggatcc ggcgcgccgt ttaaacggcc ggccaatgtg gctgtggttt cagggtc 57 <210> 13 <211> 49 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 13 aatttctaga ttaattaagc ggccgcaagg ccatgaagct ttttctttc 49 <210> 14 <211> 24 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 14 ttctcgacgt gggccttttt cttg 24 <210> 15 <211> 49 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 15 tgcagcttta aataatcggt gtcactactt tgccttcgtt tatcttgcc 49 <210> 16 <211> 49 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 16 gagcaggcaa gataaacgaa ggcaaagtag tgacaccgat tatttaaag 49 <210> 17 <211> 49 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 17 tatggaccct gaaaccacag ccacattgta accaccacga cggttgttg 49 <210> 18 <211> 49 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 18 tttagcaaca accgtcgtgg tggttacaat gtggctgtgg tttcagggt 49 <210> 19 <211> 49 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 19 ccagaaaccc tatacctgtg tggacgtaag gccatgaagc tttttcttt 49 <210> 20 <211> 49 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 20 attggaaaga aaaagcttca tggccttacg tccacacagg tatagggtt 49 <210> 21 <211> 22 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 21 cataagaaca cctttggtgg ag 22 <210> 22 <211> 22 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 22 aggattatca ttcataagtt tc 22 <210> 23 <211> 23 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 23 ttcttggagc tgggacatgt ttg 23 <210> 24 <211> 22 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 24 tgatgatatt tcataaataa tg 22 <210> 25 <211> 23 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 25 atgcgtccat ctttacagtc ctg 23 <210> 26 <211> 22 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 26 tacgtacgga ccaatcgaag tg 22 <210> 27 <211> 49 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 27 aattcgtttg agtacactac taatggcttt gttggcaata tgtttttgc 49 <210> 28 <211> 49 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 28 atatagcaaa aacatattgc caacaaagcc attagtagtg tactcaaac 49 <210> 29 <211> 49 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 29 tatggaccct gaaaccacag ccacattctt gttatttata aaaagacac 49 <210> 30 <211> 49 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 30 ctcccgtgtc tttttataaa taacaagaat gtggctgtgg tttcagggt 49 <210> 31 <211> 49 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 31 taccgtaggc gtccttagga aagatagaag gccatgaagc tttttcttt 49 <210> 32 <211> 49 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 32 attggaaaga aaaagcttca tggccttcta tctttcctaa ggacgccta 49 <210> 33 <211> 21 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 33 ttattgtttg gcatttgtag c 21 <210> 34 <211> 22 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 34 ccaagcatct cataaaccta tg 22 <210> 35 <211> 22 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 35 tgtgcagatg cagatgtgag ac 22 <210> 36 <211> 17 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 36 agttattgat accgtac 17 <210> 37 <211> 19 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 37 cgagataccg taggcgtcc 19 <210> 38 <211> 24 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 38 ttatgtatgc tcttctgact tttc 24 <210> 39 <211> 49 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 39 aataattaga gattaaatcg ctcatttttt gccagtttct tcaggcttc 49 <210> 40 <211> 49 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 40 agcctgaaga aactggcaaa aaatgagcga tttaatctct aattattag 49 <210> 41 <211> 49 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 41 tatggaccct gaaaccacag ccacattttt caatcattgg agcaatcat 49 <210> 42 <211> 49 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 42 taaaatgatt gctccaatga ttgaaaaatg tggctgtggt ttcagggtc 49 <210> 43 <211> 49 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 43 accgtaggtg ttgtttggga aagtggaagg ccatgaagct ttttctttc 49 <210> 44 <211> 49 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 44 ttggaaagaa aaagcttcat ggccttccac tttcccaaac aacacctac 49 <210> 45 <211> 23 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 45 ttattgctta gcgttggtag cag 23 <210> 46 <211> 21 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 46 tttttggtgg ttccggcttc c 21 <210> 47 <211> 22 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 47 aaagttggca tagcggaaac tt 22 <210> 48 <211> 16 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 48 gtcattgaca ccatct 16 <210> 49 <211> 19 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 49 agagataccg taggtgttg 19 <210> 50 <211> 33 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 50 aattggcgcg ccatgaaagc tctggtttat cac 33 <210> 51 <211> 49 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 51 tgaatcatga gttttatgtt aattagctca ggcagcgcct gcgttcgag 49 <210> 52 <211> 49 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 52 atcctctcga acgcaggcgc tgcctgagct aattaacata aaactcatg 49 <210> 53 <211> 34 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 53 aattgtttaa acaagtaaat aaattaatca gcat 34 <210> 54 <211> 49 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 54 acacaataca ataacaagaa gaacaaaatg aaagctctgg tttatcacg 49 <210> 55 <211> 49 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 55 agcgtataca tctgttggga aagtagaagg ccatgaagct ttttctttc 49 <210> 56 <211> 49 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 56 ttggaaagaa aaagcttcat ggccttctac tttcccaaca gatgtatac 49 <210> 57 <211> 22 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 57 ttattgttta gcgttagtag cg 22 <210> 58 <211> 72 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 58 cataatcaat ctcaaagaga acaacacaat acaataacaa gaagaacaaa atgaaagctc 60 tggtttatca cg 72 <210> 59 <211> 21 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 59 taggcataat caccgaagaa g 21 <210> 60 <211> 21 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 60 aaaatggtaa gcagctgaaa g 21 <210> 61 <211> 17 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 61 agttgttaga actgttg 17 <210> 62 <211> 19 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 62 gacgatagcg tatacatct 19 <210> 63 <211> 22 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 63 cttagcctct agccatagcc at 22 <210> 64 <211> 23 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 64 ttagttttgc tggccgcatc ttc 23 <210> 65 <211> 21 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 65 cccattaata tactattgag a 21 <210> 66 <211> 7523 <212> DNA <213> Artificial sequence <220> <223> Plasmid <400> 66 ccagcttttg ttccctttag tgagggttaa ttgcgcgctt ggcgtaatca tggtcatagc 60 tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatagga gccggaagca 120 taaagtgtaa agcctggggt gcctaatgag tgaggtaact cacattaatt gcgttgcgct 180 cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac 240 gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc 300 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt 360 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg 420 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg 480 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat 540 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta 600 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct 660 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc 720 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa 780 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg 840 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaaggacag 900 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt 960 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta 1020 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc 1080 agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca 1140 cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa 1200 cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat 1260 ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct 1320 taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt 1380 tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat 1440 ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta 1500 atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg 1560 gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt 1620 tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg 1680 cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg 1740 taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc 1800 ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa 1860 ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac 1920 cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt 1980 ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg 2040 gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa 2100 gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata 2160 aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgaacga agcatctgtg 2220 cttcattttg tagaacaaaa atgcaacgcg agagcgctaa tttttcaaac aaagaatctg 2280 agctgcattt ttacagaaca gaaatgcaac gcgaaagcgc tattttacca acgaagaatc 2340 tgtgcttcat ttttgtaaaa caaaaatgca acgcgagagc gctaattttt caaacaaaga 2400 atctgagctg catttttaca gaacagaaat gcaacgcgag agcgctattt taccaacaaa 2460 gaatctatac ttcttttttg ttctacaaaa atgcatcccg agagcgctat ttttctaaca 2520 aagcatctta gattactttt tttctccttt gtgcgctcta taatgcagtc tcttgataac 2580 tttttgcact gtaggtccgt taaggttaga agaaggctac tttggtgtct attttctctt 2640 ccataaaaaa agcctgactc cacttcccgc gtttactgat tactagcgaa gctgcgggtg 2700 cattttttca agataaaggc atccccgatt atattctata ccgatgtgga ttgcgcatac 2760 tttgtgaaca gaaagtgata gcgttgatga ttcttcattg gtcagaaaat tatgaacggt 2820 ttcttctatt ttgtctctat atactacgta taggaaatgt ttacattttc gtattgtttt 2880 cgattcactc tatgaatagt tcttactaca atttttttgt ctaaagagta atactagaga 2940 taaacataaa aaatgtagag gtcgagttta gatgcaagtt caaggagcga aaggtggatg 3000 ggtaggttat atagggatat agcacagaga tatatagcaa agagatactt ttgagcaatg 3060 tttgtggaag cggtattcgc aatattttag tagctcgtta cagtccggtg cgtttttggt 3120 tttttgaaag tgcgtcttca gagcgctttt ggttttcaaa agcgctctga agttcctata 3180 ctttctagag aataggaact tcggaatagg aacttcaaag cgtttccgaa aacgagcgct 3240 tccgaaaatg caacgcgagc tgcgcacata cagctcactg ttcacgtcgc acctatatct 3300 gcgtgttgcc tgtatatata tatacatgag aagaacggca tagtgcgtgt ttatgcttaa 3360 atgcgtactt atatgcgtct atttatgtag gatgaaaggt agtctagtac ctcctgtgat 3420 attatcccat tccatgcggg gtatcgtatg cttccttcag cactaccctt tagctgttct 3480 atatgctgcc actcctcaat tggattagtc tcatccttca atgctatcat ttcctttgat 3540 attggatcat ctaagaaacc attattatca tgacattaac ctataaaaat aggcgtatca 3600 cgaggccctt tcgtctcgcg cgtttcggtg atgacggtga aaacctctga cacatgcagc 3660 tcccggagac ggtcacagct tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg 3720 gcgcgtcagc gggtgttggc gggtgtcggg gctggcttaa ctatgcggca tcagagcaga 3780 ttgtactgag agtgcaccat aaattcccgt tttaagagct tggtgagcgc taggagtcac 3840 tgccaggtat cgtttgaaca cggcattagt cagggaagtc ataacacagt cctttcccgc 3900 aattttcttt ttctattact cttggcctcc tctagtacac tctatatttt tttatgcctc 3960 ggtaatgatt ttcatttttt tttttcccct agcggatgac tctttttttt tcttagcgat 4020 tggcattatc acataatgaa ttatacatta tataaagtaa tgtgatttct tcgaagaata 4080 tactaaaaaa tgagcaggca agataaacga aggcaaagat gacagagcag aaagccctag 4140 taaagcgtat tacaaatgaa accaagattc agattgcgat ctctttaaag ggtggtcccc 4200 tagcgataga gcactcgatc ttcccagaaa aagaggcaga agcagtagca gaacaggcca 4260 cacaatcgca agtgattaac gtccacacag gtatagggtt tctggaccat atgatacatg 4320 ctctggccaa gcattccggc tggtcgctaa tcgttgagtg cattggtgac ttacacatag 4380 acgaccatca caccactgaa gactgcggga ttgctctcgg tcaagctttt aaagaggccc 4440 tactggcgcg tggagtaaaa aggtttggat caggatttgc gcctttggat gaggcacttt 4500 ccagagcggt ggtagatctt tcgaacaggc cgtacgcagt tgtcgaactt ggtttgcaaa 4560 gggagaaagt aggagatctc tcttgcgaga tgatcccgca ttttcttgaa agctttgcag 4620 aggctagcag aattaccctc cacgttgatt gtctgcgagg caagaatgat catcaccgta 4680 gtgagagtgc gttcaaggct cttgcggttg ccataagaga agccacctcg cccaatggta 4740 ccaacgatgt tccctccacc aaaggtgttc ttatgtagtg acaccgatta tttaaagctg 4800 cagcatacga tatatataca tgtgtatata tgtataccta tgaatgtcag taagtatgta 4860 tacgaacagt atgatactga agatgacaag gtaatgcatc attctatacg tgtcattctg 4920 aacgaggcgc gctttccttt tttctttttg ctttttcttt ttttttctct tgaactcgac 4980 ggatctatgc ggtgtgaaat accgcacaga tgcgtaagga gaaaataccg catcaggaaa 5040 ttgtaaacgt taatattttg ttaaaattcg cgttaaattt ttgttaaatc agctcatttt 5100 ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag accgagatag 5160 ggttgagtgt tgttccagtt tggaacaaga gtccactatt aaagaacgtg gactccaacg 5220 tcaaagggcg aaaaaccgtc tatcagggcg atggcccact acgtgaacca tcaccctaat 5280 caagtttttt ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa gggagccccc 5340 gatttagagc ttgacgggga aagccggcga acgtggcgag aaaggaaggg aagaaagcga 5400 aaggagcggg cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta accaccacac 5460 ccgccgcgct taatgcgccg ctacagggcg cgtcgcgcca ttcgccattc aggctgcgca 5520 actgttggga agggcgatcg gtgcgggcct cttcgctatt acgccagctg gcgaaagggg 5580 gatgtgctgc aaggcgatta agttgggtaa cgccagggtt ttcccagtca cgacgttgta 5640 aaacgacggc cagtgagcgc gcgtaatacg actcactata gggcgaattg ggtaccgggc 5700 cccccctcga ggtattagaa gccgccgagc gggcgacagc cctccgacgg aagactctcc 5760 tccgtgcgtc ctcgtcttca ccggtcgcgt tcctgaaacg cagatgtgcc tcgcgccgca 5820 ctgctccgaa caataaagat tctacaatac tagcttttat ggttatgaag aggaaaaatt 5880 ggcagtaacc tggccccaca aaccttcaaa ttaacgaatc aaattaacaa ccataggatg 5940 ataatgcgat tagtttttta gccttatttc tggggtaatt aatcagcgaa gcgatgattt 6000 ttgatctatt aacagatata taaatggaaa agctgcataa ccactttaac taatactttc 6060 aacattttca gtttgtatta cttcttattc aaatgtcata aaagtatcaa caaaaaattg 6120 ttaatatacc tctatacttt aacgtcaagg agaaaaatgt ccaatttact gcccgtacac 6180 caaaatttgc ctgcattacc ggtcgatgca acgagtgatg aggttcgcaa gaacctgatg 6240 gacatgttca gggatcgcca ggcgttttct gagcatacct ggaaaatgct tctgtccgtt 6300 tgccggtcgt gggcggcatg gtgcaagttg aataaccgga aatggtttcc cgcagaacct 6360 gaagatgttc gcgattatct tctatatctt caggcgcgcg gtctggcagt aaaaactatc 6420 cagcaacatt tgggccagct aaacatgctt catcgtcggt ccgggctgcc acgaccaagt 6480 gacagcaatg ctgtttcact ggttatgcgg cggatccgaa aagaaaacgt tgatgccggt 6540 gaacgtgcaa aacaggctct agcgttcgaa cgcactgatt tcgaccaggt tcgttcactc 6600 atggaaaata gcgatcgctg ccaggatata cgtaatctgg catttctggg gattgcttat 6660 aacaccctgt tacgtatagc cgaaattgcc aggatcaggg ttaaagatat ctcacgtact 6720 gacggtggga gaatgttaat ccatattggc agaacgaaaa cgctggttag caccgcaggt 6780 gtagagaagg cacttagcct gggggtaact aaactggtcg agcgatggat ttccgtctct 6840 ggtgtagctg atgatccgaa taactacctg ttttgccggg tcagaaaaaa tggtgttgcc 6900 gcgccatctg ccaccagcca gctatcaact cgcgccctgg aagggatttt tgaagcaact 6960 catcgattga tttacggcgc taaggatgac tctggtcaga gatacctggc ctggtctgga 7020 cacagtgccc gtgtcggagc cgcgcgagat atggcccgcg ctggagtttc aataccggag 7080 atcatgcaag ctggtggctg gaccaatgta aatattgtca tgaactatat ccgtaacctg 7140 gatagtgaaa caggggcaat ggtgcgcctg ctggaagatg gcgattagga gtaagcgaat 7200 ttcttatgat ttatgatttt tattattaaa taagttataa aaaaaataag tgtatacaaa 7260 ttttaaagtg actcttaggt tttaaaacga aaattcttat tcttgagtaa ctctttcctg 7320 taggtcaggt tgctttctca ggtatagcat gaggtcgctc ttattgacca cacctctacc 7380 ggcatgccga gcaaatgcct gcaaatcgct ccccatttca cccaattgta gatatgctaa 7440 ctccagcaat gagttgatga atctcggtgt gtattttatg tcctcagagg acaacacctg 7500 tggtccgcca ccgcggtgga gct 7523 <210> 67 <211> 15456 <212> DNA <213> Artificial sequence <220> <223> Template <400> 67 aaagagtaat actagagata aacataaaaa atgtagaggt cgagtttaga tgcaagttca 60 aggagcgaaa ggtggatggg taggttatat agggatatag cacagagata tatagcaaag 120 agatactttt gagcaatgtt tgtggaagcg gtattcgcaa tattttagta gctcgttaca 180 gtccggtgcg tttttggttt tttgaaagtg cgtcttcaga gcgcttttgg ttttcaaaag 240 cgctctgaag ttcctatact ttctagagaa taggaacttc ggaataggaa cttcaaagcg 300 tttccgaaaa cgagcgcttc cgaaaatgca acgcgagctg cgcacataca gctcactgtt 360 cacgtcgcac ctatatctgc gtgttgcctg tatatatata tacatgagaa gaacggcata 420 gtgcgtgttt atgcttaaat gcgtacttat atgcgtctat ttatgtagga tgaaaggtag 480 tctagtacct cctgtgatat tatcccattc catgcggggt atcgtatgct tccttcagca 540 ctacccttta gctgttctat atgctgccac tcctcaattg gattagtctc atccttcaat 600 gctatcattt cctttgatat tggatcatac taagaaacca ttattatcat gacattaacc 660 tataaaaata ggcgtatcac gaggcccttt cgtctcgcgc gtttcggtga tgacggtgaa 720 aacctctgac acatgcagct cccggagacg gtcacagctt gtctgtaagc ggatgccggg 780 agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg ggtgtcgggg ctggcttaac 840 tatgcggcat cagagcagat tgtactgaga gtgcaccata aattcccgtt ttaagagctt 900 ggtgagcgct aggagtcact gccaggtatc gtttgaacac ggcattagtc agggaagtca 960 taacacagtc ctttcccgca attttctttt tctattactc ttggcctcct ctagtacact 1020 ctatattttt ttatgcctcg gtaatgattt tcattttttt ttttccacct agcggatgac 1080 tctttttttt tcttagcgat tggcattatc acataatgaa ttatacatta tataaagtaa 1140 tgtgatttct tcgaagaata tactaaaaaa tgagcaggca agataaacga aggcaaagat 1200 gacagagcag aaagccctag taaagcgtat tacaaatgaa accaagattc agattgcgat 1260 ctctttaaag ggtggtcccc tagcgataga gcactcgatc ttcccagaaa aagaggcaga 1320 agcagtagca gaacaggcca cacaatcgca agtgattaac gtccacacag gtatagggtt 1380 tctggaccat atgatacatg ctctggccaa gcattccggc tggtcgctaa tcgttgagtg 1440 cattggtgac ttacacatag acgaccatca caccactgaa gactgcggga ttgctctcgg 1500 tcaagctttt aaagaggccc taggggccgt gcgtggagta aaaaggtttg gatcaggatt 1560 tgcgcctttg gatgaggcac tttccagagc ggtggtagat ctttcgaaca ggccgtacgc 1620 agttgtcgaa cttggtttgc aaagggagaa agtaggagat ctctcttgcg agatgatccc 1680 gcattttctt gaaagctttg cagaggctag cagaattacc ctccacgttg attgtctgcg 1740 aggcaagaat gatcatcacc gtagtgagag tgcgttcaag gctcttgcgg ttgccataag 1800 agaagccacc tcgcccaatg gtaccaacga tgttccctcc accaaaggtg ttcttatgta 1860 gtgacaccga ttatttaaag ctgcagcata cgatatatat acatgtgtat atatgtatac 1920 ctatgaatgt cagtaagtat gtatacgaac agtatgatac tgaagatgac aaggtaatgc 1980 atcattctat acgtgtcatt ctgaacgagg cgcgctttcc ttttttcttt ttgctttttc 2040 tttttttttc tcttgaactc gacggatcta tgcggtgtga aataccgcac agatgcgtaa 2100 ggagaaaata ccgcatcagg aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa 2160 tttttgttaa atcagctcat tttttaacca ataggccgaa atcggcaaaa tcccttataa 2220 atcaaaagaa tagaccgaga tagggttgag tgttgttcca gtttggaaca agagtccact 2280 attaaagaac gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc 2340 actacgtgaa ccatcaccct aatcaagttt tttggggtcg aggtgccgta aagcactaaa 2400 tcggaaccct aaagggagcc cccgatttag agcttgacgg ggaaagccgg cgaacgtggc 2460 gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt 2520 cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg ccgctacagg gcgcgtccat 2580 tcgccattca ggctgcgcaa ctgttgggaa gggcgcggtg cgggcctctt cgctattacg 2640 ccagctggcg aaagggggat gtgctgcaag gcgattaagt tgggtaacgc cagggttttc 2700 ccagtcacga cgttgtaaaa cgacggccag tgagcgcgcg taatacgact cactataggg 2760 cgaattgggt accgggcccc ccctcgaggt cgacggcgcg ccactggtag agagcgactt 2820 tgtatgcccc aattgcgaaa cccgcgatat ccttctcgat tctttagtac ccgaccagga 2880 caaggaaaag gaggtcgaaa cgtttttgaa gaaacaagag gaactacacg gaagctctaa 2940 agatggcaac cagccagaaa ctaagaaaat gaagttgatg gatccaactg gcaccgctgg 3000 cttgaacaac aataccagcc ttccaacttc tgtaaataac ggcggtacgc cagtgccacc 3060 agtaccgtta cctttcggta tacctccttt ccccatgttt ccaatgccct tcatgcctcc 3120 aacggctact atcacaaatc ctcatcaagc tgacgcaagc cctaagaaat gaataacaat 3180 actgacagta ctaaataatt gcctacttgg cttcacatac gttgcatacg tcgatataga 3240 taataatgat aatgacagca ggattatcgt aatacgtaat agctgaaaat ctcaaaaatg 3300 tgtgggtcat tacgtaaata atgataggaa tgggattctt ctatttttcc tttttccatt 3360 ctagcagccg tcgggaaaac gtggcatcct ctctttcggg ctcaattgga gtcacgctgc 3420 cgtgagcatc ctctctttcc atatctaaca actgagcacg taaccaatgg aaaagcatga 3480 gcttagcgtt gctccaaaaa agtattggat ggttaatacc atttgtctgt tctcttctga 3540 ctttgactcc tcaaaaaaaa aaatctacaa tcaacagatc gcttcaatta cgccctcaca 3600 aaaacttttt tccttcttct tcgcccacgt taaattttat ccctcatgtt gtctaacgga 3660 tttctgcact tgatttatta taaaaagaca aagacataat acttctctat caatttcagt 3720 tattgttctt ccttgcgtta ttcttctgtt cttctttttc ttttgtcata tataaccata 3780 accaagtaat acatattcaa actagtatga ctgacaaaaa aactcttaaa gacttaagaa 3840 atcgtagttc tgtttacgat tcaatggtta aatcacctaa tcgtgctatg ttgcgtgcaa 3900 ctggtatgca agatgaagac tttgaaaaac ctatcgtcgg tgtcatttca acttgggctg 3960 aaaacacacc ttgtaatatc cacttacatg actttggtaa actagccaaa gtcggtgtta 4020 aggaagctgg tgcttggcca gttcagttcg gaacaatcac ggtttctgat ggaatcgcca 4080 tgggaaccca aggaatgcgt ttctccttga catctcgtga tattattgca gattctattg 4140 aagcagccat gggaggtcat aatgcggatg cttttgtagc cattggcggt tgtgataaaa 4200 acatgcccgg ttctgttatc gctatggcta acatggatat cccagccatt tttgcttacg 4260 gcggaacaat tgcacctggt aatttagacg gcaaagatat cgatttagtc tctgtctttg 4320 aaggtgtcgg ccattggaac cacggcgata tgaccaaaga agaagttaaa gctttggaat 4380 gtaatgcttg tcccggtcct ggaggctgcg gtggtatgta tactgctaac acaatggcga 4440 cagctattga agttttggga cttagccttc cgggttcatc ttctcacccg gctgaatccg 4500 cagaaaagaa agcagatatt gaagaagctg gtcgcgctgt tgtcaaaatg ctcgaaatgg 4560 gcttaaaacc ttctgacatt ttaacgcgtg aagcttttga agatgctatt actgtaacta 4620 tggctctggg aggttcaacc aactcaaccc ttcacctctt agctattgcc catgctgcta 4680 atgtggaatt gacacttgat gatttcaata ctttccaaga aaaagttcct catttggctg 4740 atttgaaacc ttctggtcaa tatgtattcc aagaccttta caaggtcgga ggggtaccag 4800 cagttatgaa atatctcctt aaaaatggct tccttcatgg tgaccgtatc acttgtactg 4860 gcaaaacagt cgctgaaaat ttgaaggctt ttgatgattt aacacctggt caaaaggtta 4920 ttatgccgct tgaaaatcct aaacgtgaag atggtccgct cattattctc catggtaact 4980 tggctccaga cggtgccgtt gccaaagttt ctggtgtaaa agtgcgtcgt catgtcggtc 5040 ctgctaaggt ctttaattct gaagaagaag ccattgaagc tgtcttgaat gatgatattg 5100 ttgatggtga tgttgttgtc gtacgttttg taggaccaaa gggcggtcct ggtatgcctg 5160 aaatgctttc cctttcatca atgattgttg gtaaagggca aggtgaaaaa gttgcccttc 5220 tgacagatgg ccgcttctca ggtggtactt atggtcttgt cgtgggtcat atcgctcctg 5280 aagcacaaga tggcggtcca atcgcctacc tgcaaacagg agacatagtc actattgacc 5340 aagacactaa ggaattacac tttgatatct ccgatgaaga gttaaaacat cgtcaagaga 5400 ccattgaatt gccaccgctc tattcacgcg gtatccttgg taaatatgct cacatcgttt 5460 cgtctgcttc taggggagcc gtaacagact tttggaagcc tgaagaaact ggcaaaaaat 5520 gttgtcctgg ttgctgtggt taagcggccg cgttaattca aattaattga tatagttttt 5580 taatgagtat tgaatctgtt tagaaataat ggaatattat ttttatttat ttatttatat 5640 tattggtcgg ctcttttctt ctgaaggtca atgacaaaat gatatgaagg aaataatgat 5700 ttctaaaatt ttacaacgta agatattttt acaaaagcct agctcatctt ttgtcatgca 5760 ctattttact cacgcttgaa attaacggcc agtccactgc ggagtcattt caaagtcatc 5820 ctaatcgatc tatcgttttt gatagctcat tttggagttc gcgattgtct tctgttattc 5880 acaactgttt taatttttat ttcattctgg aactcttcga gttctttgta aagtctttca 5940 tagtagctta ctttatcctc caacatattt aacttcatgt caatttcggc tcttaaattt 6000 tccacatcat caagttcaac atcatctttt aacttgaatt tattctctag ctcttccaac 6060 caagcctcat tgctccttga tttactggtg aaaagtgata cactttgcgc gcaatccagg 6120 tcaaaacttt cctgcaaaga attcaccaat ttctcgacat catagtacaa tttgttttgt 6180 tctcccatca caatttaata tacctgatgg attcttatga agcgctgggt aatggacgtg 6240 tcactctact tcgccttttt ccctactcct tttagtacgg aagacaatgc taataaataa 6300 gagggtaata ataatattat taatcggcaa aaaagattaa acgccaagcg tttaattatc 6360 agaaagcaaa cgtcgtacca atccttgaat gcttcccaat tgtatattaa gagtcatcac 6420 agcaacatat tcttgttatt aaattaatta ttattgattt ttgatattgt ataaaaaaac 6480 caaatatgta taaaaaaagt gaataaaaaa taccaagtat ggagaaatat attagaagtc 6540 tatacgttaa accacccggg ccccccctcg aggtcgacgg tatcgataag cttgatatcg 6600 aattcctgca gcccggggga tccactagtt ctagagcggc cgctctagaa ctagtaccac 6660 aggtgttgtc ctctgaggac ataaaataca caccgagatt catcaactca ttgctggagt 6720 tagcatatct acaattgggt gaaatgggga gcgatttgca ggcatttgct cggcatgccg 6780 gtagaggtgt ggtcaataag agcgacctca tgctatacct gagaaagcaa cctgacctac 6840 aggaaagagt tactcaagaa taagaatttt cgttttaaaa cctaagagtc actttaaaat 6900 ttgtatacac ttattttttt tataacttat ttaataataa aaatcataaa tcataagaaa 6960 ttcgcttact cttaattaat caggcagcgc ctgcgttcga gaggatgatc ttcatcgcct 7020 tctccttggc gccattgagg aatacctgat aggcgtgctc gatctcggcc agctcgaagc 7080 gatgggtaat catcttcttc aacggaagct tgtcggtcga ggcgaccttc atcagcatgg 7140 gcgtcgtgtt cgtgttcacc agtcccgtgg tgatcgtcag gttcttgatc cagagcttct 7200 gaatctcgaa gtcaaccttg acgccatgca cgccgacgtt ggcgatgtgc gcgccgggct 7260 tgacgatctc ctggcagatg tcccaagtcg ccggtatgcc caccgcctcg atcgcaacat 7320 cgactccctc tgccgcaatc ctatgcacgg cttcgacaac gttctccgtg ccggagttga 7380 tggtgtgcgt tgccccgagc tccttggcga gctggaggcg attctcgtcc atgtcgatca 7440 cgatgatggt cgagggggag tagaactggg cggtcaacag tacggacatg ccgacggggc 7500 ccgcgccgac aatagccacc gcatcgcccg gctggacatt cccatactgg acgccgattt 7560 cgtggccggt gggcaggatg tcgctcagca ggacggcgat ttcgtcgtca attgtctggg 7620 ggatcttgta gaggctgttg tcggcatgcg ggatgcggac gtattcggcc tgcacgccat 7680 cgatcatgta acccaggatc cacccgccgt cgcggcaatg ggagtaaagc tgcttcttgc 7740 agtagtcgca cgagccgcaa gaagtgacgc aggaaatcag gaccttgtcg cctttcttga 7800 actgcgtgac actctcgccc acttcctcga tgacgcctac cccttcatgg cccaggatgc 7860 gcccgtcggc gacctctgga ttcttgcctt tgtagatgcc gagatccgtg ccgcagatcg 7920 tggtcttcaa aacccgtact actacatccg tgggcttttg aagggtgggc ttgggcttgt 7980 cttcaagcga gatcttgtgg tcaccgtgat aaaccagagc tttcatcctc agctattgta 8040 atatgtgtgt ttgtttggat tattaagaag aataattaca aaaaaaatta caaaggaagg 8100 taattacaac agaattaaga aaggacaaga aggaggaaga gaatcagttc attatttctt 8160 ctttgttata taacaaaccc aagtagcgat ttggccatac attaaaagtt gagaaccacc 8220 ctccctggca acagccacaa ctcgttacca ttgttcatca cgatcatgaa actcgctgtc 8280 agctgaaatt tcacctcagt ggatctctct ttttattctt catcgttcca ctaacctttt 8340 tccatcagct ggcagggaac ggaaagtgga atcccattta gcgagcttcc tcttttcttc 8400 aagaaaagac gaagcttgtg tgtgggtgcg cgcgctagta tctttccaca ttaagaaata 8460 taccataaag gttacttaga catcactatg gctatatata tatatatata tatatatgta 8520 acttagcacc atcgcgcgtg catcactgca tgtgttaacc gaaaagtttg gcgaacactt 8580 caccgacacg gtcatttaga tctgtcgtct gcattgcacg tcccttagcc ttaaatccta 8640 ggcgggagca ttctcgtgta attgtgcagc ctgcgtagca actcaacata gcgtagtcta 8700 cccagttttt caagggttta tcgttagaag attctccctt ttcttcctgc tcacaaatct 8760 taaagtcata cattgcacga ctaaatgcaa gcatgcggat cccccgggct gcaggaattc 8820 gatatcaagc ttatcgatac cgtcgactgg ccattaatct ttcccatatt agatttcgcc 8880 aagccatgaa agttcaagaa aggtctttag acgaattacc cttcatttct caaactggcg 8940 tcaagggatc ctggtatggt tttatcgttt tatttctggt tcttatagca tcgttttgga 9000 cttctctgtt cccattaggc ggttcaggag ccagcgcaga atcattcttt gaaggatact 9060 tatcctttcc aattttgatt gtctgttacg ttggacataa actgtatact agaaattgga 9120 ctttgatggt gaaactagaa gatatggatc ttgataccgg cagaaaacaa gtagatttga 9180 ctcttcgtag ggaagaaatg aggattgagc gagaaacatt agcaaaaaga tccttcgtaa 9240 caagattttt acatttctgg tgttgaaggg aaagatatga gctatacagc ggaatttcca 9300 tatcactcag attttgttat ctaatttttt ccttcccacg tccgcgggaa tctgtgtata 9360 ttactgcatc tagatatatg ttatcttatc ttggcgcgta catttaattt tcaacgtatt 9420 ctataagaaa ttgcgggagt ttttttcatg tagatgatac tgactgcacg caaatatagg 9480 catgatttat aggcatgatt tgatggctgt accgatagga acgctaagag taacttcaga 9540 atcgttatcc tggcggaaaa aattcatttg taaactttaa aaaaaaaagc caatatcccc 9600 aaaattatta agagcgcctc cattattaac taaaatttca ctcagcatcc acaatgtatc 9660 aggtatctac tacagatatt acatgtggcg aaaaagacaa gaacaatgca atagcgcatc 9720 aagaaaaaac acaaagcttt caatcaatga atcgaaaatg tcattaaaat agtatataaa 9780 ttgaaactaa gtcataaagc tataaaaaga aaatttattt aaatgcaaga tttaaagtaa 9840 attcacggcc ctgcaggcct cagctcttgt tttgttctgc aaataactta cccatctttt 9900 tcaaaacttt aggtgcaccc tcctttgcta gaataagttc tatccaatac atcctatttg 9960 gatctgcttg agcttctttc atcacggata cgaattcatt ttctgttctc acaattttgg 10020 acacaactct gtcttccgtt gccccgaaac tttctggcag ttttgagtaa ttccacatag 10080 gaatgtcatt ataactctgg ttcggaccat gaatttccct ctcaaccgtg taaccatcgt 10140 tattaatgat aaagcagatt gggtttatct tctctctaat ggctagtcct aattcttgga 10200 cagtcagttg caatgatcca tctccgataa acaataaatg tctagattct ttatctgcaa 10260 tttggctgcc tagagctgcg gggaaagtgt atcctataga tccccacaag ggttgaccaa 10320 taaaatgtga tttcgatttc agaaatatag atgaggcacc gaagaaagaa gtgccttgtt 10380 cagccacgat cgtctcatta ctttgggtca aattttcgac agcttgccac agtctatctt 10440 gtgacaacag cgcgttagaa ggtacaaaat cttcttgctt tttatctatg tacttgcctt 10500 tatattcaat ttcggacaag tcaagaagag atgatatcag ggattcgaag tcgaaatttt 10560 ggattctttc gttgaaaatt ttaccttcat cgatattcaa ggaaatcatt ttattttcat 10620 taagatggtg agtaaatgca cccgtactag aatcggtaag ctttacaccc aacataagaa 10680 taaaatcagc agattccaca aattccttca agtttggctc tgacagagta ccgttgtaaa 10740 tccccaaaaa tgagggcaat gcttcatcaa cagatgattt accaaagttc aaagtagtaa 10800 taggtaactt agtctttgaa ataaactgag taacagtctt ctctaggccg aacgatataa 10860 tttcatggcc tgtgattaca attggtttct tggcattctt cagactttcc tgtattttgt 10920 tcagaatctc ttgatcagat gtattcgacg tggaattttc cttcttaaga ggcaaggatg 10980 gtttttcagc cttagcggca gctacatcta caggtaaatt gatgtaaacc ggctttcttt 11040 cctttagtaa ggcagacaac actctatcaa tttcaacagt tgcattctcg gctgtcaata 11100 aagtcctggc agcagtaacc ggttcgtgca tcttcataaa gtgcttgaaa tcaccatcag 11160 ccaacgtatg gtgaacaaac ttaccttcgt tctgcacttt cgaggtagga gatcccacga 11220 tctcaacaac aggcaggttc tcagcatagg agcccgctaa gccattaact gcggataatt 11280 cgccaacacc aaatgtagtc aagaatgccg cagccttttt cgttcttgcg tacccgtcgg 11340 ccatatagga ggcatttaac tcattagcat ttcccaccca tttcatatct ttgtgtgaaa 11400 taatttgatc tagaaattgc aaattgtagt cacctggtac tccgaatatt tcttctatac 11460 ctaattcgtg taatctgtcc aacagatagt cacctactgt atacattttg tttactagtt 11520 tatgtgtgtt tattcgaaac taagttcttg gtgttttaaa actaaaaaaa agactaacta 11580 taaaagtaga atttaagaag tttaagaaat agatttacag aattacaatc aatacctacc 11640 gtctttatat acttattagt caagtagggg aataatttca gggaactggt ttcaaccttt 11700 tttttcagct ttttccaaat cagagagagc agaaggtaat agaaggtgta agaaaatgag 11760 atagatacat gcgtgggtca attgccttgt gtcatcattt actccaggca ggttgcatca 11820 ctccattgag gttgtgcccg ttttttgcct gtttgtgccc ctgttctctg tagttgcgct 11880 aagagaatgg acctatgaac tgatggttgg tgaagaaaac aatattttgg tgctgggatt 11940 cttttttttt ctggatgcca gcttaaaaag cgggctccat tatatttagt ggatgccagg 12000 aataaactgt tcacccagac acctacgatg ttatatattc tgtgtaaccc gccccctatt 12060 ttgggcatgt acgggttaca gcagaattaa aaggctaatt ttttgactaa ataaagttag 12120 gaaaatcact actattaatt atttacgtat tctttgaaat ggcagtattg ataatgataa 12180 actcgaactg aaaaagcgtg ttttttattc aaaatgattc taactccctt acgtaatcaa 12240 ggaatctttt tgccttggcc tccgcgtcat taaacttctt gttgttgacg ctaacattca 12300 acgctagtat atattcgttt ttttcaggta agttcttttc aacgggtctt actgatgagg 12360 cagtcgcgtc tgaacctgtt aagaggtcaa atatgtcttc ttgaccgtac gtgtcttgca 12420 tgttattagc tttgggaatt tgcatcaagt cataggaaaa tttaaatctt ggctctcttg 12 480 ggctcaaggt gacaaggtcc tcgaaaatag ggcgcgcccc accgcggtgg agctccagct 12540 tttgttccct ttagtgaggg ttaattgcgc gcttggcgta atcatggtca tagctgtttc 12600 ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt 12660 gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc 12720 ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg 12780 ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct 12840 cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca 12900 cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga 12960 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc 13020 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg 13080 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat 13140 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt 13200 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc 13260 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg 13320 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg 13380 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg 13440 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg 13500 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca 13560 gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga 13620 acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga 13680 tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt 13740 ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt 13800 catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat 13860 ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag 13920 caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct 13980 ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt 14040 tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg 14100 cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca 14160 aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt 14220 tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat 14280 gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac 14340 cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa 14400 aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt 14460 tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt 14520 tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa 14580 gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt 14640 atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa 14700 taggggttcc gcgcacattt ccccgaaaag tgccacctga acgaagcatc tgtgcttcat 14760 tttgtagaac aaaaatgcaa cgcgagagcg ctaatttttc aaacaaagaa tctgagctgc 14820 atttttacag aacagaaatg caacgcgaaa gcgctatttt accaacgaag aatctgtgct 14880 tcatttttgt aaaacaaaaa tgcaacgcga gagcgctaat ttttcaaaca aagaatctga 14940 gctgcatttt tacagaacag aaatgcaacg cgagagcgct attttaccaa caaagaatct 15000 atacttcttt tttgttctac aaaaatgcat cccgagagcg ctatttttct aacaaagcat 15060 cttagattac tttttttctc ctttgtgcgc tctataatgc agtctcttga taactttttg 15120 cactgtaggt ccgttaaggt tagaagaagg ctactttggt gtctattttc tcttccataa 15180 aaaaagcctg actccacttc ccgcgtttac tgattactag cgaagctgcg ggtgcatttt 15240 ttcaagataa aggcatcccc gattatattc tataccgatg tggattgcgc atactttgtg 15300 aacagaaagt gatagcgttg atgattcttc attggtcaga aaattatgaa cggtttcttc 15360 tattttgtct ctatatacta cgtataggaa atgtttacat tttcgtattg ttttcgattc 15420 actctatgaa tagttcttac tacaattttt ttgtct 15456 <210> 68 <211> 1559 <212> DNA <213> Artificial sequence <220> <223> Template <400> 68 gcattgcgga ttacgtattc taatgttcag taccgttcgt ataatgtatg ctatacgaag 60 ttatgcagat tgtactgaga gtgcaccata ccaccttttc aattcatcat ttttttttta 120 ttcttttttt tgatttcggt ttccttgaaa tttttttgat tcggtaatct ccgaacagaa 180 ggaagaacga aggaaggagc acagacttag attggtatat atacgcatat gtagtgttga 240 agaaacatga aattgcccag tattcttaac ccaactgcac agaacaaaaa cctgcaggaa 300 acgaagataa atcatgtcga aagctacata taaggaacgt gctgctactc atcctagtcc 360 tgttgctgcc aagctattta atatcatgca cgaaaagcaa acaaacttgt gtgcttcatt 420 ggatgttcgt accaccaagg aattactgga gttagttgaa gcattaggtc ccaaaatttg 480 tttactaaaa acacatgtgg atatcttgac tgatttttcc atggagggca cagttaagcc 540 gctaaaggca ttatccgcca agtacaattt tttactcttc gaagacagaa aatttgctga 600 cattggtaat acagtcaaat tgcagtactc tgcgggtgta tacagaatag cagaatgggc 660 agacattacg aatgcacacg gtgtggtggg cccaggtatt gttagcggtt tgaagcaggc 720 ggcagaagaa gtaacaaagg aacctagagg ccttttgatg ttagcagaat tgtcatgcaa 780 gggctcccta tctactggag aatatactaa gggtactgtt gacattgcga agagcgacaa 840 agattttgtt atcggcttta ttgctcaaag agacatgggt ggaagagatg aaggttacga 900 ttggttgatt atgacacccg gtgtgggttt agatgacaag ggagacgcat tgggtcaaca 960 gtatagaacc gtggatgatg tggtctctac aggatctgac attattattg ttggaagagg 1020 actatttgca aagggaaggg atgctaaggt agagggtgaa cgttacagaa aagcaggctg 1080 ggaagcatat ttgagaagat gcggccagca aaactaaaaa actgtattat aagtaaatgc 1140 atgtatacta aactcacaaa ttagagcttc aatttaatta tatcagttat taccctatgc 1200 ggtgtgaaat accgcacaga tgcgtaagga gaaaataccg catcaggaaa ttgtaaacgt 1260 taatattttg ttaaaattcg cgttaaattt ttgttaaatc agctcatttt ttaaccaata 1320 ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag accgagatag ggttgagtgt 1380 tgttccagtt tggaacaaga gtccactatt aaagaacgtg gactccaacg tcaaagggcg 1440 aaaaaccgtc tatcagggcg atggcccact acgtgaacca tcaccctaat caagataact 1500 tcgtataatg tatgctatac gaacggtacc agtgatgata caacgagtta gccaaggtg 1559 <210> 69 <211> 1047 <212> DNA <213> Achromobacter xylosoxidans <400> 69 atgaaagctc tggtttatca cggtgaccac aagatctcgc ttgaagacaa gcccaagccc 60 acccttcaaa agcccacgga tgtagtagta cgggttttga agaccacgat ctgcggcacg 120 gatctcggca tctacaaagg caagaatcca gaggtcgccg acgggcgcat cctgggccat 180 gaaggggtag gcgtcatcga ggaagtgggc gagagtgtca cgcagttcaa gaaaggcgac 240 aaggtcctga tttcctgcgt cacttcttgc ggctcgtgcg actactgcaa gaagcagctt 300 tactcccatt gccgcgacgg cgggtggatc ctgggttaca tgatcgatgg cgtgcaggcc 360 gaatacgtcc gcatcccgca tgccgacaac agcctctaca agatccccca gacaattgac 420 gacgaaatcg ccgtcctgct gagcgacatc ctgcccaccg gccacgaaat cggcgtccag 480 tatgggaatg tccagccggg cgatgcggtg gctattgtcg gcgcgggccc cgtcggcatg 540 tccgtactgt tgaccgccca gttctactcc ccctcgacca tcatcgtgat cgacatggac 600 gagaatcgcc tccagctcgc caaggagctc ggggcaacgc acaccatcaa ctccggcacg 660 ggaacgttg tcgaagccgt gcataggatt gcggcagagg gagtcgatgt tgcgatcgag 720 gcggtgggca taccggcgac ttgggacatc tgccaggaga tcgtcaagcc cggcgcgcac 780 atcgccaacg tcggcgtgca tggcgtcaag gttgacttcg agattcagaa gctctggatc 840 aagaacctga cgatcaccac gggactggtg aacacgaaca cgacgcccat gctgatgaag 900 gtcgcctcga ccgacaagct tccgttgaag aagatgatta cccatcgctt cgagctggcc 960 gagatcgagc acgcctatca ggtattcctc aatggcgcca aggagaaggc gatgaagatc 1020 atcctctcga acgcaggcgc tgcctga 1047 <210> 70 <211> 348 <212> PRT <213> Achromobacter xylosoxidans <400> 70 Met Lys Ala Leu Val Tyr His Gly Asp His Lys Ile Ser Leu Glu Asp 1 5 10 15 Lys Pro Lys Pro Thr Leu Gln Lys Pro Thr Asp Val Val Arg Val             20 25 30 Leu Lys Thr Thr Ile Cys Gly Thr Asp Leu Gly Ile Tyr Lys Gly Lys         35 40 45 Asn Pro Glu Val Ala Asp Gly Arg Ile Leu Gly His Glu Gly Val Gly     50 55 60 Val Ile Glu Glu Val Gly Glu Ser Val Thr Gln Phe Lys Lys Gly Asp 65 70 75 80 Lys Val Leu Ile Ser Cys Val Thr Ser Cys Gly Ser Cys Asp Tyr Cys                 85 90 95 Lys Lys Gln Leu Tyr Ser His Cys Arg Asp Gly Gly Trp Ile Leu Gly             100 105 110 Tyr Met Ile Asp Gly Val Gln Ala Glu Tyr Val Arg Ile Pro His Ala         115 120 125 Asp Asn Ser Leu Tyr Lys Ile Pro Gln Thr Ile Asp Asp Glu Ile Ala     130 135 140 Val Leu Leu Ser Asp Ile Leu Pro Thr Gly His Glu Ile Gly Val Gln 145 150 155 160 Tyr Gly Asn Val Gln Pro Gly Asp Ala Val Ala Ile Val Gly Ala Gly                 165 170 175 Pro Val Gly Met Ser Val Leu Leu Thr Ala Gln Phe Tyr Ser Ser Ser             180 185 190 Thr Ile Ile Val Ile Asp Met Asp Glu Asn Arg Leu Gln Leu Ala Lys         195 200 205 Glu Leu Gly Ala Thr His Thr Ile Asn Ser Gly Thr Glu Asn Val Val     210 215 220 Glu Ala Val His Arg Ile Ala Ala Glu Gly Val Asp Val Ala Ile Glu 225 230 235 240 Ala Val Gly Ile Pro Ala Thr Trp Asp Ile Cys Gln Glu Ile Val Lys                 245 250 255 Pro Gly Ala His Ile Ala Asn Val Gly Val His Gly Val Lys Val Asp             260 265 270 Phe Glu Ile Gln Lys Leu Trp Ile Lys Asn Leu Thr Ile Thr Thr Gly         275 280 285 Leu Val Asn Thr Asn Thr Thr Pro Met Leu Met Lys Val Ala Ser Thr     290 295 300 Asp Lys Leu Pro Leu Lys Lys Met Ile Thr His Arg Phe Glu Leu Ala 305 310 315 320 Glu Ile Glu His Ala Tyr Gln Val Phe Leu Asn Gly Ala Lys Glu Lys                 325 330 335 Ala Met Lys Ile Ile Leu Ser Asn Ala Gly Ala Ala             340 345 <210> 71 <211> 1644 <212> DNA <213> artificial sequence <220> <223> codon optimized L. lactis kivD coding region for S. cerevisiae        expression <400> 71 atgtatacag taggtgacta tctgttggac agattacacg aattaggtat agaagaaata 60 ttcggagtac caggtgacta caatttgcaa tttctagatc aaattatttc acacaaagat 120 atgaaatggg tgggaaatgc taatgagtta aatgcctcct atatggccga cgggtacgca 180 agaacgaaaa aggctgcggc attcttgact acatttggtg ttggcgaatt atccgcagtt 240 aatggcttag cgggctccta tgctgagaac ctgcctgttg ttgagatcgt gggatctcct 300 acctcgaaag tgcagaacga aggtaagttt gttcaccata cgttggctga tggtgatttc 360 aagcacttta tgaagatgca cgaaccggtt actgctgcca ggactttatt gacagccgag 420 aatgcaactg ttgaaattga tagagtgttg tctgccttac taaaggaaag aaagccggtt 480 tacatcaatt tacctgtaga tgtagctgcc gctaaggctg aaaaaccatc cttgcctctt 540 aagaaggaaa attccacgtc gaatacatct gatcaagaga ttctgaacaa aatacaggaa 600 agtctgaaga atgccaagaa accaattgta atcacaggcc atgaaattat atcgttcggc 660 ctagagaaga ctgttactca gtttatttca aagactaagt tacctattac tactttgaac 720 tttggtaaat catctgttga tgaagcattg ccctcatttt tggggattta caacggtact 780 ctgtcagagc caaacttgaa ggaatttgtg gaatctgctg attttattct tatgttgggt 840 gtaaagctta ccgattctag tacgggtgca tttactcacc atcttaatga aaataaaatg 900 atttccttga atatcgatga aggtaaaatt ttcaacgaaa gaatccaaaa tttcgacttc 960 gaatccctga tatcatctct tcttgacttg tccgaaattg aatataaagg caagtacata 1020 gataaaaagc aagaagattt tgtaccttct aacgcgctgt tgtcacaaga tagactgtgg 1080 caagctgtcg aaaatttgac ccaaagtaat gagacgatcg tggctgaaca aggcacttct 1140 ttcttcggtg cctcatctat atttctgaaa tcgaaatcac attttattgg tcaacccttg 1200 tggggatcta taggatacac tttccccgca gctctaggca gccaaattgc agataaagaa 1260 tctagacatt tattgtttat cggagatgga tcattgcaac tgactgtcca agaattagga 1320 ctagccatta gagagaagat aaacccaatc tgctttatca ttaataacga tggttacacg 1380 gttgagaggg aaattcatgg tccgaaccag agttataatg acattcctat gtggaattac 1440 tcaaaactgc cagaaagttt cggggcaacg gaagacagag ttgtgtccaa aattgtgaga 1500 acagaaaatg aattcgtatc cgtgatgaaa gaagctcaag cagatccaaa taggatgtat 1560 tggatagaac ttattctagc aaaggagggt gcacctaaag ttttgaaaaa gatgggtaag 1620 ttatttgcag aacaaaacaa gagc 1644 <210> 72 <211> 753 <212> DNA <213> Saccharomyces cerevisiae <400> 72 gcatgcttgc atttagtcgt gcaatgtatg actttaagat ttgtgagcag gaagaaaagg 60 gagaatcttc taacgataaa cccttgaaaa actgggtaga ctacgctatg ttgagttgct 120 acgcaggctg cacaattaca cgagaatgct cccgcctagg atttaaggct aagggacgtg 180 caatgcagac gacagatcta aatgaccgtg tcggtgaagt gttcgccaaa cttttcggtt 240 aacacatgca gtgatgcacg cgcgatggtg ctaagttaca tatatatata tatagccata 300 gtgatgtcta agtaaccttt atggtatatt tcttaatgtg gaaagatact agcgcgcgca 360 cccacacaca agcttcgtct tttcttgaag aaaagaggaa gctcgctaaa tgggattcca 420 ctttccgttc cctgccagct gatggaaaaa ggttagtgga acgatgaaga ataaaaagag 480 agatccactg aggtgaaatt tcagctgaca gcgagtttca tgatcgtgat gaacaatggt 540 aacgagttgt ggctgttgcc agggagggtg gttctcaact tttaatgtat ggccaaatcg 600 ctacttgggt ttgttatata acaaagaaga aataatgaac tgattctctt cctccttctt 660 gtcctttctt aattctgttg taattacctt cctttgtaat tttttttgta attattcttc 720 ttaataatcc aaacaaacac acatattaca ata 753 <210> 73 <211> 316 <212> DNA <213> Saccharomyces cerevisiae <400> 73 gagtaagcga atttcttatg atttatgatt tttattatta aataagttat aaaaaaaata 60 agtgtataca aattttaaag tgactcttag gttttaaaac gaaaattctt attcttgagt 120 aactctttcc tgtaggtcag gttgctttct caggtatagc atgaggtcgc tcttattgac 180 cacacctcta ccggcatgcc gagcaaatgc ctgcaaatcg ctccccattt cacccaattg 240 tagatatgct aactccagca atgagttgat gaatctcggt gtgtatttta tgtcctcaga 300 ggacaacacc tgtggt 316 <210> 74 <211> 30 <212> DNA <213> artificial sequence <220> <223> primer <400> 74 ggaattcaca catgaaagct ctggtttatc 30 <210> 75 <211> 28 <212> DNA <213> artificial sequence <220> <223> primer <400> 75 gcgtccaggg cgtcaaagat caggcagc 28 <210> 76 <211> 55 <212> DNA <213> artificial sequence <220> <223> primer <400> 76 aaacaaacac acatattaca atagctgagg atgaaagctc tggtttatca cggtg 55 <210> 77 <211> 55 <212> DNA <213> artificial sequence <220> <223> primer <400> 77 atcataagaa attcgcttac tcttaattaa tcaggcagcg cctgcgttcg agagg 55 <210> 78 <211> 8994 <212> DNA <213> artificial sequence <220> <223> constructed plasmid <400> 78 ctagttctag agcggccgcc accgcggtgg agctccagct tttgttccct ttagtgaggg 60 ttaattgcgc gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg 120 ctcacaattc cacacaacat aggagccgga agcataaagt gtaaagcctg gggtgcctaa 180 tgagtgaggt aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac 240 ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt 300 gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga 360 gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca 420 ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg 480 ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt 540 cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc 600 ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct 660 tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc 720 gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta 780 tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca 840 gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag 900 tggtggccta actacggcta cactagaagg acagtatttg gtatctgcgc tctgctgaag 960 ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt 1020 agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa 1080 gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg 1140 attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga 1200 agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta 1260 atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc 1320 cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg 1380 ataccgcgag acccacgctc accggctcca gatttatcag caataaacca gccagccgga 1440 agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt 1500 tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt 1560 gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc 1620 caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc 1680 ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca 1740 gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag 1800 tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg 1860 tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa 1920 cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa 1980 cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga 2040 gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga 2100 atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg 2160 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt 2220 ccccgaaaag tgccacctga acgaagcatc tgtgcttcat tttgtagaac aaaaatgcaa 2280 cgcgagagcg ctaatttttc aaacaaagaa tctgagctgc atttttacag aacagaaatg 2340 caacgcgaaa gcgctatttt accaacgaag aatctgtgct tcatttttgt aaaacaaaaa 2400 tgcaacgcga gagcgctaat ttttcaaaca aagaatctga gctgcatttt tacagaacag 2460 aaatgcaacg cgagagcgct attttaccaa caaagaatct atacttcttt tttgttctac 2520 aaaaatgcat cccgagagcg ctatttttct aacaaagcat cttagattac tttttttctc 2580 ctttgtgcgc tctataatgc agtctcttga taactttttg cactgtaggt ccgttaaggt 2640 tagaagaagg ctactttggt gtctattttc tcttccataa aaaaagcctg actccacttc 2700 ccgcgtttac tgattactag cgaagctgcg ggtgcatttt ttcaagataa aggcatcccc 2760 gattatattc tataccgatg tggattgcgc atactttgtg aacagaaagt gatagcgttg 2820 atgattcttc attggtcaga aaattatgaa cggtttcttc tattttgtct ctatatacta 2880 cgtataggaa atgtttacat tttcgtattg ttttcgattc actctatgaa tagttcttac 2940 tacaattttt ttgtctaaag agtaatacta gagataaaca taaaaaatgt agaggtcgag 3000 tttagatgca agttcaagga gcgaaaggtg gatgggtagg ttatataggg atatagcaca 3060 gagatatata gcaaagagat acttttgagc aatgtttgtg gaagcggtat tcgcaatatt 3120 ttagtagctc gttacagtcc ggtgcgtttt tggttttttg aaagtgcgtc ttcagagcgc 3180 ttttggtttt caaaagcgct ctgaagttcc tatactttct agagaatagg aacttcggaa 3240 taggaacttc aaagcgtttc cgaaaacgag cgcttccgaa aatgcaacgc gagctgcgca 3300 catacagctc actgttcacg tcgcacctat atctgcgtgt tgcctgtata tatatataca 3360 tgagaagaac ggcatagtgc gtgtttatgc ttaaatgcgt acttatatgc gtctatttat 3420 gtaggatgaa aggtagtcta gtacctcctg tgatattatc ccattccatg cggggtatcg 3480 tatgcttcct tcagcactac cctttagctg ttctatatgc tgccactcct caattggatt 3540 agtctcatcc ttcaatgcta tcatttcctt tgatattgga tcatactaag aaaccattat 3600 tatcatgaca ttaacctata aaaataggcg tatcacgagg ccctttcgtc tcgcgcgttt 3660 cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca cagcttgtct 3720 gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg ttggcgggtg 3780 tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc accatatcga 3840 ctacgtcgta aggccgtttc tgacagagta aaattcttga gggaactttc accattatgg 3900 gaaatgcttc aagaaggtat tgacttaaac tccatcaaat ggtcaggtca ttgagtgttt 3960 tttatttgtt gtattttttt ttttttagag aaaatcctcc aatatcaaat taggaatcgt 4020 agtttcatga ttttctgtta cacctaactt tttgtgtggt gccctcctcc ttgtcaatat 4080 taatgttaaa gtgcaattct ttttccttat cacgttgagc cattagtatc aatttgctta 4140 cctgtattcc tttactatcc tcctttttct ccttcttgat aaatgtatgt agattgcgta 4200 tatagtttcg tctaccctat gaacatattc cattttgtaa tttcgtgtcg tttctattat 4260 gaatttcatt tataaagttt atgtacaaat atcataaaaa aagagaatct ttttaagcaa 4320 ggattttctt aacttcttcg gcgacagcat caccgacttc ggtggtactg ttggaaccac 4380 ctaaatcacc agttctgata cctgcatcca aaaccttttt aactgcatct tcaatggcct 4440 taccttcttc aggcaagttc aatgacaatt tcaacatcat tgcagcagac aagatagtgg 4500 cgatagggtc aaccttattc tttggcaaat ctggagcaga accgtggcat ggttcgtaca 4560 aaccaaatgc ggtgttcttg tctggcaaag aggccaagga cgcagatggc aacaaaccca 4620 aggaacctgg gataacggag gcttcatcgg agatgatatc accaaacatg ttgctggtga 4680 ttataatacc atttaggtgg gttgggttct taactaggat catggcggca gaatcaatca 4740 attgatgttg aaccttcaat gtagggaatt cgttcttgat ggtttcctcc acagtttttc 4800 tccataatct tgaagaggcc aaaagattag ctttatccaa ggaccaaata ggcaatggtg 4860 gctcatgttg tagggccatg aaagcggcca ttcttgtgat tctttgcact tctggaacgg 4920 tgtattgttc actatcccaa gcgacaccat caccatcgtc ttcctttctc ttaccaaagt 4980 aaatacctcc cactaattct ctgacaacaa cgaagtcagt acctttagca aattgtggct 5040 tgattggaga taagtctaaa agagagtcgg atgcaaagtt acatggtctt aagttggcgt 5100 acaattgaag ttctttacgg atttttagta aaccttgttc aggtctaaca ctaccggtac 5160 cccatttagg accagccaca gcacctaaca aaacggcatc aaccttcttg gaggcttcca 5220 gcgcctcatc tggaagtggg acacctgtag catcgatagc agcaccacca attaaatgat 5280 tttcgaaatc gaacttgaca ttggaacgaa catcagaaat agctttaaga accttaatgg 5340 cttcggctgt gatttcttga ccaacgtggt cacctggcaa aacgacgatc ttcttagggg 5400 cagacatagg ggcagacatt agaatggtat atccttgaaa tatatatata tattgctgaa 5460 atgtaaaagg taagaaaagt tagaaagtaa gacgattgct aaccacctat tggaaaaaac 5520 aataggtcct taaataatat tgtcaacttc aagtattgtg atgcaagcat ttagtcatga 5580 acgcttctct attctatatg aaaagccggt tccggcctct cacctttcct ttttctccca 5640 atttttcagt tgaaaaaggt atatgcgtca ggcgacctct gaaattaaca aaaaatttcc 5700 agtcatcgaa tttgattctg tgcgatagcg cccctgtgtg ttctcgttat gttgaggaaa 5760 aaaataatgg ttgctaagag attcgaactc ttgcatctta cgatacctga gtattcccac 5820 agttaactgc ggtcaagata tttcttgaat caggcgcctt agaccgctcg gccaaacaac 5880 caattacttg ttgagaaata gagtataatt atcctataaa tataacgttt ttgaacacac 5940 atgaacaagg aagtacagga caattgattt tgaagagaat gtggattttg atgtaattgt 6000 tgggattcca tttttaataa ggcaataata ttaggtatgt ggatatacta gaagttctcc 6060 tcgaccgtcg atatgcggtg tgaaataccg cacagatgcg taaggagaaa ataccgcatc 6120 aggaaattgt aaacgttaat attttgttaa aattcgcgtt aaatttttgt taaatcagct 6180 cattttttaa ccaataggcc gaaatcggca aaatccctta taaatcaaaa gaatagaccg 6240 agatagggtt gagtgttgtt ccagtttgga acaagagtcc actattaaag aacgtggact 6300 ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg cccactacgt gaaccatcac 6360 cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga 6420 gcccccgatt tagagcttga cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga 6480 aagcgaaagg agcgggcgct agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca 6540 ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc gcgccattcg ccattcaggc 6600 tgcgcaactg ttgggaaggg cgatcggtgc gggcctcttc gctattacgc cagctggcga 6660 aagggggatg tgctgcaagg cgattaagtt gggtaacgcc agggttttcc cagtcacgac 6720 gttgtaaaac gacggccagt gagcgcgcgt aatacgactc actatagggc gaattgggta 6780 ccgggccccc cctcgaggtc gacggtatcg ataagcttga tatcgaattc ctgcagcccg 6840 ggggatccgc atgcttgcat ttagtcgtgc aatgtatgac tttaagattt gtgagcagga 6900 agaaaaggga gaatcttcta acgataaacc cttgaaaaac tgggtagact acgctatgtt 6960 gagttgctac gcaggctgca caattacacg agaatgctcc cgcctaggat ttaaggctaa 7020 gggacgtgca atgcagacga cagatctaaa tgaccgtgtc ggtgaagtgt tcgccaaact 7080 tttcggttaa cacatgcagt gatgcacgcg cgatggtgct aagttacata tatatatata 7140 tatatatata tagccatagt gatgtctaag taacctttat ggtatatttc ttaatgtgga 7200 aagatactag cgcgcgcacc cacacacaag cttcgtcttt tcttgaagaa aagaggaagc 7260 tcgctaaatg ggattccact ttccgttccc tgccagctga tggaaaaagg ttagtggaac 7320 gatgaagaat aaaaagagag atccactgag gtgaaatttc agctgacagc gagtttcatg 7380 atcgtgatga acaatggtaa cgagttgtgg ctgttgccag ggagggtggt tctcaacttt 7440 taatgtatgg ccaaatcgct acttgggttt gttatataac aaagaagaaa taatgaactg 7500 attctcttcc tccttcttgt cctttcttaa ttctgttgta attaccttcc tttgtaattt 7560 tttttgtaat tattcttctt aataatccaa acaaacacac atattacaat agctagctga 7620 ggatgaaggc attagtttat catggggatc acaaaatttc gttagaagac aaaccaaaac 7680 ccactctgca gaaaccaaca gacgttgtgg ttagggtgtt gaaaacaaca atttgcggta 7740 ctgacttggg aatatacaaa ggtaagaatc ctgaagtggc agatggcaga atcctgggtc 7800 atgagggcgt tggcgtcatt gaagaagtgg gcgaatccgt gacacaattc aaaaaggggg 7860 ataaagtttt aatctcctgc gttactagct gtggatcgtg tgattattgc aagaagcaac 7920 tgtattcaca ctgtagagac ggtggctgga ttttaggtta catgatcgac ggtgtccaag 7980 ccgaatacgt cagaatacca catgctgaca attcattgta taagatcccg caaactatcg 8040 atgatgaaat tgcagtacta ctgtccgata ttttacctac tggacatgaa attggtgttc 8100 aatatggtaa cgttcaacca ggcgatgctg tagcaattgt aggagcaggt cctgttggaa 8160 tgtcagtttt gttaactgct caattttact cgcctagtac cattattgtt atcgacatgg 8220 acgaaaaccg tttacaatta gcgaaggagc ttggggccac acacactatt aactccggta 8280 ctgaaaatgt tgtcgaagct gtgcatcgta tagcagccga aggagtggat gtagcaatag 8340 aagctgttgg tatacccgca acctgggaca tctgtcagga aattgtaaaa cccggcgctc 8400 atattgccaa cgtgggagtt catggtgtta aggtggactt tgaaattcaa aagttgtgga 8460 ttaagaatct aaccatcacc actggtttgg ttaacactaa tactacccca atgttgatga 8520 aggtagcctc tactgataaa ttgcctttaa agaaaatgat tactcacagg tttgagttag 8580 ctgaaatcga acacgcatat caggttttct tgaatggcgc taaagaaaaa gctatgaaga 8640 ttattctatc taatgcaggt gccgcctaat taattaagag taagcgaatt tcttatgatt 8700 tatgattttt attattaaat aagttataaa aaaaataagt gtatacaaat tttaaagtga 8760 ctcttaggtt ttaaaacgaa aattcttatt cttgagtaac tctttcctgt aggtcaggtt 8820 gctttctcag gtatagcatg aggtcgctct tattgaccac acctctaccg gcatgccgag 8880 caaatgcctg caaatcgctc cccatttcac ccaattgtag atatgctaac tccagcaatg 8940 agttgatgaa tctcggtgtg tattttatgt cctcagagga caacacctgt ggta 8994 <210> 79 <211> 2145 <212> DNA <213> artificial sequence <220> <223> constructed chimeric gene <400> 79 gcatgcttgc atttagtcgt gcaatgtatg actttaagat ttgtgagcag gaagaaaagg 60 gagaatcttc taacgataaa cccttgaaaa actgggtaga ctacgctatg ttgagttgct 120 acgcaggctg cacaattaca cgagaatgct cccgcctagg atttaaggct aagggacgtg 180 caatgcagac gacagatcta aatgaccgtg tcggtgaagt gttcgccaaa cttttcggtt 240 aacacatgca gtgatgcacg cgcgatggtg ctaagttaca tatatatata tatatatata 300 tatagccata gtgatgtcta agtaaccttt atggtatatt tcttaatgtg gaaagatact 360 agcgcgcgca cccacacaca agcttcgtct tttcttgaag aaaagaggaa gctcgctaaa 420 tgggattcca ctttccgttc cctgccagct gatggaaaaa ggttagtgga acgatgaaga 480 ataaaaagag agatccactg aggtgaaatt tcagctgaca gcgagtttca tgatcgtgat 540 gaacaatggt aacgagttgt ggctgttgcc agggagggtg gttctcaact tttaatgtat 600 ggccaaatcg ctacttgggt ttgttatata acaaagaaga aataatgaac tgattctctt 660 cctccttctt gtcctttctt aattctgttg taattacctt cctttgtaat tttttttgta 720 attattcttc ttaataatcc aaacaaacac acatattaca atagctagct gaggatgaag 780 gcattagttt atcatgggga tcacaaaatt tcgttagaag acaaaccaaa acccactctg 840 cagaaaccaa cagacgttgt ggttagggtg ttgaaaacaa caatttgcgg tactgacttg 900 ggaatataca aaggtaagaa tcctgaagtg gcagatggca gaatcctggg tcatgagggc 960 gttggcgtca ttgaagaagt gggcgaatcc gtgacacaat tcaaaaaggg ggataaagtt 1020 ttaatctcct gcgttactag ctgtggatcg tgtgattatt gcaagaagca actgtattca 1080 cactgtagag acggtggctg gattttaggt tacatgatcg acggtgtcca agccgaatac 1140 gtcagaatac cacatgctga caattcattg tataagatcc cgcaaactat cgatgatgaa 1200 attgcagtac tactgtccga tattttacct actggacatg aaattggtgt tcaatatggt 1260 aacgttcaac caggcgatgc tgtagcaatt gtaggagcag gtcctgttgg aatgtcagtt 1320 ttgttaactg ctcaatttta ctcgcctagt accattattg ttatcgacat ggacgaaaac 1380 cgtttacaat tagcgaagga gcttggggcc acacacacta ttaactccgg tactgaaaat 1440 gttgtcgaag ctgtgcatcg tatagcagcc gaaggagtgg atgtagcaat agaagctgtt 1500 ggtatacccg caacctggga catctgtcag gaaattgtaa aacccggcgc tcatattgcc 1560 aacgtgggag ttcatggtgt taaggtggac tttgaaattc aaaagttgtg gattaagaat 1620 ctaaccatca ccactggttt ggttaacact aatactaccc caatgttgat gaaggtagcc 1680 tctactgata aattgccttt aaagaaaatg attactcaca ggtttgagtt agctgaaatc 1740 gaacacgcat atcaggtttt cttgaatggc gctaaagaaa aagctatgaa gattattcta 1800 tctaatgcag gtgccgccta attaattaag agtaagcgaa tttcttatga tttatgattt 1860 ttattattaa ataagttata aaaaaaataa gtgtatacaa attttaaagt gactcttagg 1920 ttttaaaacg aaaattctta ttcttgagta actctttcct gtaggtcagg ttgctttctc 1980 aggtatagca tgaggtcgct cttattgacc acacctctac cggcatgccg agcaaatgcc 2040 tgcaaatcgc tccccatttc acccaattgt agatatgcta actccagcaa tgagttgatg 2100 aatctcggtg tgtattttat gtcctcagag gacaacacct gtggt 2145 <210> 80 <211> 4280 <212> DNA <213> artificial sequence <220> <223> vector <400> 80 ggggatcctc tagagtcgac ctgcaggcat gcaagcttgg cgtaatcatg gtcatagctg 60 tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata 120 aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca 180 ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc 240 gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg 300 cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta 360 tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc 420 aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag 480 catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac 540 caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc 600 ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt 660 aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc 720 gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga 780 cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta 840 ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aaggacagta 900 tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga 960 tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg 1020 cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag 1080 tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc 1140 tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact 1200 tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt 1260 cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta 1320 ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta 1380 tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc 1440 gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat 1500 agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt 1560 atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg 1620 tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca 1680 gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta 1740 agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg 1800 cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact 1860 ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg 1920 ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt 1980 actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga 2040 ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc 2100 atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa 2160 caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtcta agaaaccatt 2220 attatcatga cattaaccta taaaaatagg cgtatcacga ggccctttcg tctcgcgcgt 2280 ttcggtgatg acggtgaaaa cctctgacac atgcagctcc cggagacggt cacagcttgt 2340 ctgtaagcgg atgccgggag cagacaagcc cgtcagggcg cgtcagcggg tgttggcggg 2400 tgtcggggct ggcttaacta tgcggcatca gagcagattg tactgagagt gcaccatatg 2460 cggtgtgaaa taccgcacag atgcgtaagg agaaaatacc gcatcaggcg ccattcgcca 2520 ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg cctcttcgct attacgccag 2580 ctggcgaaag ggggatgtgc tgcaaggcga ttaagttggg taacgccagg gttttcccag 2640 tcacgacgtt gtaaaacgac ggccagtgaa ttcgagctcg gtacccccgg ctctgagaca 2700 gtagtaggtt agtcatcgct ctaccgacgc gcaggaaaag aaagaagcat tgcggattac 2760 gtattctaat gttcagcccg cggaacgcca gcaaatcacc acccatgcgc atgatactga 2820 gtcttgtaca cgctgggctt ccagtgtact gagagtgcac cataccacag cttttcaatt 2880 caattcatca tttttttttt attctttttt ttgatttcgg tttctttgaa atttttttga 2940 ttcggtaatc tccgaacaga aggaagaacg aaggaaggag cacagactta gattggtata 3000 tatacgcata tgtagtgttg aagaaacatg aaattgccca gtattcttaa cccaactgca 3060 cagaacaaaa acctgcagga aacgaagata aatcatgtcg aaagctacat ataaggaacg 3120 tgctgctact catcctagtc ctgttgctgc caagctattt aatatcatgc acgaaaagca 3180 aacaaacttg tgtgcttcat tggatgttcg taccaccaag gaattactgg agttagttga 3240 agcattaggt cccaaaattt gtttactaaa aacacatgtg gatatcttga ctgatttttc 3300 catggagggc acagttaagc cgctaaaggc attatccgcc aagtacaatt ttttactctt 3360 cgaagacaga aaatttgctg acattggtaa tacagtcaaa ttgcagtact ctgcgggtgt 3420 atacagaata gcagaatggg cagacattac gaatgcacac ggtgtggtgg gcccaggtat 3480 tgttagcggt ttgaagcagg cggcagaaga agtaacaaag gaacctagag gccttttgat 3540 gttagcagaa ttgtcatgca agggctccct atctactgga gaatatacta agggtactgt 3600 tgacattgcg aagagcgaca aagattttgt tatcggcttt attgctcaaa gagacatggg 3660 tggaagagat gaaggttacg attggttgat tatgacaccc ggtgtgggtt tagatgacaa 3720 gggagacgca ttgggtcaac agtatagaac cgtggatgat gtggtctcta caggatctga 3780 cattattatt gttggaagag gactatttgc aaagggaagg gatgctaagg tagagggtga 3840 acgttacaga aaagcaggct gggaagcata tttgagaaga tgcggccagc aaaactaaaa 3900 aactgtatta taagtaaatg catgtatact aaactcacaa attagagctt caatttaatt 3960 atatcagtta ttaccctatg cggtgtgaaa taccgcacag atgcgtaagg agaaaatacc 4020 gcatcaggaa attgtaaacg ttaatatttt gttaaaattc gcgttaaatt tttgttaaat 4080 cagctcattt tttaaccaat aggccgaaat cggcaaaatc ttcagcccgc ggaacgccag 4140 caaatcacca cccatgcgca tgatactgag tcttgtacac gctgggcttc cagtgatgat 4200 acaacgagtt agccaaggtg agcacggatg tctaaattag aattacgttt taatatcttt 4260 ttttccatat ctagggctag 4280 <210> 81 <211> 30 <212> DNA <213> artificial sequence <220> <223> primer <400> 81 gcatgcttgc atttagtcgt gcaatgtatg 30 <210> 82 <211> 54 <212> DNA <213> artificial sequence <220> <223> primer <400> 82 gaacattaga atacgtaatc cgcaatgcac tagtaccaca ggtgttgtcc tctg 54 <210> 83 <211> 54 <212> DNA <213> artificial sequence <220> <223> primer <400> 83 cagaggacaa cacctgtggt actagtgcat tgcggattac gtattctaat gttc 54 <210> 84 <211> 28 <212> DNA <213> artificial sequence <220> <223> primer <400> 84 caccttggct aactcgttgt atcatcac 28 <210> 85 <211> 100 <212> DNA <213> artificial sequence <220> <223> primer <400> 85 ttttaagccg aatgagtgac agaaaaagcc cacaacttat caagtgatat tgaacaaagg 60 gcgaaacttc gcatgcttgc atttagtcgt gcaatgtatg 100 <210> 86 <211> 98 <212> DNA <213> artificial sequence <220> <223> primer <400> 86 cccaattggt aaatattcaa caagagacgc gcagtacgta acatgcgaat tgcgtaattc 60 acggcgataa caccttggct aactcgttgt atcatcac 98 <210> 87 <211> 29 <212> DNA <213> artificial sequence <220> <223> primer <400> 87 caaaagccca tgtcccacac caaaggatg 29 <210> 88 <211> 26 <212> DNA <213> artificial sequence <220> <223> primer <400> 88 caccatcgcg cgtgcatcac tgcatg 26 <210> 89 <211> 28 <212> DNA <213> artificial sequence <220> <223> primer <400> 89 tcggtttttg caatatgacc tgtgggcc 28 <210> 90 <211> 22 <212> DNA <213> artificial sequence <220> <223> primer <400> 90 gagaagatgc ggccagcaaa ac 22 <210> 91 <211> 2745 <212> DNA <213> artificial sequence <220> <223> constructed coding region-terminator segment <400> 91 atgactgaca aaaaaactct taaagactta agaaatcgta gttctgttta cgattcaatg 60 gttaaatcac ctaatcgtgc tatgttgcgt gcaactggta tgcaagatga agactttgaa 120 aaacctatcg tcggtgtcat ttcaacttgg gctgaaaaca caccttgtaa tatccactta 180 catgactttg gtaaactagc caaagtcggt gttaaggaag ctggtgcttg gccagttcag 240 ttcggaacaa tcacggtttc tgatggaatc gccatgggaa cccaaggaat gcgtttctcc 300 ttgacatctc gtgatattat tgcagattct attgaagcag ccatgggagg tcataatgcg 360 gatgcttttg tagccattgg cggttgtgat aaaaacatgc ccggttctgt tatcgctatg 420 gctaacatgg atatcccagc catttttgct tacggcggaa caattgcacc tggtaattta 480 gacggcaaag atatcgattt agtctctgtc tttgaaggtg tcggccattg gaaccacggc 540 gatatgacca aagaagaagt taaagctttg gaatgtaatg cttgtcccgg tcctggaggc 600 tgcggtggta tgtatactgc taacacaatg gcgacagcta ttgaagtttt gggacttagc 660 cttccgggtt catcttctca cccggctgaa tccgcagaaa agaaagcaga tattgaagaa 720 gctggtcgcg ctgttgtcaa aatgctcgaa atgggcttaa aaccttctga cattttaacg 780 cgtgaagctt ttgaagatgc tattactgta actatggctc tgggaggttc aaccaactca 840 acccttcacc tcttagctat tgcccatgct gctaatgtgg aattgacact tgatgatttc 900 aatactttcc aagaaaaagt tcctcatttg gctgatttga aaccttctgg tcaatatgta 960 ttccaagacc tttacaaggt cggaggggta ccagcagtta tgaaatatct ccttaaaaat 1020 ggcttccttc atggtgaccg tatcacttgt actggcaaaa cagtcgctga aaatttgaag 1080 gcttttgatg atttaacacc tggtcaaaag gttattatgc cgcttgaaaa tcctaaacgt 1140 gaagatggtc cgctcattat tctccatggt aacttggctc cagacggtgc cgttgccaaa 1200 gtttctggtg taaaagtgcg tcgtcatgtc ggtcctgcta aggtctttaa ttctgaagaa 1260 gaagccattg aagctgtctt gaatgatgat attgttgatg gtgatgttgt tgtcgtacgt 1320 tttgtaggac caaagggcgg tcctggtatg cctgaaatgc tttccctttc atcaatgatt 1380 gttggtaaag ggcaaggtga aaaagttgcc cttctgacag atggccgctt ctcaggtggt 1440 acttatggtc ttgtcgtggg tcatatcgct cctgaagcac aagatggcgg tccaatcgcc 1500 tacctgcaaa caggagacat agtcactatt gaccaagaca ctaaggaatt acactttgat 1560 atctccgatg aagagttaaa acatcgtcaa gagaccattg aattgccacc gctctattca 1620 cgcggtatcc ttggtaaata tgctcacatc gtttcgtctg cttctagggg agccgtaaca 1680 gacttttgga agcctgaaga aactggcaaa aaatgttgtc ctggttgctg tggttaagcg 1740 gccgcgttaa ttcaaattaa ttgatatagt tttttaatga gtattgaatc tgtttagaaa 1800 taatggaata ttatttttat ttatttattt atattattgg tcggctcttt tcttctgaag 1860 gtcaatgaca aaatgatatg aaggaaataa tgatttctaa aattttacaa cgtaagatat 1920 ttttacaaaa gcctagctca tcttttgtca tgcactattt tactcacgct tgaaattaac 1980 ggccagtcca ctgcggagtc atttcaaagt catcctaatc gatctatcgt ttttgatagc 2040 tcattttgga gttcgcgatt gtcttctgtt attcacaact gttttaattt ttatttcatt 2100 ctggaactct tcgagttctt tgtaaagtct ttcatagtag cttactttat cctccaacat 2160 atttaacttc atgtcaattt cggctcttaa attttccaca tcatcaagtt caacatcatc 2220 ttttaacttg aatttattct ctagctcttc caaccaagcc tcattgctcc ttgatttact 2280 ggtgaaaagt gatacacttt gcgcgcaatc caggtcaaaa ctttcctgca aagaattcac 2340 caatttctcg acatcatagt acaatttgtt ttgttctccc atcacaattt aatatacctg 2400 atggattctt atgaagcgct gggtaatgga cgtgtcactc tacttcgcct ttttccctac 2460 tccttttagt acggaagaca atgctaataa ataagagggt aataataata ttattaatcg 2520 gcaaaaaaga ttaaacgcca agcgtttaat tatcagaaag caaacgtcgt accaatcctt 2580 gaatgcttcc caattgtata ttaagagtca tcacagcaac atattcttgt tattaaatta 2640 attattattg atttttgata ttgtataaaa aaaccaaata tgtataaaaa aagtgaataa 2700 aaaataccaa gtatggagaa atatattaga agtctatacg ttaaa 2745 <210> 92 <211> 27 <212> DNA <213> artificial sequence <220> <223> primer <400> 92 gacttttgga agcctgaaga aactggc 27 <210> 93 <211> 20 <212> DNA <213> artificial sequence <220> <223> primer <400> 93 cttggcagca acaggactag 20 <210> 94 <211> 26 <212> DNA <213> artificial sequence <220> <223> primer <400> 94 ccaggccaat tcaacagact gtcggc 26 <210> 95 <211> 2347 <212> DNA <213> artificial sequence <220> <223> constructed URA3 marker with flanking homologous repeat sequences        for HIS gene replacement and marker excision <400> 95 gcattgcgga ttacgtattc taatgttcag gtgctggaag aagagctgct taaccgccgc 60 gcccagggtg aagatccacg ctactttacc ctgcgtcgtc tggatttcgg cggctgtcgt 120 ctttcgctgg caacgccggt tgatgaagcc tgggacggtc cgctctcctt aaacggtaaa 180 cgtatcgcca cctcttatcc tcacctgctc aagcgttatc tcgaccagaa aggcatctct 240 tttaaatcct gcttactgaa cggttctgtt gaagtcgccc cgcgtgccgg actggcggat 300 gcgatttgcg atctggtttc caccggtgcc acgctggaag ctaacggcct gcgcgaagtc 360 gaagttatct atcgctcgaa agcctgcctg attcaacgcg atggcgaaat ggaagaatcc 420 aaacagcaac tgatcgacaa actgctgacc cgtattcagg gtgtgatcca ggcgcgcgaa 480 tcaaaataca tcatgatgca cgcaccgacc gaacgtctgg atgaagtcat ggtacctact 540 gagagtgcac cataccacag cttttcaatt caattcatca tttttttttt attctttttt 600 ttgatttcgg tttctttgaa atttttttga ttcggtaatc tccgaacaga aggaagaacg 660 aaggaaggag cacagactta gattggtata tatacgcata tgtagtgttg aagaaacatg 720 aaattgccca gtattcttaa cccaactgca cagaacaaaa acctgcagga aacgaagata 780 aatcatgtcg aaagctacat ataaggaacg tgctgctact catcctagtc ctgttgctgc 840 caagctattt aatatcatgc acgaaaagca aacaaacttg tgtgcttcat tggatgttcg 900 taccaccaag gaattactgg agttagttga agcattaggt cccaaaattt gtttactaaa 960 aacacatgtg gatatcttga ctgatttttc catggagggc acagttaagc cgctaaaggc 1020 attatccgcc aagtacaatt ttttactctt cgaagacaga aaatttgctg acattggtaa 1080 tacagtcaaa ttgcagtact ctgcgggtgt atacagaata gcagaatggg cagacattac 1140 gaatgcacac ggtgtggtgg gcccaggtat tgttagcggt ttgaagcagg cggcagaaga 1200 agtaacaaag gaacctagag gccttttgat gttagcagaa ttgtcatgca agggctccct 1260 atctactgga gaatatacta agggtactgt tgacattgcg aagagcgaca aagattttgt 1320 tatcggcttt attgctcaaa gagacatggg tggaagagat gaaggttacg attggttgat 1380 tatgacaccc ggtgtgggtt tagatgacaa gggagacgca ttgggtcaac agtatagaac 1440 cgtggatgat gtggtctcta caggatctga cattattatt gttggaagag gactatttgc 1500 aaagggaagg gatgctaagg tagagggtga acgttacaga aaagcaggct gggaagcata 1560 tttgagaaga tgcggccagc aaaactaaaa aactgtatta taagtaaatg catgtatact 1620 aaactcacaa attagagctt caatttaatt atatcagtta ttaccctatg cggtgtgaaa 1680 taccgcacag atgcgtaagg agaaaatacc gcatcaggaa attgtaaacg ttaatatttt 1740 gttaaaattc gcgttaaatt tttgttaaat cagctcattt tttaaccaat aggccgaaat 1800 cggcaaaatc tctagagtgc tggaagaaga gctgcttaac cgccgcgccc agggtgaaga 1860 tccacgctac tttaccctgc gtcgtctgga tttcggcggc tgtcgtcttt cgctggcaac 1920 gccggttgat gaagcctggg acggtccgct ctccttaaac ggtaaacgta tcgccacctc 1980 ttatcctcac ctgctcaagc gttatctcga ccagaaaggc atctctttta aatcctgctt 2040 actgaacggt tctgttgaag tcgccccgcg tgccggactg gcggatgcga tttgcgatct 2100 ggtttccacc ggtgccacgc tggaagctaa cggcctgcgc gaagtcgaag ttatctatcg 2160 ctcgaaagcc tgcctgattc aacgcgatgg cgaaatggaa gaatccaaac agcaactgat 2220 cgacaaactg ctgacccgta ttcagggtgt gatccaggcg cgcgaatcaa aatacatcat 2280 gatgcacgca ccgaccgaac gtctggatga agtcatccag tgatgataca acgagttagc 2340 caaggtg 2347 <210> 96 <211> 80 <212> DNA <213> artificial sequence <220> <223> primer <400> 96 cttcgaagaa tatactaaaa aatgagcagg caagataaac gaaggcaaag gcattgcgga 60 ttacgtattc taatgttcag 80 <210> 97 <211> 81 <212> DNA <213> artificial sequence <220> <223> primer <400> 97 tatacacatg tatatatatc gtatgctgca gctttaaata atcggtgtca caccttggct 60 aactcgttgt atcatcactg g 81 <210> 98 <211> 26 <212> DNA <213> artificial sequence <220> <223> primer <400> 98 gacttgaata atgcagcggc gcttgc 26 <210> 99 <211> 30 <212> DNA <213> artificial sequence <220> <223> primer <400> 99 ccaccctctt caattagcta agatcatagc 30 <210> 100 <211> 25 <212> DNA <213> artificial sequence <220> <223> primer <400> 100 aaaaattgat tctcatcgta aatgc 25 <210> 101 <211> 20 <212> DNA <213> artificial sequence <220> <223> primer <400> 101 ctgcagcgag gagccgtaat 20 <210> 102 <211> 90 <212> DNA <213> artificial sequence <220> <223> primer <400> 102 atggttcatt taggtccaaa aaaaccacaa gccagaaagg gttccatggc cgatgtgcca 60 gcattgcgga ttacgtattc taatgttcag 90 <210> 103 <211> 91 <212> DNA <213> artificial sequence <220> <223> primer <400> 103 ttaagcaccg atgataccaa cggacttacc ttcagcaatt cttttttggg ccaaagcagc 60 caccttggct aactcgttgt atcatcactg g 91 <210> 104 <211> 24 <212> DNA <213> artificial sequence <220> <223> primer <400> 104 ctaggatgag tagcagcacg ttcc 24 <210> 105 <211> 26 <212> DNA <213> artificial sequence <220> <223> primer <400> 105 ccaattccgt gatgtctctt tgttgc 26 <210> 106 <211> 20 <212> DNA <213> artificial sequence <220> <223> primer <400> 106 gtgaacgagt tcacaaccgc 20 <210> 107 <211> 22 <212> DNA <213> artificial sequence <220> <223> primer <400> 107 gttcgttcca gaattatcac gc 22 <210> 108 <211> 28 <212> DNA <213> artificial sequence <220> <223> primer <400> 108 ggatccgcat gcttgcattt agtcgtgc 28 <210> 109 <211> 20 <212> DNA <213> artificial sequence <220> <223> primer <400> 109 gggatgcgga cgtattcggc 20 <210> 110 <211> 4506 <212> DNA <213> Saccharomyces cerevisiae <400> 110 atgtcgaata ccccttataa ttcatctgtg ccttccattg catccatgac ccagtcttcg 60 gtctcaagaa gtcctaacat gcatacagca actacgcccg gtgccaacac cagctctaac 120 tctccaccct tgcacatgtc ttcagattcg tccaagatca agaggaagcg taacagaatt 180 ccgctcagtt gcaccatttg tcggaaaagg aaagtcaaat gtgacaaact cagaccacac 240 tgccagcagt gcactaaaac tggggtagcc catctctgcc actacatgga acagacctgg 300 gcagaagagg cagagaaaga attgctgaag gacaacgaat taaagaagct tagggagcgc 360 gtaaaatctt tagaaaagac tctttctaag gtgcactctt ctccttcgtc taactccttg 420 aaaagttaca acactcccga gagcagcaac ctgtttatgg gtagcgatga acacaccacc 480 cttgttaatg caaatacagg ctctgcttcc tctgcctcgc atatgcatca gcaacaacag 540 caacagcagc aacaggaaca acaacaagac ttttccagaa gtgcgaacgc caacgcgaat 600 tcctcgtccc tttctatctc aaataaatat gacaacgatg agctggactt aactaaggac 660 tttgatcttt tgcatatcaa aagtaacgga accatccact taggtgccac ccactggttg 720 tctatcatga aaggtgaccc gtacctaaaa cttttgtggg gtcatatctt cgctatgagg 780 gaaaagttaa atgaatggta ctaccaaaaa aattcgtact ctaagctgaa gtcaagcaaa 840 tgtcccatca atcacgcgca agcgccgcct tctgccgctg ccgccgctac cagaaaatgt 900 cctgttgatc actccgcgtt ttcgtctggc atggtggccc caaaggagga gactcctctt 960 cctaggaaat gtccagttga ccacaccatg ttctcttcgg gaatgattcc tcccagagag 1020 gacacttcgt cccagaagag gtgtcccgtt gaccacacca tgtattccgc aggaatgatg 1080 ccgcccaagg acgagacacc ttccccattt tccactaaag ctatgataga ccataacaag 1140 catacaatga atccgcctca gtcaaaatgt cctgtggacc atagaaacta tatgaaggat 1200 tatccctctg acatggcaaa ttcttcttcg aacccggcaa gtcgttgccc cattgaccat 1260 tcaagcatga aaaatacagc ggccttacca gcttcaacgc acaataccat cccacaccac 1320 caaccacagt ccggatctca tgctcgttcg catcccgcac aaagcaggaa acatgattcc 1380 tacatgacag aatctgaagt cctcgcaaca ctttgtgaga tgttgccacc aaagcgcgtc 1440 atcgcattat tcatcgagaa attcttcaaa catttatacc ctgccattcc aatcttagat 1500 gaacagaatt tcaaaaatca cgtgaatcaa atgctttcgt tgtcttcgat gaatcccaca 1560 gttaacaact ttggtatgag catgccatct tcatctacac tagagaacca acccataaca 1620 caaatcaatc ttccaaaact ttccgattct tgtaacttag gtattctgat aataatcttg 1680 agattgacat ggctatccat accttctaat tcctgcgaag tcgacctggg agaagaaagt 1740 ggctcatttt tagtgcccaa cgaatctagc aatatgtctg catctgcatt gacctcgatg 1800 gctaaagaag aatcacttct gctaaagcat gagacaccgg tcgaggcact ggagctatgt 1860 caaaaatact tgattaaatt cgatgaactt tctagtattt ccaataacaa cgttaattta 1920 accacggtgc agtttgccat tttttacaac ttctatatga aaagtgcctc taatgatttg 1980 actaccttga caaataccaa caacactggc atggccaatc ctggtcacga ttccgagtct 2040 caccagatcc tattgtccaa tattactcaa atggccttta gttgtgggtt acacagagac 2100 cctgataatt ttcctcaatt aaacgctacc attccagcaa ccagccagga cgtgtctaac 2160 aacgggagca aaaaggcaaa ccctagcacc aatccaactt tgaataacaa catgtctgct 2220 gccactacca acagcagtag cagatctggc agtgctgatt caagaagtgg ttctaaccct 2280 gtgaacaaga aggaaaatca ggttagtatc gaaagattta aacacacttg gaggaaaatt 2340 tggtattaca ttgttagcat ggatgttaac caatctcttt ccctggggag ccctcgacta 2400 ctaagaaatc tgagggattt cagcgataca aagctaccaa gtgcgtcaag gattgattat 2460 gttcgcgata tcaaagagtt aatcattgtg aagaatttta ctcttttttt ccaaattgat 2520 ttgtgtatta ttgctgtatt aaatcacatt ttgaatgttt ctttagcaag aagcgtgaga 2580 aaatttgaac tggattcatt gattaattta ttgaaaaatc tgacctatgg tactgagaat 2640 gtcaatgatg tagtgagctc cttgatcaac aaagggttat taccaacttc ggaaggtggt 2700 tctgtagatt caaataatga tgaaatttac ggtctaccga aactacccga tattctaaac 2760 catggtcaac ataaccaaaa cttgtatgct gatggaagaa atacttctag tagtgatata 2820 gataagaaat tggaccttcc tcacgaatct acaacgagag ctctattctt ttccaagcat 2880 atgacaatta gaatgttgct gtacttattg aactacattt tgtttactca ttatgaacca 2940 atgggcagtg aagatcctgg tactaatatc ctagctaagg agtacgctca agaggcatta 3000 aattttgcca tggatggcta cagaaactgc atgattttct tcaacaatat cagaaacacc 3060 aattcactat tcgattacat gaatgttatc ttgtcttacc cttgtttgga cattggacat 3120 cgttctttac aatttatcgt ttgtttgatc ctgagagcta aatgtggccc attgactggt 3180 atgcgtgaat catcgatcat tactaatggt acatcaagtg gatttaatag ttcggtagaa 3240 gatgaggacg tcaaagttaa acaagaatct tctgatgaat tgaaaaaaga cgatttcatg 3300 aaagatgtaa atttggattc aggcgattca ttagcagaga ttctaatgtc aagaatgctg 3360 ctatttcaaa aactaacaaa acaactatca aagaagtaca actacgctat tcgtatgaac 3420 aaatccactg gattctttgt ctctttacta gatacacctt caaagaaatc agactcgaaa 3480 tcgggtggta gttcattcat gttgggtaat tggaaacatc caaaggtttc aaacatgagc 3540 ggatttcttg ctggtgacaa agaccaatta cagaaatgcc ccgtgtacca agatgcgctg 3600 gggtttgtta gtccaaccgg tgctaatgaa ggttctgctc cgatgcaagg catgtcctta 3660 cagggctcta ctgctaggat gggagggacc cagttgccac caattagatc atacaaacct 3720 atcacgtaca caagtagtaa tctacgtcgt atgaatgaaa cgggtgaggc agaagctaag 3780 agaagaagat ttaatgatgg ctatattgat aataatagta acaacgatat acctagagga 3840 atcagcccaa aaccttcaaa tgggctatca tcggtgcagc cactattatc gtcattttcc 3900 atgaaccagc taaacggggg taccattcca acggttccat cgttaaccaa cattacttca 3960 caaatgggag ctttaccatc tttagatagg atcaccacta atcaaataaa tttgccagac 4020 ccatctagag atgaagcatt tgacaactcc atcaagcaaa tgacgcctat gacaagtgca 4080 ttcatgaatg ctaatactac aattccaagt tcaactttaa acgggaatat gaacatgaat 4140 ggagctggaa ctgcgaatac agatacaagt gccaacggca gtgctttatc gacactgaca 4200 agcccacaag gctcagactt agcatccaat tctgctacac agtataaacc tgacttagaa 4260 gactttttga tgcaaaattc taactttaat gggctaatga taaatccttc cagtctggta 4320 gaagttgttg gtggatacaa cgatcctaat aaccttggaa gaaatgacgc ggttgatttt 4380 ctacccgttg ataatgttga aattgatggt gttggaataa aaatcaacta tcatctacta 4440 actagtattt acgttactag tatattatca tatacggtgt tagaagatga cgcaaatgat 4500 gagaaa 4506 <210> 111 <211> 99 <212> DNA <213> artificial sequence <220> <223> primer <400> 111 tcctttctca attattattt tctactcata acctcacgca aaataacaca gtcaaatcaa 60 tcaaagtatg actgacaaaa aaactcttaa agacttaag 99 <210> 112 <211> 77 <212> DNA <213> artificial sequence <220> <223> primer <400> 112 gaacattaga atacgtaatc cgcaatgctt ctttcttttc cgtttaacgt atagacttct 60 aatatatttc tccatac 77 <210> 113 <211> 45 <212> DNA <213> artificial sequence <220> <223> primer <400> 113 aaacggaaaa gaaagaagca ttgcggatta cgtattctaa tgttc 45 <210> 114 <211> 88 <212> DNA <213> artificial sequence <220> <223> primer <400> 114 tatttttcgt tacataaaaa tgcttataaa actttaacta ataattagag attaaatcgc 60 caccttggct aactcgttgt atcatcac 88 <210> 115 <211> 1713 <212> DNA <213> Streptococcus mutans <400> 115 atgactgaca aaaaaactct taaagactta agaaatcgta gttctgttta cgattcaatg 60 gttaaatcac ctaatcgtgc tatgttgcgt gcaactggta tgcaagatga agactttgaa 120 aaacctatcg tcggtgtcat ttcaacttgg gctgaaaaca caccttgtaa tatccactta 180 catgactttg gtaaactagc caaagtcggt gttaaggaag ctggtgcttg gccagttcag 240 ttcggaacaa tcacggtttc tgatggaatc gccatgggaa cccaaggaat gcgtttctcc 300 ttgacatctc gtgatattat tgcagattct attgaagcag ccatgggagg tcataatgcg 360 gatgcttttg tagccattgg cggttgtgat aaaaacatgc ccggttctgt tatcgctatg 420 gctaacatgg atatcccagc catttttgct tacggcggaa caattgcacc tggtaattta 480 gacggcaaag atatcgattt agtctctgtc tttgaaggtg tcggccattg gaaccacggc 540 gatatgacca aagaagaagt taaagctttg gaatgtaatg cttgtcccgg tcctggaggc 600 tgcggtggta tgtatactgc taacacaatg gcgacagcta ttgaagtttt gggacttagc 660 cttccgggtt catcttctca cccggctgaa tccgcagaaa agaaagcaga tattgaagaa 720 gctggtcgcg ctgttgtcaa aatgctcgaa atgggcttaa aaccttctga cattttaacg 780 cgtgaagctt ttgaagatgc tattactgta actatggctc tgggaggttc aaccaactca 840 acccttcacc tcttagctat tgcccatgct gctaatgtgg aattgacact tgatgatttc 900 aatactttcc aagaaaaagt tcctcatttg gctgatttga aaccttctgg tcaatatgta 960 ttccaagacc tttacaaggt cggaggggta ccagcagtta tgaaatatct ccttaaaaat 1020 ggcttccttc atggtgaccg tatcacttgt actggcaaaa cagtcgctga aaatttgaag 1080 gcttttgatg atttaacacc tggtcaaaag gttattatgc cgcttgaaaa tcctaaacgt 1140 gaagatggtc cgctcattat tctccatggt aacttggctc cagacggtgc cgttgccaaa 1200 gtttctggtg taaaagtgcg tcgtcatgtc ggtcctgcta aggtctttaa ttctgaagaa 1260 gaagccattg aagctgtctt gaatgatgat attgttgatg gtgatgttgt tgtcgtacgt 1320 tttgtaggac caaagggcgg tcctggtatg cctgaaatgc tttccctttc atcaatgatt 1380 gttggtaaag ggcaaggtga aaaagttgcc cttctgacag atggccgctt ctcaggtggt 1440 acttatggtc ttgtcgtggg tcatatcgct cctgaagcac aagatggcgg tccaatcgcc 1500 tacctgcaaa caggagacat agtcactatt gaccaagaca ctaaggaatt acactttgat 1560 atctccgatg aagagttaaa acatcgtcaa gagaccattg aattgccacc gctctattca 1620 cgcggtatcc ttggtaaata tgctcacatc gtttcgtctg cttctagggg agccgtaaca 1680 gacttttgga agcctgaaga aactggcaaa aaa 1713 <210> 116 <211> 571 <212> PRT <213> Streptococcus mutans <400> 116 Met Thr Asp Lys Lys Thr Leu Lys Asp Leu Arg Asn Arg Ser Ser Val 1 5 10 15 Tyr Asp Ser Met Val Lys Ser Pro Asn Arg Ala Met Leu Arg Ala Thr             20 25 30 Gly Met Gln Asp Glu Asp Phe Glu Lys Pro Ile Val Gly Val Ile Ser         35 40 45 Thr Trp Ala Glu Asn Thr Pro Cys Asn Ile His Leu His Asp Phe Gly     50 55 60 Lys Leu Ala Lys Val Gly Val Lys Glu Ala Gly Ala Trp Pro Val Gln 65 70 75 80 Phe Gly Thr Ile Thr Val Ser Asp Gly Ile Ala Met Gly Thr Gln Gly                 85 90 95 Met Arg Phe Ser Leu Thr Ser Arg Asp Ile Ile Ala Asp Ser Ile Glu             100 105 110 Ala Ala Met Gly Gly His Asn Ala Asp Ala Phe Val Ala Ile Gly Gly         115 120 125 Cys Asp Lys Asn Met Pro Gly Ser Val Ile Ala Met Ala Asn Met Asp     130 135 140 Ile Pro Ala Ile Phe Ala Tyr Gly Gly Thr Ile Ala Pro Gly Asn Leu 145 150 155 160 Asp Gly Lys Asp Ile Asp Leu Val Ser Val Phe Glu Gly Val Gly His                 165 170 175 Trp Asn His Gly Asp Met Thr Lys Glu Glu Val Lys Ala Leu Glu Cys             180 185 190 Asn Ala Cys Pro Gly Pro Gly Gly Cys Gly Gly Met Tyr Thr Ala Asn         195 200 205 Thr Met Ala Thr Ala Ile Glu Val Leu Gly Leu Ser Leu Pro Gly Ser     210 215 220 Ser Ser His Pro Ala Glu Ser Ala Glu Lys Lys Ala Asp Ile Glu Glu 225 230 235 240 Ala Gly Arg Ala Val Val Lys Met Leu Glu Met Gly Leu Lys Pro Ser                 245 250 255 Asp Ile Leu Thr Arg Glu Ala Phe Glu Asp Ala Ile Thr Val Thr Met             260 265 270 Ala Leu Gly Gly Ser Thr Asn Ser Thr Leu His Leu Leu Ala Ile Ala         275 280 285 His Ala Ala Asn Val Glu Leu Thr Leu Asp Asp Phe Asn Thr Phe Gln     290 295 300 Glu Lys Val Pro His Leu Ala Asp Leu Lys Pro Ser Gly Gln Tyr Val 305 310 315 320 Phe Gln Asp Leu Tyr Lys Val Gly Gly Val Pro Ala Val Met Lys Tyr                 325 330 335 Leu Leu Lys Asn Gly Phe Leu His Gly Asp Arg Ile Thr Cys Thr Gly             340 345 350 Lys Thr Val Ala Glu Asn Leu Lys Ala Phe Asp Asp Leu Thr Pro Gly         355 360 365 Gln Lys Val Ile Met Pro Leu Glu Asn Pro Lys Arg Glu Asp Gly Pro     370 375 380 Leu Ile Ile Leu His Gly Asn Leu Ala Pro Asp Gly Ala Val Ala Lys 385 390 395 400 Val Ser Gly Val Lys Val Arg Arg His Val Gly Pro Ala Lys Val Phe                 405 410 415 Asn Ser Glu Glu Glu Ala Ile Glu Ala Val Leu Asn Asp Asp Ile Val             420 425 430 Asp Gly Asp Val Val Val Val Arg Phe Val Gly Pro Lys Gly Gly Pro         435 440 445 Gly Met Pro Glu Met Leu Ser Leu Ser Ser Met Ile Val Gly Lys Gly     450 455 460 Gln Gly Glu Lys Val Ala Leu Leu Thr Asp Gly Arg Phe Ser Gly Gly 465 470 475 480 Thr Tyr Gly Leu Val Val Gly His Ile Ala Pro Glu Ala Gln Asp Gly                 485 490 495 Gly Pro Ile Ala Tyr Leu Gln Thr Gly Asp Ile Val Thr Ile Asp Gln             500 505 510 Asp Thr Lys Glu Leu His Phe Asp Ile Ser Asp Glu Glu Leu Lys His         515 520 525 Arg Gln Glu Thr Ile Glu Leu Pro Pro Leu Tyr Ser Arg Gly Ile Leu     530 535 540 Gly Lys Tyr Ala His Ile Val Ser Ser Ala Ser Arg Gly Ala Val Thr 545 550 555 560 Asp Phe Trp Lys Pro Glu Glu Thr Gly Lys Lys                 565 570 <210> 117 <211> 548 <212> PRT <213> Lactococcus lactis <400> 117 Met Tyr Thr Val Gly Asp Tyr Leu Leu Asp Arg Leu His Glu Leu Gly 1 5 10 15 Ile Glu Glu Ile Phe Gly Val Pro Gly Asp Tyr Asn Leu Gln Phe Leu             20 25 30 Asp Gln Ile Ile Ser His Lys Asp Met Lys Trp Val Gly Asn Ala Asn         35 40 45 Glu Leu Asn Ala Ser Tyr Met Ala Asp Gly Tyr Ala Arg Thr Lys Lys     50 55 60 Ala Ala Ala Phe Leu Thr Thr Phe Gly Val Gly Glu Leu Ser Ala Val 65 70 75 80 Asn Gly Leu Ala Gly Ser Tyr Ala Glu Asn Leu Pro Val Val Glu Ile                 85 90 95 Val Gly Ser Pro Thr Ser Lys Val Gln Asn Glu Gly Lys Phe Val His             100 105 110 His Thr Leu Ala Asp Gly Asp Phe Lys His Phe Met Lys Met His Glu         115 120 125 Pro Val Thr Ala Ala Arg Thr Leu Leu Thr Ala Glu Asn Ala Thr Val     130 135 140 Glu Ile Asp Arg Val Leu Ser Ala Leu Leu Lys Glu Arg Lys Pro Val 145 150 155 160 Tyr Ile Asn Leu Pro Val Asp Val Ala Ala Ala Lys Ala Glu Lys Pro                 165 170 175 Ser Leu Pro Leu Lys Lys Glu Asn Ser Thr Ser Asn Thr Ser Asp Gln             180 185 190 Glu Ile Leu Asn Lys Ile Gln Glu Ser Leu Lys Asn Ala Lys Lys Pro         195 200 205 Ile Val Ile Thr Gly His Glu Ile Ile Ser Phe Gly Leu Glu Lys Thr     210 215 220 Val Thr Gln Phe Ile Ser Lys Thr Lys Leu Pro Ile Thr Thr Leu Asn 225 230 235 240 Phe Gly Lys Ser Ser Val Asp Glu Ala Leu Pro Ser Phe Leu Gly Ile                 245 250 255 Tyr Asn Gly Thr Leu Ser Glu Pro Asn Leu Lys Glu Phe Val Glu Ser             260 265 270 Ala Asp Phe Ile Leu Met Leu Gly Val Lys Leu Thr Asp Ser Ser Thr         275 280 285 Gly Ala Phe Thr His His Leu Asn Glu Asn Lys Met Ile Ser Leu Asn     290 295 300 Ile Asp Glu Gly Lys Ile Phe Asn Glu Arg Ile Gln Asn Phe Asp Phe 305 310 315 320 Glu Ser Leu Ile Ser Ser Leu Leu Asp Leu Ser Glu Ile Glu Tyr Lys                 325 330 335 Gly Lys Tyr Ile Asp Lys Lys Gln Glu Asp Phe Val Pro Ser Asn Ala             340 345 350 Leu Leu Ser Gln Asp Arg Leu Trp Gln Ala Val Glu Asn Leu Thr Gln         355 360 365 Ser Asn Glu Thr Ile Val Ala Glu Gln Gly Thr Ser Phe Phe Gly Ala     370 375 380 Ser Ser Ile Phe Leu Lys Ser Lys Ser His Phe Ile Gly Gln Pro Leu 385 390 395 400 Trp Gly Ser Ile Gly Tyr Thr Phe Pro Ala Ala Leu Gly Ser Gln Ile                 405 410 415 Ala Asp Lys Glu Ser Arg His Leu Leu Phe Ile Gly Asp Gly Ser Leu             420 425 430 Gln Leu Thr Val Gln Glu Leu Gly Leu Ala Ile Arg Glu Lys Ile Asn         435 440 445 Pro Ile Cys Phe Ile Ile Asn Asn Asp Gly Tyr Thr Val Glu Arg Glu     450 455 460 Ile His Gly Pro Asn Gln Ser Tyr Asn Asp Ile Pro Met Trp Asn Tyr 465 470 475 480 Ser Lys Leu Pro Glu Ser Phe Gly Ala Thr Glu Asp Arg Val Val Ser                 485 490 495 Lys Ile Val Arg Thr Glu Asn Glu Phe Val Ser Val Met Lys Glu Ala             500 505 510 Gln Ala Asp Pro Asn Arg Met Tyr Trp Ile Glu Leu Ile Leu Ala Lys         515 520 525 Glu Gly Ala Pro Lys Val Leu Lys Lys Met Gly Lys Leu Phe Ala Glu     530 535 540 Gln asn lys ser 545 <210> 118 <211> 1125 <212> DNA <213> artificial sequence <220> <223> horse ADH coding region codon optimized for S. cerevisiae        expression <400> 118 atgtcaacag ccggtaaagt tattaagtgt aaagcggcag ttttgtggga agagaaaaag 60 ccgtttagca tagaagaagt agaagtagcg ccaccaaaag cacacgaggt tagaatcaag 120 atggttgcca ccggaatctg tagatccgac gaccatgtgg tgagtggcac tctagttact 180 cctttgccag taatcgcggg acacgaggct gccggaatcg ttgaatccat aggtgaaggt 240 gttaccactg ttcgtcctgg tgataaagtg atcccactgt tcactcctca atgtggtaag 300 tgtagagtct gcaaacatcc tgagggtaat ttctgcctta aaaatgattt gtctatgcct 360 agaggtacta tgcaggatgg tacaagcaga tttacatgca gagggaaacc tatacaccat 420 ttccttggta cttctacatt ttcccaatac acagtggtgg acgagatatc tgtcgctaaa 480 atcgatgcag cttcaccact ggaaaaagtt tgcttgatag ggtgcggatt ttccaccggt 540 tacggttccg cagttaaagt tgcaaaggtt acacagggtt cgacttgtgc agtattcggt 600 ttaggaggag taggactaag cgttattatg gggtgtaaag ctgcaggcgc agcgaggatt 660 ataggtgtag acatcaataa ggacaaattt gcaaaagcta aggaggtcgg ggctactgaa 720 tgtgttaacc ctcaagatta taagaaacca atacaagaag tccttactga aatgtcaaac 780 ggtggagttg atttctcttt tgaagttata ggccgtcttg atactatggt aactgcgttg 840 tcctgctgtc aagaggcata tggagtcagt gtgatcgtag gtgttcctcc tgattcacaa 900 aatttgtcga tgaatcctat gctgttgcta agcggtcgta catggaaggg agctatattt 960 ggcggtttta agagcaagga tagtgttcca aaacttgttg ccgactttat ggcgaagaag 1020 tttgctcttg atcctttaat tacacatgta ttgccattcg agaaaatcaa tgaagggttt 1080 gatttgttaa gaagtggtga atctattcgt acaattttaa ctttt 1125 <210> 119 <211> 375 <212> PRT <213> Equus caballus <400> 119 Met Ser Thr Ala Gly Lys Val Ile Lys Cys Lys Ala Ala Val Leu Trp 1 5 10 15 Glu Glu Lys Lys Pro Phe Ser Ile Glu Glu Val Glu Val Ala Pro Pro             20 25 30 Lys Ala His Glu Val Arg Ile Lys Met Val Ala Thr Gly Ile Cys Arg         35 40 45 Ser Asp Asp His Val Val Ser Gly Thr Leu Val Thr Pro Leu Pro Val     50 55 60 Ile Ala Gly His Glu Ala Ala Gly Ile Val Glu Ser Ile Gly Glu Gly 65 70 75 80 Val Thr Thr Val Arg Pro Gly Asp Lys Val Ile Pro Leu Phe Thr Pro                 85 90 95 Gln Cys Gly Lys Cys Arg Val Cys Lys His Pro Glu Gly Asn Phe Cys             100 105 110 Leu Lys Asn Asp Leu Ser Met Pro Arg Gly Thr Met Gln Asp Gly Thr         115 120 125 Ser Arg Phe Thr Cys Arg Gly Lys Pro Ile His His Phe Leu Gly Thr     130 135 140 Ser Thr Phe Ser Gln Tyr Thr Val Val Asp Glu Ile Ser Val Ala Lys 145 150 155 160 Ile Asp Ala Ala Ser Pro Leu Glu Lys Val Cys Leu Ile Gly Cys Gly                 165 170 175 Phe Ser Thr Gly Tyr Gly Ser Ala Val Lys Val Ala Lys Val Thr Gln             180 185 190 Gly Ser Thr Cys Ala Val Phe Gly Leu Gly Gly Val Gly Leu Ser Val         195 200 205 Ile Met Gly Cys Lys Ala Ala Gly Ala Ala Arg Ile Ile Gly Val Asp     210 215 220 Ile Asn Lys Asp Lys Phe Ala Lys Ala Lys Glu Val Gly Ala Thr Glu 225 230 235 240 Cys Val Asn Pro Gln Asp Tyr Lys Lys Pro Ile Gln Glu Val Leu Thr                 245 250 255 Glu Met Ser Asn Gly Gly Val Asp Phe Ser Phe Glu Val Ile Gly Arg             260 265 270 Leu Asp Thr Met Val Thr Ala Leu Ser Cys Cys Gln Glu Ala Tyr Gly         275 280 285 Val Ser Val Ile Val Gly Val Pro Pro Asp Ser Gln Asn Leu Ser Met     290 295 300 Asn Pro Met Leu Leu Leu Ser Gly Arg Thr Trp Lys Gly Ala Ile Phe 305 310 315 320 Gly Gly Phe Lys Ser Lys Asp Ser Val Pro Lys Leu Val Ala Asp Phe                 325 330 335 Met Ala Lys Lys Phe Ala Leu Asp Pro Leu Ile Thr His Val Leu Pro             340 345 350 Phe Glu Lys Ile Asn Glu Gly Phe Asp Leu Leu Arg Ser Gly Glu Ser         355 360 365 Ile Arg Thr Ile Leu Thr Phe     370 375 <210> 120 <211> 643 <212> DNA <213> Saccharomyces cerevisiae <400> 120 gaaatgaata acaatactga cagtactaaa taattgccta cttggcttca catacgttgc 60 atacgtcgat atagataata atgataatga cagcaggatt atcgtaatac gtaatagttg 120 aaaatctcaa aaatgtgtgg gtcattacgt aaataatgat aggaatggga ttcttctatt 180 tttccttttt ccattctagc agccgtcggg aaaacgtggc atcctctctt tcgggctcaa 240 ttggagtcac gctgccgtga gcatcctctc tttccatatc taacaactga gcacgtaacc 300 aatggaaaag catgagctta gcgttgctcc aaaaaagtat tggatggtta ataccatttg 360 tctgttctct tctgactttg actcctcaaa aaaaaaaaat ctacaatcaa cagatcgctt 420 caattacgcc ctcacaaaaa cttttttcct tcttcttcgc ccacgttaaa ttttatccct 480 catgttgtct aacggatttc tgcacttgat ttattataaa aagacaaaga cataatactt 540 ctctatcaat ttcagttatt gttcttcctt gcgttattct tctgttcttc tttttctttt 600 gtcatatata accataacca agtaatacat attcaaatct aga 643 <210> 121 <211> 9089 <212> DNA <213> artificial sequence <220> <223> constructed plasmid <400> 121 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180 accataccac agcttttcaa ttcaattcat catttttttt ttattctttt ttttgatttc 240 ggtttctttg aaattttttt gattcggtaa tctccgaaca gaaggaagaa cgaaggaagg 300 agcacagact tagattggta tatatacgca tatgtagtgt tgaagaaaca tgaaattgcc 360 cagtattctt aacccaactg cacagaacaa aaacctgcag gaaacgaaga taaatcatgt 420 cgaaagctac atataaggaa cgtgctgcta ctcatcctag tcctgttgct gccaagctat 480 ttaatatcat gcacgaaaag caaacaaact tgtgtgcttc attggatgtt cgtaccacca 540 aggaattact ggagttagtt gaagcattag gtcccaaaat ttgtttacta aaaacacatg 600 tggatatctt gactgatttt tccatggagg gcacagttaa gccgctaaag gcattatccg 660 ccaagtacaa ttttttactc ttcgaagaca gaaaatttgc tgacattggt aatacagtca 720 aattgcagta ctctgcgggt gtatacagaa tagcagaatg ggcagacatt acgaatgcac 780 acggtgtggt gggcccaggt attgttagcg gtttgaagca ggcggcagaa gaagtaacaa 840 aggaacctag aggccttttg atgttagcag aattgtcatg caagggctcc ctatctactg 900 gagaatatac taagggtact gttgacattg cgaagagcga caaagatttt gttatcggct 960 ttattgctca aagagacatg ggtggaagag atgaaggtta cgattggttg attatgacac 1020 ccggtgtggg tttagatgac aagggagacg cattgggtca acagtataga accgtggatg 1080 atgtggtctc tacaggatct gacattatta ttgttggaag aggactattt gcaaagggaa 1140 gggatgctaa ggtagagggt gaacgttaca gaaaagcagg ctgggaagca tatttgagaa 1200 gatgcggcca gcaaaactaa aaaactgtat tataagtaaa tgcatgtata ctaaactcac 1260 aaattagagc ttcaatttaa ttatatcagt tattacccta tgcggtgtga aataccgcac 1320 agatgcgtaa ggagaaaata ccgcatcagg aaattgtaaa cgttaatatt ttgttaaaat 1380 tcgcgttaaa tttttgttaa atcagctcat tttttaacca ataggccgaa atcggcaaaa 1440 tcccttataa atcaaaagaa tagaccgaga tagggttgag tgttgttcca gtttggaaca 1500 agagtccact attaaagaac gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg 1560 gcgatggccc actacgtgaa ccatcaccct aatcaagttt tttggggtcg aggtgccgta 1620 aagcactaaa tcggaaccct aaagggagcc cccgatttag agcttgacgg ggaaagccgg 1680 cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa 1740 gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg ccgctacagg 1800 gcgcgtcgcg ccattcgcca ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg 1860 cctcttcgct attacgccag ctggcgaaag ggggatgtgc tgcaaggcga ttaagttggg 1920 taacgccagg gttttcccag tcacgacgtt gtaaaacgac ggccagtgag cgcgcgtaat 1980 acgactcact atagggcgaa ttgggtaccg ggccccccct cgaggtcgac tggccattaa 2040 tctttcccat attagatttc gccaagccat gaaagttcaa gaaaggtctt tagacgaatt 2100 acccttcatt tctcaaactg gcgtcaaggg atcctggtat ggttttatcg ttttatttct 2160 ggttcttata gcatcgtttt ggacttctct gttcccatta ggcggttcag gagccagcgc 2220 agaatcattc tttgaaggat acttatcctt tccaattttg attgtctgtt acgttggaca 2280 taaactgtat actagaaatt ggactttgat ggtgaaacta gaagatatgg atcttgatac 2340 cggcagaaaa caagtagatt tgactcttcg tagggaagaa atgaggattg agcgagaaac 2400 attagcaaaa agatccttcg taacaagatt tttacatttc tggtgttgaa gggaaagata 2460 tgagctatac agcggaattt ccatatcact cagattttgt tatctaattt tttccttccc 2520 acgtccgcgg gaatctgtgt atattactgc atctagatat atgttatctt atcttggcgc 2580 gtacatttaa ttttcaacgt attctataag aaattgcggg agtttttttc atgtagatga 2640 tactgactgc acgcaaatat aggcatgatt tataggcatg atttgatggc tgtaccgata 2700 ggaacgctaa gagtaacttc agaatcgtta tcctggcgga aaaaattcat ttgtaaactt 2760 taaaaaaaaa agccaatatc cccaaaatta ttaagagcgc ctccattatt aactaaaatt 2820 tcactcagca tccacaatgt atcaggtatc tactacagat attacatgtg gcgaaaaaga 2880 caagaacaat gcaatagcgc atcaagaaaa aacacaaagc tttcaatcaa tgaatcgaaa 2940 atgtcattaa aatagtatat aaattgaaac taagtcataa agctataaaa agaaaattta 3000 tttaaatgca agatttaaag taaattcacg gccctgcagg ccctaacctg ctaggacaca 3060 acgtctttgc ctggtaaagt ttctagctga cgtgattcct tcacctgtgg atccggcaat 3120 tgtaaaggtt gtgaaaccct cagcttcata accgacacct gcaaatgact ttgcattctt 3180 aacaaagata gttgtatcaa tttcacgttc gaatctatta aggttatcga tgttcttaga 3240 ataaatgtag gcggaatgtt ttctattctg ctcagctatc ttggcgtatt taatggcttc 3300 atcaatgtcc ttcactctaa ctataggcaa aattggcatc atcaactccg tcataacgaa 3360 cggatggttt gcgttgactt cacaaataat acactttaca ttacttggtg actctacatc 3420 tatttcatcc aaaaacagtt tagcgtcctt accaacccac ttcttattaa tgaaatattc 3480 ttgagtttca ttgttctttt gaagaacaag gtctatcagc ttggatactt ggtcttcatt 3540 gataatgacg gcgttgtttt tcaacatgtt agagatcaga tcatctgcaa cgttttcaaa 3600 cacgaacact tctttttccg cgatacaagg aagattgttg tcaaacgaac aaccttcaat 3660 aatgcttctg ccggccttct cgatatctgc tgtatcgtct acaataaccg gaggattacc 3720 cgcgccagct ccgatggcct ttttaccaga attaagaagg gtttttacca tacccgggcc 3780 acccgtaccg cacaacaatt ttatggatgg atgtttgata atagcgtcta aactttccat 3840 agttgggttc tttatagtag tgacaaggtt ttcaggtcca ccacagctaa ttatggcttt 3900 gtttatcatt tctactgcga aagcgacaca ctttttggcg catgggtgac cattaaatac 3960 aactgcattc cccgcagcta tcatacctat agaattgcag ataacggttt ctgttggatt 4020 cgtgcttgga gttatagcgc cgataactcc gtatggactc atttcaacca ctgttagtcc 4080 attatcgccg gaccatgctg ttgttgtcag atcttcagtg cctggggtat acttggccac 4140 taattcatgt ttcaagattt tatcctcata ccttcccatg tgggtttcct ccaggatcat 4200 tgtggctaag acctctttat tctgtaatgc ggcttttctt atttcggtga ttattttctc 4260 tctttgttcc tttgtgtagt gtagggaaag aatcttttgt gcatgtactg cagaagaaat 4320 ggcattctca acattttcaa atactccaaa acatgaagag ttatctttgt aattctttaa 4380 gttgatgttt tcaccattag tcttcacttt caagtctttg gtggttggga ttaaggtatc 4440 tttatccatg gtgtttgttt atgtgtgttt attcgaaact aagttcttgg tgttttaaaa 4500 ctaaaaaaaa gactaactat aaaagtagaa tttaagaagt ttaagaaata gatttacaga 4560 attacaatca atacctaccg tctttatata cttattagtc aagtagggga ataatttcag 4620 ggaactggtt tcaacctttt ttttcagctt tttccaaatc agagagagca gaaggtaata 4680 gaaggtgtaa gaaaatgaga tagatacatg cgtgggtcaa ttgccttgtg tcatcattta 4740 ctccaggcag gttgcatcac tccattgagg ttgtgcccgt tttttgcctg tttgtgcccc 4800 tgttctctgt agttgcgcta agagaatgga cctatgaact gatggttggt gaagaaaaca 4860 atattttggt gctgggattc tttttttttc tggatgccag cttaaaaagc gggctccatt 4920 atatttagtg gatgccagga ataaactgtt cacccagaca cctacgatgt tatatattct 4980 gtgtaacccg ccccctattt tgggcatgta cgggttacag cagaattaaa aggctaattt 5040 tttgactaaa taaagttagg aaaatcacta ctattaatta tttacgtatt ctttgaaatg 5100 gcagtattga taatgataaa ctcgaactga aaaagcgtgt tttttattca aaatgattct 5160 aactccctta cgtaatcaag gaatcttttt gccttggcct ccgcgtcatt aaacttcttg 5220 ttgttgacgc taacattcaa cgctagtata tattcgtttt tttcaggtaa gttcttttca 5280 acgggtctta ctgatgaggc agtcgcgtct gaacctgtta agaggtcaaa tatgtcttct 5340 tgaccgtacg tgtcttgcat gttattagct ttgggaattt gcatcaagtc ataggaaaat 5400 ttaaatcttg gctctcttgg gctcaaggtg acaaggtcct cgaaaatagg gcgcgcccca 5460 ccgcggtgga gctccagctt ttgttccctt tagtgagggt taattgcgcg cttggcgtaa 5520 tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata 5580 ggagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgaggta actcacatta 5640 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa 5700 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg 5760 ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag 5820 gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa 5880 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc 5940 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca 6000 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg 6060 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct 6120 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt 6180 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag 6240 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc 6300 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac 6360 actagaagga cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga 6420 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc 6480 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg 6540 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca 6600 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt 6660 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca 6720 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg 6780 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca 6840 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt 6900 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt 6960 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca 7020 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca 7080 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga 7140 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact 7200 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga 7260 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg 7320 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc 7380 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga 7440 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat 7500 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt 7560 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt 7620 atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgaa 7680 cgaagcatct gtgcttcatt ttgtagaaca aaaatgcaac gcgagagcgc taatttttca 7740 aacaaagaat ctgagctgca tttttacaga acagaaatgc aacgcgaaag cgctatttta 7800 ccaacgaaga atctgtgctt catttttgta aaacaaaaat gcaacgcgag agcgctaatt 7860 tttcaaacaa agaatctgag ctgcattttt acagaacaga aatgcaacgc gagagcgcta 7920 ttttaccaac aaagaatcta tacttctttt ttgttctaca aaaatgcatc ccgagagcgc 7980 tatttttcta acaaagcatc ttagattact ttttttctcc tttgtgcgct ctataatgca 8040 gtctcttgat aactttttgc actgtaggtc cgttaaggtt agaagaaggc tactttggtg 8100 tctattttct cttccataaa aaaagcctga ctccacttcc cgcgtttact gattactagc 8160 gaagctgcgg gtgcattttt tcaagataaa ggcatccccg attatattct ataccgatgt 8220 ggattgcgca tactttgtga acagaaagtg atagcgttga tgattcttca ttggtcagaa 8280 aattatgaac ggtttcttct attttgtctc tatatactac gtataggaaa tgtttacatt 8340 ttcgtattgt tttcgattca ctctatgaat agttcttact acaatttttt tgtctaaaga 8400 gtaatactag agataaacat aaaaaatgta gaggtcgagt ttagatgcaa gttcaaggag 8460 cgaaaggtgg atgggtaggt tatataggga tatagcacag agatatatag caaagagata 8520 cttttgagca atgtttgtgg aagcggtatt cgcaatattt tagtagctcg ttacagtccg 8580 gtgcgttttt ggttttttga aagtgcgtct tcagagcgct tttggttttc aaaagcgctc 8640 tgaagttcct atactttcta gagaatagga acttcggaat aggaacttca aagcgtttcc 8700 gaaaacgagc gcttccgaaa atgcaacgcg agctgcgcac atacagctca ctgttcacgt 8760 cgcacctata tctgcgtgtt gcctgtatat atatatacat gagaagaacg gcatagtgcg 8820 tgtttatgct taaatgcgta cttatatgcg tctatttatg taggatgaaa ggtagtctag 8880 tacctcctgt gatattatcc cattccatgc ggggtatcgt atgcttcctt cagcactacc 8940 ctttagctgt tctatatgct gccactcctc aattggatta gtctcatcct tcaatgctat 9000 catttccttt gatattggat catactaaga aaccattatt atcatgacat taacctataa 9060 aaataggcgt atcacgaggc cctttcgtc 9089 <210> 122 <211> 1023 <212> DNA <213> Saccharomyces cerevisiae <400> 122 caccgcggtg gggcgcgccc tattttcgag gaccttgtca ccttgagccc aagagagcca 60 agatttaaat tttcctatga cttgatgcaa attcccaaag ctaataacat gcaagacacg 120 tacggtcaag aagacatatt tgacctctta acaggttcag acgcgactgc ctcatcagta 180 agacccgttg aaaagaactt acctgaaaaa aacgaatata tactagcgtt gaatgttagc 240 gtcaacaaca agaagtttaa tgacgcggag gccaaggcaa aaagattcct tgattacgta 300 agggagttag aatcattttg aataaaaaac acgctttttc agttcgagtt tatcattatc 360 aatactgcca tttcaaagaa tacgtaaata attaatagta gtgattttcc taactttatt 420 tagtcaaaaa attagccttt taattctgct gtaacccgta catgcccaaa atagggggcg 480 ggttacacag aatatataac atcgtaggtg tctgggtgaa cagtttattc ctggcatcca 540 ctaaatataa tggagcccgc tttttaagct ggcatccaga aaaaaaaaga atcccagcac 600 caaaatattg ttttcttcac caaccatcag ttcataggtc cattctctta gcgcaactac 660 agagaacagg ggcacaaaca ggcaaaaaac gggcacaacc tcaatggagt gatgcaacct 720 gcctggagta aatgatgaca caaggcaatt gacccacgca tgtatctatc tcattttctt 780 acaccttcta ttaccttctg ctctctctga tttggaaaaa gctgaaaaaa aaggttgaaa 840 ccagttccct gaaattattc ccctacttga ctaataagta tataaagacg gtaggtattg 900 attgtaattc tgtaaatcta tttcttaaac ttcttaaatt ctacttttat agttagtctt 960 ttttttagtt ttaaaacacc aagaacttag tttcgaataa acacacataa actagtaaac 1020 aaa 1023 <210> 123 <211> 21 <212> DNA <213> artificial sequence <220> <223> primer <400> 123 caaaagctga gctccaccgc g 21 <210> 124 <211> 44 <212> DNA <213> artificial sequence <220> <223> primer <400> 124 gtttactagt ttatgtgtgt ttattcgaaa ctaagttctt ggtg 44 <210> 125 <400> 125 000 <210> 126 <211> 39 <212> DNA <213> artificial sequence <220> <223> primer <400> 126 cacacatatt acaatagcta gctgaggatg aaagctctg 39 <210> 127 <211> 39 <212> DNA <213> artificial sequence <220> <223> primer <400> 127 cagagctttc atcctcagct agctattgta atatgtgtg 39 <210> 128 <211> 9491 <212> DNA <213> artificial sequence <220> <223> constructed plasmid <400> 128 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180 accataaatt cccgttttaa gagcttggtg agcgctagga gtcactgcca ggtatcgttt 240 gaacacggca ttagtcaggg aagtcataac acagtccttt cccgcaattt tctttttcta 300 ttactcttgg cctcctctag tacactctat atttttttat gcctcggtaa tgattttcat 360 tttttttttt cccctagcgg atgactcttt ttttttctta gcgattggca ttatcacata 420 atgaattata cattatataa agtaatgtga tttcttcgaa gaatatacta aaaaatgagc 480 aggcaagata aacgaaggca aagatgacag agcagaaagc cctagtaaag cgtattacaa 540 atgaaaccaa gattcagatt gcgatctctt taaagggtgg tcccctagcg atagagcact 600 cgatcttccc agaaaaagag gcagaagcag tagcagaaca ggccacacaa tcgcaagtga 660 ttaacgtcca cacaggtata gggtttctgg accatatgat acatgctctg gccaagcatt 720 ccggctggtc gctaatcgtt gagtgcattg gtgacttaca catagacgac catcacacca 780 ctgaagactg cgggattgct ctcggtcaag cttttaaaga ggccctactg gcgcgtggag 840 taaaaaggtt tggatcagga tttgcgcctt tggatgaggc actttccaga gcggtggtag 900 atctttcgaa caggccgtac gcagttgtcg aacttggttt gcaaagggag aaagtaggag 960 atctctcttg cgagatgatc ccgcattttc ttgaaagctt tgcagaggct agcagaatta 1020 ccctccacgt tgattgtctg cgaggcaaga atgatcatca ccgtagtgag agtgcgttca 1080 aggctcttgc ggttgccata agagaagcca cctcgcccaa tggtaccaac gatgttccct 1140 ccaccaaagg tgttcttatg tagtgacacc gattatttaa agctgcagca tacgatatat 1200 atacatgtgt atatatgtat acctatgaat gtcagtaagt atgtatacga acagtatgat 1260 actgaagatg acaaggtaat gcatcattct atacgtgtca ttctgaacga ggcgcgcttt 1320 ccttttttct ttttgctttt tctttttttt tctcttgaac tcgacggatc tatgcggtgt 1380 gaaataccgc acagatgcgt aaggagaaaa taccgcatca ggaaattgta aacgttaata 1440 ttttgttaaa attcgcgtta aatttttgtt aaatcagctc attttttaac caataggccg 1500 aaatcggcaa aatcccttat aaatcaaaag aatagaccga gatagggttg agtgttgttc 1560 cagtttggaa caagagtcca ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa 1620 ccgtctatca gggcgatggc ccactacgtg aaccatcacc ctaatcaagt tttttggggt 1680 cgaggtgccg taaagcacta aatcggaacc ctaaagggag cccccgattt agagcttgac 1740 ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta 1800 gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg 1860 cgccgctaca gggcgcgtcg cgccattcgc cattcaggct gcgcaactgt tgggaagggc 1920 gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa agggggatgt gctgcaaggc 1980 gattaagttg ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg acggccagtg 2040 agcgcgcgta atacgactca ctatagggcg aattgggtac cgggcccccc ctcgaggtcg 2100 acggcgcgcc actggtagag agcgactttg tatgccccaa ttgcgaaacc cgcgatatcc 2160 ttctcgattc tttagtaccc gaccaggaca aggaaaagga ggtcgaaacg tttttgaaga 2220 aacaagagga actacacgga agctctaaag atggcaacca gccagaaact aagaaaatga 2280 agttgatgga tccaactggc accgctggct tgaacaacaa taccagcctt ccaacttctg 2340 taaataacgg cggtacgcca gtgccaccag taccgttacc tttcggtata cctcctttcc 2400 ccatgtttcc aatgcccttc atgcctccaa cggctactat cacaaatcct catcaagctg 2460 acgcaagccc taagaaatga ataacaatac tgacagtact aaataattgc ctacttggct 2520 tcacatacgt tgcatacgtc gatatagata ataatgataa tgacagcagg attatcgtaa 2580 tacgtaatag ttgaaaatct caaaaatgtg tgggtcatta cgtaaataat gataggaatg 2640 ggattcttct atttttcctt tttccattct agcagccgtc gggaaaacgt ggcatcctct 2700 ctttcgggct caattggagt cacgctgccg tgagcatcct ctctttccat atctaacaac 2760 tgagcacgta accaatggaa aagcatgagc ttagcgttgc tccaaaaaag tattggatgg 2820 ttaataccat ttgtctgttc tcttctgact ttgactcctc aaaaaaaaaa aatctacaat 2880 caacagatcg cttcaattac gccctcacaa aaactttttt ccttcttctt cgcccacgtt 2940 aaattttatc cctcatgttg tctaacggat ttctgcactt gatttattat aaaaagacaa 3000 agacataata cttctctatc aatttcagtt attgttcttc cttgcgttat tcttctgttc 3060 ttctttttct tttgtcatat ataaccataa ccaagtaata catattcaaa ctagtatgac 3120 tgacaaaaaa actcttaaag acttaagaaa tcgtagttct gtttacgatt caatggttaa 3180 atcacctaat cgtgctatgt tgcgtgcaac tggtatgcaa gatgaagact ttgaaaaacc 3240 tatcgtcggt gtcatttcaa cttgggctga aaacacacct tgtaatatcc acttacatga 3300 ctttggtaaa ctagccaaag tcggtgttaa ggaagctggt gcttggccag ttcagttcgg 3360 aacaatcacg gtttctgatg gaatcgccat gggaacccaa ggaatgcgtt tctccttgac 3420 atctcgtgat attattgcag attctattga agcagccatg ggaggtcata atgcggatgc 3480 ttttgtagcc attggcggtt gtgataaaaa catgcccggt tctgttatcg ctatggctaa 3540 catggatatc ccagccattt ttgcttacgg cggaacaatt gcacctggta atttagacgg 3600 caaagatatc gatttagtct ctgtctttga aggtgtcggc cattggaacc acggcgatat 3660 gaccaaagaa gaagttaaag ctttggaatg taatgcttgt cccggtcctg gaggctgcgg 3720 tggtatgtat actgctaaca caatggcgac agctattgaa gttttgggac ttagccttcc 3780 gggttcatct tctcacccgg ctgaatccgc agaaaagaaa gcagatattg aagaagctgg 3840 tcgcgctgtt gtcaaaatgc tcgaaatggg cttaaaacct tctgacattt taacgcgtga 3900 agcttttgaa gatgctatta ctgtaactat ggctctggga ggttcaacca actcaaccct 3960 tcacctctta gctattgccc atgctgctaa tgtggaattg acacttgatg atttcaatac 4020 tttccaagaa aaagttcctc atttggctga tttgaaacct tctggtcaat atgtattcca 4080 agacctttac aaggtcggag gggtaccagc agttatgaaa tatctcctta aaaatggctt 4140 ccttcatggt gaccgtatca cttgtactgg caaaacagtc gctgaaaatt tgaaggcttt 4200 tgatgattta acacctggtc aaaaggttat tatgccgctt gaaaatccta aacgtgaaga 4260 tggtccgctc attattctcc atggtaactt ggctccagac ggtgccgttg ccaaagtttc 4320 tggtgtaaaa gtgcgtcgtc atgtcggtcc tgctaaggtc tttaattctg aagaagaagc 4380 cattgaagct gtcttgaatg atgatattgt tgatggtgat gttgttgtcg tacgttttgt 4440 aggaccaaag ggcggtcctg gtatgcctga aatgctttcc ctttcatcaa tgattgttgg 4500 taaagggcaa ggtgaaaaag ttgcccttct gacagatggc cgcttctcag gtggtactta 4560 tggtcttgtc gtgggtcata tcgctcctga agcacaagat ggcggtccaa tcgcctacct 4620 gcaaacagga gacatagtca ctattgacca agacactaag gaattacact ttgatatctc 4680 cgatgaagag ttaaaacatc gtcaagagac cattgaattg ccaccgctct attcacgcgg 4740 tatccttggt aaatatgctc acatcgtttc gtctgcttct aggggagccg taacagactt 4800 ttggaagcct gaagaaactg gcaaaaaatg ttgtcctggt tgctgtggtt aagcggccgc 4860 gttaattcaa attaattgat atagtttttt aatgagtatt gaatctgttt agaaataatg 4920 gaatattatt tttatttatt tatttatatt attggtcggc tcttttcttc tgaaggtcaa 4980 tgacaaaatg atatgaagga aataatgatt tctaaaattt tacaacgtaa gatattttta 5040 caaaagccta gctcatcttt tgtcatgcac tattttactc acgcttgaaa ttaacggcca 5100 gtccactgcg gagtcatttc aaagtcatcc taatcgatct atcgtttttg atagctcatt 5160 ttggagttcg cgattgtctt ctgttattca caactgtttt aatttttatt tcattctgga 5220 actcttcgag ttctttgtaa agtctttcat agtagcttac tttatcctcc aacatattta 5280 acttcatgtc aatttcggct cttaaatttt ccacatcatc aagttcaaca tcatctttta 5340 acttgaattt attctctagc tcttccaacc aagcctcatt gctccttgat ttactggtga 5400 aaagtgatac actttgcgcg caatccaggt caaaactttc ctgcaaagaa ttcaccaatt 5460 tctcgacatc atagtacaat ttgttttgtt ctcccatcac aatttaatat acctgatgga 5520 ttcttatgaa gcgctgggta atggacgtgt cactctactt cgcctttttc cctactcctt 5580 ttagtacgga agacaatgct aataaataag agggtaataa taatattatt aatcggcaaa 5640 aaagattaaa cgccaagcgt ttaattatca gaaagcaaac gtcgtaccaa tccttgaatg 5700 cttcccaatt gtatattaag agtcatcaca gcaacatatt cttgttatta aattaattat 5760 tattgatttt tgatattgta taaaaaaacc aaatatgtat aaaaaaagtg aataaaaaat 5820 accaagtatg gagaaatata ttagaagtct atacgttaaa ccaccgcggt ggagctccag 5880 cttttgttcc ctttagtgag ggttaattgc gcgcttggcg taatcatggt catagctgtt 5940 tcctgtgtga aattgttatc cgctcacaat tccacacaac ataggagccg gaagcataaa 6000 gtgtaaagcc tggggtgcct aatgagtgag gtaactcaca ttaattgcgt tgcgctcact 6060 gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc 6120 ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg 6180 ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc 6240 cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag 6300 gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca 6360 tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca 6420 ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg 6480 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag 6540 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt 6600 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca 6660 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg 6720 cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa ggacagtatt 6780 tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc 6840 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg 6900 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg 6960 gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta 7020 gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg 7080 gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg 7140 ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc 7200 atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc 7260 agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc 7320 ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag 7380 tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat 7440 ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg 7500 caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt 7560 gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag 7620 atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg 7680 accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt 7740 aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct 7800 gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac 7860 tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat 7920 aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat 7980 ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca 8040 aataggggtt ccgcgcacat ttccccgaaa agtgccacct gaacgaagca tctgtgcttc 8100 attttgtaga acaaaaatgc aacgcgagag cgctaatttt tcaaacaaag aatctgagct 8160 gcatttttac agaacagaaa tgcaacgcga aagcgctatt ttaccaacga agaatctgtg 8220 cttcattttt gtaaaacaaa aatgcaacgc gagagcgcta atttttcaaa caaagaatct 8280 gagctgcatt tttacagaac agaaatgcaa cgcgagagcg ctattttacc aacaaagaat 8340 ctatacttct tttttgttct acaaaaatgc atcccgagag cgctattttt ctaacaaagc 8400 atcttagatt actttttttc tcctttgtgc gctctataat gcagtctctt gataactttt 8460 tgcactgtag gtccgttaag gttagaagaa ggctactttg gtgtctattt tctcttccat 8520 aaaaaaagcc tgactccact tcccgcgttt actgattact agcgaagctg cgggtgcatt 8580 ttttcaagat aaaggcatcc ccgattatat tctataccga tgtggattgc gcatactttg 8640 tgaacagaaa gtgatagcgt tgatgattct tcattggtca gaaaattatg aacggtttct 8700 tctattttgt ctctatatac tacgtatagg aaatgtttac attttcgtat tgttttcgat 8760 tcactctatg aatagttctt actacaattt ttttgtctaa agagtaatac tagagataaa 8820 cataaaaaat gtagaggtcg agtttagatg caagttcaag gagcgaaagg tggatgggta 8880 ggttatatag ggatatagca cagagatata tagcaaagag atacttttga gcaatgtttg 8940 tggaagcggt attcgcaata ttttagtagc tcgttacagt ccggtgcgtt tttggttttt 9000 tgaaagtgcg tcttcagagc gcttttggtt ttcaaaagcg ctctgaagtt cctatacttt 9060 ctagagaata ggaacttcgg aataggaact tcaaagcgtt tccgaaaacg agcgcttccg 9120 aaaatgcaac gcgagctgcg cacatacagc tcactgttca cgtcgcacct atatctgcgt 9180 gttgcctgta tatatatata catgagaaga acggcatagt gcgtgtttat gcttaaatgc 9240 gtacttatat gcgtctattt atgtaggatg aaaggtagtc tagtacctcc tgtgatatta 9300 tcccattcca tgcggggtat cgtatgcttc cttcagcact accctttagc tgttctatat 9360 gctgccactc ctcaattgga ttagtctcat ccttcaatgc tatcatttcc tttgatattg 9420 gatcatctaa gaaaccatta ttatcatgac attaacctat aaaaataggc gtatcacgag 9480 gccctttcgt c 9491 <210> 129 <211> 1000 <212> DNA <213> Saccharymoces cerevisiae <400> 129 gttaattcaa attaattgat atagtttttt aatgagtatt gaatctgttt agaaataatg 60 gaatattatt tttatttatt tatttatatt attggtcggc tcttttcttc tgaaggtcaa 120 tgacaaaatg atatgaagga aataatgatt tctaaaattt tacaacgtaa gatattttta 180 caaaagccta gctcatcttt tgtcatgcac tattttactc acgcttgaaa ttaacggcca 240 gtccactgcg gagtcatttc aaagtcatcc taatcgatct atcgtttttg atagctcatt 300 ttggagttcg cgattgtctt ctgttattca caactgtttt aatttttatt tcattctgga 360 actcttcgag ttctttgtaa agtctttcat agtagcttac tttatcctcc aacatattta 420 acttcatgtc aatttcggct cttaaatttt ccacatcatc aagttcaaca tcatctttta 480 acttgaattt attctctagc tcttccaacc aagcctcatt gctccttgat ttactggtga 540 aaagtgatac actttgcgcg caatccaggt caaaactttc ctgcaaagaa ttcaccaatt 600 tctcgacatc atagtacaat ttgttttgtt ctcccatcac aatttaatat acctgatgga 660 ttcttatgaa gcgctgggta atggacgtgt cactctactt cgcctttttc cctactcctt 720 ttagtacgga agacaatgct aataaataag agggtaataa taatattatt aatcggcaaa 780 aaagattaaa cgccaagcgt ttaattatca gaaagcaaac gtcgtaccaa tccttgaatg 840 cttcccaatt gtatattaag agtcatcaca gcaacatatt cttgttatta aattaattat 900 tattgatttt tgatattgta taaaaaaacc aaatatgtat aaaaaaagtg aataaaaaat 960 accaagtatg gagaaatata ttagaagtct atacgttaaa 1000 <210> 130 <211> 16387 <212> DNA <213> Artificial sequence <220> <223> Plasmid <400> 130 gatcctctag tttctcggta ctatgcatat gatccaatat caaaggaaat gatagcattg 60 aaggatgaga ctaatccaat tgaggagtgg cagcatatag aacagctaaa gggtagtgct 120 gaaggaagca tacgataccc cgcatggaat gggataatat cacaggaggt actagactac 180 ctttcatcct acataaatag acgcatataa gtacgcattt aagcataaac acgcactatg 240 ccgttcttct catgtatata tatatacagg caacacgcag atataggtgc gacgtgaaca 300 gtgagctgta tgtgcgcagc tcgcgttgca ttttcggaag cgctcgtttt cggaaacgct 360 ttgaagttcc tattccgaag ttcctattct ctagaaagta taggaacttc agagcgcttt 420 tgaaaaccaa aagcgctctg aagacgcact ttcaaaaaac caaaaacgca ccggactgta 480 acgagctact aaaatattgc gaataccgct tccacaaaca ttgctcaaaa gtatctcttt 540 gctatatatc tctgtgctat atccctatat aacctaccca tccacctttc gctccttgaa 600 cttgcatcta aactcgacct ctacattttt tatgtttatc tctagtatta ctctttagac 660 aaaaaaattg tagtaagaac tattcataga gtgaatcgaa aacaatacga aaatgtaaac 720 atttcctata cgtagtatat agagacaaaa tagaagaaac cgttcataat tttctgacca 780 atgaagaatc atcaacgcta tcactttctg ttcacaaagt atgcgcaatc cacatcggta 840 tagaatataa tcggggatgc ctttatcttg aaaaaatgca cccgcagctt cgctagtaat 900 cagtaaacgc gggaagtgga gtcaggcttt ttttatggaa gagaaaatag acaccaaagt 960 agccttcttc taaccttaac ggacctacag tgcaaaaagt tatcaagaga ctgcattata 1020 gagcgcacaa aggagaaaaa aagtaatcta agatgctttg ttagaaaaat agcgctctcg 1080 ggatgcattt ttgtagaaca aaaaagaagt atagattctt tgttggtaaa atagcgctct 1140 cgcgttgcat ttctgttctg taaaaatgca gctcagattc tttgtttgaa aaattagcgc 1200 tctcgcgttg catttttgtt ttacaaaaat gaagcacaga ttcttcgttg gtaaaatagc 1260 gctttcgcgt tgcatttctg ttctgtaaaa atgcagctca gattctttgt ttgaaaaatt 1320 agcgctctcg cgttgcattt ttgttctaca aaatgaagca cagatgcttc gttaacaaag 1380 atatgctatt gaagtgcaag atggaaacgc agaaaatgaa ccggggatgc gacgtgcaag 1440 attacctatg caatagatgc aatagtttct ccaggaaccg aaatacatac attgtcttcc 1500 gtaaagcgct agactatata ttattataca ggttcaaata tactatctgt ttcagggaaa 1560 actcccaggt tcggatgttc aaaattcaat gatgggtaac aagtacgatc gtaaatctgt 1620 aaaacagttt gtcggatatt aggctgtatc tcctcaaagc gtattcgaat atcattgaga 1680 agctgcattt tttttttttt tttttttttt tttttttata tatatttcaa ggatatacca 1740 ttgtaatgtc tgcccctaag aagatcgtcg ttttgccagg tgaccacgtt ggtcaagaaa 1800 tcacagccga agccattaag gttcttaaag ctatttctga tgttcgttcc aatgtcaagt 1860 tcgatttcga aaatcattta attggtggtg ctgctatcga tgctacaggt gttccacttc 1920 cagatgaggc gctggaagcc tccaagaagg ctgatgccgt tttgttaggt gctgtgggtg 1980 gtcctaaatg gggtaccggt agtgttagac ctgaacaagg tttactaaaa atccgtaaag 2040 aacttcaatt gtacgccaac ttaagaccat gtaactttgc atccgactct cttttagact 2100 tatctccaat caagccacaa tttgctaaag gtactgactt cgttgttgtt agagaattag 2160 tgggaggtat ttactttggt aagagaaagg aagacgatgg tgatggtgtc gcttgggata 2220 gtgaacaata caccgttcca gaagtgcaaa gaatcacaag aatggccgct ttcatggccc 2280 tacaacatga gccaccattg cctatttggt ccttggataa agctaatgtt ttggcctctt 2340 caagattatg gagaaaaact gtggaggaaa ccatcaagaa cgaattccct acattgaaag 2400 ttcaacatca attgattgat tctgccgcca tgatcctagt taagaaccca acccacctaa 2460 atggtattat aatcaccagc aacatgtttg gtgatatcat ctccgatgaa gcctccgtta 2520 tcccaggctc cttgggtttg ttgccatctg cgtccttggc ctctttgcca gacaagaaca 2580 ccgcatttgg tttgtacgaa ccatgccatg gttccgctcc agatttgcca aagaataagg 2640 tcaaccctat cgccactatc ttgtctgctg caatgatgtt gaaattgtca ttgaacttgc 2700 ctgaagaagg taaagccatt gaagatgcag ttaaaaaggt tttggatgca ggtatcagaa 2760 ctggtgattt aggtggttcc aacagtacca ccgaagtcgg tgatgctgtc gccgaagaag 2820 ttaagaaaat ccttgcttaa aaagattctc tttttttatg atatttgtac aaaaaaaaaa 2880 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aatgcagcgt cacatcggat 2940 aataatgatg gcagccattg tagaagtgcc ttttgcattt ctagtctctt tctcggtcta 3000 gctagtttta ctacatcgcg aagatagaat cttagatcac actgcctttg ctgagctgga 3060 tcaatagagt aacaaaagag tggtaaggcc tcgttaaagg acaaggacct gagcggaagt 3120 gtatcgtaca gtagacggag tatactagag tcgacctgca ggcatgcaag cttttcaatt 3180 catcattttt tttttattct tttttttgat ttcggtttcc ttgaaatttt tttgattcgg 3240 taatctccga acagaaggaa gaacgaagga aggagcacag acttagattg gtatatatac 3300 gcatatgtag tgttgaagaa acatgaaatt gcccagtatt cttaacccaa ctgcacagaa 3360 caaaaacctg caggaaacga agataaatca tgtcgaaagc tacatataag gaacgtgctg 3420 ctactcatcc tagtcctgtt gctgccaagc tatttaatat catgcacgaa aagcaaacaa 3480 acttgtgtgc ttcattggat gttcgtacca ccaaggaatt actggagtta gttgaagcat 3540 taggtcccaa aatttgttta ctaaaaacac atgtggatat cttgactgat ttttccatgg 3600 agggcacagt taagccgcta aaggcattat ccgccaagta caatttttta ctcttcgaag 3660 acagaaaatt tgctgacatt ggtaatacag tcaaattgca gtactctgcg ggtgtataca 3720 gaatagcaga atgggcagac attacgaatg cacacggtgt ggtgggccca ggtattgtta 3780 gcggtttgaa gcaggcggca gaagaagtaa caaaggaacc tagaggcctt ttgatgttag 3840 cagaattgtc atgcaagggc tccctatcta ctggagaata tactaagggt actgttgaca 3900 ttgcgaagag cgacaaagat tttgttatcg gctttattgc tcaaagagac atgggtggaa 3960 gagatgaagg ttacgattgg ttgattatga cacccggtgt gggtttagat gacaagggag 4020 acgcattggg tcaacagtat agaaccgtgg atgatgtggt ctctacagga tctgacatta 4080 ttattgttgg aagaggacta tttgcaaagg gaagggatgc taaggtagag ggtgaacgtt 4140 acagaaaagc aggctgggaa gcatatttga gaagatgcgg ccagcaaaac taaaaaactg 4200 tattataagt aaatgcatgt atactaaact cacaaattag agcttcaatt taattatatc 4260 agttattacc cgggaatctc ggtcgtaatg atttttataa tgacgaaaaa aaaaaaattg 4320 gaaagaaaaa gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg 4380 ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa 4440 tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac 4500 ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt 4560 gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga 4620 gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca 4680 ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg 4740 ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt 4800 cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc 4860 ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct 4920 tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc 4980 gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta 5040 tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca 5100 gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag 5160 tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag 5220 ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt 5280 agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa 5340 gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg 5400 attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga 5460 agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta 5520 atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc 5580 cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg 5640 ataccgcgag acccacgctc accggctcca gatttatcag caataaacca gccagccgga 5700 agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt 5760 tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt 5820 gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc 5880 caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc 5940 ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca 6000 gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag 6060 tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg 6120 tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa 6180 cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa 6240 cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga 6300 gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga 6360 atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg 6420 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt 6480 ccccgaaaag tgccacctga cgtctaagaa accattatta tcatgacatt aacctataaa 6540 aataggcgta tcacgaggcc ctttcgtctc gcgcgtttcg gtgatgacgg tgaaaacctc 6600 tgacacatgc agctcccgga gacggtcaca gcttgtctgt aagcggatgc cgggagcaga 6660 caagcccgtc agggcgcgtc agcgggtgtt ggcgggtgtc ggggctggct taactatgcg 6720 gcatcagagc agattgtact gagagtgcac cataaaattg taaacgttaa tattttgtta 6780 aaattcgcgt taaatttttg ttaaatcagc tcatttttta accaatagac cgaaatcggc 6840 aaaatccctt ataaatcaaa agaatagccc gagatagagt tgagtgttgt tccagtttgg 6900 aacaagagtc cactattaaa gaacgtggac tccaacgtca aagggcgaaa aaccgtctat 6960 cagggcgatg gcccactacg tgaaccatca cccaaatcaa gttttttggg gtcgaggtgc 7020 cgtaaagcac taaatcggaa ccctaaaggg agcccccgat ttagagcttg acggggaaag 7080 ccggcgaacg tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc taaggcgctg 7140 gcaagtgtag cggtcacgct gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta 7200 cagggcgcgt actatggttg ctttgacgta tgcggtgtga aataccgcac agatgcgtaa 7260 ggagaaaata ccgcatcagg cgccattcgc cattcaggct gcgcaactgt tgggaagggc 7320 gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa agggggatgt gctgcaaggc 7380 gattaagttg ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg acggccagtg 7440 aattcgagct ccaccgcgga tagatctgaa atgaataaca atactgacag tactaaataa 7500 ttgcctactt ggcttcacat acgttgcata cgtcgatata gataataatg ataatgacag 7560 caggattatc gtaatacgta atagttgaaa atctcaaaaa tgtgtgggtc attacgtaaa 7620 taatgatagg aatgggattc ttctattttt cctttttcca ttctagcagc cgtcgggaaa 7680 acgtggcatc ctctctttcg ggctcaattg gagtcacgct gccgtgagca tcctctcttt 7740 ccatatctaa caactgagca cgtaaccaat ggaaaagcat gagcttagcg ttgctccaaa 7800 aaagtattgg atggttaata ccatttgtct gttctcttct gactttgact cctcaaaaaa 7860 aaaaaatcta caatcaacag atcgcttcaa ttacgccctc acaaaaactt ttttccttct 7920 tcttcgccca cgttaaattt tatccctcat gttgtctaac ggatttctgc acttgattta 7980 ttataaaaag acaaagacat aatacttctc tatcaatttc agttattgtt cttccttgcg 8040 ttattcttct gttcttcttt ttcttttgtc atatataacc ataaccaagt aatacatatt 8100 caaatctaga gctgaggatg ttgaagcaaa tcaacttcgg tggtactgtt gaaaccgtct 8160 acgaaagagc tgactggcca agagaaaagt tgttggacta cttcaagaac gacacttttg 8220 ctttgatcgg ttacggttcc caaggttacg gtcaaggttt gaacttgaga gacaacggtt 8280 tgaacgttat cattggtgtc cgtaaagatg gtgcttcttg gaaggctgcc atcgaagacg 8340 gttgggttcc aggcaagaac ttgttcactg ttgaagatgc tatcaagaga ggtagttacg 8400 ttatgaactt gttgtccgat gccgctcaat cagaaacctg gcctgctatc aagccattgt 8460 tgaccaaggg taagactttg tacttctccc acggtttctc cccagtcttc aaggacttga 8520 ctcacgttga accaccaaag gacttagatg ttatcttggt tgctccaaag ggttccggta 8580 gaactgtcag atctttgttc aaggaaggtc gtggtattaa ctcttcttac gccgtctgga 8640 acgatgtcac cggtaaggct cacgaaaagg cccaagcttt ggccgttgcc attggttccg 8700 gttacgttta ccaaaccact ttcgaaagag aagtcaactc tgacttgtac ggtgaaagag 8760 gttgtttaat gggtggtatc cacggtatgt tcttggctca atacgacgtc ttgagagaaa 8820 acggtcactc cccatctgaa gctttcaacg aaaccgtcga agaagctacc caatctctat 8880 acccattgat cggtaagtac ggtatggatt acatgtacga tgcttgttcc accaccgcca 8940 gaagaggtgc tttggactgg tacccaatct tcaagaatgc tttgaagcct gttttccaag 9000 acttgtacga atctaccaag aacggtaccg aaaccaagag atctttggaa ttcaactctc 9060 aacctgacta cagagaaaag ctagaaaagg aattagacac catcagaaac atggaaatct 9120 ggaaggttgg taaggaagtc agaaagttga gaccagaaaa ccaataatta attaatcatg 9180 taattagtta tgtcacgctt acattcacgc cctcccccca catccgctct aaccgaaaag 9240 gaaggagtta gacaacctga agtctaggtc cctatttatt tttttatagt tatgttagta 9300 ttaagaacgt tatttatatt tcaaattttt cttttttttc tgtacagacg cgtgtacgca 9360 tgtaacatta tactgaaaac cttgcttgag aaggttttgg gacgctcgaa ggctttaatt 9420 tgcgggcggc cgctctagag agttgttagc aaccttttgt ttcttttgag ctggttcaga 9480 cattatgtac acgtatatgt gacgagttcg agaagtattt tactatcgta ctaaatttta 9540 cctgaaaaat tatatactcg agaaagagga agccaagaat tgagaaaaaa gaaaaacccg 9600 cgagtaagga aattaaatac aggtgtacac atacacgcac acatatatat atatatatat 9660 atgtatatgt gtatatagga agcgcgcgca tgttagtata tacgattcgt tggaaagggg 9720 ccgtccacca aacgtgactt gacgagttga caaattgacc tcaatatggc tcagtcagta 9780 atttttagtt ccgctttatt cccgccatct ttcaggccac gagggtagct cataacgccg 9840 cgctaatgcc gctgcgtcac agcaaccagt agctcagcca aaaccgaaag agaaatcgta 9900 gctgtcccga tgaggactta tacacttgtc accatctaaa taaattattt attcgcgttt 9960 cggttcttgt tttcgattta attagattgt tcattgaatc ataataaata tgtaaaaaat 10020 atatatattt gaagctgctt cagaaaaaca gggcttccta gtgtacagat gtatgtcgga 10080 tgaaaaaaaa aaaatcttaa atgtgaaatt gggtcaattc aattgactat gacttgatgt 10140 tgcaaaaatt ccaagagaaa aagtttccag cacttgatat tattttcctc tttaattttt 10200 cgccttgtct acgatcttat tagcaccgat ccagggcatc atagacctta actgttcacc 10260 aataatttcg ataccatgtg ctgcattgtt tcttctttta gcagtcatac tcgggtaacc 10320 cgtagcgcct tcacttatga acatcttagc gtattcaccg tcctggatac gtttcaaggc 10380 atttctcatg gcttgtcttg attctgcgtt aatgacttca ggtccggtga catactcacc 10440 atattctgca ttatttgaaa tggaatagtt catattagct ataccacctt catacattaa 10500 gtctactatc aacttcaatt catgtagaca ttcgaagtat gccatttcgg gagcgtaccc 10560 tgcttcgaca agcgtctcaa agcctgcttt aaccaattca acagttcctc cgcacagaac 10620 cgcttgttct ccaaataaat ctgtctcagt ctcgtcttta aaagtggttt ctattatacc 10680 cgttctcccg ccaccaactc ctgctgcgta gcttaaagct acattcttag cgtttccgct 10740 tgcgtcttgg tatatagcga tcaaatctgg aataccacca cccttaacaa attcgctcct 10800 aacagtatgc cccggagcct taggtgcaat cataataacg tccaaatctg ccctggggac 10860 tacttgattg taatgaatgg caaatccatg actgaaggcc aaggtagcgc ccttcttaat 10920 gtttggttct atttcatttt tgtacaattg cgattgaaat tcatctggcg ttaaaatcat 10980 gactaaatca gcgccggcaa cagccgctgc aacatctgtg actttcaagc catgtgcttc 11040 agcctttgca acggtagcac taccttttct cagacctact gtcacgtcga ccccagaatc 11100 tttcaagtta caggcttgtg cgtgtccttg ggaaccatat cctataatag caaccttctt 11160 tccctggatg atgctcagat cgcagtcttt atcgtaaaac accttcatgt tttatttttt 11220 acttatattg ctggtagggt aaaaaaatat aactcctagg aataggttgt ctatatgttt 11280 ttgtcttgct tctataattg taacaaacaa ggaaagggaa aatactgggt gtaaaagcca 11340 ttgagtcaag ttaggtcatc ccttttatac aaaatttttc aatttttttt ccaagattct 11400 tgtacgatta attatttttt ttttgcgtcc tacagcgtga tgaaaatttc cgcctgctgc 11460 aagatgagcg ggaacgggcg aaatgtgcac gcgcacaact tacgaaacgc ggatgagtca 11520 ctgacagcca ccgcagaggt tctgactcct actgagctct attggaggtg gcagaaccgg 11580 taccggagga gaccgctata accggtttga atttattgtc acagtgtcac atcagcggca 11640 actcagaagt ttgacagcaa gcaagttcat cattcgaact agccttattg ttttagttca 11700 gtgacagcga actgccgtac tcgatgcttt atttctcacg gtagagcgga agaacagata 11760 ggggcagcgt gagaagagtt agaaagtaaa tttttatcac gtctgaagta ttcttattca 11820 taggaaattt tgcaaggttt tttagctcaa taacgggcta agttatataa ggtgttcacg 11880 cgattttctt gttatgtata cctcttctct gaggaatggt actactgtcc tgatgtaggc 11940 tccttaaatt ggtgggcaag aataacttat cgatattttg tatattggtc ttggagttca 12000 ccacgtaatg cctgtttaag accatcagtt aactctagta ttatttggtc ttggctactg 12060 gccgtttgct attattcaag tcttttgtgc cttcccgtcg ggtaagggag ttatttaggg 12120 atacagaatc taacgaaaac taaatctcaa tgattaactc catttaatcc ttttttgaaa 12180 ggcaaaagag gtcccttgtt cacttacaac gttcttagcc aaattcgctt atcacttact 12240 acttcacgat atacagaagt aaaaacatat aaaaagatgt ctgtttgttt agccatcaca 12300 aaaggtatcg cagtttcttc tataggcctc tactctggtc ttttggcttc cgcttcattg 12360 attacatcta ctactccact agaggtttta acaggatctc taaaaacatc gatatcgtct 12420 ctgcgttcca atcctacggt gaatatattt ccaagcaatt cactgaagaa gaaagagaag 12480 atgttgtgga acatgcatgc ccaggtcctg gttcttgtgg tggtatgtat actgccaaca 12540 caatggcttc tgccgctgaa gtgctaggtt tgaccattcc aaactcctct tccttcccag 12600 ccgtttccaa ggagaagtta gctgagtgtg acaacattgg tgaatacatc aagaagacaa 12660 tggaattggg tattttacct cgtgatatcc tcacaaaaga ggcttttgaa aacgccatta 12720 cttatgtcgt tgcaaccggt gggtccacta atgctgtttt gcatttggtg gctgttgctc 12780 actctgcggg tgtcaagttg tcaccagatg atttccaaag aatcagtgat actacaccat 12840 tgatcggtga cttcaaacct tctggtaaat acgtcatggc cgatttgatt aacgttggtg 12900 gtacccaatc tgtgattaag tatctatatg aaaacaacat gttgcacggt aacacaatga 12960 ctgttaccgg tgacactttg gcagaacgtg caaagaaagc accaagccta cctgaaggac 13020 aagagattat taagccactc tcccacccaa tcaaggccaa cggtcacttg caaattctgt 13080 acggttcatt ggcaccaggt ggagctgtgg gtaaaattac cggtaaggaa ggtacttact 13140 tcaagggtag agcacgtgtg ttcgaagagg aaggtgcctt tattgaagcc ttggaaagag 13200 gtgaaatcaa gaagggtgaa aaaaccgttg ttgttatcag atatgaaggt ccaagaggtg 13260 caccaggtat gcctgaaatg ctaaagcctt cctctgctct gatgggttac ggtttgggta 13320 aagatgttgc attgttgact gatggtagat tctctggtgg ttctcacggg ttcttaatcg 13380 gccacattgt tcccgaagcc gctgaaggtg gtcctatcgg gttggtcaga gacggcgatg 13440 agattatcat tgatgctgat aataacaaga ttgacctatt agtctctgat aaggaaatgg 13500 ctcaacgtaa acaaagttgg gttgcacctc cacctcgtta cacaagaggt actctatcca 13560 agtatgctaa gttggtttcc aacgcttcca acggttgtgt tttagatgct tgattaatta 13620 agagtaagcg aatttcttat gatttatgat ttttattatt aaataagtta taaaaaaaat 13680 aagtgtatac aaattttaaa gtgactctta ggttttaaaa cgaaaattct tattcttgag 13740 taactctttc ctgtaggtca ggttgctttc tcaggtatag catgaggtcg ctcttattga 13800 ccacacctct accggcatgc cgagcaaatg cctgcaaatc gctccccatt tcacccaatt 13860 gtagatatgc taactccagc aatgagttga tgaatctcgg tgtgtatttt atgtcctcag 13920 aggacaacac ctgtggtact agttctagag cggccgcccg caaattaaag ccttcgagcg 13980 tcccaaaacc ttctcaagca aggttttcag tataatgtta catgcgtaca cgcgtctgta 14040 cagaaaaaaa agaaaaattt gaaatataaa taacgttctt aatactaaca taactataaa 14100 aaaataaata gggacctaga cttcaggttg tctaactcct tccttttcgg ttagagcgga 14160 tgtgggggga gggcgtgaat gtaagcgtga cataactaat tacatgatta attaactaga 14220 gagctttcgt tttcatgagt tccccgaatt ctttcggaag cttgtcactt gctaaattaa 14280 tgttatcact gtagtcaacc gggacatcga tgatgacagg accttcagcg ttcatgcctt 14340 gacgcagaac atctgccagc tggtctggtg attctacgcg caagccagtt gctccgaagc 14400 tttccgcata tttcacgata tcgatatttc cgaaatcgac cgcagatgta cggttatatt 14460 ttttcaattg ctggaatgca accatgtcat atgtgctgtc gttccataca atgtgtacaa 14520 ttggtgcttt tagtcgaact gctgtctcta attccattgc tgagaataag aaaccgccgt 14580 caccagagac agaaaccact ttttctcccg gtttcaccaa tgaagcgccg attgcccaag 14640 gaagcgcaac gccgagtgtt tgcataccgt tactgatcat taatgttaac ggctcgtagc 14700 tgcggaaata acgtgacatc caaatggcgt gcgaaccgat atcgcaagtt actgtaacat 14760 gatcatcgac tgcattacgc aactctttaa cgatttcaag agggtgcgct ctgtctgatt 14820 tccaatctgc aggcacctgc tcaccttcat gcatatattg ttttaaatca gaaaggattt 14880 tctgctcacg ctctgcaaat tccactttca cagcatcgtg ttcgatatga ttgatcgtgg 14940 acggaatgtc accgatcaat tcaagatcag gctggtaagc atgatcaatg tcagcgataa 15000 tctcgtctaa atggataatt gtccggtctc cattgatatt ccagaatttc ggatcatatt 15060 caatcgggtc atagccgatc gtcagaacaa catctgcctg ctctagcagt aaatcgccag 15120 gctggttgcg gaacaaaccg atacggccaa aatattgatc ctctaaatct ctagaaaggg 15180 taccggcagc ttgatatgtt tcaacaaatg gaagctgaac ctttttcaaa agcttgcgaa 15240 ccgctttaat tgcttccggt cttccgcctt tcatgccgac caaaacgaca ggaagttttg 15300 ctgtttggat ttttgctatg gccgcactga ttgcatcatc tgctgcagga ccgagttttg 15360 gcgctgcaac agcacgcacg tttttcgtat ttgtgacttc attcacaaca tcttgcggaa 15420 agctcacaaa agcggcccca gcctgccctg ctgacgctat cctaaatgca tttgtaacag 15480 cttccggtat attttttaca tcttgaactt ctacactgta ttttgtaatc ggctggaata 15540 gcgccgcatt atccaaagat tgatgtgtcc gttttaaacg atctgcacgg atcacgtttc 15600 cagcaagcgc aacgacaggg tctccttcag tgttcgctgt cagcaggcct gttgccaagt 15660 tagaggcacc cggtcctgat gtgactaaca cgactcccgg ttttccagtt aaacggccga 15720 ctgcttgggc catgaatgct gcgttttgtt cgtgccgggc aacgataatt tcaggtcctt 15780 tatcttgtaa agcgtcaaat accgcatcaa tttttgcacc tggaatgcca aatacatgtg 15840 tgacaccttg ctccactaag caatcaacaa caagctccgc ccctctgttt ttcacaaggg 15900 atttttgttc ttttgttgct tttgtcaaca tcctcagcga tgattgattg attgattgta 15960 cagtttgttt ttcttaatat ctatttcgat gacttctata tgatattgca ctaacaagaa 16020 gatattataa tgcaattgat acaagacaag gagttatttg cttctctttt atatgattct 16080 gacaatccat attgcgttgg tagtcttttt tgctggaacg gttcagcgga aaagacgcat 16140 cgctcttttt gcttctagaa gaaatgccag caaaagaatc tcttgacagt gactgacagc 16200 aaaaatgtct ttttctaact agtaacaagg ctaagatatc agcctgaaat aaagggtggt 16260 gaagtaataa ttaaatcatc cgtataaacc tatacacata tatgaggaaa aataatacaa 16320 aagtgtttta aatacagata catacatgaa catatgcacg tatagcgccc aaatgtcggt 16380 aatggga 16387 <210> 131 <211> 1188 <212> DNA <213> Saccharomyces cerevisiae <400> 131 atgttgagaa ctcaagccgc cagattgatc tgcaactccc gtgtcatcac tgctaagaga 60 acctttgctt tggccacccg tgctgctgct tacagcagac cagctgcccg tttcgttaag 120 ccaatgatca ctacccgtgg tttgaagcaa atcaacttcg gtggtactgt tgaaaccgtc 180 tacgaaagag ctgactggcc aagagaaaag ttgttggact acttcaagaa cgacactttt 240 gctttgatcg gttacggttc ccaaggttac ggtcaaggtt tgaacttgag agacaacggt 300 ttgaacgtta tcattggtgt ccgtaaagat ggtgcttctt ggaaggctgc catcgaagac 360 ggttgggttc caggcaagaa cttgttcact gttgaagatg ctatcaagag aggtagttac 420 gttatgaact tgttgtccga tgccgctcaa tcagaaacct ggcctgctat caagccattg 480 ttgaccaagg gtaagacttt gtacttctcc cacggtttct ccccagtctt caaggacttg 540 actcacgttg aaccaccaaa ggacttagat gttatcttgg ttgctccaaa gggttccggt 600 agaactgtca gatctttgtt caaggaaggt cgtggtatta actcttctta cgccgtctgg 660 aacgatgtca ccggtaaggc tcacgaaaag gcccaagctt tggccgttgc cattggttcc 720 ggttacgttt accaaaccac tttcgaaaga gaagtcaact ctgacttgta cggtgaaaga 780 ggttgtttaa tgggtggtat ccacggtatg ttcttggctc aatacgacgt cttgagagaa 840 aacggtcact ccccatctga agctttcaac gaaaccgtcg aagaagctac ccaatctcta 900 tacccattga tcggtaagta cggtatggat tacatgtacg atgcttgttc caccaccgcc 960 agaagaggtg ctttggactg gtacccaatc ttcaagaatg ctttgaagcc tgttttccaa 1020 gacttgtacg aatctaccaa gaacggtacc gaaaccaaga gatctttgga attcaactct 1080 caacctgact acagagaaaa gctagaaaag gaattagaca ccatcagaaa catggaaatc 1140 tggaaggttg gtaaggaagt cagaaagttg agaccagaaa accaataa 1188 <210> 132 <211> 1014 <212> DNA <213> Pseudomonas fluorescens <400> 132 atgaaagttt tctacgataa agactgcgac ctgtcgatca tccaaggtaa gaaagttgcc 60 atcatcggct acggttccca gggccacgct caagcatgca acctgaagga ttccggcgta 120 gacgtgactg ttggcctgcg taaaggctcg gctaccgttg ccaaggctga agcccacggc 180 ttgaaagtga ccgacgttgc tgcagccgtt gccggtgccg acttggtcat gatcctgacc 240 ccggacgagt tccagtccca gctgtacaag aacgaaatcg agccgaacat caagaagggc 300 gccactctgg ccttctccca cggcttcgcg atccactaca accaggttgt gcctcgtgcc 360 gacctcgacg tgatcatgat cgcgccgaag gctccaggcc acaccgtacg ttccgagttc 420 gtcaagggcg gtggtattcc tgacctgatc gcgatctacc aggacgcttc cggcaacgcc 480 aagaacgttg ccctgtccta cgccgcaggc gtgggcggcg gccgtaccgg catcatcgaa 540 accaccttca aggacgagac tgaaaccgac ctgttcggtg agcaggctgt tctgtgtggc 600 ggtaccgtcg agctggtcaa agccggtttc gaaaccctgg ttgaagctgg ctacgctcca 660 gaaatggcct acttcgagtg cctgcacgaa ctgaagctga tcgttgacct catgtacgaa 720 ggcggtatcg ccaacatgaa ctactcgatc tccaacaacg ctgaatacgg cgagtacgtg 780 actggtccag aagtcatcaa cgccgaatcc cgtcaggcca tgcgcaatgc tctgaagcgc 840 atccaggacg gcgaatacgc gaagatgttc atcagcgaag gcgctaccgg ctacccatcg 900 atgaccgcca agcgtcgtaa caacgctgct cacggtatcg aaatcatcgg cgagcaactg 960 cgctcgatga tgccttggat cggtgccaac aaaatcgtcg acaaagccaa gaac 1014 <210> 133 <211> 250 <212> DNA <213> Saccharomyces cerevisiae <400> 133 ccgcaaatta aagccttcga gcgtcccaaa accttctcaa gcaaggtttt cagtataatg 60 ttacatgcgt acacgcgtct gtacagaaaa aaaagaaaaa tttgaaatat aaataacgtt 120 cttaatacta acataactat aaaaaaataa atagggacct agacttcagg ttgtctaact 180 ccttcctttt cggttagagc ggatgtgggg ggagggcgtg aatgtaagcg tgacataact 240 aattacatga 250 <210> 134 <211> 1181 <212> DNA <213> Saccharomyces cerevisiae <400> 134 taaaacctct agtggagtag tagatgtaat caatgaagcg gaagccaaaa gaccagagta 60 gaggcctata gaagaaactg cgataccttt tgtgatggct aaacaaacag acatcttttt 120 atatgttttt acttctgtat atcgtgaagt agtaagtgat aagcgaattt ggctaagaac 180 gttgtaagtg aacaagggac ctcttttgcc tttcaaaaaa ggattaaatg gagttaatca 240 ttgagattta gttttcgtta gattctgtat ccctaaataa ctcccttacc cgacgggaag 300 gcacaaaaga cttgaataat agcaaacggc cagtagccaa gaccaaataa tactagagtt 360 aactgatggt cttaaacagg cattacgtgg tgaactccaa gaccaatata caaaatatcg 420 ataagttatt cttgcccacc aatttaagga gcctacatca ggacagtagt accattcctc 480 agagaagagg tatacataac aagaaaatcg cgtgaacacc ttatataact tagcccgtta 540 ttgagctaaa aaaccttgca aaatttccta tgaataagaa tacttcagac gtgataaaaa 600 tttactttct aactcttctc acgctgcccc tatctgttct tccgctctac cgtgagaaat 660 aaagcatcga gtacggcagt tcgctgtcac tgaactaaaa caataaggct agttcgaatg 720 atgaacttgc ttgctgtcaa acttctgagt tgccgctgat gtgacactgt gacaataaat 780 tcaaaccggt tatagcggtc tcctccggta ccggttctgc cacctccaat agagctcagt 840 aggagtcaga acctctgcgg tggctgtcag tgactcatcc gcgtttcgta agttgtgcgc 900 gtgcacattt cgcccgttcc cgctcatctt gcagcaggcg gaaattttca tcacgctgta 960 ggacgcaaaa aaaaaataat taatcgtaca agaatcttgg aaaaaaaatt gaaaaatttt 1020 gtataaaagg gatgacctaa cttgactcaa tggcttttac acccagtatt ttccctttcc 1080 ttgtttgtta caattataga agcaagacaa aaacatatag acaacctatt cctaggagtt 1140 atattttttt accctaccag caatataagt aaaaaactag t 1181 <210> 135 <211> 759 <212> DNA <213> Saccharomyces cerevisiae <400> 135 ggccctgcag gcctatcaag tgctggaaac tttttctctt ggaatttttg caacatcaag 60 tcatagtcaa ttgaattgac ccaatttcac atttaagatt tttttttttt catccgacat 120 acatctgtac actaggaagc cctgtttttc tgaagcagct tcaaatatat atatttttta 180 catatttatt atgattcaat gaacaatcta attaaatcga aaacaagaac cgaaacgcga 240 ataaataatt tatttagatg gtgacaagtg tataagtcct catcgggaca gctacgattt 300 ctctttcggt tttggctgag ctactggttg ctgtgacgca gcggcattag cgcggcgtta 360 tgagctaccc tcgtggcctg aaagatggcg ggaataaagc ggaactaaaa attactgact 420 gagccatatt gaggtcaatt tgtcaactcg tcaagtcacg tttggtggac ggcccctttc 480 caacgaatcg tatatactaa catgcgcgcg cttcctatat acacatatac atatatatat 540 atatatatat gtgtgcgtgt atgtgtacac ctgtatttaa tttccttact cgcgggtttt 600 tcttttttct caattcttgg cttcctcttt ctcgagtata taatttttca ggtaaaattt 660 agtacgatag taaaatactt ctcgaactcg tcacatatac gtgtacataa tgtctgaacc 720 agctcaaaag aaacaaaagg ttgctaacaa ctctctaga 759 <210> 136 <211> 643 <212> DNA <213> Saccharomyces cerevisiae <400> 136 gaaatgaata acaatactga cagtactaaa taattgccta cttggcttca catacgttgc 60 atacgtcgat atagataata atgataatga cagcaggatt atcgtaatac gtaatagttg 120 aaaatctcaa aaatgtgtgg gtcattacgt aaataatgat aggaatggga ttcttctatt 180 tttccttttt ccattctagc agccgtcggg aaaacgtggc atcctctctt tcgggctcaa 240 ttggagtcac gctgccgtga gcatcctctc tttccatatc taacaactga gcacgtaacc 300 aatggaaaag catgagctta gcgttgctcc aaaaaagtat tggatggtta ataccatttg 360 tctgttctct tctgactttg actcctcaaa aaaaaaaaat ctacaatcaa cagatcgctt 420 caattacgcc ctcacaaaaa cttttttcct tcttcttcgc ccacgttaaa ttttatccct 480 catgttgtct aacggatttc tgcacttgat ttattataaa aagacaaaga cataatactt 540 ctctatcaat ttcagttatt gttcttcctt gcgttattct tctgttcttc tttttctttt 600 gtcatatata accataacca agtaatacat attcaaatct aga 643 <210> 137 <211> 1716 <212> DNA <213> Bacillus subtilis <400> 137 atgttgacaa aagcaacaaa agaacaaaaa tcccttgtga aaaacagagg ggcggagctt 60 gttgttgatt gcttagtgga gcaaggtgtc acacatgtat ttggcattcc aggtgcaaaa 120 attgatgcgg tatttgacgc tttacaagat aaaggacctg aaattatcgt tgcccggcac 180 gaacaaaacg cagcattcat ggcccaagca gtcggccgtt taactggaaa accgggagtc 240 gtgttagtca catcaggacc gggtgcctct aacttggcaa caggcctgct gacagcgaac 300 actgaaggag accctgtcgt tgcgcttgct ggaaacgtga tccgtgcaga tcgtttaaaa 360 cggacacatc aatctttgga taatgcggcg ctattccagc cgattacaaa atacagtgta 420 gaagttcaag atgtaaaaaa tataccggaa gctgttacaa atgcatttag gatagcgtca 480 gcagggcagg ctggggccgc ttttgtgagc tttccgcaag atgttgtgaa tgaagtcaca 540 aatacgaaaa acgtgcgtgc tgttgcagcg ccaaaactcg gtcctgcagc agatgatgca 600 atcagtgcgg ccatagcaaa aatccaaaca gcaaaacttc ctgtcgtttt ggtcggcatg 660 aaaggcggaa gaccggaagc aattaaagcg gttcgcaagc ttttgaaaaa ggttcagctt 720 ccatttgttg aaacatatca agctgccggt accctttcta gagatttaga ggatcaatat 780 tttggccgta tcggtttgtt ccgcaaccag cctggcgatt tactgctaga gcaggcagat 840 gttgttctga cgatcggcta tgacccgatt gaatatgatc cgaaattctg gaatatcaat 900 ggagaccgga caattatcca tttagacgag attatcgctg acattgatca tgcttaccag 960 cctgatcttg aattgatcgg tgacattccg tccacgatca atcatatcga acacgatgct 1020 gtgaaagtgg aatttgcaga gcgtgagcag aaaatccttt ctgatttaaa acaatatatg 1080 catgaaggtg agcaggtgcc tgcagattgg aaatcagaca gagcgcaccc tcttgaaatc 1140 gttaaagagt tgcgtaatgc agtcgatgat catgttacag taacttgcga tatcggttcg 1200 cacgccattt ggatgtcacg ttatttccgc agctacgagc cgttaacatt aatgatcagt 1260 aacggtatgc aaacactcgg cgttgcgctt ccttgggcaa tcggcgcttc attggtgaaa 1320 ccgggagaaa aagtggtttc tgtctctggt gacggcggtt tcttattctc agcaatggaa 1380 ttagagacag cagttcgact aaaagcacca attgtacaca ttgtatggaa cgacagcaca 1440 tatgacatgg ttgcattcca gcaattgaaa aaatataacc gtacatctgc ggtcgatttc 1500 ggaaatatcg atatcgtgaa atatgcggaa agcttcggag caactggctt gcgcgtagaa 1560 tcaccagacc agctggcaga tgttctgcgt caaggcatga acgctgaagg tcctgtcatc 1620 atcgatgtcc cggttgacta cagtgataac attaatttag caagtgacaa gcttccgaaa 1680 gaattcgggg aactcatgaa aacgaaagct ctctag 1716 <210> 138 <211> 571 <212> PRT <213> Bacillus subtilis <400> 138 Met Leu Thr Lys Ala Thr Lys Glu Gln Lys Ser Leu Val Lys Asn Arg 1 5 10 15 Gly Ala Glu Leu Val Val Asp Cys Leu Val Glu Gln Gly Val Thr His             20 25 30 Val Phe Gly Ile Pro Gly Ala Lys Ile Asp Ala Val Phe Asp Ala Leu         35 40 45 Gln Asp Lys Gly Pro Glu Ile Ile Val Ala Arg His Glu Gln Asn Ala     50 55 60 Ala Phe Met Ala Gln Ala Val Gly Arg Leu Thr Gly Lys Pro Gly Val 65 70 75 80 Val Leu Val Thr Ser Gly Pro Gly Ala Ser Asn Leu Ala Thr Gly Leu                 85 90 95 Leu Thr Ala Asn Thr Glu Gly Asp Pro Val Val Ala Leu Ala Gly Asn             100 105 110 Val Ile Arg Ala Asp Arg Leu Lys Arg Thr His Gln Ser Leu Asp Asn         115 120 125 Ala Ala Leu Phe Gln Pro Ile Thr Lys Tyr Ser Val Glu Val Gln Asp     130 135 140 Val Lys Asn Ile Pro Glu Ala Val Thr Asn Ala Phe Arg Ile Ala Ser 145 150 155 160 Ala Gly Gln Ala Gly Ala Ala Phe Val Ser Phe Pro Gln Asp Val Val                 165 170 175 Asn Glu Val Thr Asn Thr Lys Asn Val Arg Ala Val Ala Ala Pro Lys             180 185 190 Leu Gly Pro Ala Ala Asp Asp Ala Ile Ser Ala Ala Ile Ala Lys Ile         195 200 205 Gln Thr Ala Lys Leu Pro Val Val Leu Val Gly Met Lys Gly Gly Arg     210 215 220 Pro Glu Ala Ile Lys Ala Val Arg Lys Leu Leu Lys Lys Val Gln Leu 225 230 235 240 Pro Phe Val Glu Thr Tyr Gln Ala Ala Gly Thr Leu Ser Arg Asp Leu                 245 250 255 Glu Asp Gln Tyr Phe Gly Arg Ile Gly Leu Phe Arg Asn Gln Pro Gly             260 265 270 Asp Leu Leu Leu Glu Gln Ala Asp Val Val Leu Thr Ile Gly Tyr Asp         275 280 285 Pro Ile Glu Tyr Asp Pro Lys Phe Trp Asn Ile Asn Gly Asp Arg Thr     290 295 300 Ile Ile His Leu Asp Glu Ile Ile Ala Asp Ile Asp His Ala Tyr Gln 305 310 315 320 Pro Asp Leu Glu Leu Ile Gly Asp Ile Pro Ser Thr Ile Asn His Ile                 325 330 335 Glu His Asp Ala Val Lys Val Glu Phe Ala Glu Arg Glu Gln Lys Ile             340 345 350 Leu Ser Asp Leu Lys Gln Tyr Met His Glu Gly Glu Gln Val Pro Ala         355 360 365 Asp Trp Lys Ser Asp Arg Ala His Pro Leu Glu Ile Val Lys Glu Leu     370 375 380 Arg Asn Ala Val Asp Asp His Val Thr Val Thr Cys Asp Ile Gly Ser 385 390 395 400 His Ala Ile Trp Met Ser Arg Tyr Phe Arg Ser Tyr Glu Pro Leu Thr                 405 410 415 Leu Met Ile Ser Asn Gly Met Gln Thr Leu Gly Val Ala Leu Pro Trp             420 425 430 Ala Ile Gly Ala Ser Leu Val Lys Pro Gly Glu Lys Val Val Ser Val         435 440 445 Ser Gly Asp Gly Gly Phe Leu Phe Ser Ala Met Glu Leu Glu Thr Ala     450 455 460 Val Arg Leu Lys Ala Pro Ile Val His Ile Val Trp Asn Asp Ser Thr 465 470 475 480 Tyr Asp Met Val Ala Phe Gln Gln Leu Lys Lys Tyr Asn Arg Thr Ser                 485 490 495 Ala Val Asp Phe Gly Asn Ile Asp Ile Val Lys Tyr Ala Glu Ser Phe             500 505 510 Gly Ala Thr Gly Leu Arg Val Glu Ser Pro Asp Gln Leu Ala Asp Val         515 520 525 Leu Arg Gln Gly Met Asn Ala Glu Gly Pro Val Ile Ile Asp Val Pro     530 535 540 Val Asp Tyr Ser Asp Asn Ile Asn Leu Ala Ser Asp Lys Leu Pro Lys 545 550 555 560 Glu Phe Gly Glu Leu Met Lys Thr Lys Ala Leu                 565 570 <210> 139 <211> 448 <212> DNA <213> Saccharomyces cerevisiae <400> 139 cccattaccg acatttgggc gctatacgtg catatgttca tgtatgtatc tgtatttaaa 60 acacttttgt attatttttc ctcatatatg tgtataggtt tatacggatg atttaattat 120 tacttcacca ccctttattt caggctgata tcttagcctt gttactagtt agaaaaagac 180 atttttgctg tcagtcactg tcaagagatt cttttgctgg catttcttct agaagcaaaa 240 agagcgatgc gtcttttccg ctgaaccgtt ccagcaaaaa agactaccaa cgcaatatgg 300 attgtcagaa tcatataaaa gagaagcaaa taactccttg tcttgtatca attgcattat 360 aatatcttct tgttagtgca atatcatata gaagtcatcg aaatagatat taagaaaaac 420 aaactgtaca atcaatcaat caatcatc 448 <210> 140 <211> 16387 <212> DNA <213> artificial sequence <220> <223> plasmid construct <400> 140 tcccattacc gacatttggg cgctatacgt gcatatgttc atgtatgtat ctgtatttaa 60 aacacttttg tattattttt cctcatatat gtgtataggt ttatacggat gatttaatta 120 ttacttcacc accctttatt tcaggctgat atcttagcct tgttactagt tagaaaaaga 180 catttttgct gtcagtcact gtcaagagat tcttttgctg gcatttcttc tagaagcaaa 240 aagagcgatg cgtcttttcc gctgaaccgt tccagcaaaa aagactacca acgcaatatg 300 gattgtcaga atcatataaa agagaagcaa ataactcctt gtcttgtatc aattgcatta 360 taatatcttc ttgttagtgc aatatcatat agaagtcatc gaaatagata ttaagaaaaa 420 caaactgtac aatcaatcaa tcaatcatcg ctgaggatgt tgacaaaagc aacaaaagaa 480 caaaaatccc ttgtgaaaaa cagaggggcg gagcttgttg ttgattgctt agtggagcaa 540 ggtgtcacac atgtatttgg cattccaggt gcaaaaattg atgcggtatt tgacgcttta 600 caagataaag gacctgaaat tatcgttgcc cggcacgaac aaaacgcagc attcatggcc 660 caagcagtcg gccgtttaac tggaaaaccg ggagtcgtgt tagtcacatc aggaccgggt 720 gcctctaact tggcaacagg cctgctgaca gcgaacactg aaggagaccc tgtcgttgcg 780 cttgctggaa acgtgatccg tgcagatcgt ttaaaacgga cacatcaatc tttggataat 840 gcggcgctat tccagccgat tacaaaatac agtgtagaag ttcaagatgt aaaaaatata 900 ccggaagctg ttacaaatgc atttaggata gcgtcagcag ggcaggctgg ggccgctttt 960 gtgagctttc cgcaagatgt tgtgaatgaa gtcacaaata cgaaaaacgt gcgtgctgtt 1020 gcagcgccaa aactcggtcc tgcagcagat gatgcaatca gtgcggccat agcaaaaatc 1080 caaacagcaa aacttcctgt cgttttggtc ggcatgaaag gcggaagacc ggaagcaatt 1140 aaagcggttc gcaagctttt gaaaaaggtt cagcttccat ttgttgaaac atatcaagct 1200 gccggtaccc tttctagaga tttagaggat caatattttg gccgtatcgg tttgttccgc 1260 aaccagcctg gcgatttact gctagagcag gcagatgttg ttctgacgat cggctatgac 1320 ccgattgaat atgatccgaa attctggaat atcaatggag accggacaat tatccattta 1380 gacgagatta tcgctgacat tgatcatgct taccagcctg atcttgaatt gatcggtgac 1440 attccgtcca cgatcaatca tatcgaacac gatgctgtga aagtggaatt tgcagagcgt 1500 gagcagaaaa tcctttctga tttaaaacaa tatatgcatg aaggtgagca ggtgcctgca 1560 gattggaaat cagacagagc gcaccctctt gaaatcgtta aagagttgcg taatgcagtc 1620 gatgatcatg ttacagtaac ttgcgatatc ggttcgcacg ccatttggat gtcacgttat 1680 ttccgcagct acgagccgtt aacattaatg atcagtaacg gtatgcaaac actcggcgtt 1740 gcgcttcctt gggcaatcgg cgcttcattg gtgaaaccgg gagaaaaagt ggtttctgtc 1800 tctggtgacg gcggtttctt attctcagca atggaattag agacagcagt tcgactaaaa 1860 gcaccaattg tacacattgt atggaacgac agcacatatg acatggttgc attccagcaa 1920 ttgaaaaaat ataaccgtac atctgcggtc gatttcggaa atatcgatat cgtgaaatat 1980 gcggaaagct tcggagcaac tggcttgcgc gtagaatcac cagaccagct ggcagatgtt 2040 ctgcgtcaag gcatgaacgc tgaaggtcct gtcatcatcg atgtcccggt tgactacagt 2100 gataacatta atttagcaag tgacaagctt ccgaaagaat tcggggaact catgaaaacg 2160 aaagctctct agttaattaa tcatgtaatt agttatgtca cgcttacatt cacgccctcc 2220 ccccacatcc gctctaaccg aaaaggaagg agttagacaa cctgaagtct aggtccctat 2280 ttattttttt atagttatgt tagtattaag aacgttattt atatttcaaa tttttctttt 2340 ttttctgtac agacgcgtgt acgcatgtaa cattatactg aaaaccttgc ttgagaaggt 2400 tttgggacgc tcgaaggctt taatttgcgg gcggccgctc tagaactagt accacaggtg 2460 ttgtcctctg aggacataaa atacacaccg agattcatca actcattgct ggagttagca 2520 tatctacaat tgggtgaaat ggggagcgat ttgcaggcat ttgctcggca tgccggtaga 2580 ggtgtggtca ataagagcga cctcatgcta tacctgagaa agcaacctga cctacaggaa 2640 agagttactc aagaataaga attttcgttt taaaacctaa gagtcacttt aaaatttgta 2700 tacacttatt ttttttataa cttatttaat aataaaaatc ataaatcata agaaattcgc 2760 ttactcttaa ttaatcaagc atctaaaaca caaccgttgg aagcgttgga aaccaactta 2820 gcatacttgg atagagtacc tcttgtgtaa cgaggtggag gtgcaaccca actttgttta 2880 cgttgagcca tttccttatc agagactaat aggtcaatct tgttattatc agcatcaatg 2940 ataatctcat cgccgtctct gaccaacccg ataggaccac cttcagcggc ttcgggaaca 3000 atgtggccga ttaagaaccc gtgagaacca ccagagaatc taccatcagt caacaatgca 3060 acatctttac ccaaaccgta acccatcaga gcagaggaag gctttagcat ttcaggcata 3120 cctggtgcac ctcttggacc ttcatatctg ataacaacaa cggttttttc acccttcttg 3180 atttcacctc tttccaaggc ttcaataaag gcaccttcct cttcgaacac acgtgctcta 3240 cccttgaagt aagtaccttc cttaccggta attttaccca cagctccacc tggtgccaat 3300 gaaccgtaca gaatttgcaa gtgaccgttg gccttgattg ggtgggagag tggcttaata 3360 atctcttgtc cttcaggtag gcttggtgct ttctttgcac gttctgccaa agtgtcaccg 3420 gtaacagtca ttgtgttacc gtgcaacatg ttgttttcat atagatactt aatcacagat 3480 tgggtaccac caacgttaat caaatcggcc atgacgtatt taccagaagg tttgaagtca 3540 ccgatcaatg gtgtagtatc actgattctt tggaaatcat ctggtgacaa cttgacaccc 3600 gcagagtgag caacagccac caaatgcaaa acagcattag tggacccacc ggttgcaacg 3660 acataagtaa tggcgttttc aaaagcctct tttgtgagga tatcacgagg taaaataccc 3720 aattccattg tcttcttgat gtattcacca atgttgtcac actcagctaa cttctccttg 3780 gaaacggctg ggaaggaaga ggagtttgga atggtcaaac ctagcacttc agcggcagaa 3840 gccattgtgt tggcagtata cataccacca caagaaccag gacctgggca tgcatgttcc 3900 acaacatctt ctctttcttc ttcagtgaat tgcttggaaa tatattcacc gtaggattgg 3960 aacgcagaga cgatatcgat gtttttagag atcctgttaa aacctctagt ggagtagtag 4020 atgtaatcaa tgaagcggaa gccaaaagac cagagtagag gcctatagaa gaaactgcga 4080 taccttttgt gatggctaaa caaacagaca tctttttata tgtttttact tctgtatatc 4140 gtgaagtagt aagtgataag cgaatttggc taagaacgtt gtaagtgaac aagggacctc 4200 ttttgccttt caaaaaagga ttaaatggag ttaatcattg agatttagtt ttcgttagat 4260 tctgtatccc taaataactc ccttacccga cgggaaggca caaaagactt gaataatagc 4320 aaacggccag tagccaagac caaataatac tagagttaac tgatggtctt aaacaggcat 4380 tacgtggtga actccaagac caatatacaa aatatcgata agttattctt gcccaccaat 4440 ttaaggagcc tacatcagga cagtagtacc attcctcaga gaagaggtat acataacaag 4500 aaaatcgcgt gaacacctta tataacttag cccgttattg agctaaaaaa ccttgcaaaa 4560 tttcctatga ataagaatac ttcagacgtg ataaaaattt actttctaac tcttctcacg 4620 ctgcccctat ctgttcttcc gctctaccgt gagaaataaa gcatcgagta cggcagttcg 4680 ctgtcactga actaaaacaa taaggctagt tcgaatgatg aacttgcttg ctgtcaaact 4740 tctgagttgc cgctgatgtg acactgtgac aataaattca aaccggttat agcggtctcc 4800 tccggtaccg gttctgccac ctccaataga gctcagtagg agtcagaacc tctgcggtgg 4860 ctgtcagtga ctcatccgcg tttcgtaagt tgtgcgcgtg cacatttcgc ccgttcccgc 4920 tcatcttgca gcaggcggaa attttcatca cgctgtagga cgcaaaaaaa aaataattaa 4980 tcgtacaaga atcttggaaa aaaaattgaa aaattttgta taaaagggat gacctaactt 5040 gactcaatgg cttttacacc cagtattttc cctttccttg tttgttacaa ttatagaagc 5100 aagacaaaaa catatagaca acctattcct aggagttata tttttttacc ctaccagcaa 5160 tataagtaaa aaactagtat gaaggtgttt tacgataaag actgcgatct gagcatcatc 5220 cagggaaaga aggttgctat tataggatat ggttcccaag gacacgcaca agccttgaac 5280 ttgaaagatt ctggggtcga cgtgacagta ggtctgtata aaggtgctgc tgatgcagca 5340 aaggctgaag cacatggctt taaagtcaca gatgttgcag cggctgttgc tggcgctgat 5400 ttagtcatga ttttaattcc agatgaattt caatcgcaat tgtacaaaaa tgaaatagaa 5460 ccaaacatta agaagggcgc taccttggcc ttcagtcatg gatttgccat tcattacaat 5520 caagtagtcc ccagggcaga tttggacgtt attatgattg cacctaaggc tccggggcat 5580 actgttagga gcgaatttgt taagggtggt ggtattccag atttgatcgc tatataccaa 5640 gacgttagcg gaaacgctaa gaatgtagct ttaagctacg cagcaggagt tggtggcggg 5700 agaacgggta taatagaaac cacttttaaa gacgagactg agacagattt atttggagaa 5760 caagcggttc tgtgcggagg aactgttgaa ttggttaaag caggctttga gacgcttgtc 5820 gaagcagggt acgctcccga aatggcatac ttcgaatgtc tacatgaatt gaagttgata 5880 gtagacttaa tgtatgaagg tggtatagct aatatgaact attccatttc aaataatgca 5940 gaatatggtg agtatgtcac cggacctgaa gtcattaacg cagaatcaag acaagccatg 6000 agaaatgcct tgaaacgtat ccaggacggt gaatacgcta agatgttcat aagtgaaggc 6060 gctacgggtt acccgagtat gactgctaaa agaagaaaca atgcagcaca tggtatcgaa 6120 attattggtg aacagttaag gtctatgatg ccctggatcg gtgctaataa gatcgtagac 6180 aaggcgaaaa attaaggccc tgcaggccta tcaagtgctg gaaacttttt ctcttggaat 6240 ttttgcaaca tcaagtcata gtcaattgaa ttgacccaat ttcacattta agattttttt 6300 tttttcatcc gacatacatc tgtacactag gaagccctgt ttttctgaag cagcttcaaa 6360 tatatatatt ttttacatat ttattatgat tcaatgaaca atctaattaa atcgaaaaca 6420 agaaccgaaa cgcgaataaa taatttattt agatggtgac aagtgtataa gtcctcatcg 6480 ggacagctac gatttctctt tcggttttgg ctgagctact ggttgctgtg acgcagcggc 6540 attagcgcgg cgttatgagc taccctcgtg gcctgaaaga tggcgggaat aaagcggaac 6600 taaaaattac tgactgagcc atattgaggt caatttgtca actcgtcaag tcacgtttgg 6660 tggacggccc ctttccaacg aatcgtatat actaacatgc gcgcgcttcc tatatacaca 6720 tatacatata tatatatata tatatgtgtg cgtgtatgtg tacacctgta tttaatttcc 6780 ttactcgcgg gtttttcttt tttctcaatt cttggcttcc tctttctcga gtatataatt 6840 tttcaggtaa aatttagtac gatagtaaaa tacttctcga actcgtcaca tatacgtgta 6900 cataatgtct gaaccagctc aaaagaaaca aaaggttgct aacaactctc tagagcggcc 6960 gcccgcaaat taaagccttc gagcgtccca aaaccttctc aagcaaggtt ttcagtataa 7020 tgttacatgc gtacacgcgt ctgtacagaa aaaaaagaaa aatttgaaat ataaataacg 7080 ttcttaatac taacataact ataaaaaaat aaatagggac ctagacttca ggttgtctaa 7140 ctccttcctt ttcggttaga gcggatgtgg ggggagggcg tgaatgtaag cgtgacataa 7200 ctaattacat gattaattaa ttattggttt tctggtctca actttctgac ttccttacca 7260 accttccaga tttccatgtt tctgatggtg tctaattcct tttctagctt ttctctgtag 7320 tcaggttgag agttgaattc caaagatctc ttggtttcgg taccgttctt ggtagattcg 7380 tacaagtctt ggaaaacagg cttcaaagca ttcttgaaga ttgggtacca gtccaaagca 7440 cctcttctgg cggtggtgga acaagcatcg tacatgtaat ccataccgta cttaccgatc 7500 aatgggtata gagattgggt agcttcttcg acggtttcgt tgaaagcttc agatggggag 7560 tgaccgtttt ctctcaagac gtcgtattga gccaagaaca taccgtggat accacccatt 7620 aaacaacctc tttcaccgta caagtcagag ttgacttctc tttcgaaagt ggtttggtaa 7680 acgtaaccgg aaccaatggc aacggccaaa gcttgggcct tttcgtgagc cttaccggtg 7740 acatcgttcc agacggcgta agaagagtta ataccacgac cttccttgaa caaagatctg 7800 acagttctac cggaaccctt tggagcaacc aagataacat ctaagtcctt tggtggttca 7860 acgtgagtca agtccttgaa gactggggag aaaccgtggg agaagtacaa agtcttaccc 7920 ttggtcaaca atggcttgat agcaggccag gtttctgatt gagcggcatc ggacaacaag 7980 ttcataacgt aactacctct cttgatagca tcttcaacag tgaacaagtt cttgcctgga 8040 acccaaccgt cttcgatggc agccttccaa gaagcaccat ctttacggac accaatgata 8100 acgttcaaac cgttgtctct caagttcaaa ccttgaccgt aaccttggga accgtaaccg 8160 atcaaagcaa aagtgtcgtt cttgaagtag tccaacaact tttctcttgg ccagtcagct 8220 ctttcgtaga cggtttcaac agtaccaccg aagttgattt gcttcaacat cctcagctct 8280 agatttgaat atgtattact tggttatggt tatatatgac aaaagaaaaa gaagaacaga 8340 agaataacgc aaggaagaac aataactgaa attgatagag aagtattatg tctttgtctt 8400 tttataataa atcaagtgca gaaatccgtt agacaacatg agggataaaa tttaacgtgg 8460 gcgaagaaga aggaaaaaag tttttgtgag ggcgtaattg aagcgatctg ttgattgtag 8520 attttttttt tttgaggagt caaagtcaga agagaacaga caaatggtat taaccatcca 8580 atactttttt ggagcaacgc taagctcatg cttttccatt ggttacgtgc tcagttgtta 8640 gatatggaaa gagaggatgc tcacggcagc gtgactccaa ttgagcccga aagagaggat 8700 gccacgtttt cccgacggct gctagaatgg aaaaaggaaa aatagaagaa tcccattcct 8760 atcattattt acgtaatgac ccacacattt ttgagatttt caactattac gtattacgat 8820 aatcctgctg tcattatcat tattatctat atcgacgtat gcaacgtatg tgaagccaag 8880 taggcaatta tttagtactg tcagtattgt tattcatttc agatctatcc gcggtggagc 8940 tcgaattcac tggccgtcgt tttacaacgt cgtgactggg aaaaccctgg cgttacccaa 9000 cttaatcgcc ttgcagcaca tccccctttc gccagctggc gtaatagcga agaggcccgc 9060 accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg aatggcgcct gatgcggtat 9120 tttctcctta cgcatctgtg cggtatttca caccgcatac gtcaaagcaa ccatagtacg 9180 cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta 9240 cacttgccag cgccttagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt 9300 tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg 9360 ctttacggca cctcgacccc aaaaaacttg atttgggtga tggttcacgt agtgggccat 9420 cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac 9480 tcttgttcca aactggaaca acactcaact ctatctcggg ctattctttt gatttataag 9540 ggattttgcc gatttcggtc tattggttaa aaaatgagct gatttaacaa aaatttaacg 9600 cgaattttaa caaaatatta acgtttacaa ttttatggtg cactctcagt acaatctgct 9660 ctgatgccgc atagttaagc cagccccgac acccgccaac acccgctgac gcgccctgac 9720 gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca 9780 tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac 9840 gcctattttt ataggttaat gtcatgataa taatggtttc ttagacgtca ggtggcactt 9900 ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt 9960 atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta 10020 tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg 10080 tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac 10140 gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg 10200 aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc 10260 gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg 10320 ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat 10380 gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg 10440 gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg 10500 atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc 10560 ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt 10620 cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct 10680 cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc 10740 gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca 10800 cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct 10860 cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt 10920 taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga 10980 ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca 11040 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac 11100 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg 11160 taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag 11220 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac 11280 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt 11340 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg 11400 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc 11460 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc 11520 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc 11580 acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa 11640 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt 11700 tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg 11760 ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag 11820 agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc 11880 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc 11940 tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa 12000 ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac gccaagcttt 12060 ttctttccaa tttttttttt ttcgtcatta taaaaatcat tacgaccgag attcccgggt 12120 aataactgat ataattaaat tgaagctcta atttgtgagt ttagtataca tgcatttact 12180 tataatacag ttttttagtt ttgctggccg catcttctca aatatgcttc ccagcctgct 12240 tttctgtaac gttcaccctc taccttagca tcccttccct ttgcaaatag tcctcttcca 12300 acaataataa tgtcagatcc tgtagagacc acatcatcca cggttctata ctgttgaccc 12360 aatgcgtctc ccttgtcatc taaacccaca ccgggtgtca taatcaacca atcgtaacct 12420 tcatctcttc cacccatgtc tctttgagca ataaagccga taacaaaatc tttgtcgctc 12480 ttcgcaatgt caacagtacc cttagtatat tctccagtag atagggagcc cttgcatgac 12540 aattctgcta acatcaaaag gcctctaggt tcctttgtta cttcttctgc cgcctgcttc 12600 aaaccgctaa caatacctgg gcccaccaca ccgtgtgcat tcgtaatgtc tgcccattct 12660 gctattctgt atacacccgc agagtactgc aatttgactg tattaccaat gtcagcaaat 12720 tttctgtctt cgaagagtaa aaaattgtac ttggcggata atgcctttag cggcttaact 12780 gtgccctcca tggaaaaatc agtcaagata tccacatgtg tttttagtaa acaaattttg 12840 ggacctaatg cttcaactaa ctccagtaat tccttggtgg tacgaacatc caatgaagca 12900 cacaagtttg tttgcttttc gtgcatgata ttaaatagct tggcagcaac aggactagga 12960 tgagtagcag cacgttcctt atatgtagct ttcgacatga tttatcttcg tttcctgcag 13020 gtttttgttc tgtgcagttg ggttaagaat actgggcaat ttcatgtttc ttcaacacta 13080 catatgcgta tatataccaa tctaagtctg tgctccttcc ttcgttcttc cttctgttcg 13140 gagattaccg aatcaaaaaa atttcaagga aaccgaaatc aaaaaaaaga ataaaaaaaa 13200 aatgatgaat tgaaaagctt gcatgcctgc aggtcgactc tagtatactc cgtctactgt 13260 acgatacact tccgctcagg tccttgtcct ttaacgaggc cttaccactc ttttgttact 13320 ctattgatcc agctcagcaa aggcagtgtg atctaagatt ctatcttcgc gatgtagtaa 13380 aactagctag accgagaaag agactagaaa tgcaaaaggc acttctacaa tggctgccat 13440 cattattatc cgatgtgacg ctgcattttt tttttttttt tttttttttt tttttttttt 13500 tttttttttt tttttttgta caaatatcat aaaaaaagag aatcttttta agcaaggatt 13560 ttcttaactt cttcggcgac agcatcaccg acttcggtgg tactgttgga accacctaaa 13620 tcaccagttc tgatacctgc atccaaaacc tttttaactg catcttcaat ggctttacct 13680 tcttcaggca agttcaatga caatttcaac atcattgcag cagacaagat agtggcgata 13740 gggttgacct tattctttgg caaatctgga gcggaaccat ggcatggttc gtacaaacca 13800 aatgcggtgt tcttgtctgg caaagaggcc aaggacgcag atggcaacaa acccaaggag 13860 cctgggataa cggaggcttc atcggagatg atatcaccaa acatgttgct ggtgattata 13920 ataccattta ggtgggttgg gttcttaact aggatcatgg cggcagaatc aatcaattga 13980 tgttgaactt tcaatgtagg gaattcgttc ttgatggttt cctccacagt ttttctccat 14040 aatcttgaag aggccaaaac attagcttta tccaaggacc aaataggcaa tggtggctca 14100 tgttgtaggg ccatgaaagc ggccattctt gtgattcttt gcacttctgg aacggtgtat 14160 tgttcactat cccaagcgac accatcacca tcgtcttcct ttctcttacc aaagtaaata 14220 cctcccacta attctctaac aacaacgaag tcagtacctt tagcaaattg tggcttgatt 14280 ggagataagt ctaaaagaga gtcggatgca aagttacatg gtcttaagtt ggcgtacaat 14340 tgaagttctt tacggatttt tagtaaacct tgttcaggtc taacactacc ggtaccccat 14400 ttaggaccac ccacagcacc taacaaaacg gcatcagcct tcttggaggc ttccagcgcc 14460 tcatctggaa gtggaacacc tgtagcatcg atagcagcac caccaattaa atgattttcg 14520 aaatcgaact tgacattgga acgaacatca gaaatagctt taagaacctt aatggcttcg 14580 gctgtgattt cttgaccaac gtggtcacct ggcaaaacga cgatcttctt aggggcagac 14640 attacaatgg tatatccttg aaatatatat aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 14700 tgcagcttct caatgatatt cgaatacgct ttgaggagat acagcctaat atccgacaaa 14760 ctgttttaca gatttacgat cgtacttgtt acccatcatt gaattttgaa catccgaacc 14820 tgggagtttt ccctgaaaca gatagtatat ttgaacctgt ataataatat atagtctagc 14880 gctttacgga agacaatgta tgtatttcgg ttcctggaga aactattgca tctattgcat 14940 aggtaatctt gcacgtcgca tccccggttc attttctgcg tttccatctt gcacttcaat 15000 agcatatctt tgttaacgaa gcatctgtgc ttcattttgt agaacaaaaa tgcaacgcga 15060 gagcgctaat ttttcaaaca aagaatctga gctgcatttt tacagaacag aaatgcaacg 15120 cgaaagcgct attttaccaa cgaagaatct gtgcttcatt tttgtaaaac aaaaatgcaa 15180 cgcgagagcg ctaatttttc aaacaaagaa tctgagctgc atttttacag aacagaaatg 15240 caacgcgaga gcgctatttt accaacaaag aatctatact tcttttttgt tctacaaaaa 15300 tgcatcccga gagcgctatt tttctaacaa agcatcttag attacttttt ttctcctttg 15360 tgcgctctat aatgcagtct cttgataact ttttgcactg taggtccgtt aaggttagaa 15420 gaaggctact ttggtgtcta ttttctcttc cataaaaaaa gcctgactcc acttcccgcg 15480 tttactgatt actagcgaag ctgcgggtgc attttttcaa gataaaggca tccccgatta 15540 tattctatac cgatgtggat tgcgcatact ttgtgaacag aaagtgatag cgttgatgat 15600 tcttcattgg tcagaaaatt atgaacggtt tcttctattt tgtctctata tactacgtat 15660 aggaaatgtt tacattttcg tattgttttc gattcactct atgaatagtt cttactacaa 15720 tttttttgtc taaagagtaa tactagagat aaacataaaa aatgtagagg tcgagtttag 15780 atgcaagttc aaggagcgaa aggtggatgg gtaggttata tagggatata gcacagagat 15840 atatagcaaa gagatacttt tgagcaatgt ttgtggaagc ggtattcgca atattttagt 15900 agctcgttac agtccggtgc gtttttggtt ttttgaaagt gcgtcttcag agcgcttttg 15960 gttttcaaaa gcgctctgaa gttcctatac tttctagaga ataggaactt cggaatagga 16020 acttcaaagc gtttccgaaa acgagcgctt ccgaaaatgc aacgcgagct gcgcacatac 16080 agctcactgt tcacgtcgca cctatatctg cgtgttgcct gtatatatat atacatgaga 16140 agaacggcat agtgcgtgtt tatgcttaaa tgcgtactta tatgcgtcta tttatgtagg 16200 atgaaaggta gtctagtacc tcctgtgata ttatcccatt ccatgcgggg tatcgtatgc 16260 ttccttcagc actacccttt agctgttcta tatgctgcca ctcctcaatt ggattagtct 16320 catccttcaa tgctatcatt tcctttgata ttggatcata tgcatagtac cgagaaacta 16380 gaggatc 16387 <210> 141 <211> 2237 <212> DNA <213> Artificial sequence <220> <223> Fragment <400> 141 ttatgtatgc tcttctgact tttcgtgtga tgaggctcgt ggaaaaaatg aataatttat 60 gaatttgaga acaattttgt gttgttacgg tattttacta tggaataatc aatcaattga 120 ggattttatg caaatatcgt ttgaatattt ttccgaccct ttgagtactt ttcttcataa 180 ttgcataata ttgtccgctg cccctttttc tgttagacgg tgtcttgatc tacttgctat 240 cgttcaacac caccttattt tctaactatt ttttttttag ctcatttgaa tcagcttatg 300 gtgatggcac atttttgcat aaacctagct gtcctcgttg aacataggaa aaaaaaatat 360 ataaacaagg ctctttcact ctccttgcaa tcagatttgg gtttgttccc tttattttca 420 tatttcttgt catattcctt tctcaattat tattttctac tcataacctc acgcaaaata 480 acacagtcaa atcaatcaaa atgactgaca aaaaaactct taaagactta agaaatcgta 540 gttctgttta cgattcaatg gttaaatcac ctaatcgtgc tatgttgcgt gcaactggta 600 tgcaagatga agactttgaa aaacctatcg tcggtgtcat ttcaacttgg gctgaaaaca 660 caccttgtaa tatccactta catgactttg gtaaactagc caaagtcggt gttaaggaag 720 ctggtgcttg gccagttcag ttcggaacaa tcacggtttc tgatggaatc gccatgggaa 780 cccaaggaat gcgtttctcc ttgacatctc gtgatattat tgcagattct attgaagcag 840 ccatgggagg tcataatgcg gatgcttttg tagccattgg cggttgtgat aaaaacatgc 900 ccggttctgt tatcgctatg gctaacatgg atatcccagc catttttgct tacggcggaa 960 caattgcacc tggtaattta gacggcaaag atatcgattt agtctctgtc tttgaaggtg 1020 tcggccattg gaaccacggc gatatgacca aagaagaagt taaagctttg gaatgtaatg 1080 cttgtcccgg tcctggaggc tgcggtggta tgtatactgc taacacaatg gcgacagcta 1140 ttgaagtttt gggacttagc cttccgggtt catcttctca cccggctgaa tccgcagaaa 1200 agaaagcaga tattgaagaa gctggtcgcg ctgttgtcaa aatgctcgaa atgggcttaa 1260 aaccttctga cattttaacg cgtgaagctt ttgaagatgc tattactgta actatggctc 1320 tgggaggttc aaccaactca acccttcacc tcttagctat tgcccatgct gctaatgtgg 1380 aattgacact tgatgatttc aatactttcc aagaaaaagt tcctcatttg gctgatttga 1440 aaccttctgg tcaatatgta ttccaagacc tttacaaggt cggaggggta ccagcagtta 1500 tgaaatatct ccttaaaaat ggcttccttc atggtgaccg tatcacttgt actggcaaaa 1560 cagtcgctga aaatttgaag gcttttgatg atttaacacc tggtcaaaag gttattatgc 1620 cgcttgaaaa tcctaaacgt gaagatggtc cgctcattat tctccatggt aacttggctc 1680 cagacggtgc cgttgccaaa gtttctggtg taaaagtgcg tcgtcatgtc ggtcctgcta 1740 aggtctttaa ttctgaagaa gaagccattg aagctgtctt gaatgatgat attgttgatg 1800 gtgatgttgt tgtcgtacgt tttgtaggac caaagggcgg tcctggtatg cctgaaatgc 1860 tttccctttc atcaatgatt gttggtaaag ggcaaggtga aaaagttgcc cttctgacag 1920 atggccgctt ctcaggtggt acttatggtc ttgtcgtggg tcatatcgct cctgaagcac 1980 aagatggcgg tccaatcgcc tacctgcaaa caggagacat agtcactatt gaccaagaca 2040 ctaaggaatt acactttgat atctccgatg aagagttaaa acatcgtcaa gagaccattg 2100 aattgccacc gctctattca cgcggtatcc ttggtaaata tgctcacatc gtttcgtctg 2160 cttctagggg agccgtaaca gacttttgga agcctgaaga aactggcaaa aaatgagcga 2220 tttaatctct aattatt 2237 <210> 142 <211> 4420 <212> DNA <213> Artificial sequence <220> <223> Cassette <400> 142 ttatgtatgc tcttctgact tttcgtgtga tgaggctcgt ggaaaaaatg aataatttat 60 gaatttgaga acaattttgt gttgttacgg tattttacta tggaataatc aatcaattga 120 ggattttatg caaatatcgt ttgaatattt ttccgaccct ttgagtactt ttcttcataa 180 ttgcataata ttgtccgctg cccctttttc tgttagacgg tgtcttgatc tacttgctat 240 cgttcaacac caccttattt tctaactatt ttttttttag ctcatttgaa tcagcttatg 300 gtgatggcac atttttgcat aaacctagct gtcctcgttg aacataggaa aaaaaaatat 360 ataaacaagg ctctttcact ctccttgcaa tcagatttgg gtttgttccc tttattttca 420 tatttcttgt catattcctt tctcaattat tattttctac tcataacctc acgcaaaata 480 acacagtcaa atcaatcaaa atgactgaca aaaaaactct taaagactta agaaatcgta 540 gttctgttta cgattcaatg gttaaatcac ctaatcgtgc tatgttgcgt gcaactggta 600 tgcaagatga agactttgaa aaacctatcg tcggtgtcat ttcaacttgg gctgaaaaca 660 caccttgtaa tatccactta catgactttg gtaaactagc caaagtcggt gttaaggaag 720 ctggtgcttg gccagttcag ttcggaacaa tcacggtttc tgatggaatc gccatgggaa 780 cccaaggaat gcgtttctcc ttgacatctc gtgatattat tgcagattct attgaagcag 840 ccatgggagg tcataatgcg gatgcttttg tagccattgg cggttgtgat aaaaacatgc 900 ccggttctgt tatcgctatg gctaacatgg atatcccagc catttttgct tacggcggaa 960 caattgcacc tggtaattta gacggcaaag atatcgattt agtctctgtc tttgaaggtg 1020 tcggccattg gaaccacggc gatatgacca aagaagaagt taaagctttg gaatgtaatg 1080 cttgtcccgg tcctggaggc tgcggtggta tgtatactgc taacacaatg gcgacagcta 1140 ttgaagtttt gggacttagc cttccgggtt catcttctca cccggctgaa tccgcagaaa 1200 agaaagcaga tattgaagaa gctggtcgcg ctgttgtcaa aatgctcgaa atgggcttaa 1260 aaccttctga cattttaacg cgtgaagctt ttgaagatgc tattactgta actatggctc 1320 tgggaggttc aaccaactca acccttcacc tcttagctat tgcccatgct gctaatgtgg 1380 aattgacact tgatgatttc aatactttcc aagaaaaagt tcctcatttg gctgatttga 1440 aaccttctgg tcaatatgta ttccaagacc tttacaaggt cggaggggta ccagcagtta 1500 tgaaatatct ccttaaaaat ggcttccttc atggtgaccg tatcacttgt actggcaaaa 1560 cagtcgctga aaatttgaag gcttttgatg atttaacacc tggtcaaaag gttattatgc 1620 cgcttgaaaa tcctaaacgt gaagatggtc cgctcattat tctccatggt aacttggctc 1680 cagacggtgc cgttgccaaa gtttctggtg taaaagtgcg tcgtcatgtc ggtcctgcta 1740 aggtctttaa ttctgaagaa gaagccattg aagctgtctt gaatgatgat attgttgatg 1800 gtgatgttgt tgtcgtacgt tttgtaggac caaagggcgg tcctggtatg cctgaaatgc 1860 tttccctttc atcaatgatt gttggtaaag ggcaaggtga aaaagttgcc cttctgacag 1920 atggccgctt ctcaggtggt acttatggtc ttgtcgtggg tcatatcgct cctgaagcac 1980 aagatggcgg tccaatcgcc tacctgcaaa caggagacat agtcactatt gaccaagaca 2040 ctaaggaatt acactttgat atctccgatg aagagttaaa acatcgtcaa gagaccattg 2100 aattgccacc gctctattca cgcggtatcc ttggtaaata tgctcacatc gtttcgtctg 2160 cttctagggg agccgtaaca gacttttgga agcctgaaga aactggcaaa aaatgagcga 2220 tttaatctct aattattagt taaagtttta taagcatttt tatgtaacga aaaataaatt 2280 ggttcatatt attactgcac tgtcacttac catggaaaga ccagacaaga agttgccgac 2340 agtctgttga attggcctgg ttaggcttaa gtctgggtcc gcttctttac aaatttggag 2400 aatttctctt aaacgatatg tatattcttt tcgttggaaa agatgtcttc caaaaaaaaa 2460 accgatgaat tagtggaacc aaggaaaaaa aaagaggtat ccttgattaa ggaacactgt 2520 ttaaacagtg tggtttccaa aaccctgaaa ctgcattagt gtaatagaag actagacacc 2580 tcgatacaaa taatggttac tcaattcaaa actgccagcg aattcgactc tgcaattgct 2640 caagacaagc tagttgtcgt agatttctac gccacttggt gcggtccatg taaaatgatt 2700 gctccaatga ttgaaaaatg tggctgtggt ttcagggtcc ataaagcttt tcaattcatc 2760 tttttttttt ttgttctttt ttttgattcc ggtttctttg aaattttttt gattcggtaa 2820 tctccgagca gaaggaagaa cgaaggaagg agcacagact tagattggta tatatacgca 2880 tatgtggtgt tgaagaaaca tgaaattgcc cagtattctt aacccaactg cacagaacaa 2940 aaacctgcag gaaacgaaga taaatcatgt cgaaagctac atataaggaa cgtgctgcta 3000 ctcatcctag tcctgttgct gccaagctat ttaatatcat gcacgaaaag caaacaaact 3060 tgtgtgcttc attggatgtt cgtaccacca aggaattact ggagttagtt gaagcattag 3120 gtcccaaaat ttgtttacta aaaacacatg tggatatctt gactgatttt tccatggagg 3180 gcacagttaa gccgctaaag gcattatccg ccaagtacaa ttttttactc ttcgaagaca 3240 gaaaatttgc tgacattggt aatacagtca aattgcagta ctctgcgggt gtatacagaa 3300 tagcagaatg ggcagacatt acgaatgcac acggtgtggt gggcccaggt attgttagcg 3360 gtttgaagca ggcggcggaa gaagtaacaa aggaacctag aggccttttg atgttagcag 3420 aattgtcatg caagggctcc ctagctactg gagaatatac taagggtact gttgacattg 3480 cgaagagcga caaagatttt gttatcggct ttattgctca aagagacatg ggtggaagag 3540 atgaaggtta cgattggttg attatgacac ccggtgtggg tttagatgac aagggagacg 3600 cattgggtca acagtataga accgtggatg atgtggtctc tacaggatct gacattatta 3660 ttgttggaag aggactattt gcaaagggaa gggatgctaa ggtagagggt gaacgttaca 3720 gaaaagcagg ctgggaagca tatttgagaa gatgcggcca gcaaaactaa aaaactgtat 3780 tataagtaaa tgcatgtata ctaaactcac aaattagagc ttcaatttaa ttatatcagt 3840 tattacccgg gaatctcggt cgtaatgatt tctataatga cgaaaaaaaa aaaattggaa 3900 agaaaaagct tcatggcctt ccactttccc aaacaacacc tacggtatct ctcaagtctt 3960 atggggttcc attggtttca ccactggtgc taccttgggt gctgctttcg ctgctgaaga 4020 aattgatcca aagaagagag ttatcttatt cattggtgac ggttctttgc aattgactgt 4080 tcaagaaatc tccaccatga tcagatgggg cttgaagcca tacttgttcg tcttgaacaa 4140 cgatggttac accattgaaa agttgattca cggtccaaag gctcaataca acgaaattca 4200 aggttgggac cacctatcct tgttgccaac tttcggtgct aaggactatg aaacccacag 4260 agtcgctacc accggtgaat gggacaagtt gacccaagac aagtctttca acgacaactc 4320 taagatcaga atgattgaaa tcatgttgcc agtcttcgat gctccacaaa acttggttga 4380 acaagctaag ttgactgctg ctaccaacgc taagcaataa 4420 <210> 143 <211> 3521 <212> DNA <213> Artificial sequence <220> <223> Cassette <400> 143 aaggaaataa agcaaataac aataacacca ttattttaat tttttttcta ttactgtcgc 60 taacacctgt atggttgcaa ccaggtgaga atccttctga tgcatacttt atgcgtttat 120 gcgttttgcg ccccttggaa aaaaattgat tctcatcgta aatgcatact acatgcgttt 180 atgggaaaag cctccatatc caaaggtcgc gtttctttta gaaaaactaa tacgtaaacc 240 tgcattaagg taagattata tcagaaaatg tgttgcaaga aatgcattat gcaatttttt 300 gattatgaca atctctcgaa agaaatttca tatgatgaga cttgaataat gcagcggcgc 360 ttgctaaaag aacttgtata taagagctgc cattctcgat caatatactg tagtaagtcc 420 tttcctctct ttcttattac acttatttca cataatcaat ctcaaagaga acaacacaat 480 acaataacaa gaagaacaaa atgaaagctc tggtttatca cggtgaccac aagatctcgc 540 ttgaagacaa gcccaagccc acccttcaaa agcccacgga tgtagtagta cgggttttga 600 agaccacgat ctgcggcacg gatctcggca tctacaaagg caagaatcca gaggtcgccg 660 acgggcgcat cctgggccat gaaggggtag gcgtcatcga ggaagtgggc gagagtgtca 720 cgcagttcaa gaaaggcgac aaggtcctga tttcctgcgt cacttcttgc ggctcgtgcg 780 actactgcaa gaagcagctt tactcccatt gccgcgacgg cgggtggatc ctgggttaca 840 tgatcgatgg cgtgcaggcc gaatacgtcc gcatcccgca tgccgacaac agcctctaca 900 agatccccca gacaattgac gacgaaatcg ccgtcctgct gagcgacatc ctgcccaccg 960 gccacgaaat cggcgtccag tatgggaatg tccagccggg cgatgcggtg gctattgtcg 1020 gcgcgggccc cgtcggcatg tccgtactgt tgaccgccca gttctactcc ccctcgacca 1080 tcatcgtgat cgacatggac gagaatcgcc tccagctcgc caaggagctc ggggcaacgc 1140 acaccatcaa ctccggcacg gagaacgttg tcgaagccgt gcataggatt gcggcagagg 1200 gagtcgatgt tgcgatcgag gcggtgggca taccggcgac ttgggacatc tgccaggaga 1260 tcgtcaagcc cggcgcgcac atcgccaacg tcggcgtgca tggcgtcaag gttgacttcg 1320 agattcagaa gctctggatc aagaacctga cgatcaccac gggactggtg aacacgaaca 1380 cgacgcccat gctgatgaag gtcgcctcga ccgacaagct tccgttgaag aagatgatta 1440 cccatcgctt cgagctggcc gagatcgagc acgcctatca ggtattcctc aatggcgcca 1500 aggagaaggc gatgaagatc atcctctcga acgcaggcgc tgcctgagct aattaacata 1560 aaactcatga ttcaacgttt gtgtattttt ttacttttga aggttataga tgtttaggta 1620 aataattggc atagatatag ttttagtata ataaatttct gatttggttt aaaatatcaa 1680 ctattttttt tcacatatgt tcttgtaatt acttttctgt cctgtcttcc aggttaaaga 1740 ttagcttcta atattttagg tggtttatta tttaatttta tgctgattaa tttatttact 1800 tgtttaaacg gccggccaat gtggctgtgg tttcagggtc cataaagctt ttcaattcat 1860 cttttttttt tttgttcttt tttttgattc cggtttcttt gaaatttttt tgattcggta 1920 atctccgagc agaaggaaga acgaaggaag gagcacagac ttagattggt atatatacgc 1980 atatgtggtg ttgaagaaac atgaaattgc ccagtattct taacccaact gcacagaaca 2040 aaaacctgca ggaaacgaag ataaatcatg tcgaaagcta catataagga acgtgctgct 2100 actcatccta gtcctgttgc tgccaagcta tttaatatca tgcacgaaaa gcaaacaaac 2160 ttgtgtgctt cattggatgt tcgtaccacc aaggaattac tggagttagt tgaagcatta 2220 ggtcccaaaa tttgtttact aaaaacacat gtggatatct tgactgattt ttccatggag 2280 ggcacagtta agccgctaaa ggcattatcc gccaagtaca attttttact cttcgaagac 2340 agaaaatttg ctgacattgg taatacagtc aaattgcagt actctgcggg tgtatacaga 2400 atagcagaat gggcagacat tacgaatgca cacggtgtgg tgggcccagg tattgttagc 2460 ggtttgaagc aggcggcgga agaagtaaca aaggaaccta gaggcctttt gatgttagca 2520 gaattgtcat gcaagggctc cctagctact ggagaatata ctaagggtac tgttgacatt 2580 gcgaagagcg acaaagattt tgttatcggc tttattgctc aaagagacat gggtggaaga 2640 gatgaaggtt acgattggtt gattatgaca cccggtgtgg gtttagatga caagggagac 2700 gcattgggtc aacagtatag aaccgtggat gatgtggtct ctacaggatc tgacattatt 2760 attgttggaa gaggactatt tgcaaaggga agggatgcta aggtagaggg tgaacgttac 2820 agaaaagcag gctgggaagc atatttgaga agatgcggcc agcaaaacta aaaaactgta 2880 ttataagtaa atgcatgtat actaaactca caaattagag cttcaattta attatatcag 2940 ttattacccg ggaatctcgg tcgtaatgat ttctataatg acgaaaaaaa aaaaattgga 3000 aagaaaaagc ttcatggcct tctactttcc caacagatgt atacgctatc gtccaagtct 3060 tgtggggttc cattggtttc acagtcggcg ctctattggg tgctactatg gccgctgaag 3120 aacttgatcc aaagaagaga gttattttat tcattggtga cggttctcta caattgactg 3180 ttcaagaaat ctctaccatg attagatggg gtttgaagcc atacattttt gtcttgaata 3240 acaacggtta caccattgaa aaattgattc acggtcctca tgccgaatat aatgaaattc 3300 aaggttggga ccacttggcc ttattgccaa cttttggtgc tagaaactac gaaacccaca 3360 gagttgctac cactggtgaa tgggaaaagt tgactcaaga caaggacttc caagacaact 3420 ctaagattag aatgattgaa gttatgttgc cagtctttga tgctccacaa aacttggtta 3480 aacaagctca attgactgcc gctactaacg ctaaacaata a 3521 <210> 144 <211> 571 <212> PRT <213> Bacillus subtilis <400> 144 Met Leu Thr Lys Ala Thr Lys Glu Gln Lys Ser Leu Val Lys Asn Arg 1 5 10 15 Gly Ala Glu Leu Val Val Asp Cys Leu Val Glu Gln Gly Val Thr His             20 25 30 Val Phe Gly Ile Pro Gly Ala Lys Ile Asp Ala Val Phe Asp Ala Leu         35 40 45 Gln Asp Lys Gly Pro Glu Ile Ile Val Ala Arg His Glu Gln Asn Ala     50 55 60 Ala Phe Met Ala Gln Ala Val Gly Arg Leu Thr Gly Lys Pro Gly Val 65 70 75 80 Val Leu Val Thr Ser Gly Pro Gly Ala Ser Asn Leu Ala Thr Gly Leu                 85 90 95 Leu Thr Ala Asn Thr Glu Gly Asp Pro Val Val Ala Leu Ala Gly Asn             100 105 110 Val Ile Arg Ala Asp Arg Leu Lys Arg Thr His Gln Ser Leu Asp Asn         115 120 125 Ala Ala Leu Phe Gln Pro Ile Thr Lys Tyr Ser Val Glu Val Gln Asp     130 135 140 Val Lys Asn Ile Pro Glu Ala Val Thr Asn Ala Phe Arg Ile Ala Ser 145 150 155 160 Ala Gly Gln Ala Gly Ala Ala Phe Val Ser Phe Pro Gln Asp Val Val                 165 170 175 Asn Glu Val Thr Asn Thr Lys Asn Val Arg Ala Val Ala Ala Pro Lys             180 185 190 Leu Gly Pro Ala Ala Asp Asp Ala Ile Ser Ala Ala Ile Ala Lys Ile         195 200 205 Gln Thr Ala Lys Leu Pro Val Val Leu Val Gly Met Lys Gly Gly Arg     210 215 220 Pro Glu Ala Ile Lys Ala Val Arg Lys Leu Leu Lys Lys Val Gln Leu 225 230 235 240 Pro Phe Val Glu Thr Tyr Gln Ala Ala Gly Thr Leu Ser Arg Asp Leu                 245 250 255 Glu Asp Gln Tyr Phe Gly Arg Ile Gly Leu Phe Arg Asn Gln Pro Gly             260 265 270 Asp Leu Leu Leu Glu Gln Ala Asp Val Val Leu Thr Ile Gly Tyr Asp         275 280 285 Pro Ile Glu Tyr Asp Pro Lys Phe Trp Asn Ile Asn Gly Asp Arg Thr     290 295 300 Ile Ile His Leu Asp Glu Ile Ile Ala Asp Ile Asp His Ala Tyr Gln 305 310 315 320 Pro Asp Leu Glu Leu Ile Gly Asp Ile Pro Ser Thr Ile Asn His Ile                 325 330 335 Glu His Asp Ala Val Lys Val Glu Phe Ala Glu Arg Glu Gln Lys Ile             340 345 350 Leu Ser Asp Leu Lys Gln Tyr Met His Glu Gly Glu Gln Val Pro Ala         355 360 365 Asp Trp Lys Ser Asp Arg Ala His Pro Leu Glu Ile Val Lys Glu Leu     370 375 380 Arg Asn Ala Val Asp Asp His Val Thr Val Thr Cys Asp Ile Gly Ser 385 390 395 400 His Ala Ile Trp Met Ser Arg Tyr Phe Arg Ser Tyr Glu Pro Leu Thr                 405 410 415 Leu Met Ile Ser Asn Gly Met Gln Thr Leu Gly Val Ala Leu Pro Trp             420 425 430 Ala Ile Gly Ala Ser Leu Val Lys Pro Gly Glu Lys Val Val Ser Val         435 440 445 Ser Gly Asp Gly Gly Phe Leu Phe Ser Ala Met Glu Leu Glu Thr Ala     450 455 460 Val Arg Leu Lys Ala Pro Ile Val His Ile Val Trp Asn Asp Ser Thr 465 470 475 480 Tyr Asp Met Val Ala Phe Gln Gln Leu Lys Lys Tyr Asn Arg Thr Ser                 485 490 495 Ala Val Asp Phe Gly Asn Ile Asp Ile Val Lys Tyr Ala Glu Ser Phe             500 505 510 Gly Ala Thr Gly Leu Arg Val Glu Ser Pro Asp Gln Leu Ala Asp Val         515 520 525 Leu Arg Gln Gly Met Asn Ala Glu Gly Pro Val Ile Ile Asp Val Pro     530 535 540 Val Asp Tyr Ser Asp Asn Ile Asn Leu Ala Ser Asp Lys Leu Pro Lys 545 550 555 560 Glu Phe Gly Glu Leu Met Lys Thr Lys Ala Leu                 565 570 <210> 145 <211> 340 <212> PRT <213> Lactococcus lactis <400> 145 Met Ala Val Thr Met Tyr Tyr Glu Asp Asp Val Glu Val Ser Ala Leu 1 5 10 15 Ala Gly Lys Gln Ile Ala Val Ile Gly Tyr Gly Ser Gln Gly His Ala             20 25 30 His Ala Gln Asn Leu Arg Asp Ser Gly His Asn Val Ile Ile Gly Val         35 40 45 Arg His Gly Lys Ser Phe Asp Lys Ala Lys Glu Asp Gly Phe Glu Thr     50 55 60 Phe Glu Val Gly Glu Ala Val Ala Lys Ala Asp Val Ile Met Val Leu 65 70 75 80 Ala Pro Asp Glu Leu Gln Gln Ser Ile Tyr Glu Glu Asp Ile Lys Pro                 85 90 95 Asn Leu Lys Ala Gly Ser Ala Leu Gly Phe Ala His Gly Phe Asn Ile             100 105 110 His Phe Gly Tyr Ile Lys Val Pro Glu Asp Val Asp Val Phe Met Val         115 120 125 Ala Pro Lys Ala Pro Gly His Leu Val Arg Arg Thr Tyr Thr Glu Gly     130 135 140 Phe Gly Thr Pro Ala Leu Phe Val Ser His Gln Asn Ala Ser Gly His 145 150 155 160 Ala Arg Glu Ile Ala Met Asp Trp Ala Lys Gly Ile Gly Cys Ala Arg                 165 170 175 Val Gly Ile Ile Glu Thr Thr Phe Lys Glu Glu Thr Glu Glu Asp Leu             180 185 190 Phe Gly Glu Gln Ala Val Leu Cys Gly Gly Leu Thr Ala Leu Val Glu         195 200 205 Ala Gly Phe Glu Thr Leu Thr Glu Ala Gly Tyr Ala Gly Glu Leu Ala     210 215 220 Tyr Phe Glu Val Leu His Glu Met Lys Leu Ile Val Asp Leu Met Tyr 225 230 235 240 Glu Gly Gly Phe Thr Lys Met Arg Gln Ser Ile Ser Asn Thr Ala Glu                 245 250 255 Phe Gly Asp Tyr Val Thr Gly Pro Arg Ile Ile Thr Asp Glu Val Lys             260 265 270 Lys Asn Met Lys Leu Val Leu Ala Asp Ile Gln Ser Gly Lys Phe Ala         275 280 285 Gln Asp Phe Val Asp Asp Phe Lys Ala Gly Arg Pro Lys Leu Ile Ala     290 295 300 Tyr Arg Glu Ala Ala Lys Asn Leu Glu Ile Glu Lys Ile Gly Ala Glu 305 310 315 320 Leu Arg Gln Ala Met Pro Phe Thr Gln Ser Gly Asp Asp Asp Ala Phe                 325 330 335 Lys Ile Tyr Gln             340 <210> 146 <211> 571 <212> PRT <213> Streptococcus mutans <400> 146 Met Thr Asp Lys Lys Thr Leu Lys Asp Leu Arg Asn Arg Ser Ser Val 1 5 10 15 Tyr Asp Ser Met Val Lys Ser Pro Asn Arg Ala Met Leu Arg Ala Thr             20 25 30 Gly Met Gln Asp Glu Asp Phe Glu Lys Pro Ile Val Gly Val Ile Ser         35 40 45 Thr Trp Ala Glu Asn Thr Pro Cys Asn Ile His Leu His Asp Phe Gly     50 55 60 Lys Leu Ala Lys Val Gly Val Lys Glu Ala Gly Ala Trp Pro Val Gln 65 70 75 80 Phe Gly Thr Ile Thr Val Ser Asp Gly Ile Ala Met Gly Thr Gln Gly                 85 90 95 Met Arg Phe Ser Leu Thr Ser Arg Asp Ile Ile Ala Asp Ser Ile Glu             100 105 110 Ala Ala Met Gly Gly His Asn Ala Asp Ala Phe Val Ala Ile Gly Gly         115 120 125 Cys Asp Lys Asn Met Pro Gly Ser Val Ile Ala Met Ala Asn Met Asp     130 135 140 Ile Pro Ala Ile Phe Ala Tyr Gly Gly Thr Ile Ala Pro Gly Asn Leu 145 150 155 160 Asp Gly Lys Asp Ile Asp Leu Val Ser Val Phe Glu Gly Val Gly His                 165 170 175 Trp Asn His Gly Asp Met Thr Lys Glu Glu Val Lys Ala Leu Glu Cys             180 185 190 Asn Ala Cys Pro Gly Pro Gly Gly Cys Gly Gly Met Tyr Thr Ala Asn         195 200 205 Thr Met Ala Thr Ala Ile Glu Val Leu Gly Leu Ser Leu Pro Gly Ser     210 215 220 Ser Ser His Pro Ala Glu Ser Ala Glu Lys Lys Ala Asp Ile Glu Glu 225 230 235 240 Ala Gly Arg Ala Val Val Lys Met Leu Glu Met Gly Leu Lys Pro Ser                 245 250 255 Asp Ile Leu Thr Arg Glu Ala Phe Glu Asp Ala Ile Thr Val Thr Met             260 265 270 Ala Leu Gly Gly Ser Thr Asn Ser Thr Leu His Leu Leu Ala Ile Ala         275 280 285 His Ala Ala Asn Val Glu Leu Thr Leu Asp Asp Phe Asn Thr Phe Gln     290 295 300 Glu Lys Val Pro His Leu Ala Asp Leu Lys Pro Ser Gly Gln Tyr Val 305 310 315 320 Phe Gln Asp Leu Tyr Lys Val Gly Gly Val Pro Ala Val Met Lys Tyr                 325 330 335 Leu Leu Lys Asn Gly Phe Leu His Gly Asp Arg Ile Thr Cys Thr Gly             340 345 350 Lys Thr Val Ala Glu Asn Leu Lys Ala Phe Asp Asp Leu Thr Pro Gly         355 360 365 Gln Lys Val Ile Met Pro Leu Glu Asn Pro Lys Arg Glu Asp Gly Pro     370 375 380 Leu Ile Ile Leu His Gly Asn Leu Ala Pro Asp Gly Ala Val Ala Lys 385 390 395 400 Val Ser Gly Val Lys Val Arg Arg His Val Gly Pro Ala Lys Val Phe                 405 410 415 Asn Ser Glu Glu Glu Ala Ile Glu Ala Val Leu Asn Asp Asp Ile Val             420 425 430 Asp Gly Asp Val Val Val Val Arg Phe Val Gly Pro Lys Gly Gly Pro         435 440 445 Gly Met Pro Glu Met Leu Ser Leu Ser Ser Met Ile Val Gly Lys Gly     450 455 460 Gln Gly Glu Lys Val Ala Leu Leu Thr Asp Gly Arg Phe Ser Gly Gly 465 470 475 480 Thr Tyr Gly Leu Val Val Gly His Ile Ala Pro Glu Ala Gln Asp Gly                 485 490 495 Gly Pro Ile Ala Tyr Leu Gln Thr Gly Asp Ile Val Thr Ile Asp Gln             500 505 510 Asp Thr Lys Glu Leu His Phe Asp Ile Ser Asp Glu Glu Leu Lys His         515 520 525 Arg Gln Glu Thr Ile Glu Leu Pro Pro Leu Tyr Ser Arg Gly Ile Leu     530 535 540 Gly Lys Tyr Ala His Ile Val Ser Ser Ala Ser Arg Gly Ala Val Thr 545 550 555 560 Asp Phe Trp Lys Pro Glu Glu Thr Gly Lys Lys                 565 570 <210> 147 <211> 548 <212> PRT <213> Lactococcus lactis <400> 147 Met Tyr Thr Val Gly Asp Tyr Leu Leu Asp Arg Leu His Glu Leu Gly 1 5 10 15 Ile Glu Glu Ile Phe Gly Val Pro Gly Asp Tyr Asn Leu Gln Phe Leu             20 25 30 Asp Gln Ile Ile Ser His Lys Asp Met Lys Trp Val Gly Asn Ala Asn         35 40 45 Glu Leu Asn Ala Ser Tyr Met Ala Asp Gly Tyr Ala Arg Thr Lys Lys     50 55 60 Ala Ala Ala Phe Leu Thr Thr Phe Gly Val Gly Glu Leu Ser Ala Val 65 70 75 80 Asn Gly Leu Ala Gly Ser Tyr Ala Glu Asn Leu Pro Val Val Glu Ile                 85 90 95 Val Gly Ser Pro Thr Ser Lys Val Gln Asn Glu Gly Lys Phe Val His             100 105 110 His Thr Leu Ala Asp Gly Asp Phe Lys His Phe Met Lys Met His Glu         115 120 125 Pro Val Thr Ala Ala Arg Thr Leu Leu Thr Ala Glu Asn Ala Thr Val     130 135 140 Glu Ile Asp Arg Val Leu Ser Ala Leu Leu Lys Glu Arg Lys Pro Val 145 150 155 160 Tyr Ile Asn Leu Pro Val Asp Val Ala Ala Ala Lys Ala Glu Lys Pro                 165 170 175 Ser Leu Pro Leu Lys Lys Glu Asn Ser Thr Ser Asn Thr Ser Asp Gln             180 185 190 Glu Ile Leu Asn Lys Ile Gln Glu Ser Leu Lys Asn Ala Lys Lys Pro         195 200 205 Ile Val Ile Thr Gly His Glu Ile Ile Ser Phe Gly Leu Glu Lys Thr     210 215 220 Val Thr Gln Phe Ile Ser Lys Thr Lys Leu Pro Ile Thr Thr Leu Asn 225 230 235 240 Phe Gly Lys Ser Ser Val Asp Glu Ala Leu Pro Ser Phe Leu Gly Ile                 245 250 255 Tyr Asn Gly Thr Leu Ser Glu Pro Asn Leu Lys Glu Phe Val Glu Ser             260 265 270 Ala Asp Phe Ile Leu Met Leu Gly Val Lys Leu Thr Asp Ser Ser Thr         275 280 285 Gly Ala Phe Thr His His Leu Asn Glu Asn Lys Met Ile Ser Leu Asn     290 295 300 Ile Asp Glu Gly Lys Ile Phe Asn Glu Arg Ile Gln Asn Phe Asp Phe 305 310 315 320 Glu Ser Leu Ile Ser Ser Leu Leu Asp Leu Ser Glu Ile Glu Tyr Lys                 325 330 335 Gly Lys Tyr Ile Asp Lys Lys Gln Glu Asp Phe Val Pro Ser Asn Ala             340 345 350 Leu Leu Ser Gln Asp Arg Leu Trp Gln Ala Val Glu Asn Leu Thr Gln         355 360 365 Ser Asn Glu Thr Ile Val Ala Glu Gln Gly Thr Ser Phe Phe Gly Ala     370 375 380 Ser Ser Ile Phe Leu Lys Ser Lys Ser His Phe Ile Gly Gln Pro Leu 385 390 395 400 Trp Gly Ser Ile Gly Tyr Thr Phe Pro Ala Ala Leu Gly Ser Gln Ile                 405 410 415 Ala Asp Lys Glu Ser Arg His Leu Leu Phe Ile Gly Asp Gly Ser Leu             420 425 430 Gln Leu Thr Val Gln Glu Leu Gly Leu Ala Ile Arg Glu Lys Ile Asn         435 440 445 Pro Ile Cys Phe Ile Ile Asn Asn Asp Gly Tyr Thr Val Glu Arg Glu     450 455 460 Ile His Gly Pro Asn Gln Ser Tyr Asn Asp Ile Pro Met Trp Asn Tyr 465 470 475 480 Ser Lys Leu Pro Glu Ser Phe Gly Ala Thr Glu Asp Arg Val Val Ser                 485 490 495 Lys Ile Val Arg Thr Glu Asn Glu Phe Val Ser Val Met Lys Glu Ala             500 505 510 Gln Ala Asp Pro Asn Arg Met Tyr Trp Ile Glu Leu Ile Leu Ala Lys         515 520 525 Glu Gly Ala Pro Lys Val Leu Lys Lys Met Gly Lys Leu Phe Ala Glu     530 535 540 Gln asn lys ser 545 <210> 148 <211> 375 <212> PRT <213> Equus caballus <400> 148 Met Ser Thr Ala Gly Lys Val Ile Lys Cys Lys Ala Ala Val Leu Trp 1 5 10 15 Glu Glu Lys Lys Pro Phe Ser Ile Glu Glu Val Glu Val Ala Pro Pro             20 25 30 Lys Ala His Glu Val Arg Ile Lys Met Val Ala Thr Gly Ile Cys Arg         35 40 45 Ser Asp Asp His Val Val Ser Gly Thr Leu Val Thr Pro Leu Pro Val     50 55 60 Ile Ala Gly His Glu Ala Ala Gly Ile Val Glu Ser Ile Gly Glu Gly 65 70 75 80 Val Thr Thr Val Arg Pro Gly Asp Lys Val Ile Pro Leu Phe Thr Pro                 85 90 95 Gln Cys Gly Lys Cys Arg Val Cys Lys His Pro Glu Gly Asn Phe Cys             100 105 110 Leu Lys Asn Asp Leu Ser Met Pro Arg Gly Thr Met Gln Asp Gly Thr         115 120 125 Ser Arg Phe Thr Cys Arg Gly Lys Pro Ile His His Phe Leu Gly Thr     130 135 140 Ser Thr Phe Ser Gln Tyr Thr Val Val Asp Glu Ile Ser Val Ala Lys 145 150 155 160 Ile Asp Ala Ala Ser Pro Leu Glu Lys Val Cys Leu Ile Gly Cys Gly                 165 170 175 Phe Ser Thr Gly Tyr Gly Ser Ala Val Lys Val Ala Lys Val Thr Gln             180 185 190 Gly Ser Thr Cys Ala Val Phe Gly Leu Gly Gly Val Gly Leu Ser Val         195 200 205 Ile Met Gly Cys Lys Ala Ala Gly Ala Ala Arg Ile Ile Gly Val Asp     210 215 220 Ile Asn Lys Asp Lys Phe Ala Lys Ala Lys Glu Val Gly Ala Thr Glu 225 230 235 240 Cys Val Asn Pro Gln Asp Tyr Lys Lys Pro Ile Gln Glu Val Leu Thr                 245 250 255 Glu Met Ser Asn Gly Gly Val Asp Phe Ser Phe Glu Val Ile Gly Arg             260 265 270 Leu Asp Thr Met Val Thr Ala Leu Ser Cys Cys Gln Glu Ala Tyr Gly         275 280 285 Val Ser Val Ile Val Gly Val Pro Pro Asp Ser Gln Asn Leu Ser Met     290 295 300 Asn Pro Met Leu Leu Leu Ser Gly Arg Thr Trp Lys Gly Ala Ile Phe 305 310 315 320 Gly Gly Phe Lys Ser Lys Asp Ser Val Pro Lys Leu Val Ala Asp Phe                 325 330 335 Met Ala Lys Lys Phe Ala Leu Asp Pro Leu Ile Thr His Val Leu Pro             340 345 350 Phe Glu Lys Ile Asn Glu Gly Phe Asp Leu Leu Arg Ser Gly Glu Ser         355 360 365 Ile Arg Thr Ile Leu Thr Phe     370 375 <210> 149 <211> 348 <212> PRT <213> Achromobacter xylosoxidans <400> 149 Met Lys Ala Leu Val Tyr His Gly Asp His Lys Ile Ser Leu Glu Asp 1 5 10 15 Lys Pro Lys Pro Thr Leu Gln Lys Pro Thr Asp Val Val Arg Val             20 25 30 Leu Lys Thr Thr Ile Cys Gly Thr Asp Leu Gly Ile Tyr Lys Gly Lys         35 40 45 Asn Pro Glu Val Ala Asp Gly Arg Ile Leu Gly His Glu Gly Val Gly     50 55 60 Val Ile Glu Glu Val Gly Glu Ser Val Thr Gln Phe Lys Lys Gly Asp 65 70 75 80 Lys Val Leu Ile Ser Cys Val Thr Ser Cys Gly Ser Cys Asp Tyr Cys                 85 90 95 Lys Lys Gln Leu Tyr Ser His Cys Arg Asp Gly Gly Trp Ile Leu Gly             100 105 110 Tyr Met Ile Asp Gly Val Gln Ala Glu Tyr Val Arg Ile Pro His Ala         115 120 125 Asp Asn Ser Leu Tyr Lys Ile Pro Gln Thr Ile Asp Asp Glu Ile Ala     130 135 140 Val Leu Leu Ser Asp Ile Leu Pro Thr Gly His Glu Ile Gly Val Gln 145 150 155 160 Tyr Gly Asn Val Gln Pro Gly Asp Ala Val Ala Ile Val Gly Ala Gly                 165 170 175 Pro Val Gly Met Ser Val Leu Leu Thr Ala Gln Phe Tyr Ser Ser Ser             180 185 190 Thr Ile Ile Val Ile Asp Met Asp Glu Asn Arg Leu Gln Leu Ala Lys         195 200 205 Glu Leu Gly Ala Thr His Thr Ile Asn Ser Gly Thr Glu Asn Val Val     210 215 220 Glu Ala Val His Arg Ile Ala Ala Glu Gly Val Asp Val Ala Ile Glu 225 230 235 240 Ala Val Gly Ile Pro Ala Thr Trp Asp Ile Cys Gln Glu Ile Val Lys                 245 250 255 Pro Gly Ala His Ile Ala Asn Val Gly Val His Gly Val Lys Val Asp             260 265 270 Phe Glu Ile Gln Lys Leu Trp Ile Lys Asn Leu Thr Ile Thr Thr Gly         275 280 285 Leu Val Asn Thr Asn Thr Thr Pro Met Leu Met Lys Val Ala Ser Thr     290 295 300 Asp Lys Leu Pro Leu Lys Lys Met Ile Thr His Arg Phe Glu Leu Ala 305 310 315 320 Glu Ile Glu His Ala Tyr Gln Val Phe Leu Asn Gly Ala Lys Glu Lys                 325 330 335 Ala Met Lys Ile Ile Leu Ser Asn Ala Gly Ala Ala             340 345 <210> 150 <211> 2686 <212> DNA <213> Artificial sequence <220> <223> Plasmid <400> 150 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 240 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 300 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 360 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt cgagctcggt acccggggat 420 cctctagagt cgacctgcag gcatgcaagc ttggcgtaat catggtcata gctgtttcct 480 gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt 540 aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc 600 gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg 660 agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg 720 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca 780 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac 840 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac 900 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg 960 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac 1020 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat 1080 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag 1140 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac 1200 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt 1260 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaaggac agtatttggt 1320 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc 1380 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga 1440 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac 1500 gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc 1560 cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct 1620 gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca 1680 tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct 1740 ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca 1800 ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc 1860 atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg 1920 cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct 1980 tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa 2040 aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta 2100 tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc 2160 ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg 2220 agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa 2280 gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg 2340 agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc 2400 accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg 2460 gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat 2520 cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata 2580 ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tctaagaaac cattattatc 2640 atgacattaa cctataaaaa taggcgtatc acgaggccct ttcgtc 2686 <210> 151 <211> 1685 <212> DNA <213> Artificial sequence <220> <223> Cassette <400> 151 gtattttggt agattcaatt ctctttccct ttccttttcc ttcgctcccc ttccttatca 60 gcattgcgga ttacgtattc taatgttcag ataacttcgt atagcataca ttatacgaag 120 ttatgcagat tgtactgaga gtgcaccata ccacagcttt tcaattcaat tcatcatttt 180 ttttttattc ttttttttga tttcggtttc tttgaaattt ttttgattcg gtaatctccg 240 aacagaagga agaacgaagg aaggagcaca gacttagatt ggtatatata cgcatatgta 300 gtgttgaaga aacatgaaat tgcccagtat tcttaaccca actgcacaga acaaaaacct 360 gcaggaaacg aagataaatc atgtcgaaag ctacatataa ggaacgtgct gctactcatc 420 ctagtcctgt tgctgccaag ctatttaata tcatgcacga aaagcaaaca aacttgtgtg 480 cttcattgga tgttcgtacc accaaggaat tactggagtt agttgaagca ttaggtccca 540 aaatttgttt actaaaaaca catgtggata tcttgactga tttttccatg gagggcacag 600 ttaagccgct aaaggcatta tccgccaagt acaatttttt actcttcgaa gacagaaaat 660 ttgctgacat tggtaataca gtcaaattgc agtactctgc gggtgtatac agaatagcag 720 aatgggcaga cattacgaat gcacacggtg tggtgggccc aggtattgtt agcggtttga 780 agcaggcggc agaagaagta acaaaggaac ctagaggcct tttgatgtta gcagaattgt 840 catgcaaggg ctccctatct actggagaat atactaaggg tactgttgac attgcgaaga 900 gcgacaaaga ttttgttatc ggctttattg ctcaaagaga catgggtgga agagatgaag 960 gttacgattg gttgattatg acacccggtg tgggtttaga tgacaaggga gacgcattgg 1020 gtcaacagta tagaaccgtg gatgatgtgg tctctacagg atctgacatt attattgttg 1080 gaagaggact atttgcaaag ggaagggatg ctaaggtaga gggtgaacgt tacagaaaag 1140 caggctggga agcatatttg agaagatgcg gccagcaaaa ctaaaaaact gtattataag 1200 taaatgcatg tatactaaac tcacaaatta gagcttcaat ttaattatat cagttattac 1260 cctatgcggt gtgaaatacc gcacagatgc gtaaggagaa aataccgcat caggaaattg 1320 taaacgttaa tattttgtta aaattcgcgt taaatttttg ttaaatcagc tcatttttta 1380 accaataggc cgaaatcggc aaaatccctt ataaatcaaa agaatagacc gagatagggt 1440 tgagtgttgt tccagtttgg aacaagagtc cactattaaa gaacgtggac tccaacgtca 1500 aagggcgaaa aaccgtctat cagggcgatg gcccactacg tgaaccatca ccctaatcaa 1560 gataacttcg tatagcatac attatacgaa gttatccagt gatgatacaa cgagttagcc 1620 aaggtgacac tctccccccc cctccccctc tgatctttcc tgttgcctct ttttccccca 1680 accaa 1685 <210> 152 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Primer oBP594 <400> 152 agctgtctcg tgttgtgggt tt 22 <210> 153 <211> 49 <212> DNA <213> Artificial Sequence <220> <223> Primer oBP595 <400> 153 cttaataata gaacaatatc atcctttacg ggcatcttat agtgtcgtt 49 <210> 154 <211> 49 <212> DNA <213> Artificial Sequence <220> <223> Primer oBP596 <400> 154 gcgccaacga cactataaga tgcccgtaaa ggatgatatt gttctatta 49 <210> 155 <211> 49 <212> DNA <213> Artificial Sequence <220> <223> Primer oBP597 <400> 155 tatggaccct gaaaccacag ccacattgca acgacgacaa tgccaaacc 49 <210> 156 <211> 49 <212> DNA <213> Artificial Sequence <220> <223> Primer oBP598 <400> 156 tccttggttt ggcattgtcg tcgttgcaat gtggctgtgg tttcagggt 49 <210> 157 <211> 49 <212> DNA <213> Artificial Sequence <220> <223> Primer oBP599 <400> 157 atcctctcgc ggagtccctg ttcagtaaag gccatgaagc tttttcttt 49 <210> 158 <211> 49 <212> DNA <213> Artificial Sequence <220> <223> Primer oBP600 <400> 158 attggaaaga aaaagcttca tggcctttac tgaacaggga ctccgcgag 49 <210> 159 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Primer oBP601 <400> 159 tcataccaca atcttagacc at 22 <210> 160 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Primer oBP602 <400> 160 tgttcaaacc cctaaccaac c 21 <210> 161 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Primer oBP603 <400> 161 tgttcccaca atctattacc ta 22 <210> 162 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Primer oBP605 <400> 162 tactgaacag ggactccgcg a 21 <210> 163 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Primer oBP606 <400> 163 tcataccaca atcttagacc a 21 <210> 164 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Primer oBP562 <400> 164 aattgtttaa acatgtatac agtaggtgac tatc 34 <210> 165 <211> 49 <212> DNA <213> Artificial Sequence <220> <223> Primer oBP563 <400> 165 aatcataaat cataagaaat tcgcttatca gctcttgttt tgttctgca 49 <210> 166 <211> 49 <212> DNA <213> Artificial Sequence <220> <223> Primer oBP564 <400> 166 ttatttgcag aacaaaacaa gagctgataa gcgaatttct tatgattta 49 <210> 167 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Primer oBP565 <400> 167 aattggccgg ccaaaaaaag catgcacgta taca 34 <210> 168 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Primer oBP505 <400> 168 aattgagctc actgtagccc tagacttgat ag 32 <210> 169 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Primer oBP506 <400> 169 aattggcgcg cctgtatatg agatagttga ttgta 35 <210> 170 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Primer oBP507 <400> 170 aattttaatt aagtctaggt tctttggctg ttcaa 35 <210> 171 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Primer oBP508 <400> 171 aattgtcgac tttagaagtg tcaacaacgt atc 33 <210> 172 <211> 8366 <212> DNA <213> Artificial Sequence <220> <223> pRS316-UAS (PGK1) -PFBA1-GUS <400> 172 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180 accacgcttt tcaattcaat tcatcatttt ttttttattc ttttttttga tttcggtttc 240 tttgaaattt ttttgattcg gtaatctccg aacagaagga agaacgaagg aaggagcaca 300 gacttagatt ggtatatata cgcatatgta gtgttgaaga aacatgaaat tgcccagtat 360 tcttaaccca actgcacaga acaaaaacct gcaggaaacg aagataaatc atgtcgaaag 420 ctacatataa ggaacgtgct gctactcatc ctagtcctgt tgctgccaag ctatttaata 480 tcatgcacga aaagcaaaca aacttgtgtg cttcattgga tgttcgtacc accaaggaat 540 tactggagtt agttgaagca ttaggtccca aaatttgttt actaaaaaca catgtggata 600 tcttgactga tttttccatg gagggcacag ttaagccgct aaaggcatta tccgccaagt 660 acaatttttt actcttcgaa gacagaaaat ttgctgacat tggtaataca gtcaaattgc 720 agtactctgc gggtgtatac agaatagcag aatgggcaga cattacgaat gcacacggtg 780 tggtgggccc aggtattgtt agcggtttga agcaggcggc agaagaagta acaaaggaac 840 ctagaggcct tttgatgtta gcagaattgt catgcaaggg ctccctatct actggagaat 900 atactaaggg tactgttgac attgcgaaga gcgacaaaga ttttgttatc ggctttattg 960 ctcaaagaga catgggtgga agagatgaag gttacgattg gttgattatg acacccggtg 1020 tgggtttaga tgacaaggga gacgcattgg gtcaacagta tagaaccgtg gatgatgtgg 1080 tctctacagg atctgacatt attattgttg gaagaggact atttgcaaag ggaagggatg 1140 ctaaggtaga gggtgaacgt tacagaaaag caggctggga agcatatttg agaagatgcg 1200 gccagcaaaa ctaaaaaact gtattataag taaatgcatg tatactaaac tcacaaatta 1260 gagcttcaat ttaattatat cagttattac cctgcggtgt gaaataccgc acagatgcgt 1320 aaggagaaaa taccgcatca ggaaattgta aacgttaata ttttgttaaa attcgcgtta 1380 aatttttgtt aaatcagctc attttttaac caataggccg aaatcggcaa aatcccttat 1440 aaatcaaaag aatagaccga gatagggttg agtgttgttc cagtttggaa caagagtcca 1500 ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc 1560 ccactacgtg aaccatcacc ctaatcaagt tttttggggt cgaggtgccg taaagcacta 1620 aatcggaacc ctaaagggag cccccgattt agagcttgac ggggaaagcc ggcgaacgtg 1680 gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg 1740 gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcg 1800 cgccattcgc cattcaggct gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg 1860 ctattacgcc agctggcgaa ggggggatgt gctgcaaggc gattaagttg ggtaacgcca 1920 gggttttccc agtcacgacg ttgtaaaacg acggccagtg aattgtaata cgactcacta 1980 tagggcgaat tggagctcca ccgcggtggt ttaacgtata gacttctaat atatttctcc 2040 atacttggta ttttttattc acttttttta tacatatttg gtttttttat acaatatcaa 2100 aaatcaataa taattaattt aataacaaga atatgttgct gtgatgactc ttaatataca 2160 attgggaagc attcaaggat tggtacgacg tttgctttct gataattaaa cgcttggcgt 2220 ttaatctttt ttgccgatta ataatattat tattaccctc ttatttatta gcattgtctt 2280 ccgtactaaa aggagtaggg aaaaaggcga agtagagtga cacgtccatt acccagcgct 2340 tcataagaat ccatcaggta tattaaattg tgatgggaga acaaaacaaa ttgtactatg 2400 atgtcgagaa attggtgaat tctttgcagg aaagttttga cctggattgc gcgcaaagtg 2460 tatcactttt caccagtaaa tcaaggagca atgaggcttg gttggaagag ctagagaata 2520 aattcaagtt aaaagatgat gttgaacttg atgatgtgga aaatttaaga gccgaaattg 2580 acatgaagtt aaatatgttg gaggataaag taagctacta tgaaagactt tacaaagaac 2640 tcgaagagtt ccagaatgaa ataaaaatta aaacagttgt gaataacaga agacaatcgc 2700 gaactccaaa atgagctatc aaaaacgata gatcgattag gatgactttg aaatgactcc 2760 gcagtggact ggccgttaat ttcaagcgtg agtaaaatag tgcatgacaa aagatgagct 2820 aggcttttgt aaaaatatct tacgttgtaa aattttagaa atcattattt ccttcatatc 2880 attttgtcat tgaccttcag aagaaaagag ccgaccaata atataaataa ataaataaaa 2940 ataatattcc attatttcta aacagattca atactcatta aaaaactata tcaattaatt 3000 tgaattaacg cggccgctct agttaattaa tcattgtttg cctccctgct gcggtttttc 3060 accgaagttc atgccagtcc agcgtttttg cagcagaaaa gccgccgact tcggtttgcg 3120 gtcgcgagtg aagatccctt tcttgttacc gccaacgcgc aatatgcctt gcgaggtcgc 3180 aaaatcggcg aaattccata cctgttcacc gacgacggcg ctgacgcgat caaagacgcg 3240 gtgatacata tccagccatg cacactgata ctcttcactc cacatgtcgg tgtacattga 3300 gtgcagcccg gctaacgtat ccacgccgta ttcggtgatg ataatcggct gatgcagttt 3360 ctcctgccag gccagaagtt ctttttccag taccttctct gccgtttcca aatcgccgct 3420 ttggacatac catccgtaat aacggttcag gcacagcaca tcaaagagat cgctgatggt 3480 atcggtgtga gcgtcgcaga acattacatt gacgcaggtg atcggacgcg tcgggtcgag 3540 tttacgcgtt gcttccgcca gtggcgcgaa atattcccgt gcaccttgcg gacgggtatc 3600 cggttcgttg gcaatactcc acatcaccac gcttgggtgg tttttgtcac gcgctatcag 3660 ctctttaatc gcctgtaagt gcgcttgctg agtttccccg ttgactgcct cttcgctgta 3720 cagttctttc ggcttgttgc ccgcttcgaa accaatgcct aaagagaggt taaagccgac 3780 agcagcagtt tcatcaatca ccacgatgcc atgttcatct gcccagtcga gcatctcttc 3840 agcgtaaggg taatgcgagg tacggtagga gttggcccca atccagtcca ttaatgcgtg 3900 gtcgtgcacc atcagcacgt tatcgaatcc tttgccacgc aagtccgcat cttcatgacg 3960 accaaagcca gtaaagtaga acggtttgtg gttaatcagg aactgttcgc ccttcactgc 4020 cactgaccgg atgccgacgc gaagcgggta gatatcacac tctgtctggc ttttggctgt 4080 gacgcacagt tcatagagat aaccttcacc cggttgccag aggtgcggat tcaccacttg 4140 caaagtcccg ctagtgcctt gtccagttgc aaccacctgt tgatccgcat cacgcagttc 4200 aacgctgaca tcaccattgg ccaccacctg ccagtcaaca gacgcgtggt tacagtcttg 4260 cgcgacatgc gtcaccacgg tgatatcgtc cacccaggtg ttcggcgtgg tgtagagcat 4320 tacgctgcga tggattccgg catagttaaa gaaatcatgg aagtaagact gctttttctt 4380 gccgttttcg tcggtaatca ccattcccgg cgggatagtc tgccagttca gttcgttgtt 4440 cacacaaacg gtgatacgta cacttttccc ggcaataaca tacggcgtga catcggcttc 4500 aaatggagta tagccgccct gatgctccat cacttcctga ttattgaccc acactttgcc 4560 gtaatgagtg accgcatcga aacgcagcac gatacgctgg cctgcccaac ctttcggtat 4620 aaagacttcg cgctgatacc agacgttgcc cgcataatta cgaatatctg catcggcgaa 4680 ctgatcgtta aaactgcctg gcacagcaat tgcccggctt tcttgtaacg cgctttccca 4740 ccaacgctga tcaattccac agttttcgcg atccagactg aatgcccaca ggccgtcgag 4800 ttttttgatt tcacgggttg gggtttctac aggacgtacc atactagttt gaatatgtat 4860 tacttggtta tggttatata tgacaaaaga aaaagaagaa cagaagaata acgcaaggaa 4920 gaacaataac tgaaattgat agagaagtat tatgtctttg tctttttata ataaatcaag 4980 tgcagaaatc cgttagacaa catgagggat aaaatttaac gtgggcgaag aagaaggaaa 5040 aaagtttttg tgagggcgta attgaagcga tctgttgatt gtagattttt tttttttgag 5100 gagtcaaagt cagaagagaa cagacaaatg gtattaacca tccaatactt ttttggagca 5160 acgctaagct catgcttttc cattggttac gtgctcagtt gttagatatg gaaagagagg 5220 atgctcacgg cagcgtgact ccaattgagc ccgaaagaga ggatgccacg ttttcccgac 5280 ggctgctaga atggaaaaag gaaaaataga agaatcccat tcctatcatt atttacgtaa 5340 tgacccacac atttttgaga ttttcaacta ttacgtatta cgataatcct gctgtcatta 5400 tcattattat ctatatcgac gtatgcaacg tatgtgaagc caagtaggcg tcgacaggac 5460 cttgttgtgt gacgaaattg gaagctgcaa tcaataggaa gacaggaagt cgagcgtgtc 5520 tgggtttttt cagttttgtt ctttttgcaa acaaatcacg agcgacggta attggtaccc 5580 agcttttgtt ccctttagtg agggttaatt ccgagcttgg cgtaatcatg gtcatagctg 5640 tttcctgtgt gaaattgtta tccgctcaca attccacaca acataggagc cggaagcata 5700 aagtgtaaag cctggggtgc ctaatgagtg aggtaactca cattaattgc gttgcgctca 5760 ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc 5820 gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg 5880 cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta 5940 tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc 6000 aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctcggccc ccctgacgag 6060 catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac 6120 caggcgttcc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc 6180 ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcaatg ctcacgctgt 6240 aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc 6300 gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga 6360 cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta 6420 ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aaggacagta 6480 tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga 6540 tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg 6600 cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag 6660 tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc 6720 tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact 6780 tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt 6840 cgttcatcca tagttgcctg actgcccgtc gtgtagataa ctacgatacg ggagggctta 6900 ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta 6960 tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc 7020 gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat 7080 agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt 7140 atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg 7200 tgaaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca 7260 gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta 7320 agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg 7380 cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact 7440 ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg 7500 ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt 7560 actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga 7620 ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc 7680 atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa 7740 caaatagggg ttccgcgcac atttccccga aaagtgccac ctgggtcctt ttcatcacgt 7800 gctataaaaa taattataat ttaaattttt taatataaat atataaatta aaaatagaaa 7860 gtaaaaaaag aaattaaaga aaaaatagtt tttgttttcc gaagatgtaa aagactctag 7920 ggggatcgcc aacaaatact accttttatc ttgctcttcc tgctctcagg tattaatgcc 7980 gaattgtttc atcttgtctg tgtagaagac cacacacgaa aatcctgtga ttttacattt 8040 tacttatcgt taatcgaatg tatatctatt taatctgctt ttcttgtcta ataaatatat 8100 atgtaaagta cgctttttgt tgaaattttt taaacctttg tttatttttt tttcttcatt 8160 ccgtaactct tctaccttct ttatttactt tctaaaatcc aaatacaaaa cataaaaata 8220 aataaacaca gagtaaattc ccaaattatt ccatcattaa aagatacgag gcgcgtgtaa 8280 gttacaggca agcgatccgt cctaagaaac cattattatc atgacattaa cctataaaaa 8340 taggcgtatc acgaggccct ttcgtc 8366 <210> 173 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Primer oBP674 <400> 173 aattggcgcg ccaattaccg tcgctcgtga tttg 34 <210> 174 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Primer oBP675 <400> 174 aattgtttaa acttgaatat gtattacttg gtta 34 <175> 175 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Primer oBP495 <400> 175 ggagatatac aatagaacag at 22 <210> 176 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Primer oBP496 <400> 176 tagcaatggg gtttttttca gt 22 <210> 177 <211> 725 <212> DNA <213> Artificial Sequence <220> <223> UAS (PGK1) -FBA1p <400> 177 aattaccgtc gctcgtgatt tgtttgcaaa aagaacaaaa ctgaaaaaac ccagacacgc 60 tcgacttcct gtcttcctat tgattgcagc ttccaatttc gtcacacaac aaggtcctgt 120 cgacgcctac ttggcttcac atacgttgca tacgtcgata tagataataa tgataatgac 180 agcaggatta tcgtaatacg taatagttga aaatctcaaa aatgtgtggg tcattacgta 240 aataatgata ggaatgggat tcttctattt ttcctttttc cattctagca gccgtcggga 300 aaacgtggca tcctctcttt cgggctcaat tggagtcacg ctgccgtgag catcctctct 360 ttccatatct aacaactgag cacgtaacca atggaaaagc atgagcttag cgttgctcca 420 aaaaagtatt ggatggttaa taccatttgt ctgttctctt ctgactttga ctcctcaaaa 480 aaaaaaaatc tacaatcaac agatcgcttc aattacgccc tcacaaaaac ttttttcctt 540 cttcttcgcc cacgttaaat tttatccctc atgttgtcta acggatttct gcacttgatt 600 tattataaaa agacaaagac ataatacttc tctatcaatt tcagttattg ttcttccttg 660 cgttattctt ctgttcttct ttttcttttg tcatatataa ccataaccaa gtaatacata 720 ttcaa 725 <210> 178 <211> 47 <212> DNA <213> Artificial Sequence <220> Primer T-FBA1 (SalI) <400> 178 attctacgta cgtcgacgcc tacttggctt cacatacgtt gcatacg 47 <210> 179 <211> 49 <212> DNA <213> Artificial Sequence <220> Primer B-FBA1 (SpeI) <400> 179 gtatcaaata ctagtttgaa tatgtattac ttggttatgg ttatatatg 49 <210> 180 <211> 10494 <212> DNA <213> Artificial Sequence <220> <223> pWS358-PGK1p-GUS <400> 180 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180 accataaatt cccgttttaa gagcttggtg agcgctagga gtcactgcca ggtatcgttt 240 gaacacggca ttagtcaggg aagtcataac acagtccttt cccgcaattt tctttttcta 300 ttactcttgg cctcctctag tacactctat atttttttat gcctcggtaa tgattttcat 360 tttttttttt cccctagcgg atgactcttt ttttttctta gcgattggca ttatcacata 420 atgaattata cattatataa agtaatgtga tttcttcgaa gaatatacta aaaaatgagc 480 aggcaagata aacgaaggca aagatgacag agcagaaagc cctagtaaag cgtattacaa 540 atgaaaccaa gattcagatt gcgatctctt taaagggtgg tcccctagcg atagagcact 600 cgatcttccc agaaaaagag gcagaagcag tagcagaaca ggccacacaa tcgcaagtga 660 ttaacgtcca cacaggtata gggtttctgg accatatgct agggattcat aaccattttc 720 tcaatcgaat tacacagaac acaccgtaca aacctctcta tcataactac ttaatagtca 780 cacacgtact cgtctaaata cacatcatcg tcctacaagt tcatcaaagt gttggacaga 840 caactatacc agcatggatc tcttgtatcg gttcttttct cccgctctct cgcaataaca 900 atgaacactg ggtcaatcat agcctacaca ggtgaacaga gtagcgttta tacagggttt 960 atacggtgat tcctacggca aaaatttttc atttctaaaa aaaaaaagaa aaatttttct 1020 ttccaacgct agaaggaaaa gaaaaatcta attaaattga tttggtgatt ttctgagagt 1080 tccctttttc atatatcgaa ttttgaatat aaaaggagat cgaaaaaatt tttctattca 1140 atctgttttc tggttttatt tgatagtttt tttgtgtatt attattatgg attagtactg 1200 gtttatatgg gtttttctgt ataacttctt tttattttag tttgtttaat cttattttga 1260 gttacattat agttccctaa ctgcaagaga agtaacatta aaactcgaga tgggtaagga 1320 aaagactcac gtttcgaggc cgcgattaaa ttccaacatg gatgctgatt tatatgggta 1380 taaatgggct cgcgataatg tcgggcaatc aggtgcgaca atctatcgat tgtatgggaa 1440 gcccgatgcg ccagagttgt ttctgaaaca tggcaaaggt agcgttgcca atgatgttac 1500 agatgagatg gtcagactaa actggctgac ggaatttatg cctcttccga ccatcaagca 1560 ttttatccgt actcctgatg atgcatggtt actcaccact gcgatccccg gcaaaacagc 1620 attccaggta ttagaagaat atcctgattc aggtgaaaat attgttgatg cgctggcagt 1680 gttcctgcgc cggttgcatt cgattcctgt ttgtaattgt ccttttaaca gcgatcgcgt 1740 atttcgtctc gctcaggcgc aatcacgaat gaataacggt ttggttgatg cgagtgattt 1800 tgatgacgag cgtaatggct ggcctgttga acaagtctgg aaagaaatgc ataagctttt 1860 gccattctca ccggattcag tcgtcactca tggtgatttc tcacttgata accttatttt 1920 tgacgagggg aaattaatag gttgtattga tgttggacga gtcggaatcg cagaccgata 1980 ccaggatctt gccatcctat ggaactgcct cggtgagttt tctccttcat tacagaaacg 2040 gctttttcaa aaatatggta ttgataatcc tgatatgaat aaattgcagt ttcatttgat 2100 gctcgatgag tttttctaag tttaacttga tactactaga ttttttctct tcatttataa 2160 aatttttggt tataattgaa gctttagaag tatgaaaaaa tccttttttt tcattctttg 2220 caaccaaaat aagaagcttc ttttattcat tgaaatgatg aatataaacc taacaaaaga 2280 aaaagactcg aatatcaaac attaaaaaaa aataaaagag gttatctgtt ttcccattta 2340 gttggagttt gcattttcta atagatagaa ctctcaatta atgtggattt agtttctctg 2400 ttcgctgcag catacgatat atatacatgt gtatatatgt atacctatga atgtcagtaa 2460 gtatgtatac gaacagtatg atactgaaga tgacaaggta atgcatcatt ctatacgtgt 2520 cattctgaac gaggcgcgct ttcctttttt ctttttgctt tttctttttt tttctcttga 2580 actcgacgga tctatgcggt gtgaaatacc gcacagatgc gtaaggagaa aataccgcat 2640 caggaaattg taaacgttaa tattttgtta aaattcgcgt taaatttttg ttaaatcagc 2700 tcatttttta accaataggc cgaaatcggc aaaatccctt ataaatcaaa agaatagacc 2760 gagatagggt tgagtgttgt tccagtttgg aacaagagtc cactattaaa gaacgtggac 2820 tccaacgtca aagggcgaaa aaccgtctat cagggcgatg gcccactacg tgaaccatca 2880 ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg 2940 agcccccgat ttagagcttg acggggaaag ccggcgaacg tggcgagaaa ggaagggaag 3000 aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct gcgcgtaacc 3060 accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt cgcgccattc gccattcagg 3120 ctgcgcaact gttgggaagg gcgatcggtg cgggcctctt cgctattacg ccagctggcg 3180 aaagggggat gtgctgcaag gcgattaagt tgggtaacgc cagggttttc ccagtcacga 3240 cgttgtaaaa cgacggccag tgagcgcgcg taatacgact cactataggg cgaattgggt 3300 accgggcccc ccctcgaggt cgacgtgagt aaggaaagag tgaggaacta tcgcatacct 3360 gcatttaaag atgccgattt gggcgcgaat cctttatttt ggcttcaccc tcatactatt 3420 atcagggcca gaaaaaggaa gtgtttccct ccttcttgaa ttgatgttac cctcataaag 3480 cacgtggcct cttatcgaga aagaaattac cgtcgctcgt gatttgtttg caaaaagaac 3540 aaaactgaaa aaacccagac acgctcgact tcctgtcttc ctattgattg cagcttccaa 3600 tttcgtcaca caacaaggtc ctagcgacgg ctcacaggtt ttgtaacaag caatcgaagg 3660 ttctggaatg gcgggaaagg gtttagtacc acatgctatg atgcccactg tgatctccag 3720 agcaaagttc gttcgatcgt actgttactc tctctctttc aaacagaatt gtccgaatcg 3780 tgtgacaaca acagcctgtt ctcacacact cttttcttct aaccaagggg gtggtttagt 3840 ttagtagaac ctcgtgaaac ttacatttac atatatataa acttgcataa attggtcaat 3900 gcaagaaata catatttggt cttttctaat tcgtagtttt tcaagttctt agatgctttc 3960 tttttctctt ttttacagat catcaaggaa gtaattatct actttttaca acaaatataa 4020 aacaactagt atggtacgtc ctgtagaaac cccaacccgt gaaatcaaaa aactcgacgg 4080 cctgtgggca ttcagtctgg atcgcgaaaa ctgtggaatt gatcagcgtt ggtgggaaag 4140 cgcgttacaa gaaagccggg caattgctgt gccaggcagt tttaacgatc agttcgccga 4200 tgcagatatt cgtaattatg cgggcaacgt ctggtatcag cgcgaagtct ttataccgaa 4260 aggttgggca ggccagcgta tcgtgctgcg tttcgatgcg gtcactcatt acggcaaagt 4320 gtgggtcaat aatcaggaag tgatggagca tcagggcggc tatactccat ttgaagccga 4380 tgtcacgccg tatgttattg ccgggaaaag tgtacgtatc accgtttgtg tgaacaacga 4440 actgaactgg cagactatcc cgccgggaat ggtgattacc gacgaaaacg gcaagaaaaa 4500 gcagtcttac ttccatgatt tctttaacta tgccggaatc catcgcagcg taatgctcta 4560 caccacgccg aacacctggg tggacgatat caccgtggtg acgcatgtcg cgcaagactg 4620 taaccacgcg tctgttgact ggcaggtggt ggccaatggt gatgtcagcg ttgaactgcg 4680 tgatgcggat caacaggtgg ttgcaactgg acaaggcact agcgggactt tgcaagtggt 4740 gaatccgcac ctctggcaac cgggtgaagg ttatctctat gaactgtgcg tcacagccaa 4800 aagccagaca gagtgtgata tctacccgct tcgcgtcggc atccggtcag tggcagtgaa 4860 gggcgaacag ttcctgatta accacaaacc gttctacttt actggctttg gtcgtcatga 4920 agatgcggac ttgcgtggca aaggattcga taacgtgctg atggtgcacg accacgcatt 4980 aatggactgg attggggcca actcctaccg tacctcgcat tacccttacg ctgaagagat 5040 gctcgactgg gcagatgaac atggcatcgt ggtgattgat gaaactgctg ctgtcggctt 5100 taacctctct ttaggcattg gtttcgaagc gggcaacaag ccgaaagaac tgtacagcga 5160 agaggcagtc aacggggaaa ctcagcaagc gcacttacag gcgattaaag agctgatagc 5220 gcgtgacaaa aaccacccaa gcgtggtgat gtggagtatt gccaacgaac cggatacccg 5280 tccgcaaggt gcacgggaat atttcgcgcc actggcggaa gcaacgcgta aactcgaccc 5340 gacgcgtccg atcacctgcg tcaatgtaat gttctgcgac gctcacaccg ataccatcag 5400 cgatctcttt gatgtgctgt gcctgaaccg ttattacgga tggtatgtcc aaagcggcga 5460 tttggaaacg gcagagaagg tactggaaaa agaacttctg gcctggcagg agaaactgca 5520 tcagccgatt atcatcaccg aatacggcgt ggatacgtta gccgggctgc actcaatgta 5580 caccgacatg tggagtgaag agtatcagtg tgcatggctg gatatgtatc accgcgtctt 5640 tgatcgcgtc agcgccgtcg tcggtgaaca ggtatggaat ttcgccgatt ttgcgacctc 5700 gcaaggcata ttgcgcgttg gcggtaacaa gaaagggatc ttcactcgcg accgcaaacc 5760 gaagtcggcg gcttttctgc tgcaaaaacg ctggactggc atgaacttcg gtgaaaaacc 5820 gcagcaggga ggcaaacaat gattaattaa ctagagcggc cgcgttaatt caaattaatt 5880 gatatagttt tttaatgagt attgaatctg tttagaaata atggaatatt atttttattt 5940 atttatttat attattggtc ggctcttttc ttctgaaggt caatgacaaa atgatatgaa 6000 ggaaataatg atttctaaaa ttttacaacg taagatattt ttacaaaagc ctagctcatc 6060 ttttgtcatg cactatttta ctcacgcttg aaattaacgg ccagtccact gcggagtcat 6120 ttcaaagtca tcctaatcga tctatcgttt ttgatagctc attttggagt tcgcgattgt 6180 cttctgttat tcacaactgt tttaattttt atttcattct ggaactcttc gagttctttg 6240 taaagtcttt catagtagct tactttatcc tccaacatat ttaacttcat gtcaatttcg 6300 gctcttaaat tttccacatc atcaagttca acatcatctt ttaacttgaa tttattctct 6360 agctcttcca accaagcctc attgctcctt gatttactgg tgaaaagtga tacactttgc 6420 gcgcaatcca ggtcaaaact ttcctgcaaa gaattcacca atttctcgac atcatagtac 6480 aatttgtttt gttctcccat cacaatttaa tatacctgat ggattcttat gaagcgctgg 6540 gtaatggacg tgtcactcta cttcgccttt ttccctactc cttttagtac ggaagacaat 6600 gctaataaat aagagggtaa taataatatt attaatcggc aaaaaagatt aaacgccaag 6660 cgtttaatta tcagaaagca aacgtcgtac caatccttga atgcttccca attgtatatt 6720 aagagtcatc acagcaacat attcttgtta ttaaattaat tattattgat ttttgatatt 6780 gtataaaaaa accaaatatg tataaaaaaa gtgaataaaa aataccaagt atggagaaat 6840 atattagaag tctatacgtt aaaccaccgc ggtggagctc cagcttttgt tccctttagt 6900 gagggttaat tgcgcgcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt 6960 atccgctcac aattccacac aacataggag ccggaagcat aaagtgtaaa gcctggggtg 7020 cctaatgagt gaggtaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg 7080 gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc 7140 gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc 7200 ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata 7260 acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg 7320 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct 7380 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa 7440 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc 7500 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt 7560 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg 7620 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg 7680 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct 7740 tgaagtggtg gcctaactac ggctacacta gaaggacagt atttggtatc tgcgctctgc 7800 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg 7860 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc 7920 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt 7980 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa 8040 aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat 8100 gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct 8160 gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg 8220 caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag 8280 ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta 8340 attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg 8400 ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg 8460 gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct 8520 ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta 8580 tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg 8640 gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc 8700 cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg 8760 gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga 8820 tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg 8880 ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat 8940 gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc 9000 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca 9060 catttccccg aaaagtgcca cctgaacgaa gcatctgtgc ttcattttgt agaacaaaaa 9120 tgcaacgcga gagcgctaat ttttcaaaca aagaatctga gctgcatttt tacagaacag 9180 aaatgcaacg cgaaagcgct attttaccaa cgaagaatct gtgcttcatt tttgtaaaac 9240 aaaaatgcaa cgcgagagcg ctaatttttc aaacaaagaa tctgagctgc atttttacag 9300 aacagaaatg caacgcgaga gcgctatttt accaacaaag aatctatact tcttttttgt 9360 tctacaaaaa tgcatcccga gagcgctatt tttctaacaa agcatcttag attacttttt 9420 ttctcctttg tgcgctctat aatgcagtct cttgataact ttttgcactg taggtccgtt 9480 aaggttagaa gaaggctact ttggtgtcta ttttctcttc cataaaaaaa gcctgactcc 9540 acttcccgcg tttactgatt actagcgaag ctgcgggtgc attttttcaa gataaaggca 9600 tccccgatta tattctatac cgatgtggat tgcgcatact ttgtgaacag aaagtgatag 9660 cgttgatgat tcttcattgg tcagaaaatt atgaacggtt tcttctattt tgtctctata 9720 tactacgtat aggaaatgtt tacattttcg tattgttttc gattcactct atgaatagtt 9780 cttactacaa tttttttgtc taaagagtaa tactagagat aaacataaaa aatgtagagg 9840 tcgagtttag atgcaagttc aaggagcgaa aggtggatgg gtaggttata tagggatata 9900 gcacagagat atatagcaaa gagatacttt tgagcaatgt ttgtggaagc ggtattcgca 9960 atattttagt agctcgttac agtccggtgc gtttttggtt ttttgaaagt gcgtcttcag 10020 agcgcttttg gttttcaaaa gcgctctgaa gttcctatac tttctagaga ataggaactt 10080 cggaatagga acttcaaagc gtttccgaaa acgagcgctt ccgaaaatgc aacgcgagct 10140 gcgcacatac agctcactgt tcacgtcgca cctatatctg cgtgttgcct gtatatatat 10200 atacatgaga agaacggcat agtgcgtgtt tatgcttaaa tgcgtactta tatgcgtcta 10260 tttatgtagg atgaaaggta gtctagtacc tcctgtgata ttatcccatt ccatgcgggg 10320 tatcgtatgc ttccttcagc actacccttt agctgttcta tatgctgcca ctcctcaatt 10380 ggattagtct catccttcaa tgctatcatt tcctttgata ttggatcatc taagaaacca 10440 ttattatcat gacattaacc tataaaaata ggcgtatcac gaggcccttt cgtc 10494 <210> 181 <211> 43 <212> DNA <213> Artificial Sequence <220> <223> Primer T-U / PGK1 (KpnI) <400> 181 actacagatg gtaccaatta ccgtcgctcg tgatttgttt gca 43 <210> 182 <211> 45 <212> DNA <213> Artificial Sequence <220> Primer B-U / PGK1 (SalI) <400> 182 agcatccttg tcgacaggac cttgttgtgt gacgaaattg gaagc 45 <210> 183 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer N98SeqF1 <400> 183 cgtgttagtc acatcaggac 20 <210> 184 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Primer N99SeqR2 <400> 184 catcgactgc attacgcaac tc 22 <210> 185 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer N98SeqF4 <400> 185 ggtttctgtc tctggtgacg 20 <210> 186 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Primer N1111 <400> 186 tatttgtatc gaggtgtcta gtcttctatt 30 <210> 187 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Primer N1110 <400> 187 gcgatttaat ctctaattat tagttaaagt 30 <210> 188 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> Primer BK468 <400> 188 gcctcgagtt ttaatgttac ttctcttgca gttaggga 38 <210> 189 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Primer N160SeqF5 <400> 189 cctgaagtct aggtccctat tt 22 <210> 190 <211> 7589 <212> DNA <213> Artificial Sequence <220> <223> pUC19-kan :: pdc1 :: FBA-alsS :: TRX1 (clone A) <400> 190 tatttgtatc gaggtgtcta gtcttctatt acactaatgc agtttcaggg ttttggaaac 60 cacactgttt aaacagtgtt ccttaatcaa ggatacctct ttttttttcc ttggttccac 120 taattcatcg gttttttttt tggaagacat cttttccaac gaaaagaata tacatatcgt 180 ttaagagaaa ttctccaaat ttgtaaagaa gcggacccag acttaaggcc gcccgcaaat 240 taaagccttc gagcgtccca aaaccttctc aagcaaggtt ttcagtataa tgttacatgc 300 gtacacgcgt ctgtacagaa aaaaaagaaa aatttgaaat ataaataacg ttcttaatac 360 taacataact ataaaaaaat aaatagggac ctagacttca ggttgtctaa ctccttcctt 420 ttcggttaga gcggatgtgg ggggagggcg tgaatgtaag cgtgacataa ctaattacat 480 gattaattaa ctagagagct ttcgttttca tgagttcccc gaattctttc ggaagcttgt 540 cacttgctaa attaatgtta tcactgtagt caaccgggac atcgatgatg acaggacctt 600 cagcgttcat gccttgacgc agaacatctg ccagctggtc tggtgattct acgcgcaagc 660 cagttgctcc gaagctttcc gcatatttca cgatatcgat atttccgaaa tcgaccgcag 720 atgtacggtt atattttttc aattgctgga atgcaaccat gtcatatgtg ctgtcgttcc 780 atacaatgtg tacaattggt gcttttagtc gaactgctgt ctctaattcc attgctgaga 840 ataagaaacc gccgtcacca gagacagaaa ccactttttc tcccggtttc accaatgaag 900 cgccgattgc ccaaggaagc gcaacgccga gtgtttgcat accgttactg atcattaatg 960 ttaacggctc gtagctgcgg aaataacgtg acatccaaat ggcgtgcgaa ccgatatcgc 1020 aagttactgt aacatgatca tcgactgcat tacgcaactc tttaacgatt tcaagagggt 1080 gcgctctgtc tgatttccaa tctgcaggca cctgctcacc ttcatgcata tattgtttta 1140 aatcagaaag gattttctgc tcacgctctg caaattccac tttcacagca tcgtgttcga 1200 tatgattgat cgtggacgga atgtcaccga tcaattcaag atcaggctgg taagcatgat 1260 caatgtcagc gataatctcg tctaaatgga taattgtccg gtctccattg atattccaga 1320 atttcggatc atattcaatc gggtcatagc cgatcgtcag aacaacatct gcctgctcta 1380 gcagtaaatc gccaggctgg ttgcggaaca aaccgatacg gccaaaatat tgatcctcta 1440 aatctctaga aagggtaccg gcagcttgat atgtttcaac aaatggaagc tgaacctttt 1500 tcaaaagctt gcgaaccgct ttaattgctt ccggtcttcc gcctttcatg ccgaccaaaa 1560 cgacaggaag ttttgctgtt tggatttttg ctatggccgc actgattgca tcatctgctg 1620 caggaccgag ttttggcgct gcaacagcac gcacgttttt cgtatttgtg acttcattca 1680 caacatcttg cggaaagctc acaaaagcgg ccccagcctg ccctgctgac gctatcctaa 1740 atgcatttgt aacagcttcc ggtatatttt ttacatcttg aacttctaca ctgtattttg 1800 taatcggctg gaatagcgcc gcattatcca aagattgatg tgtccgtttt aaacgatctg 1860 cacggatcac gtttccagca agcgcaacga cagggtctcc ttcagtgttc gctgtcagca 1920 ggcctgttgc caagttagag gcacccggtc ctgatgtgac taacacgact cccggttttc 1980 cagttaaacg gccgactgct tgggccatga atgctgcgtt ttgttcgtgc cgggcaacga 2040 taatttcagg tcctttatct tgtaaagcgt caaataccgc atcaattttt gcacctggaa 2100 tgccaaatac atgtgtgaca ccttgctcca ctaagcaatc aacaacaagc tccgcccctc 2160 tgtttttcac aagggatttt tgttcttttg ttgcttttgt caacatcctc agctctagat 2220 ttgaatatgt attacttggt tatggttata tatgacaaaa gaaaaagaag aacagaagaa 2280 taacgcaagg aagaacaata actgaaattg atagagaagt attatgtctt tgtcttttta 2340 taataaatca agtgcagaaa tccgttagac aacatgaggg ataaaattta acgtgggcga 2400 agaagaagga aaaaagtttt tgtgagggcg taattgaagc gatctgttga ttgtagattt 2460 tttttttttg aggagtcaaa gtcagaagag aacagacaaa tggtattaac catccaatac 2520 ttttttggag caacgctaag ctcatgcttt tccattggtt acgtgctcag ttgttagata 2580 tggaaagaga ggatgctcac ggcagcgtga ctccaattga gcccgaaaga gaggatgcca 2640 cgttttcccg acggctgcta gaatggaaaa aggaaaaata gaagaatccc attcctatca 2700 ttatttacgt aatgacccac acatttttga gattttcaac tattacgtat tacgataatc 2760 ctgctgtcat tatcattatt atctatatcg acgtatgcaa cgtatgtgaa gccaagtagg 2820 caattattta gtactgtcag tattgttatt catttcagat cttaagccta accaggccaa 2880 ttcaacagac tgtcggcaac ttcttgtctg gtctttccat ggtaagtgac agtgcagtaa 2940 taatatgaac caatttattt ttcgttacat aaaaatgctt ataaaacttt aactaataat 3000 tagagattaa atcgcaaacg gccgactcta gaggatcccc caccttggct aactcgttgt 3060 atcatcactg gataacttcg tatagcatac attatacgaa gttatctagg gattcataac 3120 cattttctca atcgaattac acagaacaca ccgtacaaac ctctctatca taactactta 3180 atagtcacac acgtactcgt ctaaatacac atcatcgtcc tacaagttca tcaaagtgtt 3240 ggacagacaa ctataccagc atggatctct tgtatcggtt cttttctccc gctctctcgc 3300 aataacaatg aacactgggt caatcatagc ctacacaggt gaacagagta gcgtttatac 3360 agggtttata cggtgattcc tacggcaaaa atttttcatt tctaaaaaaa aaaagaaaaa 3420 tttttctttc caacgctaga aggaaaagaa aaatctaatt aaattgattt ggtgattttc 3480 tgagagttcc ctttttcata tatcgaattt tgaatataaa aggagatcga aaaaattttt 3540 ctattcaatc tgttttctgg ttttatttga tagttttttt gtgtattatt attatggatt 3600 agtactggtt tatatgggtt tttctgtata acttcttttt attttagttt gtttaatctt 3660 attttgagtt acattatagt tccctaactg caagagaagt aacattaaaa ctcgagatgg 3720 gtaaggaaaa gactcacgtt tcgaggccgc gattaaattc caacatggat gctgatttat 3780 atgggtataa atgggctcgc gataatgtcg ggcaatcagg tgcgacaatc tatcgattgt 3840 atgggaagcc cgatgcgcca gagttgtttc tgaaacatgg caaaggtagc gttgccaatg 3900 atgttacaga tgagatggtc agactaaact ggctgacgga atttatgcct cttccgacca 3960 tcaagcattt tatccgtact cctgatgatg catggttact caccactgcg atccccggca 4020 aaacagcatt ccaggtatta gaagaatatc ctgattcagg tgaaaatatt gttgatgcgc 4080 tggcagtgtt cctgcgccgg ttgcattcga ttcctgtttg taattgtcct tttaacagcg 4140 atcgcgtatt tcgtctcgct caggcgcaat cacgaatgaa taacggtttg gttgatgcga 4200 gtgattttga tgacgagcgt aatggctggc ctgttgaaca agtctggaaa gaaatgcata 4260 agcttttgcc attctcaccg gattcagtcg tcactcatgg tgatttctca cttgataacc 4320 ttatttttga cgaggggaaa ttaataggtt gtattgatgt tggacgagtc ggaatcgcag 4380 accgatacca ggatcttgcc atcctatgga actgcctcgg tgagttttct ccttcattac 4440 agaaacggct ttttcaaaaa tatggtattg ataatcctga tatgaataaa ttgcagtttc 4500 atttgatgct cgatgagttt ttctaagttt aacttgatac tactagattt tttctcttca 4560 tttataaaat ttttggttat aattgaagct ttagaagtat gaaaaaatcc ttttttttca 4620 ttctttgcaa ccaaaataag aagcttcttt tattcattga aatgatgaat ataaacctaa 4680 caaaagaaaa agactcgaat atcaaacatt aaaaaaaaat aaaagaggtt atctgttttc 4740 ccatttagtt ggagtttgca ttttctaata gatagaactc tcaattaatg tggatttagt 4800 ttctctgttc gataacttcg tatagcatac attatacgaa gttatctgaa cattagaata 4860 cgtaatccgc aatgcggggc cgcttaatta atctagagtc gacctgcagg catgcaagct 4920 tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac 4980 acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac 5040 tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc 5100 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg 5160 cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc 5220 actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt 5280 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc 5340 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa 5400 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc 5460 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg 5520 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc 5580 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc 5640 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca 5700 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact 5760 acggctacac tagaaggaca gtatttggta tctgcgctct gctgaagcca gttaccttcg 5820 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt 5880 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct 5940 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga 6000 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa 6060 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac 6120 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga 6180 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc 6240 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca 6300 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta 6360 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg 6420 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc 6480 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg 6540 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt 6600 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt 6660 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata 6720 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc 6780 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac 6840 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa 6900 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct 6960 tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat 7020 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc 7080 cacctgacgt ctaagaaacc attattatca tgacattaac ctataaaaat aggcgtatca 7140 cgaggccctt tcgtctcgcg cgtttcggtg atgacggtga aaacctctga cacatgcagc 7200 tcccggagac ggtcacagct tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg 7260 gcgcgtcagc gggtgttggc gggtgtcggg gctggcttaa ctatgcggca tcagagcaga 7320 ttgtactgag agtgcaccat atgcggtgtg aaataccgca cagatgcgta aggagaaaat 7380 accgcatcag gcgccattcg ccattcaggc tgcgcaactg ttgggaaggg cgatcggtgc 7440 gggcctcttc gctattacgc cagctggcga aagggggatg tgctgcaagg cgattaagtt 7500 gggtaacgcc agggttttcc cagtcacgac gttgtaaaac gacggccagt gaattcgagc 7560 tcggtacccg gggatccggc gcgccgttt 7589 <210> 191 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> Primer N191 <400> 191 atccgcggat agatctccca ttaccgacat ttgggcgc 38 <210> 192 <211> 9593 <212> DNA <213> Artificial Sequence <220> <223> pYZ090 alsS <400> 192 ggccgcacct ggtaaaacct ctagtggagt agtagatgta atcaatgaag cggaagccaa 60 aagaccagag tagaggccta tagaagaaac tgcgatacct tttgtgatgg ctaaacaaac 120 agacatcttt ttatatgttt ttacttctgt atatcgtgaa gtagtaagtg ataagcgaat 180 ttggctaaga acgttgtaag tgaacaaggg acctcttttg cctttcaaaa aaggattaaa 240 tggagttaat cattgagatt tagttttcgt tagattctgt atccctaaat aactccctta 300 cccgacggga aggcacaaaa gacttgaata atagcaaacg gccagtagcc aagaccaaat 360 aatactagag ttaactgatg gtcttaaaca ggcattacgt ggtgaactcc aagaccaata 420 tacaaaatat cgataagtta ttcttgccca ccaatttaag gagcctacat caggacagta 480 gtaccattcc tcagagaaga ggtatacata acaagaaaat cgcgtgaaca ccttatataa 540 cttagcccgt tattgagcta aaaaaccttg caaaatttcc tatgaataag aatacttcag 600 acgtgataaa aatttacttt ctaactcttc tcacgctgcc cctatctgtt cttccgctct 660 accgtgagaa ataaagcatc gagtacggca gttcgctgtc actgaactaa aacaataagg 720 ctagttcgaa tgatgaactt gcttgctgtc aaacttctga gttgccgctg atgtgacact 780 gtgacaataa attcaaaccg gttatagcgg tctcctccgg taccggttct gccacctcca 840 atagagctca gtaggagtca gaacctctgc ggtggctgtc agtgactcat ccgcgtttcg 900 taagttgtgc gcgtgcacat ttcgcccgtt cccgctcatc ttgcagcagg cggaaatttt 960 catcacgctg taggacgcaa aaaaaaaata attaatcgta caagaatctt ggaaaaaaaa 1020 ttgaaaaatt ttgtataaaa gggatgacct aacttgactc aatggctttt acacccagta 1080 ttttcccttt ccttgtttgt tacaattata gaagcaagac aaaaacatat agacaaccta 1140 ttcctaggag ttatattttt ttaccctacc agcaatataa gtaaaaaact gtttaaacag 1200 tatggcagtt acaatgtatt atgaagatga tgtagaagta tcagcacttg ctggaaagca 1260 aattgcagta atcggttatg gttcacaagg acatgctcac gcacagaatt tgcgtgattc 1320 tggtcacaac gttatcattg gtgtgcgcca cggaaaatct tttgataaag caaaagaaga 1380 tggctttgaa acatttgaag taggagaagc agtagctaaa gctgatgtta ttatggtttt 1440 ggcaccagat gaacttcaac aatccattta tgaagaggac atcaaaccaa acttgaaagc 1500 aggttcagca cttggttttg ctcacggatt taatatccat tttggctata ttaaagtacc 1560 agaagacgtt gacgtcttta tggttgcgcc taaggctcca ggtcaccttg tccgtcggac 1620 ttatactgaa ggttttggta caccagcttt gtttgtttca caccaaaatg caagtggtca 1680 tgcgcgtgaa atcgcaatgg attgggccaa aggaattggt tgtgctcgag tgggaattat 1740 tgaaacaact tttaaagaag aaacagaaga agatttgttt ggagaacaag ctgttctatg 1800 tggaggtttg acagcacttg ttgaagccgg ttttgaaaca ctgacagaag ctggatacgc 1860 tggcgaattg gcttactttg aagttttgca cgaaatgaaa ttgattgttg acctcatgta 1920 tgaaggtggt tttactaaaa tgcgtcaatc catctcaaat actgctgagt ttggcgatta 1980 tgtgactggt ccacggatta ttactgacga agttaaaaag aatatgaagc ttgttttggc 2040 tgatattcaa tctggaaaat ttgctcaaga tttcgttgat gacttcaaag cggggcgtcc 2100 aaaattaata gcctatcgcg aagctgcaaa aaatcttgaa attgaaaaaa ttggggcaga 2160 gctacgtcaa gcaatgccat tcacacaatc tggtgatgac gatgccttta aaatctatca 2220 gtaaggccct gcaggccaga ggaaaataat atcaagtgct ggaaactttt tctcttggaa 2280 tttttgcaac atcaagtcat agtcaattga attgacccaa tttcacattt aagatttttt 2340 ttttttcatc cgacatacat ctgtacacta ggaagccctg tttttctgaa gcagcttcaa 2400 atatatatat tttttacata tttattatga ttcaatgaac aatctaatta aatcgaaaac 2460 aagaaccgaa acgcgaataa ataatttatt tagatggtga caagtgtata agtcctcatc 2520 gggacagcta cgatttctct ttcggttttg gctgagctac tggttgctgt gacgcagcgg 2580 cattagcgcg gcgttatgag ctaccctcgt ggcctgaaag atggcgggaa taaagcggaa 2640 ctaaaaatta ctgactgagc catattgagg tcaatttgtc aactcgtcaa gtcacgtttg 2700 gtggacggcc cctttccaac gaatcgtata tactaacatg cgcgcgcttc ctatatacac 2760 atatacatat atatatatat atatatgtgt gcgtgtatgt gtacacctgt atttaatttc 2820 cttactcgcg ggtttttctt ttttctcaat tcttggcttc ctctttctcg agcggaccgg 2880 atcctccgcg gtgccggcag atctatttaa atggcgcgcc gacgtcaggt ggcacttttc 2940 ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc 3000 cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga 3060 gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt 3120 ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag 3180 tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag 3240 aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta 3300 ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg 3360 agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca 3420 gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag 3480 gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc 3540 gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg 3600 tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc 3660 ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg 3720 cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg 3780 gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga 3840 cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac 3900 tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa 3960 aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca 4020 aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag 4080 gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac 4140 cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa 4200 ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc 4260 accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag 4320 tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac 4380 cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc 4440 gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc 4500 ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca 4560 cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc 4620 tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg 4680 ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct 4740 ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata 4800 ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc 4860 gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc agctggcacg 4920 acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc aattaatgtg agttagctca 4980 ctcattaggc accccaggct ttacacttta tgcttccggc tcgtatgttg tgtggaattg 5040 tgagcggata acaatttcac acaggaaaca gctatgacca tgattacgcc aagctttttc 5100 tttccaattt tttttttttc gtcattataa aaatcattac gaccgagatt cccgggtaat 5160 aactgatata attaaattga agctctaatt tgtgagttta gtatacatgc atttacttat 5220 aatacagttt tttagttttg ctggccgcat cttctcaaat atgcttccca gcctgctttt 5280 ctgtaacgtt caccctctac cttagcatcc cttccctttg caaatagtcc tcttccaaca 5340 ataataatgt cagatcctgt agagaccaca tcatccacgg ttctatactg ttgacccaat 5400 gcgtctccct tgtcatctaa acccacaccg ggtgtcataa tcaaccaatc gtaaccttca 5460 tctcttccac ccatgtctct ttgagcaata aagccgataa caaaatcttt gtcgctcttc 5520 gcaatgtcaa cagtaccctt agtatattct ccagtagata gggagccctt gcatgacaat 5580 tctgctaaca tcaaaaggcc tctaggttcc tttgttactt cttctgccgc ctgcttcaaa 5640 ccgctaacaa tacctgggcc caccacaccg tgtgcattcg taatgtctgc ccattctgct 5700 attctgtata cacccgcaga gtactgcaat ttgactgtat taccaatgtc agcaaatttt 5760 ctgtcttcga agagtaaaaa attgtacttg gcggataatg cctttagcgg cttaactgtg 5820 ccctccatgg aaaaatcagt caagatatcc acatgtgttt ttagtaaaca aattttggga 5880 cctaatgctt caactaactc cagtaattcc ttggtggtac gaacatccaa tgaagcacac 5940 aagtttgttt gcttttcgtg catgatatta aatagcttgg cagcaacagg actaggatga 6000 gtagcagcac gttccttata tgtagctttc gacatgattt atcttcgttt cctgcaggtt 6060 tttgttctgt gcagttgggt taagaatact gggcaatttc atgtttcttc aacactacat 6120 atgcgtatat ataccaatct aagtctgtgc tccttccttc gttcttcctt ctgttcggag 6180 attaccgaat caaaaaaatt tcaaggaaac cgaaatcaaa aaaaagaata aaaaaaaaat 6240 gatgaattga aaagcttgca tgcctgcagg tcgactctag tatactccgt ctactgtacg 6300 atacacttcc gctcaggtcc ttgtccttta acgaggcctt accactcttt tgttactcta 6360 ttgatccagc tcagcaaagg cagtgtgatc taagattcta tcttcgcgat gtagtaaaac 6420 tagctagacc gagaaagaga ctagaaatgc aaaaggcact tctacaatgg ctgccatcat 6480 tattatccga tgtgacgctg catttttttt tttttttttt tttttttttt tttttttttt 6540 tttttttttt ttttgtacaa atatcataaa aaaagagaat ctttttaagc aaggattttc 6600 ttaacttctt cggcgacagc atcaccgact tcggtggtac tgttggaacc acctaaatca 6660 ccagttctga tacctgcatc caaaaccttt ttaactgcat cttcaatggc tttaccttct 6720 tcaggcaagt tcaatgacaa tttcaacatc attgcagcag acaagatagt ggcgataggg 6780 ttgaccttat tctttggcaa atctggagcg gaaccatggc atggttcgta caaaccaaat 6840 gcggtgttct tgtctggcaa agaggccaag gacgcagatg gcaacaaacc caaggagcct 6900 gggataacgg aggcttcatc ggagatgata tcaccaaaca tgttgctggt gattataata 6960 ccatttaggt gggttgggtt cttaactagg atcatggcgg cagaatcaat caattgatgt 7020 tgaactttca atgtagggaa ttcgttcttg atggtttcct ccacagtttt tctccataat 7080 cttgaagagg ccaaaacatt agctttatcc aaggaccaaa taggcaatgg tggctcatgt 7140 tgtagggcca tgaaagcggc cattcttgtg attctttgca cttctggaac ggtgtattgt 7200 tcactatccc aagcgacacc atcaccatcg tcttcctttc tcttaccaaa gtaaatacct 7260 cccactaatt ctctaacaac aacgaagtca gtacctttag caaattgtgg cttgattgga 7320 gataagtcta aaagagagtc ggatgcaaag ttacatggtc ttaagttggc gtacaattga 7380 agttctttac ggatttttag taaaccttgt tcaggtctaa cactaccggt accccattta 7440 ggaccaccca cagcacctaa caaaacggca tcagccttct tggaggcttc cagcgcctca 7500 tctggaagtg gaacacctgt agcatcgata gcagcaccac caattaaatg attttcgaaa 7560 tcgaacttga cattggaacg aacatcagaa atagctttaa gaaccttaat ggcttcggct 7620 gtgatttctt gaccaacgtg gtcacctggc aaaacgacga tcttcttagg ggcagacatt 7680 acaatggtat atccttgaaa tatatataaa aaaaaaaaaa aaaaaaaaaa aaaaaaatgc 7740 agcttctcaa tgatattcga atacgctttg aggagataca gcctaatatc cgacaaactg 7800 ttttacagat ttacgatcgt acttgttacc catcattgaa ttttgaacat ccgaacctgg 7860 gagttttccc tgaaacagat agtatatttg aacctgtata ataatatata gtctagcgct 7920 ttacggaaga caatgtatgt atttcggttc ctggagaaac tattgcatct attgcatagg 7980 taatcttgca cgtcgcatcc ccggttcatt ttctgcgttt ccatcttgca cttcaatagc 8040 atatctttgt taacgaagca tctgtgcttc attttgtaga acaaaaatgc aacgcgagag 8100 cgctaatttt tcaaacaaag aatctgagct gcatttttac agaacagaaa tgcaacgcga 8160 aagcgctatt ttaccaacga agaatctgtg cttcattttt gtaaaacaaa aatgcaacgc 8220 gagagcgcta atttttcaaa caaagaatct gagctgcatt tttacagaac agaaatgcaa 8280 cgcgagagcg ctattttacc aacaaagaat ctatacttct tttttgttct acaaaaatgc 8340 atcccgagag cgctattttt ctaacaaagc atcttagatt actttttttc tcctttgtgc 8400 gctctataat gcagtctctt gataactttt tgcactgtag gtccgttaag gttagaagaa 8460 ggctactttg gtgtctattt tctcttccat aaaaaaagcc tgactccact tcccgcgttt 8520 actgattact agcgaagctg cgggtgcatt ttttcaagat aaaggcatcc ccgattatat 8580 tctataccga tgtggattgc gcatactttg tgaacagaaa gtgatagcgt tgatgattct 8640 tcattggtca gaaaattatg aacggtttct tctattttgt ctctatatac tacgtatagg 8700 aaatgtttac attttcgtat tgttttcgat tcactctatg aatagttctt actacaattt 8760 ttttgtctaa agagtaatac tagagataaa cataaaaaat gtagaggtcg agtttagatg 8820 caagttcaag gagcgaaagg tggatgggta ggttatatag ggatatagca cagagatata 8880 tagcaaagag atacttttga gcaatgtttg tggaagcggt attcgcaata ttttagtagc 8940 tcgttacagt ccggtgcgtt tttggttttt tgaaagtgcg tcttcagagc gcttttggtt 9000 ttcaaaagcg ctctgaagtt cctatacttt ctagagaata ggaacttcgg aataggaact 9060 tcaaagcgtt tccgaaaacg agcgcttccg aaaatgcaac gcgagctgcg cacatacagc 9120 tcactgttca cgtcgcacct atatctgcgt gttgcctgta tatatatata catgagaaga 9180 acggcatagt gcgtgtttat gcttaaatgc gtacttatat gcgtctattt atgtaggatg 9240 aaaggtagtc tagtacctcc tgtgatatta tcccattcca tgcggggtat cgtatgcttc 9300 cttcagcact accctttagc tgttctatat gctgccactc ctcaattgga ttagtctcat 9360 ccttcaatgc tatcatttcc tttgatattg gatcatatgc atagtaccga gaaactagag 9420 gatctcccat taccgacatt tgggcgctat acgtgcatat gttcatgtat gtatctgtat 9480 ttaaaacact tttgtattat ttttcctcat atatgtgtat aggtttatac ggatgattta 9540 attattactt caccaccctt tatttcaggc tgatatctta gccttgttac tag 9593 <210> 193 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Primer oBP556 <400> 193 tattttcgag gaccttgtca cc 22 <210> 194 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Primer oBP561 <400> 194 tggccattaa tctttcccat attag 25 <210> 195 <211> 12896 <212> DNA <213> Artificial Sequence <220> <223> pBP915 <400> 195 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180 accataaatt cccgttttaa gagcttggtg agcgctagga gtcactgcca ggtatcgttt 240 gaacacggca ttagtcaggg aagtcataac acagtccttt cccgcaattt tctttttcta 300 ttactcttgg cctcctctag tacactctat atttttttat gcctcggtaa tgattttcat 360 tttttttttt ccacctagcg gatgactctt tttttttctt agcgattggc attatcacat 420 aatgaattat acattatata aagtaatgtg atttcttcga agaatatact aaaaaatgag 480 caggcaagat aaacgaaggc aaagatgaca gagcagaaag ccctagtaaa gcgtattaca 540 aatgaaacca agattcagat tgcgatctct ttaaagggtg gtcccctagc gatagagcac 600 tcgatcttcc cagaaaaaga ggcagaagca gtagcagaac aggccacaca atcgcaagtg 660 attaacgtcc acacaggtat agggtttctg gaccatatga tacatgctct ggccaagcat 720 tccggctggt cgctaatcgt tgagtgcatt ggtgacttac acatagacga ccatcacacc 780 actgaagact gcgggattgc tctcggtcaa gcttttaaag aggccctagg ggccgtgcgt 840 ggagtaaaaa ggtttggatc aggatttgcg cctttggatg aggcactttc cagagcggtg 900 gtagatcttt cgaacaggcc gtacgcagtt gtcgaacttg gtttgcaaag ggagaaagta 960 ggagatctct cttgcgagat gatcccgcat tttcttgaaa gctttgcaga ggctagcaga 1020 attaccctcc acgttgattg tctgcgaggc aagaatgatc atcaccgtag tgagagtgcg 1080 ttcaaggctc ttgcggttgc cataagagaa gccacctcgc ccaatggtac caacgatgtt 1140 ccctccacca aaggtgttct tatgtagtga caccgattat ttaaagctgc agcatacgat 1200 atatatacat gtgtatatat gtatacctat gaatgtcagt aagtatgtat acgaacagta 1260 tgatactgaa gatgacaagg taatgcatca ttctatacgt gtcattctga acgaggcgcg 1320 ctttcctttt ttctttttgc tttttctttt tttttctctt gaactcgacg gatctatgcg 1380 gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggaaat tgtaagcgtt 1440 aatattttgt taaaattcgc gttaaatttt tgttaaatca gctcattttt taaccaatag 1500 gccgaaatcg gcaaaatccc ttataaatca aaagaataga ccgagatagg gttgagtgtt 1560 gttccagttt ggaacaagag tccactatta aagaacgtgg actccaacgt caaagggcga 1620 aaaaccgtct atcagggcga tggcccacta cgtgaaccat caccctaatc aagttttttg 1680 gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag ggagcccccg atttagagct 1740 tgacggggaa agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc 1800 gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt 1860 aatgcgccgc tacagggcgc gtccattcgc cattcaggct gcgcaactgt tgggaagggc 1920 gcggtgcggg cctcttcgct attacgccag ctggcgaaag ggggatgtgc tgcaaggcga 1980 ttaagttggg taacgccagg gttttcccag tcacgacgtt gtaaaacgac ggccagtgag 2040 cgcgcgtaat acgactcact atagggcgaa ttgggtaccg ggccccccct cgaggtcgac 2100 ggcgcgccac tggtagagag cgactttgta tgccccaatt gcgaaacccg cgatatcctt 2160 ctcgattctt tagtacccga ccaggacaag gaaaaggagg tcgaaacgtt tttgaagaaa 2220 caagaggaac tacacggaag ctctaaagat ggcaaccagc cagaaactaa gaaaatgaag 2280 ttgatggatc caactggcac cgctggcttg aacaacaata ccagccttcc aacttctgta 2340 aataacggcg gtacgccagt gccaccagta ccgttacctt tcggtatacc tcctttcccc 2400 atgtttccaa tgcccttcat gcctccaacg gctactatca caaatcctca tcaagctgac 2460 gcaagcccta agaaatgaat aacaatactg acagtactaa ataattgcct acttggcttc 2520 acatacgttg catacgtcga tatagataat aatgataatg acagcaggat tatcgtaata 2580 cgtaatagct gaaaatctca aaaatgtgtg ggtcattacg taaataatga taggaatggg 2640 attcttctat ttttcctttt tccattctag cagccgtcgg gaaaacgtgg catcctctct 2700 ttcgggctca attggagtca cgctgccgtg agcatcctct ctttccatat ctaacaactg 2760 agcacgtaac caatggaaaa gcatgagctt agcgttgctc caaaaaagta ttggatggtt 2820 aataccattt gtctgttctc ttctgacttt gactcctcaa aaaaaaaaat ctacaatcaa 2880 cagatcgctt caattacgcc ctcacaaaaa cttttttcct tcttcttcgc ccacgttaaa 2940 ttttatccct catgttgtct aacggatttc tgcacttgat ttattataaa aagacaaaga 3000 cataatactt ctctatcaat ttcagttatt gttcttcctt gcgttattct tctgttcttc 3060 tttttctttt gtcatatata accataacca agtaatacat attcaaacta gtatgactga 3120 caaaaaaact cttaaagact taagaaatcg tagttctgtt tacgattcaa tggttaaatc 3180 acctaatcgt gctatgttgc gtgcaactgg tatgcaagat gaagactttg aaaaacctat 3240 cgtcggtgtc atttcaactt gggctgaaaa cacaccttgt aatatccact tacatgactt 3300 tggtaaacta gccaaagtcg gtgttaagga agctggtgct tggccagttc agttcggaac 3360 aatcacggtt tctgatggaa tcgccatggg aacccaagga atgcgtttct ccttgacatc 3420 tcgtgatatt attgcagatt ctattgaagc agccatggga ggtcataatg cggatgcttt 3480 tgtagccatt ggcggttgtg ataaaaacat gcccggttct gttatcgcta tggctaacat 3540 ggatatccca gccatttttg cttacggcgg aacaattgca cctggtaatt tagacggcaa 3600 agatatcgat ttagtctctg tctttgaagg tgtcggccat tggaaccacg gcgatatgac 3660 caaagaagaa gttaaagctt tggaatgtaa tgcttgtccc ggtcctggag gctgcggtgg 3720 tatgtatact gctaacacaa tggcgacagc tattgaagtt ttgggactta gccttccggg 3780 ttcatcttct cacccggctg aatccgcaga aaagaaagca gatattgaag aagctggtcg 3840 cgctgttgtc aaaatgctcg aaatgggctt aaaaccttct gacattttaa cgcgtgaagc 3900 ttttgaagat gctattactg taactatggc tctgggaggt tcaaccaact caacccttca 3960 cctcttagct attgcccatg ctgctaatgt ggaattgaca cttgatgatt tcaatacttt 4020 ccaagaaaaa gttcctcatt tggctgattt gaaaccttct ggtcaatatg tattccaaga 4080 cctttacaag gtcggagggg taccagcagt tatgaaatat ctccttaaaa atggcttcct 4140 tcatggtgac cgtatcactt gtactggcaa aacagtcgct gaaaatttga aggcttttga 4200 tgatttaaca cctggtcaaa aggttattat gccgcttgaa aatcctaaac gtgaagatgg 4260 tccgctcatt attctccatg gtaacttggc tccagacggt gccgttgcca aagtttctgg 4320 tgtaaaagtg cgtcgtcatg tcggtcctgc taaggtcttt aattctgaag aagaagccat 4380 tgaagctgtc ttgaatgatg atattgttga tggtgatgtt gttgtcgtac gttttgtagg 4440 accaaagggc ggtcctggta tgcctgaaat gctttccctt tcatcaatga ttgttggtaa 4500 agggcaaggt gaaaaagttg cccttctgac agatggccgc ttctcaggtg gtacttatgg 4560 tcttgtcgtg ggtcatatcg ctcctgaagc acaagatggc ggtccaatcg cctacctgca 4620 aacaggagac atagtcacta ttgaccaaga cactaaggaa ttacactttg atatctccga 4680 tgaagagtta aaacatcgtc aagagaccat tgaattgcca ccgctctatt cacgcggtat 4740 ccttggtaaa tatgctcaca tcgtttcgtc tgcttctagg ggagccgtaa cagacttttg 4800 gaagcctgaa gaaactggca aaaaatgttg tcctggttgc tgtggttaag cggccgcgtt 4860 aattcaaatt aattgatata gttttttaat gagtattgaa tctgtttaga aataatggaa 4920 tattattttt atttatttat ttatattatt ggtcggctct tttcttctga aggtcaatga 4980 caaaatgata tgaaggaaat aatgatttct aaaattttac aacgtaagat atttttacaa 5040 aagcctagct catcttttgt catgcactat tttactcacg cttgaaatta acggccagtc 5100 cactgcggag tcatttcaaa gtcatcctaa tcgatctatc gtttttgata gctcattttg 5160 gagttcgcga ttgtcttctg ttattcacaa ctgttttaat ttttatttca ttctggaact 5220 cttcgagttc tttgtaaagt ctttcatagt agcttacttt atcctccaac atatttaact 5280 tcatgtcaat ttcggctctt aaattttcca catcatcaag ttcaacatca tcttttaact 5340 tgaatttatt ctctagctct tccaaccaag cctcattgct ccttgattta ctggtgaaaa 5400 gtgatacact ttgcgcgcaa tccaggtcaa aactttcctg caaagaattc accaatttct 5460 cgacatcata gtacaatttg ttttgttctc ccatcacaat ttaatatacc tgatggattc 5520 ttatgaagcg ctgggtaatg gacgtgtcac tctacttcgc ctttttccct actcctttta 5580 gtacggaaga caatgctaat aaataagagg gtaataataa tattattaat cggcaaaaaa 5640 gattaaacgc caagcgttta attatcagaa agcaaacgtc gtaccaatcc ttgaatgctt 5700 cccaattgta tattaagagt catcacagca acatattctt gttattaaat taattattat 5760 tgatttttga tattgtataa aaaaaccaaa tatgtataaa aaaagtgaat aaaaaatacc 5820 aagtatggag aaatatatta gaagtctata cgttaaacca cccgggcccc ccctcgaggt 5880 cgacggtatc gataagcttg atatcgaatt cctgcagccc gggggatcca ctagttctag 5940 agcggccgct ctagaactag taccacaggt gttgtcctct gaggacataa aatacacacc 6000 gagattcatc aactcattgc tggagttagc atatctacaa ttgggtgaaa tggggagcga 6060 tttgcaggca tttgctcggc atgccggtag aggtgtggtc aataagagcg acctcatgct 6120 atacctgaga aagcaacctg acctacagga aagagttact caagaataag aattttcgtt 6180 ttaaaaccta agagtcactt taaaatttgt atacacttat tttttttata acttatttaa 6240 taataaaaat cataaatcat aagaaattcg cttactctta attaatcaaa aagttaaaat 6300 tgtacgaata gattcaccac ttcttaacaa atcaaaccct tcattgattt tctcgaatgg 6360 caatacatgt gtaattaaag gatcaagagc aaacttcttc gccataaagt cggcaacaag 6420 ttttggaaca ctatccttgc tcttaaaacc gccaaatata gctcccttcc atgtacgacc 6480 gcttagcaac agcataggat tcatcgacaa attttgtgaa tcaggaggaa cacctacgat 6540 cacactgact ccatatgcct cttgacagca ggacaacgca gttaccatag tatcaagacg 6600 gcctataact tcaaaagaga aatcaactcc accgtttgac atttcagtaa ggacttcttg 6660 tattggtttc ttataatctt gagggttaac acattcagta gccccgacct ccttagcttt 6720 tgcaaatttg tccttattga tgtctacacc tataatcctc gctgcgcctg cagctttaca 6780 ccccataata acgcttagtc ctactcctcc taaaccgaat actgcacaag tcgaaccctg 6840 tgtaaccttt gcaactttaa ctgcggaacc gtaaccggtg gaaaatccgc accctatcaa 6900 gcaaactttt tccagtggtg aagctgcatc gattttagcg acagatatct cgtccaccac 6960 tgtgtattgg gaaaatgtag aagtaccaag gaaatggtgt ataggtttcc ctctgcatgt 7020 aaatctgctt gtaccatcct gcatagtacc tctaggcata gacaaatcat ttttaaggca 7080 gaaattaccc tcaggatgtt tgcagactct acacttacca cattgaggag tgaacagtgg 7140 gatcacttta tcaccaggac gaacagtggt aacaccttca cctatggatt caacgattcc 7200 ggcagcctcg tgtcccgcga ttactggcaa aggagtaact agagtgccac tcaccacatg 7260 gtcgtcggat ctacagattc cggtggcaac catcttgatt ctaacctcgt gtgcttttgg 7320 tggcgctact tctacttctt ctatgctaaa cggctttttc tcttcccaca aaactgccgc 7380 tttacactta ataactttac cggctgttga catcctcagc tagctattgt aatatgtgtg 7440 tttgtttgga ttattaagaa gaataattac aaaaaaaatt acaaaggaag gtaattacaa 7500 cagaattaag aaaggacaag aaggaggaag agaatcagtt cattatttct tctttgttat 7560 ataacaaacc caagtagcga tttggccata cattaaaagt tgagaaccac cctccctggc 7620 aacagccaca actcgttacc attgttcatc acgatcatga aactcgctgt cagctgaaat 7680 ttcacctcag tggatctctc tttttattct tcatcgttcc actaaccttt ttccatcagc 7740 tggcagggaa cggaaagtgg aatcccattt agcgagcttc ctcttttctt caagaaaaga 7800 cgaagcttgt gtgtgggtgc gcgcgctagt atctttccac attaagaaat ataccataaa 7860 ggttacttag acatcactat ggctatatat atatatatat atatatgtaa cttagcacca 7920 tcgcgcgtgc atcactgcat gtgttaaccg aaaagtttgg cgaacacttc accgacacgg 7980 tcatttagat ctgtcgtctg cattgcacgt cccttagcct taaatcctag gcgggagcat 8040 tctcgtgtaa ttgtgcagcc tgcgtagcaa ctcaacatag cgtagtctac ccagtttttc 8100 aagggtttat cgttagaaga ttctcccttt tcttcctgct cacaaatctt aaagtcatac 8160 attgcacgac taaatgcaag catgcggatc ccccgggctg caggaattcg atatcaagct 8220 tatcgatacc gtcgactggc cattaatctt tcccatatta gatttcgcca agccatgaaa 8280 gttcaagaaa ggtctttaga cgaattaccc ttcatttctc aaactggcgt caagggatcc 8340 tggtatggtt ttatcgtttt atttctggtt cttatagcat cgttttggac ttctctgttc 8400 ccattaggcg gttcaggagc cagcgcagaa tcattctttg aaggatactt atcctttcca 8460 attttgattg tctgttacgt tggacataaa ctgtatacta gaaattggac tttgatggtg 8520 aaactagaag atatggatct tgataccggc agaaaacaag tagatttgac tcttcgtagg 8580 gaagaaatga ggattgagcg agaaacatta gcaaaaagat ccttcgtaac aagattttta 8640 catttctggt gttgaaggga aagatatgag ctatacagcg gaatttccat atcactcaga 8700 ttttgttatc taattttttc cttcccacgt ccgcgggaat ctgtgtatat tactgcatct 8760 agatatatgt tatcttatct tggcgcgtac atttaatttt caacgtattc tataagaaat 8820 tgcgggagtt tttttcatgt agatgatact gactgcacgc aaatataggc atgatttata 8880 ggcatgattt gatggctgta ccgataggaa cgctaagagt aacttcagaa tcgttatcct 8940 ggcggaaaaa attcatttgt aaactttaaa aaaaaaagcc aatatcccca aaattattaa 9000 gagcgcctcc attattaact aaaatttcac tcagcatcca caatgtatca ggtatctact 9060 acagatatta catgtggcga aaaagacaag aacaatgcaa tagcgcatca agaaaaaaca 9120 caaagctttc aatcaatgaa tcgaaaatgt cattaaaata gtatataaat tgaaactaag 9180 tcataaagct ataaaaagaa aatttattta aatcttggct ctcttgggct caaggtgaca 9240 aggtcctcga aaatagggcg cgccccaccg cggtggagct ccagcttttg ttccctttag 9300 tgagggttaa ttgcgcgctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt 9360 tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt 9420 gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg 9480 ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg 9540 cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg 9600 cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat 9660 aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc 9720 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc 9780 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga 9840 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt 9900 ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg 9960 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc 10020 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg 10080 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc 10140 ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg 10200 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc 10260 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct 10320 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt 10380 taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa 10440 aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa 10500 tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc 10560 tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct 10620 gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca 10680 gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt 10740 aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt 10800 gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc 10860 ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc 10920 tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt 10980 atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact 11040 ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc 11100 ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt 11160 ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg 11220 atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct 11280 gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa 11340 tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt 11400 ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc 11460 acatttcccc gaaaagtgcc acctgaacga agcatctgtg cttcattttg tagaacaaaa 11520 atgcaacgcg agagcgctaa tttttcaaac aaagaatctg agctgcattt ttacagaaca 11580 gaaatgcaac gcgaaagcgc tattttacca acgaagaatc tgtgcttcat ttttgtaaaa 11640 caaaaatgca acgcgagagc gctaattttt caaacaaaga atctgagctg catttttaca 11700 gaacagaaat gcaacgcgag agcgctattt taccaacaaa gaatctatac ttcttttttg 11760 ttctacaaaa atgcatcccg agagcgctat ttttctaaca aagcatctta gattactttt 11820 tttctccttt gtgcgctcta taatgcagtc tcttgataac tttttgcact gtaggtccgt 11880 taaggttaga agaaggctac tttggtgtct attttctctt ccataaaaaa agcctgactc 11940 cacttcccgc gtttactgat tactagcgaa gctgcgggtg cattttttca agataaaggc 12000 atccccgatt atattctata ccgatgtgga ttgcgcatac tttgtgaaca gaaagtgata 12060 gcgttgatga ttcttcattg gtcagaaaat tatgaacggt ttcttctatt ttgtctctat 12120 atactacgta taggaaatgt ttacattttc gtattgtttt cgattcactc tatgaatagt 12180 tcttactaca atttttttgt ctaaagagta atactagaga taaacataaa aaatgtagag 12240 gtcgagttta gatgcaagtt caaggagcga aaggtggatg ggtaggttat atagggatat 12300 agcacagaga tatatagcaa agagatactt ttgagcaatg tttgtggaag cggtattcgc 12360 aatattttag tagctcgtta cagtccggtg cgtttttggt tttttgaaag tgcgtcttca 12420 gagcgctttt ggttttcaaa agcgctctga agttcctata ctttctagag aataggaact 12480 tcggaatagg aacttcaaag cgtttccgaa aacgagcgct tccgaaaatg caacgcgagc 12540 tgcgcacata cagctcactg ttcacgtcgc acctatatct gcgtgttgcc tgtatatata 12600 tatacatgag aagaacggca tagtgcgtgt ttatgcttaa atgcgtactt atatgcgtct 12660 atttatgtag gatgaaaggt agtctagtac ctcctgtgat attatcccat tccatgcggg 12720 gtatcgtatg cttccttcag cactaccctt tagctgttct atatgctgcc actcctcaat 12780 tggattagtc tcatccttca atgctatcat ttcctttgat attggatcat actaagaaac 12840 cattattatc atgacattaa cctataaaaa taggcgtatc acgaggccct ttcgtc 12896 <210> 196 <211> 3015 <212> DNA <213> Artificial Sequence <220> <223> FBA1-als S-CYCt in Chr. XII; Upstream region = nt 1-154; FBA1        promoter = nt 155-802; alsS CDS = nt; CYC1 terminator = nt        2534-2788; Downstream region = nt 2790-3015 <400> 196 gcgatttaat ctctaattat tagttaaagt tttataagca tttttatgta acgaaaaata 60 aattggttca tattattact gcactgtcac ttaccatgga aagaccagac aagaagttgc 120 cgacagtctg ttgaattggc ctggttaggc ttaagatctg aaatgaataa caatactgac 180 agtactaaat aattgcctac ttggcttcac atacgttgca tacgtcgata tagataataa 240 tgataatgac agcaggatta tcgtaatacg taatagttga aaatctcaaa aatgtgtggg 300 tcattacgta aataatgata ggaatgggat tcttctattt ttcctttttc cattctagca 360 gccgtcggga aaacgtggca tcctctcttt cgggctcaat tggagtcacg ctgccgtgag 420 catcctctct ttccatatct aacaactgag cacgtaacca atggaaaagc atgagcttag 480 cgttgctcca aaaaagtatt ggatggttaa taccatttgt ctgttctctt ctgactttga 540 ctcctcaaaa aaaaaaaatc tacaatcaac agatcgcttc aattacgccc tcacaaaaac 600 ttttttcctt cttcttcgcc cacgttaaat tttatccctc atgttgtcta acggatttct 660 gcacttgatt tattataaaa agacaaagac ataatacttc tctatcaatt tcagttattg 720 ttcttccttg cgttattctt ctgttcttct ttttcttttg tcatatataa ccataaccaa 780 gtaatacata ttcaaatcta gagctgagga tgttgacaaa agcaacaaaa gaacaaaaat 840 cccttgtgaa aaacagaggg gcggagcttg ttgttgattg cttagtggag caaggtgtca 900 cacatgtatt tggcattcca ggtgcaaaaa ttgatgcggt atttgacgct ttacaagata 960 aaggacctga aattatcgtt gcccggcacg aacaaaacgc agcattcatg gcccaagcag 1020 tcggccgttt aactggaaaa ccgggagtcg tgttagtcac atcaggaccg ggtgcctcta 1080 acttggcaac aggcctgctg acagcgaaca ctgaaggaga ccctgtcgtt gcgcttgctg 1140 gaaacgtgat ccgtgcagat cgtttaaaac ggacacatca atctttggat aatgcggcgc 1200 tattccagcc gattacaaaa tacagtgtag aagttcaaga tgtaaaaaat ataccggaag 1260 ctgttacaaa tgcatttagg atagcgtcag cagggcaggc tggggccgct tttgtgagct 1320 ttccgcaaga tgttgtgaat gaagtcacaa atacgaaaaa cgtgcgtgct gttgcagcgc 1380 caaaactcgg tcctgcagca gatgatgcaa tcagtgcggc catagcaaaa atccaaacag 1440 caaaacttcc tgtcgttttg gtcggcatga aaggcggaag accggaagca attaaagcgg 1500 ttcgcaagct tttgaaaaag gttcagcttc catttgttga aacatatcaa gctgccggta 1560 ccctttctag agatttagag gatcaatatt ttggccgtat cggtttgttc cgcaaccagc 1620 ctggcgattt actgctagag caggcagatg ttgttctgac gatcggctat gacccgattg 1680 aatatgatcc gaaattctgg aatatcaatg gagaccggac aattatccat ttagacgaga 1740 ttatcgctga cattgatcat gcttaccagc ctgatcttga attgatcggt gacattccgt 1800 ccacgatcaa tcatatcgaa cacgatgctg tgaaagtgga atttgcagag cgtgagcaga 1860 aaatcctttc tgatttaaaa caatatatgc atgaaggtga gcaggtgcct gcagattgga 1920 aatcagacag agcgcaccct cttgaaatcg ttaaagagtt gcgtaatgca gtcgatgatc 1980 atgttacagt aacttgcgat atcggttcgc acgccatttg gatgtcacgt tatttccgca 2040 gctacgagcc gttaacatta atgatcagta acggtatgca aacactcggc gttgcgcttc 2100 cttgggcaat cggcgcttca ttggtgaaac cgggagaaaa agtggtttct gtctctggtg 2160 acggcggttt cttattctca gcaatggaat tagagacagc agttcgacta aaagcaccaa 2220 ttgtacacat tgtatggaac gacagcacat atgacatggt tgcattccag caattgaaaa 2280 aatataaccg tacatctgcg gtcgatttcg gaaatatcga tatcgtgaaa tatgcggaaa 2340 gcttcggagc aactggcttg cgcgtagaat caccagacca gctggcagat gttctgcgtc 2400 aaggcatgaa cgctgaaggt cctgtcatca tcgatgtccc ggttgactac agtgataaca 2460 ttaatttagc aagtgacaag cttccgaaag aattcgggga actcatgaaa acgaaagctc 2520 tctagttaat taatcatgta attagttatg tcacgcttac attcacgccc tccccccaca 2580 tccgctctaa ccgaaaagga aggagttaga caacctgaag tctaggtccc tatttatttt 2640 tttatagtta tgttagtatt aagaacgtta tttatatttc aaatttttct tttttttctg 2700 tacagacgcg tgtacgcatg taacattata ctgaaaacct tgcttgagaa ggttttggga 2760 cgctcgaagg ctttaatttg cgggcggcct taagtctggg tccgcttctt tacaaatttg 2820 gagaatttct cttaaacgat atgtatattc ttttcgttgg aaaagatgtc ttccaaaaaa 2880 aaaaccgatg aattagtgga accaaggaaa aaaaaagagg tatccttgat taaggaacac 2940 tgtttaaaca gtgtggtttc caaaaccctg aaactgcatt agtgtaatag aagactagac 3000 acctcgatac aaata 3015 <210> 197 <211> 2900 <212> DNA <213> Artificial Sequence <220> Fra2delta :: PDC1-hADH-ADHt; Upstream region = nt 1-300; PDC1        promoter = nt 309-1178; hADH coding region = nt 1179-2306; ADH1        terminator = nt 2315-2630; Downstream region = nt 2639-2900 <400> 197 gttagcatcc acagcttctt gagcaactga cttcgtgagg tctacagtca aaaggacctt 60 tgttttagcg tttgcatcgg cagtggtgac ttgtgcagtg gaacaatcga tgagaagacc 120 cgtattgtcc cagctcttgt ctgcgtactt ttgagggtag aacttggtaa tgctacgcac 180 aagtttgtcc aattgtgctc tagtaatagc tctgctcatt gcgtttcggt ggactcttgt 240 tctgtttcct tccgactgtg tattggaata agtttttcgg tgttatatat atacatatat 300 ggcgcgcccc gcacgccgaa atgcatgcaa gtaacctatt caaagtaata tctcatacat 360 gtttcatgag ggtaacaaca tgcgactggg tgagcatatg ttccgctgat gtgatgtgca 420 agataaacaa gcaaggcaga aactaacttc ttcttcatgt aataaacaca ccccgcgttt 480 atttacctat ctctaaactt caacacctta tatcataact aatatttctt gagataagca 540 cactgcaccc ataccttcct taaaaacgta gcttccagtt tttggtggtt ccggcttcct 600 tcccgattcc gcccgctaaa cgcatatttt tgttgcctgg tggcatttgc aaaatgcata 660 acctatgcat ttaaaagatt atgtatgctc ttctgacttt tcgtgtgatg aggctcgtgg 720 aaaaaatgaa taatttatga atttgagaac aattttgtgt tgttacggta ttttactatg 780 gaataatcaa tcaattgagg attttatgca aatatcgttt gaatattttt ccgacccttt 840 gagtactttt cttcataatt gcataatatt gtccgctgcc cctttttctg ttagacggtg 900 tcttgatcta cttgctatcg ttcaacacca ccttattttc taactatttt ttttttagct 960 catttgaatc agcttatggt gatggcacat ttttgcataa acctagctgt cctcgttgaa 1020 cataggaaaa aaaaatatat aaacaaggct ctttcactct ccttgcaatc agatttgggt 1080 ttgttccctt tattttcata tttcttgtca tattcctttc tcaattatta ttttctactc 1140 ataacctcac gcaaaataac acagtcaaat caatcaaaat gtcaacagcc ggtaaagtta 1200 ttaagtgtaa agcggcagtt ttgtgggaag agaaaaagcc gtttagcata gaagaagtag 1260 aagtagcgcc accaaaagca cacgaggtta gaatcaagat ggttgccacc ggaatctgta 1320 gatccgacga ccatgtggtg agtggcactc tagttactcc tttgccagta atcgcgggac 1380 acgaggctgc cggaatcgtt gaatccatag gtgaaggtgt taccactgtt cgtcctggtg 1440 ataaagtgat cccactgttc actcctcaat gtggtaagtg tagagtctgc aaacatcctg 1500 agggtaattt ctgccttaaa aatgatttgt ctatgcctag aggtactatg caggatggta 1560 caagcagatt tacatgcaga gggaaaccta tacaccattt ccttggtact tctacatttt 1620 cccaatacac agtggtggac gagatatctg tcgctaaaat cgatgcagct tcaccactgg 1680 aaaaagtttg cttgataggg tgcggatttt ccaccggtta cggttccgca gttaaagttg 1740 caaaggttac acagggttcg acttgtgcag tattcggttt aggaggagta ggactaagcg 1800 ttattatggg gtgtaaagct gcaggcgcag cgaggattat aggtgtagac atcaataagg 1860 acaaatttgc aaaagctaag gaggtcgggg ctactgaatg tgttaaccct caagattata 1920 agaaaccaat acaagaagtc cttactgaaa tgtcaaacgg tggagttgat ttctcttttg 1980 aagttatagg ccgtcttgat actatggtaa ctgcgttgtc ctgctgtcaa gaggcatatg 2040 gagtcagtgt gatcgtaggt gttcctcctg attcacaaaa tttgtcgatg aatcctatgc 2100 tgttgctaag cggtcgtaca tggaagggag ctatatttgg cggttttaag agcaaggata 2160 gtgttccaaa acttgttgcc gactttatgg cgaagaagtt tgctcttgat cctttaatta 2220 cacatgtatt gccattcgag aaaatcaatg aagggtttga tttgttaaga agtggtgaat 2280 ctattcgtac aattttaact ttttgattaa ttaagagtaa gcgaatttct tatgatttat 2340 gatttttatt attaaataag ttataaaaaa aataagtgta tacaaatttt aaagtgactc 2400 ttaggtttta aaacgaaaat tcttattctt gagtaactct ttcctgtagg tcaggttgct 2460 ttctcaggta tagcatgagg tcgctcttat tgaccacacc tctaccggca tgccgagcaa 2520 atgcctgcaa atcgctcccc atttcaccca attgtagata tgctaactcc agcaatgagt 2580 tgatgaatct cggtgtgtat tttatgtcct cagaggacaa cacctgtggt gtttaaacaa 2640 aggatgatat tgttctatta ttaagtttcg aaaggagacg gcattgaatc tttttggcat 2700 agacgatttt gctttcttac gttatttaca tcggagagta tgtatttgtg tagttatgta 2760 cttagatatg taacttaatt taatgatatg gtggggggct atcctccatg ccccatttcc 2820 agttatttga aggttcttgc gagttcaaac ctatgaagta aaacggtttc tttttagatg 2880 tatctttctt ggagtccaaa 2900 <210> 198 <211> 2656 <212> DNA <213> Artificial Sequence <220> Yprcdelta15delta :: PDC5-hADH-ADHt; Upstream region = nt 1-150;        PDC5 promoter = nt 159-696; hADH coding region = nt 697-1824;        ADH1 terminator = nt 1833-2148; Downstream region = nt 2157-2656 <400> 198 ccaaaaggaa tattgggtca gatgaatgga cgcgaatgca agacagaagt ccaaatcacg 60 tcaagacaaa gaaagaaaga aagaaaaact aacacattaa tgtagtttta aaatttcaaa 120 tccgaacaac agagcatagg gtttcgcaaa ggcgcgccta tttgtaatac gtatacgaat 180 tccttcaaca aaggccaagg aaataaagca aataacaata acaccattat tttaattttt 240 tttctattac tgtcgctaac acctgtatgg ttgcaaccag gtgagaatcc ttctgatgca 300 tactttatgc gtttatgcgt tttgcgcccc ttggaaaaaa attgattctc atcgtaaatg 360 catactacat gcgtttatgg gaaaagcctc catatccaaa ggtcgcgttt cttttagaaa 420 aactaatacg taaacctgca ttaaggtaag attatatcag aaaatgtgtt gcaagaaatg 480 cattatgcaa ttttttgatt atgacaatct ctcgaaagaa atttcatatg atgagacttg 540 aataatgcag cggcgcttgc taaaagaact tgtatataag agctgccatt ctcgatcaat 600 atactgtagt aagtcctttc ctctctttct tattacactt atttcacata atcaatctca 660 aagagaacaa cacaatacaa taacaagaag aacaaaatgt caacagccgg taaagttatt 720 aagtgtaaag cggcagtttt gtgggaagag aaaaagccgt ttagcataga agaagtagaa 780 gtagcgccac caaaagcaca cgaggttaga atcaagatgg ttgccaccgg aatctgtaga 840 tccgacgacc atgtggtgag tggcactcta gttactcctt tgccagtaat cgcgggacac 900 gaggctgccg gaatcgttga atccataggt gaaggtgtta ccactgttcg tcctggtgat 960 aaagtgatcc cactgttcac tcctcaatgt ggtaagtgta gagtctgcaa acatcctgag 1020 ggtaatttct gccttaaaaa tgatttgtct atgcctagag gtactatgca ggatggtaca 1080 agcagattta catgcagagg gaaacctata caccatttcc ttggtacttc tacattttcc 1140 caatacacag tggtggacga gatatctgtc gctaaaatcg atgcagcttc accactggaa 1200 aaagtttgct tgatagggtg cggattttcc accggttacg gttccgcagt taaagttgca 1260 aaggttacac agggttcgac ttgtgcagta ttcggtttag gaggagtagg actaagcgtt 1320 attatggggt gtaaagctgc aggcgcagcg aggattatag gtgtagacat caataaggac 1380 aaatttgcaa aagctaagga ggtcggggct actgaatgtg ttaaccctca agattataag 1440 aaaccaatac aagaagtcct tactgaaatg tcaaacggtg gagttgattt ctcttttgaa 1500 gttataggcc gtcttgatac tatggtaact gcgttgtcct gctgtcaaga ggcatatgga 1560 gtcagtgtga tcgtaggtgt tcctcctgat tcacaaaatt tgtcgatgaa tcctatgctg 1620 ttgctaagcg gtcgtacatg gaagggagct atatttggcg gttttaagag caaggatagt 1680 gttccaaaac ttgttgccga ctttatggcg aagaagtttg ctcttgatcc tttaattaca 1740 catgtattgc cattcgagaa aatcaatgaa gggtttgatt tgttaagaag tggtgaatct 1800 attcgtacaa ttttaacttt ttgattaatt aagagtaagc gaatttctta tgatttatga 1860 tttttattat taaataagtt ataaaaaaaa taagtgtata caaattttaa agtgactctt 1920 aggttttaaa acgaaaattc ttattcttga gtaactcttt cctgtaggtc aggttgcttt 1980 ctcaggtata gcatgaggtc gctcttattg accacacctc taccggcatg ccgagcaaat 2040 gcctgcaaat cgctccccat ttcacccaat tgtagatatg ctaactccag caatgagttg 2100 atgaatctcg gtgtgtattt tatgtcctca gaggacaaca cctgtggtgt ttaaacaatg 2160 gaaggtcggg atgagcatat acaagcacta agaagaacaa tacagaactc tacacggtat 2220 tattgtgcta caagctcgag taaaaccgag tgttttgacg atactaacgt tgttaagaaa 2280 gtaacttgtt atcaaactca ttaccaactt gtgattaatt ggtgaataat atgataattg 2340 tcgaaattcc attgttggta aagcctataa tattatgtat acagattata ctagaaattc 2400 tctcgagaat ataagaatcc ccaaaattga atcggtattt ctacatacta atattaccat 2460 tacttctcct ttcgttttat atgtttcatt cctattacat tatcgatctt tgcatttcag 2520 cttccattat atttgatgtc tgttttatgt ccccacgtta caccgcatgt gacagtatac 2580 tagtaacatg agtgctaccg aatagatgac attttagact ttcattccaa caacttggtt 2640 gacagaatgt tacgta 2656 <210> 199 <211> 451 <212> DNA <213> Artificial Sequence <220> Ymr226c delta; Upstream region = nt 1-250; Downstream region = nt        251-451 <400> 199 gatgatgcta tttggtgcag agggtgatga gtaagttctc tttatcatct atatattact 60 cttatacact atacgactct actctatgac gtatagcatt gatatatatt aaggcacgac 120 accgattttt cttatcggag cgatataaaa agctgaagaa aggaggatag atgaaacagc 180 atggcgcata gaaagtgttc aagctcacta gtaaaggcgg gaaatagaac attgagaacg 240 tattttgata acctagctaa actaagtaaa tctgtatact ttttatacat gtaagacttt 300 ttagctattt cattcttccc tgaaccgttt tgcgcgattc tacggaatat accggcgaaa 360 taaaagagat aaacttatgg atcctcaaat agaggatgaa cagatgtgaa gaagaaacga 420 gcttaattat atcttcgcca tgataactaa a 451 <210> 200 <211> 1128 <212> DNA <213> Artificial Sequence <220> Ald6 delta :: loxP; Upstream region = nt 1-500; loxP site = nt        551-584; Downstream region = nt 678-1128 <400> 200 tgatctgatg cgctttgcat atctcatatt ccttcactag cataaaaatc caaaaaaaaa 60 gaatatttag gccgaatgga attattcgta acgtcatacg aaaaaagttt caattcgtac 120 aatgcctggc atgttcattc gaatataagg ccgccgcctt ccagtcaggg tagccaaaag 180 tataatcccg ggtggaaact aaactaaaaa ccgtactcac aactttccgc ggacgctaac 240 agacaaatag acacactatc aggtcaggaa ctgccgtcac atacgacact gcccctcacg 300 taagggcatg atagaattgg attatgtaaa aggtgaagat accattgtag aagcaaccag 360 cacgtcgccg tggctgatga ggtctcctct tgcccgggcc gcagaaaaga ggggcagtgg 420 cctgtttttc gacataaatg aggggcatgg ccagcaccga gacgtcattg ttgcatatgg 480 cgtatccaag ccgaaacggc ggtaccgcaa gggcgaattc accttggcta actcgttgta 540 tcatcactgg ataacttcgt ataatgtatg ctatacgaag ttatctgaac attagaatac 600 gtaatccgca atgcggatcc tctagagtcg acctgcaggc atgctcgagc ggccgccagt 660 gtgatggata tctgcagaat tcgcccttgc cgatcgtgta ccaacctgca tttctttccg 720 tcatatacac aaaatacttt catataaact tacttggtct tacgtcataa ataaatatgt 780 atacatataa attaaaaaat ttggttttat atttttacaa aaagaatcgt ttacttcatt 840 tctccctttt aagcgataca atccatgaaa aaagagaaaa agagagaaca ggcttgtgcc 900 ttctttaaaa catcccacac aaaatcatat tgaattgaat tttacatctt aagctagtgt 960 acaacaactg ctatatccaa agaaaactaa cgtggaccgc ttttagagtt gagaaaaagg 1020 tttgaaaaaa atagcaatac aaagacttgt ttcatatata aaatacaggg agcacattga 1080 gctaatataa cataaacact gcgaaccaat tccaatcaaa aggtacac 1128 <210> 201 <211> 9612 <212> DNA <213> Artificial Sequence <220> <223> pLH702 <400> 201 aaacagtatg gaagaatgta agatggctaa gatttactac caagaagact gtaacttgtc 60 cttgttggat ggtaagacta tcgccgttat cggttacggt tctcaaggtc acgctcatgc 120 cctgaatgct aaggaatccg gttgtaacgt tatcattggt ttatacgaag gtgctaagga 180 ttggaaaaga gctgaagaac aaggtttcga agtctacacc gctgctgaag ctgctaagaa 240 ggctgacatc attatgatct tgatcaacga tgaaaagcag gctaccatgt acaaaaacga 300 catcgaacca aacttggaag ccggtaacat gttgatgttc gctcacggtt tcaacatcca 360 tttcggttgt attgttccac caaaggacgt tgatgtcact atgatcgctc caaagggtcc 420 aggtcacacc gttagatccg aatacgaaga aggtaaaggt gtcccatgct tggttgctgt 480 cgaacaagac gctactggca aggctttgga tatggctttg gcctacgctt tagccatcgg 540 tggtgctaga gccggtgtct tggaaactac cttcagaacc gaaactgaaa ccgacttgtt 600 cggtgaacaa gctgttttat gtggtggtgt ctgcgctttg atgcaggccg gttttgaaac 660 cttggttgaa gccggttacg acccaagaaa cgcttacttc gaatgtatcc acgaaatgaa 720 gttgatcgtt gacttgatct accaatctgg tttctccggt atgcgttact ctatctccaa 780 cactgctgaa tacggtgact acattaccgg tccaaagatc attactgaag ataccaagaa 840 ggctatgaag aagattttgt ctgacattca agatggtacc tttgccaagg acttcttggt 900 tgacatgtct gatgctggtt cccaggtcca cttcaaggct atgagaaagt tggcctccga 960 acacccagct gaagttgtcg gtgaagaaat tagatccttg tactcctggt ccgacgaaga 1020 caagttgatt aacaactgat attttcctct ggccctgcag gcctatcaag tgctggaaac 1080 tttttctctt ggaatttttg caacatcaag tcatagtcaa ttgaattgac ccaatttcac 1140 atttaagatt tttttttttt catccgacat acatctgtac actaggaagc cctgtttttc 1200 tgaagcagct tcaaatatat atatttttta catatttatt atgattcaat gaacaatcta 1260 attaaatcga aaacaagaac cgaaacgcga ataaataatt tatttagatg gtgacaagtg 1320 tataagtcct catcgggaca gctacgattt ctctttcggt tttggctgag ctactggttg 1380 ctgtgacgca gcggcattag cgcggcgtta tgagctaccc tcgtggcctg aaagatggcg 1440 ggaataaagc ggaactaaaa attactgact gagccatatt gaggtcaatt tgtcaactcg 1500 tcaagtcacg tttggtggac ggcccctttc caacgaatcg tatatactaa catgcgcgcg 1560 cttcctatat acacatatac atatatatat atatatatat gtgtgcgtgt atgtgtacac 1620 ctgtatttaa tttccttact cgcgggtttt tcttttttct caattcttgg cttcctcttt 1680 ctcgagcgga ccggatcctc cgcggtgccg gcagatctat ttaaatggcg cgccgacgtc 1740 aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca 1800 ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa 1860 aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt 1920 ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca 1980 gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag 2040 ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc 2100 ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca 2160 gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt 2220 aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct 2280 gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt 2340 aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga 2400 caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact 2460 tactctagct tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc 2520 acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga 2580 gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt 2640 agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga 2700 gataggtgcc tcactgatta agcattggta actgtcagac caagtttact catatatact 2760 ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga 2820 taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt 2880 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca 2940 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct 3000 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta 3060 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct 3120 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc 3180 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca 3240 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga 3300 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg 3360 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt 3420 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag 3480 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt 3540 tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt 3600 tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga 3660 ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc cgattcatta 3720 atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca acgcaattaa 3780 tgtgagttag ctcactcatt aggcacccca ggctttacac tttatgcttc cggctcgtat 3840 gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg accatgatta 3900 cgccaagctt tttctttcca attttttttt tttcgtcatt ataaaaatca ttacgaccga 3960 gattcccggg taataactga tataattaaa ttgaagctct aatttgtgag tttagtatac 4020 atgcatttac ttataataca gttttttagt tttgctggcc gcatcttctc aaatatgctt 4080 cccagcctgc ttttctgtaa cgttcaccct ctaccttagc atcccttccc tttgcaaata 4140 gtcctcttcc aacaataata atgtcagatc ctgtagagac cacatcatcc acggttctat 4200 actgttgacc caatgcgtct cccttgtcat ctaaacccac accgggtgtc ataatcaacc 4260 aatcgtaacc ttcatctctt ccacccatgt ctctttgagc aataaagccg ataacaaaat 4320 ctttgtcgct cttcgcaatg tcaacagtac ccttagtata ttctccagta gatagggagc 4380 ccttgcatga caattctgct aacatcaaaa ggcctctagg ttcctttgtt acttcttctg 4440 ccgcctgctt caaaccgcta acaatacctg ggcccaccac accgtgtgca ttcgtaatgt 4500 ctgcccattc tgctattctg tatacacccg cagagtactg caatttgact gtattaccaa 4560 tgtcagcaaa ttttctgtct tcgaagagta aaaaattgta cttggcggat aatgccttta 4620 gcggcttaac tgtgccctcc atggaaaaat cagtcaagat atccacatgt gtttttagta 4680 aacaaatttt gggacctaat gcttcaacta actccagtaa ttccttggtg gtacgaacat 4740 ccaatgaagc acacaagttt gtttgctttt cgtgcatgat attaaatagc ttggcagcaa 4800 caggactagg atgagtagca gcacgttcct tatatgtagc tttcgacatg atttatcttc 4860 gtttcctgca ggtttttgtt ctgtgcagtt gggttaagaa tactgggcaa tttcatgttt 4920 cttcaacact acatatgcgt atatatacca atctaagtct gtgctccttc cttcgttctt 4980 ccttctgttc ggagattacc gaatcaaaaa aatttcaagg aaaccgaaat caaaaaaaag 5040 aataaaaaaa aaatgatgaa ttgaaaagct tgcatgcctg caggtcgact ctagtatact 5100 ccgtctactg tacgatacac ttccgctcag gtccttgtcc tttaacgagg ccttaccact 5160 cttttgttac tctattgatc cagctcagca aaggcagtgt gatctaagat tctatcttcg 5220 cgatgtagta aaactagcta gaccgagaaa gagactagaa atgcaaaagg cacttctaca 5280 atggctgcca tcattattat ccgatgtgac gctgcatttt tttttttttt tttttttttt 5340 tttttttttt tttttttttt ttttttttgt acaaatatca taaaaaaaga gaatcttttt 5400 aagcaaggat tttcttaact tcttcggcga cagcatcacc gacttcggtg gtactgttgg 5460 aaccacctaa atcaccagtt ctgatacctg catccaaaac ctttttaact gcatcttcaa 5520 tggctttacc ttcttcaggc aagttcaatg acaatttcaa catcattgca gcagacaaga 5580 tagtggcgat agggttgacc ttattctttg gcaaatctgg agcggaacca tggcatggtt 5640 cgtacaaacc aaatgcggtg ttcttgtctg gcaaagaggc caaggacgca gatggcaaca 5700 aacccaagga gcctgggata acggaggctt catcggagat gatatcacca aacatgttgc 5760 tggtgattat aataccattt aggtgggttg ggttcttaac taggatcatg gcggcagaat 5820 caatcaattg atgttgaact ttcaatgtag ggaattcgtt cttgatggtt tcctccacag 5880 tttttctcca taatcttgaa gaggccaaaa cattagcttt atccaaggac caaataggca 5940 atggtggctc atgttgtagg gccatgaaag cggccattct tgtgattctt tgcacttctg 6000 gaacggtgta ttgttcacta tcccaagcga caccatcacc atcgtcttcc tttctcttac 6060 caaagtaaat acctcccact aattctctaa caacaacgaa gtcagtacct ttagcaaatt 6120 gtggcttgat tggagataag tctaaaagag agtcggatgc aaagttacat ggtcttaagt 6180 tggcgtacaa ttgaagttct ttacggattt ttagtaaacc ttgttcaggt ctaacactac 6240 cggtacccca tttaggacca cccacagcac ctaacaaaac ggcatcagcc ttcttggagg 6300 cttccagcgc ctcatctgga agtggaacac ctgtagcatc gatagcagca ccaccaatta 6360 aatgattttc gaaatcgaac ttgacattgg aacgaacatc agaaatagct ttaagaacct 6420 taatggcttc ggctgtgatt tcttgaccaa cgtggtcacc tggcaaaacg acgatcttct 6480 taggggcaga cattacaatg gtatatcctt gaaatatata taaaaaaaaa aaaaaaaaaa 6540 aaaaaaaaaa atgcagcttc tcaatgatat tcgaatacgc tttgaggaga tacagcctaa 6600 tatccgacaa actgttttac agatttacga tcgtacttgt tacccatcat tgaattttga 6660 acatccgaac ctgggagttt tccctgaaac agatagtata tttgaacctg tataataata 6720 tatagtctag cgctttacgg aagacaatgt atgtatttcg gttcctggag aaactattgc 6780 atctattgca taggtaatct tgcacgtcgc atccccggtt cattttctgc gtttccatct 6840 tgcacttcaa tagcatatct ttgttaacga agcatctgtg cttcattttg tagaacaaaa 6900 atgcaacgcg agagcgctaa tttttcaaac aaagaatctg agctgcattt ttacagaaca 6960 gaaatgcaac gcgaaagcgc tattttacca acgaagaatc tgtgcttcat ttttgtaaaa 7020 caaaaatgca acgcgagagc gctaattttt caaacaaaga atctgagctg catttttaca 7080 gaacagaaat gcaacgcgag agcgctattt taccaacaaa gaatctatac ttcttttttg 7140 ttctacaaaa atgcatcccg agagcgctat ttttctaaca aagcatctta gattactttt 7200 tttctccttt gtgcgctcta taatgcagtc tcttgataac tttttgcact gtaggtccgt 7260 taaggttaga agaaggctac tttggtgtct attttctctt ccataaaaaa agcctgactc 7320 cacttcccgc gtttactgat tactagcgaa gctgcgggtg cattttttca agataaaggc 7380 atccccgatt atattctata ccgatgtgga ttgcgcatac tttgtgaaca gaaagtgata 7440 gcgttgatga ttcttcattg gtcagaaaat tatgaacggt ttcttctatt ttgtctctat 7500 atactacgta taggaaatgt ttacattttc gtattgtttt cgattcactc tatgaatagt 7560 tcttactaca atttttttgt ctaaagagta atactagaga taaacataaa aaatgtagag 7620 gtcgagttta gatgcaagtt caaggagcga aaggtggatg ggtaggttat atagggatat 7680 agcacagaga tatatagcaa agagatactt ttgagcaatg tttgtggaag cggtattcgc 7740 aatattttag tagctcgtta cagtccggtg cgtttttggt tttttgaaag tgcgtcttca 7800 gagcgctttt ggttttcaaa agcgctctga agttcctata ctttctagag aataggaact 7860 tcggaatagg aacttcaaag cgtttccgaa aacgagcgct tccgaaaatg caacgcgagc 7920 tgcgcacata cagctcactg ttcacgtcgc acctatatct gcgtgttgcc tgtatatata 7980 tatacatgag aagaacggca tagtgcgtgt ttatgcttaa atgcgtactt atatgcgtct 8040 atttatgtag gatgaaaggt agtctagtac ctcctgtgat attatcccat tccatgcggg 8100 gtatcgtatg cttccttcag cactaccctt tagctgttct atatgctgcc actcctcaat 8160 tggattagtc tcatccttca atgctatcat ttcctttgat attggatcat atgcatagta 8220 ccgagaaact agaggatctc ccattaccga catttgggcg ctatacgtgc atatgttcat 8280 gtatgtatct gtatttaaaa cacttttgta ttatttttcc tcatatatgt gtataggttt 8340 atacggatga tttaattatt acttcaccac cctttatttc aggctgatat cttagccttg 8400 ttactagtca ccggtggcgg ccgcacctgg taaaacctct agtggagtag tagatgtaat 8460 caatgaagcg gaagccaaaa gaccagagta gaggcctata gaagaaactg cgataccttt 8520 tgtgatggct aaacaaacag acatcttttt atatgttttt acttctgtat atcgtgaagt 8580 agtaagtgat aagcgaattt ggctaagaac gttgtaagtg aacaagggac ctcttttgcc 8640 tttcaaaaaa ggattaaatg gagttaatca ttgagattta gttttcgtta gattctgtat 8700 ccctaaataa ctcccttacc cgacgggaag gcacaaaaga cttgaataat agcaaacggc 8760 cagtagccaa gaccaaataa tactagagtt aactgatggt cttaaacagg cattacgtgg 8820 tgaactccaa gaccaatata caaaatatcg ataagttatt cttgcccacc aatttaagga 8880 gcctacatca ggacagtagt accattcctc agagaagagg tatacataac aagaaaatcg 8940 cgtgaacacc ttatataact tagcccgtta ttgagctaaa aaaccttgca aaatttccta 9000 tgaataagaa tacttcagac gtgataaaaa tttactttct aactcttctc acgctgcccc 9060 tatctgttct tccgctctac cgtgagaaat aaagcatcga gtacggcagt tcgctgtcac 9120 tgaactaaaa caataaggct agttcgaatg atgaacttgc ttgctgtcaa acttctgagt 9180 tgccgctgat gtgacactgt gacaataaat tcaaaccggt tatagcggtc tcctccggta 9240 ccggttctgc cacctccaat agagctcagt aggagtcaga acctctgcgg tggctgtcag 9300 tgactcatcc gcgtttcgta agttgtgcgc gtgcacattt cgcccgttcc cgctcatctt 9360 gcagcaggcg gaaattttca tcacgctgta ggacgcaaaa aaaaaataat taatcgtaca 9420 agaatcttgg aaaaaaaatt gaaaaatttt gtataaaagg gatgacctaa cttgactcaa 9480 tggcttttac acccagtatt ttccctttcc ttgtttgtta caattataga agcaagacaa 9540 aaacatatag acaacctatt cctaggagtt atattttttt accctaccag caatataagt 9600 aaaaaactgt tt 9612 <210> 202 <211> 7938 <212> DNA <213> Artificial Sequence <220> <223> pYZ067DkivDDhADH <400> 202 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180 accataaatt cccgttttaa gagcttggtg agcgctagga gtcactgcca ggtatcgttt 240 gaacacggca ttagtcaggg aagtcataac acagtccttt cccgcaattt tctttttcta 300 ttactcttgg cctcctctag tacactctat atttttttat gcctcggtaa tgattttcat 360 tttttttttt ccacctagcg gatgactctt tttttttctt agcgattggc attatcacat 420 aatgaattat acattatata aagtaatgtg atttcttcga agaatatact aaaaaatgag 480 caggcaagat aaacgaaggc aaagatgaca gagcagaaag ccctagtaaa gcgtattaca 540 aatgaaacca agattcagat tgcgatctct ttaaagggtg gtcccctagc gatagagcac 600 tcgatcttcc cagaaaaaga ggcagaagca gtagcagaac aggccacaca atcgcaagtg 660 attaacgtcc acacaggtat agggtttctg gaccatatga tacatgctct ggccaagcat 720 tccggctggt cgctaatcgt tgagtgcatt ggtgacttac acatagacga ccatcacacc 780 actgaagact gcgggattgc tctcggtcaa gcttttaaag aggccctagg ggccgtgcgt 840 ggagtaaaaa ggtttggatc aggatttgcg cctttggatg aggcactttc cagagcggtg 900 gtagatcttt cgaacaggcc gtacgcagtt gtcgaacttg gtttgcaaag ggagaaagta 960 ggagatctct cttgcgagat gatcccgcat tttcttgaaa gctttgcaga ggctagcaga 1020 attaccctcc acgttgattg tctgcgaggc aagaatgatc atcaccgtag tgagagtgcg 1080 ttcaaggctc ttgcggttgc cataagagaa gccacctcgc ccaatggtac caacgatgtt 1140 ccctccacca aaggtgttct tatgtagtga caccgattat ttaaagctgc agcatacgat 1200 atatatacat gtgtatatat gtatacctat gaatgtcagt aagtatgtat acgaacagta 1260 tgatactgaa gatgacaagg taatgcatca ttctatacgt gtcattctga acgaggcgcg 1320 ctttcctttt ttctttttgc tttttctttt tttttctctt gaactcgacg gatctatgcg 1380 gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggaaat tgtaagcgtt 1440 aatattttgt taaaattcgc gttaaatttt tgttaaatca gctcattttt taaccaatag 1500 gccgaaatcg gcaaaatccc ttataaatca aaagaataga ccgagatagg gttgagtgtt 1560 gttccagttt ggaacaagag tccactatta aagaacgtgg actccaacgt caaagggcga 1620 aaaaccgtct atcagggcga tggcccacta cgtggccggc ttcacatacg ttgcatacgt 1680 cgatatagat aataatgata atgacagcag gattatcgta atacgtaata gctgaaaatc 1740 tcaaaaatgt gtgggtcatt acgtaaataa tgataggaat gggattcttc tatttttcct 1800 ttttccattc tagcagccgt cgggaaaacg tggcatcctc tctttcgggc tcaattggag 1860 tcacgctgcc gtgagcatcc tctctttcca tatctaacaa ctgagcacgt aaccaatgga 1920 aaagcatgag cttagcgttg ctccaaaaaa gtattggatg gttaatacca tttgtctgtt 1980 ctcttctgac tttgactcct caaaaaaaaa aatctacaat caacagatcg cttcaattac 2040 gccctcacaa aaactttttt ccttcttctt cgcccacgtt aaattttatc cctcatgttg 2100 tctaacggat ttctgcactt gatttattat aaaaagacaa agacataata cttctctatc 2160 aatttcagtt attgttcttc cttgcgttat tcttctgttc ttctttttct tttgtcatat 2220 ataaccataa ccaagtaata catattcaaa cacgtgagta tgactgacaa aaaaactctt 2280 aaagacttaa gaaatcgtag ttctgtttac gattcaatgg ttaaatcacc taatcgtgct 2340 atgttgcgtg caactggtat gcaagatgaa gactttgaaa aacctatcgt cggtgtcatt 2400 tcaacttggg ctgaaaacac accttgtaat atccacttac atgactttgg taaactagcc 2460 aaagtcggtg ttaaggaagc tggtgcttgg ccagttcagt tcggaacaat cacggtttct 2520 gatggaatcg ccatgggaac ccaaggaatg cgtttctcct tgacatctcg tgatattatt 2580 gcagattcta ttgaagcagc catgggaggt cataatgcgg atgcttttgt agccattggc 2640 ggttgtgata aaaacatgcc cggttctgtt atcgctatgg ctaacatgga tatcccagcc 2700 atttttgctt acggcggaac aattgcacct ggtaatttag acggcaaaga tatcgattta 2760 gtctctgtct ttgaaggtgt cggccattgg aaccacggcg atatgaccaa agaagaagtt 2820 aaagctttgg aatgtaatgc ttgtcccggt cctggaggct gcggtggtat gtatactgct 2880 aacacaatgg cgacagctat tgaagttttg ggacttagcc ttccgggttc atcttctcac 2940 ccggctgaat ccgcagaaaa gaaagcagat attgaagaag ctggtcgcgc tgttgtcaaa 3000 atgctcgaaa tgggcttaaa accttctgac attttaacgc gtgaagcttt tgaagatgct 3060 attactgtaa ctatggctct gggaggttca accaactcaa cccttcacct cttagctatt 3120 gcccatgctg ctaatgtgga attgacactt gatgatttca atactttcca agaaaaagtt 3180 cctcatttgg ctgatttgaa accttctggt caatatgtat tccaagacct ttacaaggtc 3240 ggaggggtac cagcagttat gaaatatctc cttaaaaatg gcttccttca tggtgaccgt 3300 atcacttgta ctggcaaaac agtcgctgaa aatttgaagg cttttgatga tttaacacct 3360 ggtcaaaagg ttattatgcc gcttgaaaat cctaaacgtg aagatggtcc gctcattatt 3420 ctccatggta acttggctcc agacggtgcc gttgccaaag tttctggtgt aaaagtgcgt 3480 cgtcatgtcg gtcctgctaa ggtctttaat tctgaagaag aagccattga agctgtcttg 3540 aatgatgata ttgttgatgg tgatgttgtt gtcgtacgtt ttgtaggacc aaagggcggt 3600 cctggtatgc ctgaaatgct ttccctttca tcaatgattg ttggtaaagg gcaaggtgaa 3660 aaagttgccc ttctgacaga tggccgcttc tcaggtggta cttatggtct tgtcgtgggt 3720 catatcgctc ctgaagcaca agatggcggt ccaatcgcct acctgcaaac aggagacata 3780 gtcactattg accaagacac taaggaatta cactttgata tctccgatga agagttaaaa 3840 catcgtcaag agaccattga attgccaccg ctctattcac gcggtatcct tggtaaatat 3900 gctcacatcg tttcgtctgc ttctagggga gccgtaacag acttttggaa gcctgaagaa 3960 actggcaaaa aatgttgtcc tggttgctgt ggttaagcgg ccgcgttaat tcaaattaat 4020 tgatatagtt ttttaatgag tattgaatct gtttagaaat aatggaatat tatttttatt 4080 tatttattta tattattggt cggctctttt cttctgaagg tcaatgacaa aatgatatga 4140 aggaaataat gatttctaaa attttacaac gtaagatatt tttacaaaag cctagctcat 4200 cttttgtcat gcactatttt actcacgctt gaaattaacg gccagtccac tgcggagtca 4260 tttcaaagtc atcctaatcg atctatcgtt tttgatagct cattttggag ttcgcgagga 4320 tcccagcttt tgttcccttt agtgagggtt aattgcgcgc ttggcgtaat catggtcata 4380 gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag 4440 cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg 4500 ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca 4560 acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc 4620 gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg 4680 gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa 4740 ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga 4800 cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag 4860 ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct 4920 taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg 4980 ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc 5040 ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt 5100 aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta 5160 tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac 5220 agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc 5280 ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat 5340 tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc 5400 tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt 5460 cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta 5520 aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct 5580 atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg 5640 cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga 5700 tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt 5760 atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt 5820 taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt 5880 tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat 5940 gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc 6000 cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc 6060 cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat 6120 gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag 6180 aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt 6240 accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc 6300 ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa 6360 gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg 6420 aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa 6480 taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgaac gaagcatctg 6540 tgcttcattt tgtagaacaa aaatgcaacg cgagagcgct aatttttcaa acaaagaatc 6600 tgagctgcat ttttacagaa cagaaatgca acgcgaaagc gctattttac caacgaagaa 6660 tctgtgcttc atttttgtaa aacaaaaatg caacgcgaga gcgctaattt ttcaaacaaa 6720 gaatctgagc tgcattttta cagaacagaa atgcaacgcg agagcgctat tttaccaaca 6780 aagaatctat acttcttttt tgttctacaa aaatgcatcc cgagagcgct atttttctaa 6840 caaagcatct tagattactt tttttctcct ttgtgcgctc tataatgcag tctcttgata 6900 actttttgca ctgtaggtcc gttaaggtta gaagaaggct actttggtgt ctattttctc 6960 ttccataaaa aaagcctgac tccacttccc gcgtttactg attactagcg aagctgcggg 7020 tgcatttttt caagataaag gcatccccga ttatattcta taccgatgtg gattgcgcat 7080 actttgtgaa cagaaagtga tagcgttgat gattcttcat tggtcagaaa attatgaacg 7140 gtttcttcta ttttgtctct atatactacg tataggaaat gtttacattt tcgtattgtt 7200 ttcgattcac tctatgaata gttcttacta caattttttt gtctaaagag taatactaga 7260 gataaacata aaaaatgtag aggtcgagtt tagatgcaag ttcaaggagc gaaaggtgga 7320 tgggtaggtt atatagggat atagcacaga gatatatagc aaagagatac ttttgagcaa 7380 tgtttgtgga agcggtattc gcaatatttt agtagctcgt tacagtccgg tgcgtttttg 7440 gttttttgaa agtgcgtctt cagagcgctt ttggttttca aaagcgctct gaagttccta 7500 tactttctag agaataggaa cttcggaata ggaacttcaa agcgtttccg aaaacgagcg 7560 cttccgaaaa tgcaacgcga gctgcgcaca tacagctcac tgttcacgtc gcacctatat 7620 ctgcgtgttg cctgtatata tatatacatg agaagaacgg catagtgcgt gtttatgctt 7680 aaatgcgtac ttatatgcgt ctatttatgt aggatgaaag gtagtctagt acctcctgtg 7740 atattatccc attccatgcg gggtatcgta tgcttccttc agcactaccc tttagctgtt 7800 ctatatgctg ccactcctca attggattag tctcatcctt caatgctatc atttcctttg 7860 atattggatc atactaagaa accattatta tcatgacatt aacctataaa aataggcgta 7920 tcacgaggcc ctttcgtc 7938

Claims (197)

Contacting butanol produced in the fermentation process with at least one carboxylic acid and at least one catalyst capable of esterifying the carboxylic acid with butanol to produce a butyl ester of carboxylic acid;
Wherein the carboxylic acid in the fermentation process is present at a concentration sufficient to produce a biphasic mixture. The process of claim 1 wherein butanol production and butyl ester production occur simultaneously or sequentially. The method of claim 1, wherein the feedstock in the fermentation process comprises one or more fermentable sugars. 2. The feedstock of the fermentation process according to claim 1, wherein the feedstocks in the fermentation process are corn residues, corn cobs, crop residues such as corn husk, corn cobs, grass, wheat, rye, straw, barley, barley straw, hay, rice straw, switchgrass. ), Waste paper, sugarcane waste, sorghum, sugarcane, soybeans, ingredients obtained from milling of grains, cellulosic substances, lignocellulosic substances, trees, branches, roots, leaves, wood chips, sawdust, shrubs and bushes, vegetables At least one fermentable sugar derived from fruit, flower, animal compost, and mixtures thereof. The method of claim 1, further comprising providing a native oil to contact the natural oil with one or more enzymes to convert at least some of the natural oil into carboxylic acids. The method of claim 1 wherein the carboxylic acid comprises a fatty acid. The method of claim 1 wherein the carboxylic acid comprises 12 to 22 carbons. The method of claim 1 wherein the carboxylic acid is a mixture of carboxylic acids. The method of claim 1 wherein the butyl ester of carboxylic acid is a butyl ester of fatty acid. The method of claim 1 wherein the catalyst is an enzyme capable of esterifying a carboxylic acid with butanol to produce a butyl ester of carboxylic acid. The method of claim 10, wherein the enzyme is an esterase, lipase, phospholipase, or lysophospholipase. (a) providing a feedstock;
(b) liquefying the feedstock to produce a liquefied biomass comprising oligosaccharides;
(c) separating the feedstock slurry to produce a product comprising an aqueous stream comprising oligosaccharides, an oil stream, and a solid;
(d) adding the aqueous stream to a fermentation vessel comprising fermentation broth;
(e) saccharifying oligosaccharides in the aqueous stream; And
(f) fermenting the oligosaccharide saccharification product present in the aqueous stream to produce butanol, while simultaneously contacting butanol with at least one carboxylic acid and at least one catalyst capable of esterifying the carboxylic acid with butanol, thereby Producing a butyl ester of a carboxylic acid present in a concentration sufficient to produce a biphasic mixture;
A process for producing butanol and butyl esters from a feedstock, wherein optionally steps (e) and (f) occur simultaneously. 13. The method of claim 12, further comprising the step of obtaining oil from the oil stream and converting at least some oil into carboxylic acids. 13. The feedstock slurry of claim 12, wherein the feedstock slurry is decanter bowl centrifugation, tricanter centrifugation, disk stack centrifugation, filtration centrifugation, decanter centrifugation, filtration, vacuum filtration, belt. Filters, pressure filtration, screen filtration, screen separation, grating, porous grating, floatation, hydroclone, filter press, screw press, gravity settler, vortex separator, Or separated by a combination thereof. 13. The method of claim 12 wherein the carboxylic acid comprises a fatty acid. 13. The method of claim 12, wherein the carboxylic acid comprises 12 to 22 carbons. 13. The method of claim 12, further comprising adding oil to the fermentation vessel prior to converting at least some of the oil to carboxylic acid. 13. The method of claim 12,
Adding further carboxylic acid to the fermentation vessel. The method of claim 18, wherein the oil is converted to the carboxylic acid after the step of adding additional carboxylic acid. 19. The method of claim 18, wherein the carboxylic acid is corn oil fatty acid, soybean oil fatty acid, or a mixture of corn oil fatty acid and soybean oil fatty acid. 13. The process of claim 12, wherein the oil obtained from the oil stream comprises glycerides and at least one catalyst hydrolyzes the glycerides to fatty acids. 13. The process of claim 12, wherein the butyl ester of carboxylic acid is butyl ester of fatty acid. 13. The process of claim 12 wherein the catalyst is an enzyme capable of esterifying a carboxylic acid with butanol to produce a butyl ester of carboxylic acid. The method of claim 23, wherein the enzyme is an esterase, lipase, phospholipase, or lysophospholipase. 13. The method of claim 12, further comprising washing the solid with a solvent. The method of claim 25, wherein the solvent is selected from petroleum distillates such as hexane, isobutanol, isohexane, ethanol, petroleum ether, or mixtures thereof. 27. The method of claim 26, wherein the solids are processed to produce an animal feed product. 13. The method of claim 12, wherein the solids are processed to produce an animal feed product. 29. The animal feed product of claim 28, wherein the animal feed product comprises at least one crude protein, crude fat, triglycerides, fatty acids, fatty acid isobutyl esters, lysine, neutral detergent insoluble fiber (NDF), and acid resistant fiber (ADF). ). The method of claim 29, wherein the animal feed product further comprises one or more vitamins, minerals, flavors, or colorants. 30. The animal feed product of claim 29, wherein the animal feed product comprises 20 to 35 wt% crude protein, 1 to 20 wt% crude fat, 0 to 5 wt% triglycerides, 4 to 10 wt% fatty acids, and 2 to 6 wt% fatty acid isobutyl esters. How to include. 13. The method of claim 12, wherein separating the solids from the feedstock slurry increases the butanol production efficiency by increasing the liquid-liquid mass transfer coefficient of butanol from the fermentation broth to the extractant; Use of an extractant to increase butanol extraction efficiency to increase butanol production efficiency; Increasing the rate of phase separation between the fermentation broth and the extractant to increase butanol production efficiency; Increased recovery and recycle of the extractant to increase butanol production efficiency; Reducing the flow rate of extractant to increase butanol production efficiency. (a) providing a feedstock;
(b) liquefying the feedstock to produce a liquefied biomass comprising oligosaccharides;
(c) separating the feedstock slurry to produce a stream comprising oligosaccharides and oil, and a solid;
(d) adding the stream to a fermentation vessel comprising fermentation broth;
(e) saccharifying oligosaccharides of the stream; And
(f) fermenting the oligosaccharide saccharification product present in the stream to produce butanol, while simultaneously contacting butanol with at least one carboxylic acid and at least one catalyst capable of esterifying the carboxylic acid with butanol, Producing a butyl ester, wherein the carboxylic acid is present at a concentration sufficient to produce a biphasic mixture;
A process for producing butanol and butyl esters from a feedstock, wherein optionally steps (e) and (f) occur simultaneously. 34. The method of claim 33,
And converting at least some oil into carboxylic acids. 34. The feedstock slurry of claim 33, wherein the feedstock slurry is decanter ball centrifuge, tricanter centrifuge, disc stack centrifuge, filtration centrifuge, decanter centrifuge, filtration, vacuum filtration, belt filter, pressure filtration, screen filtration, screen separation, Separation by grating, porous grating, floatation, hydroclone, filter press, screw press, gravity settling tank, vortex separator, or a combination thereof. The method of claim 33, wherein the carboxylic acid comprises a fatty acid. 34. The method of claim 33, wherein the carboxylic acid comprises 12 to 22 carbons. 34. The method of claim 33, further comprising adding oil to the fermentation vessel. 34. The method of claim 33, further comprising adding additional carboxylic acid to the fermentation vessel. 34. The method of claim 33, wherein the oil is converted to the carboxylic acid after adding additional carboxylic acid. 34. The method of claim 33, wherein the carboxylic acid is corn oil fatty acid, soybean oil fatty acid, or a mixture of corn oil fatty acid and soybean oil fatty acid. The method of claim 33, wherein the oil comprises glycerides and the one or more catalysts hydrolyze the glycerides into fatty acids. 34. The method of claim 33, wherein the butyl ester of carboxylic acid is butyl ester of fatty acid. 34. The method of claim 33, wherein the catalyst is an enzyme capable of esterifying a carboxylic acid with butanol to produce a butyl ester of carboxylic acid. 45. The method of claim 44, wherein the enzyme is an esterase, lipase, phospholipase, or lysophospholipase. 34. The method of claim 33, further comprising washing the solid with a solvent. 47. The method of claim 46, wherein the solvent is selected from petroleum distillates such as hexane, isobutanol, isohexane, ethanol, petroleum ether, or mixtures thereof. 47. The method of claim 46, wherein the solids are processed to produce an animal feed product. 34. The method of claim 33, wherein the solids are processed to produce an animal feed product. The method of claim 49, wherein the animal feed product comprises one or more crude proteins, crude fats, triglycerides, fatty acids, fatty acid isobutyl esters, lysine, neutral detergent insoluble fiber (NDF), and acid resistant fiber (ADF). 51. The method of claim 50, wherein the animal feed product further comprises one or more vitamins, minerals, flavors, or colorants. 51. The animal feed product of claim 50, wherein the animal feed product comprises 20 to 35 wt% crude protein, 1 to 20 wt% crude fat, 0 to 5 wt% triglycerides, 4 to 10 wt% fatty acids, and 2 to 6 wt% fatty acid isobutyl esters. How to include. 34. The method of claim 33, wherein separating the solids from the feedstock slurry increases the butanol production efficiency by increasing the liquid-liquid mass transfer coefficient of butanol from the fermentation broth to the extractant; Use of an extractant to increase butanol extraction efficiency to increase butanol production efficiency; Increasing the rate of phase separation between the fermentation broth and the extractant to increase butanol production efficiency; Increased recovery and recycle of the extractant to increase butanol production efficiency; Reducing the flow rate of extractant to increase butanol production efficiency. (a) contacting butanol produced in the fermentation process with at least one carboxylic acid and at least one catalyst capable of esterifying the carboxylic acid with butanol to produce a butyl ester of carboxylic acid
The carboxylic acid in the fermentation process is present at a concentration sufficient to produce a biphasic mixture comprising an aqueous phase and a butyl ester containing organic phase;
(b) separating the butyl ester containing organic phase from the aqueous phase; And
(c) recovering butanol from a butyl ester. 55. The method of claim 54, wherein recovering butanol from the butyl ester comprises hydrolyzing the ester to carboxylic acid and butanol. 56. The process of claim 55, wherein the butyl ester is hydrolyzed in the presence of a hydrolysis catalyst. 56. The process of claim 55, wherein the butyl ester is hydrolyzed in the presence of water and the hydrolysis catalyst comprises an acid catalyst, organic acid, inorganic acid, water soluble acid, or water insoluble acid. 59. The method of claim 56, wherein the hydrolysis catalyst comprises an enzyme capable of hydrolyzing butyl esters to produce carboxylic acids and butanol. The method of claim 58, wherein the enzyme is an esterase, lipase, phospholipase, or lysophospholipase. 60. The method of claim 59, wherein the enzymatic reaction conditions support enzymatic hydrolysis over esterification. 61. The method of claim 60, wherein the enzymatic reaction conditions comprise a cosolvent. 62. The method of claim 61, wherein the fatty acid butyl esters, fatty acids, isobutanol, and water are dissolved in the cosolvent and the free fatty acids do not react with the cosolvent. 63. The method of claim 62, wherein the cosolvent is selected from acetone, tert-butanol, 2-Me-2-butanol, 2-Me-2-pentanol, and 3-Me-3-pentanol. 61. The method of claim 60, wherein the enzymatic reaction conditions comprise final product removal. 65. The method of claim 64, wherein the final product is isobutanol or fatty acid. 67. The method of claim 65, wherein the isobutanol is removed by vacuum distillation, pervaporartion, permselective filtration, or gas sparging. 67. The method of claim 66, wherein the fatty acids are removed by precipitation, selective permeation filtration, or electrophoresis. The method of claim 55, wherein the hydrolysis reaction occurs in a reaction vessel. 55. The method of claim 54, wherein recovering butanol from a butyl ester comprises transesterifying the butyl ester with butanol and fatty acid alkyl esters or acyl glycerides. 70. The method of claim 69, wherein the fatty acid alkyl ester comprises a fatty acid methyl ester, a fatty acid ethyl ester, or a fatty acid propyl ester. 55. The method of claim 54, further comprising providing a natural oil to contact the natural oil with one or more enzymes to convert at least some natural oils to carboxylic acids. 72. The method of claim 71, wherein the enzyme is an enzyme capable of hydrolyzing or transesterifying butyl esters to produce butanol. The method of claim 72, wherein the enzyme is an esterase, lipase, phospholipase, or lysophospholipase. 55. The method of claim 54, wherein the carboxylic acid comprises a fatty acid. 55. The method of claim 54, wherein the carboxylic acid has a carbon chain length of 12 to 22 carbons. 55. The method of claim 54, wherein at least about 10% butanol is recovered from the butyl ester. 55. The method of claim 54, wherein at least about 50% of butanol is recovered from the butyl ester. 55. The method of claim 54, wherein at least about 90% of butanol is recovered from the butyl ester. 55. The method of claim 54, wherein the carboxylic acid is recovered from the butyl ester. 55. The method of claim 54, further comprising: removing butanol from the fermentation vessel as extractant stream; And
Adding the extractant stream to at least two distillation columns. 81. The method of claim 80, wherein the distillation column is a superatmospheric distillation column having a steam heated reboiler. 81. The method of claim 80, further comprising recovering water and solvent from the distillation column; and recycling the water and solvent. 81. The method of claim 80, further comprising: recovering heat from the distillation process; And recycling the heat to evaporate the water. (a) providing a feedstock;
(b) liquefying the feedstock to produce a feedstock slurry;
(c) separating the feedstock slurry to produce a product comprising an aqueous stream, an oil stream, and a solid;
(d) adding the aqueous stream to a fermentation vessel comprising fermentation broth;
(e) saccharifying the aqueous stream;
(f) fermenting the glycated aqueous stream to produce butanol, while simultaneously contacting butanol with at least one carboxylic acid and at least one catalyst capable of esterifying the carboxylic acid with butanol, thereby butyl ester of carboxylic acid Producing, the carboxylic acid is present at a concentration sufficient to produce a biphasic mixture;
(g) separating the butyl ester containing organic phase from the aqueous phase; And
(h) recovering butanol from the butyl ester;
A process for producing butanol from a feedstock, wherein optionally steps (e) and (f) occur simultaneously. 85. The method of claim 84, further comprising the step of obtaining oil from the oil stream and converting at least some oil into carboxylic acids. 85. The method of claim 84, wherein the feedstock slurry is separated by centrifugation, filtration, or decantation. 85. The method of claim 84, wherein the carboxylic acid comprises a fatty acid. 85. The method of claim 84, wherein the carboxylic acid has a carbon chain length of 12 to 22 carbons. 85. The method of claim 84, further comprising adding oil to the fermentation vessel prior to converting at least some oil into carboxylic acid. 85. The method of claim 84, further comprising adding additional carboxylic acid to the fermentation vessel. 91. The method of claim 90, wherein the oil is converted to the carboxylic acid after the step of adding additional carboxylic acid. 85. The method of claim 84, wherein the carboxylic acid is corn oil fatty acid, soybean oil fatty acid, or a mixture of corn oil fatty acid and soybean oil fatty acid. 85. The method of claim 84, wherein the oil obtained from the oil stream comprises glycerides and at least one catalyst hydrolyzes the glycerides to fatty acids. 85. The method of claim 84, wherein the butyl ester of carboxylic acid is butyl ester of fatty acid. 85. The method of claim 84, wherein the catalyst is an enzyme capable of esterifying a carboxylic acid with butanol to produce a butyl ester of carboxylic acid. 97. The method of claim 95, wherein the enzyme is an esterase, lipase, phospholipase, or lysophospholipase. 85. The method of claim 84, wherein the solids are processed to produce an animal feed product. 85. The method of claim 84, wherein recovering butanol from the butyl ester comprises hydrolyzing the ester to carboxylic acid and butanol. 99. The method of claim 98, wherein the butyl ester is hydrolyzed in the presence of a hydrolysis catalyst. 107. The process of claim 99, wherein the butyl ester is hydrolyzed in the presence of water and the hydrolysis catalyst comprises an acid catalyst, organic acid, inorganic acid, water soluble acid, or water insoluble acid. 107. The method of claim 99, wherein the hydrolysis catalyst comprises an enzyme capable of hydrolyzing the butyl ester to produce carboxylic acid and butanol. 102. The method of claim 101, wherein the enzyme is an esterase, lipase, phospholipase, or lysophospholipase. 99. The method of claim 98, wherein the hydrolysis reaction occurs in a reaction vessel. 85. The method of claim 84, wherein recovering butanol from the butyl ester comprises transesterifying the butyl ester with butanol and fatty acid alkyl esters or acyl glycerides. 107. The method of claim 104, wherein the fatty acid alkyl ester comprises a fatty acid methyl ester, fatty acid ethyl ester, or fatty acid propyl ester. 85. The method of claim 84, further comprising providing a natural oil to contact the natural oil with one or more enzymes to convert at least some natural oils to carboxylic acids. 107. The method of claim 106, wherein the enzyme is an enzyme capable of producing butanol by hydrolyzing or transesterifying butyl esters. 107. The method of claim 107, wherein the enzyme is an esterase, lipase, phospholipase, or lysophospholipase. (a) providing a feedstock;
(b) liquefying the feedstock to produce a liquefied biomass comprising oligosaccharides;
(c) separating the feedstock slurry to produce an aqueous stream comprising oligosaccharides and oils, and solids;
(d) adding the aqueous stream to a fermentation vessel comprising fermentation broth;
(e) saccharifying oligosaccharides in the aqueous stream;
(f) fermenting the oligosaccharide saccharification product present in the aqueous stream to produce butanol, while simultaneously contacting butanol with at least one carboxylic acid and at least one catalyst capable of esterifying the carboxylic acid with butanol, thereby Producing a butyl ester of carboxylic acid present in a concentration sufficient to produce a biphasic mixture;
(g) separating the butyl ester containing organic phase from the aqueous phase; And
(h) recovering butanol from the butyl ester;
A process for producing butanol and butyl esters from a feedstock, wherein optionally steps (e) and (f) occur simultaneously. 109. The method of claim 109, further comprising converting at least some oil into carboxylic acids. 109. The method of claim 109, wherein the feedstock slurry is separated by centrifugation, filtration, or decantation. 109. The method of claim 109, wherein the carboxylic acid comprises a fatty acid. 109. The method of claim 109, wherein the carboxylic acid comprises 12 to 22 carbons. 109. The method of claim 109, further comprising adding oil to the fermentation vessel. 109. The method of claim 109, further comprising adding additional carboxylic acid to the fermentation vessel. 116. The method of claim 115, wherein the oil is converted to the carboxylic acid after adding the additional carboxylic acid. 109. The method of claim 109, wherein the carboxylic acid is corn oil fatty acid or soybean oil fatty acid. 109. The method of claim 109, wherein the carboxylic acid is a mixture of corn oil fatty acid and soybean oil fatty acid. 109. The method of claim 109, wherein the oil comprises glycerides and the one or more catalysts hydrolyze glycerides to fatty acids. 109. The process of claim 109, wherein the butyl ester of carboxylic acid is butyl ester of fatty acid. 109. The process of claim 109, wherein the catalyst is an enzyme capable of esterifying carboxylic acids with butanol to produce butyl esters of carboxylic acids. 126. The method of claim 121, wherein the enzyme is an esterase, lipase, phospholipase, or lysophospholipase. 109. The method of claim 109, wherein the solids are processed to produce an animal feed product. 109. The process of claim 109, wherein recovering butanol from the butyl ester comprises hydrolyzing the ester to carboxylic acid and butanol. 124. The process of claim 124, wherein the butyl ester is hydrolyzed in the presence of a hydrolysis catalyst. 126. The process of claim 125, wherein the butyl ester is hydrolyzed in the presence of water and the hydrolysis catalyst comprises an acid catalyst, organic acid, water soluble acid, or water insoluble acid. 126. The method of claim 125, wherein the hydrolysis catalyst comprises an enzyme capable of hydrolyzing the butyl ester to produce carboxylic acid and butanol. 127. The method of claim 127, wherein the enzyme is an esterase, lipase, phospholipase, or lysophospholipase. 126. The method of claim 124, wherein the hydrolysis reaction occurs in a reaction vessel. 109. The method of claim 109, wherein recovering butanol from butyl esters comprises transesterifying the butyl esters with butanol and other fatty acid alkyl esters or acyl glycerides. 133. The method of claim 130, wherein the other fatty acid alkyl ester comprises a fatty acid methyl ester, a fatty acid ethyl ester, or a fatty acid propyl ester. 109. The method of claim 109, further comprising providing a natural oil to contact the natural oil with one or more enzymes to convert at least some natural oils to carboxylic acids. 134. The method of claim 132, wherein the enzyme is an enzyme capable of hydrolyzing or transesterifying butyl esters to produce butanol. 134. The method of claim 133, wherein the enzyme is an esterase, lipase, phospholipase, or lysophospholipase. Contacting the alcohol produced in the fermentation process with at least one carboxylic acid and at least one catalyst capable of esterifying the carboxylic acid with an alcohol to produce an alcohol ester of carboxylic acid;
Wherein the carboxylic acid in the fermentation process is present at a concentration sufficient to produce a biphasic mixture. 137. The method of claim 135, wherein alcohol production and alcohol ester production occur simultaneously or sequentially. 136. The method of claim 135, wherein the feedstock in the fermentation process comprises one or more fermentable sugars. 136. The process of claim 135, wherein the feedstock in the fermentation process comprises: crop residues such as corn kernels, corn cobs, corn husks, corn cobs, grass, wheat, rye, straw, barley, barley straw, hay, rice straw, wax grass, waste paper. , Sugarcane waste, sorghum, sugarcane, soybean, ingredients obtained from milling of cereals, cellulosic substances, lignocellulosic substances, trees, branches, roots, leaves, wood chips, sawdust, shrubs and bushes, vegetables, fruits, A method comprising one or more fermentable sugars derived from flowers, animal compost, and mixtures thereof. 137. The method of claim 135, further comprising providing a natural oil to contact the natural oil with one or more enzymes to convert at least some natural oils to carboxylic acids. 136. The method of claim 135, wherein the carboxylic acid comprises a fatty acid. 137. The method of claim 135, wherein the carboxylic acid has a carbon chain length of 12 to 22 carbons. 137. The method of claim 135, wherein the alcohol ester of carboxylic acid is an alcohol ester of fatty acid. 136. The process of claim 135, wherein the catalyst is an enzyme capable of esterifying a carboxylic acid with butanol to produce a butyl ester of carboxylic acid. 143. The method of claim 143, wherein the enzyme is an esterase, lipase, phospholipase, or lysophospholipase. 137. The method of claim 135, wherein the alcohol is a C ₂ to C ₈ alkyl alcohol. 145. The method of claim 145, wherein the C ₂ to C ₈ alkyl alcohol is ethanol, propanol, butanol, pentanol, or 2-methyl-1 butanol. (a) contacting the alcohol produced in the fermentation process with at least one carboxylic acid and at least one catalyst capable of esterifying the carboxylic acid with an alcohol to produce an alcohol ester of carboxylic acid
The carboxylic acid in the fermentation process is present in a concentration sufficient to produce a biphasic mixture comprising an aqueous phase and an alcohol ester containing organic phase;
(b) separating the alcohol ester containing organic phase from the aqueous phase; And
(c) recovering the alcohol from the alcohol ester. 147. The method of claim 147, wherein recovering the alcohol from the alcohol ester comprises hydrolyzing the ester to carboxylic acid and alcohol. 148. The process of claim 148, wherein the alcohol ester is hydrolyzed in the presence of a hydrolysis catalyst. 148. The process of claim 148, wherein the alcohol ester is hydrolyzed in the presence of water and the hydrolysis catalyst comprises an acid catalyst, organic acid, water soluble acid, or water insoluble acid. 158. The method of claim 149, wherein the hydrolysis catalyst comprises an enzyme capable of hydrolyzing the butyl ester to produce carboxylic acid and butanol. 151. The method of claim 151, wherein the enzyme is an esterase, lipase, phospholipase, or lysophospholipase. 148. The method of claim 148, wherein the hydrolysis reaction occurs in a reaction vessel. 147. The method of claim 147, wherein recovering the alcohol from the alcohol ester comprises transesterifying the alcohol ester with an alcohol and a fatty acid alkyl ester or acyl glyceride. 154. The method of claim 154, wherein the fatty acid alkyl ester comprises a fatty acid methyl ester, a fatty acid ethyl ester, or a fatty acid propyl ester. 147. The method of claim 147, further comprising providing a natural oil to contact the natural oil with one or more enzymes to convert at least some natural oils to carboxylic acids. 158. The method of claim 156, wherein the enzyme is an enzyme capable of producing alcohols by hydrolyzing or transesterifying alcohol esters. 158. The method of claim 157, wherein the enzyme is an esterase, lipase, phospholipase, or lysophospholipase. 147. The method of claim 147, wherein the carboxylic acid comprises a fatty acid. 147. The method of claim 147, wherein the carboxylic acid has a carbon chain length of 12 to 22 carbons. 147. The method of claim 147, wherein the alcohol is a C ₂ to C ₈ alkyl alcohol. 162. The method of claim 161, wherein the C ₂ to C ₈ alkyl alcohol is ethanol, propanol, butanol, pentanol, or 2-methyl-1 butanol. 158. The method of claim 148, wherein the carboxylic acid is recovered from the butyl ester. (a) recombinant microorganisms capable of producing product alcohols;
(b) fermentable carbon substrates;
(c) a catalyst capable of extracellular esterification of fatty acids with fatty alcohol esters with product alcohols; And
(d) fatty acid alcohol esters, wherein fatty alcohol esters are produced upon fermentation. 175. The fermentation broth of claim 164, further comprising at least one of acyl glycerides, fatty acids, product alcohols, or oleic acid. 175. The fermentation broth according to claim 164, further comprising a catalyst for esterifying a fatty acid with an alcohol to a fatty alcohol ester and for hydrolyzing the triglyceride to a free fatty acid. 167. The fermentation broth of claim 166, wherein the catalyst is one or more lipase enzymes. 167. The fermentation broth of claim 167, further comprising glycosylating enzymes capable of converting oligosaccharides into fermentable sugars. 168. The fermentation broth of claim 168, wherein the glycosylating enzyme comprises glucoamylase. 179. The fermentation broth of claim 169, wherein the fermentable sugar comprises monomeric glucose. 175. The fermentation broth of claim 164, wherein the recombinant microorganism is capable of producing butanol. 177. The fermentation broth of claim 164, wherein the fatty acid alcohol ester is a fatty acid butyl ester. 175. The fermentation broth according to claim 164, further comprising isobutanol. At least one enzyme catalyzing the conversion of each of the following substrates to a product, wherein at least one of the enzymes of iii) or the enzymes of v) comprises an isobutanol biosynthetic pathway encoded by a heterologous polynucleotide integrated into a chromosome Recombinant yeast cells capable of producing isobutanol,
i) conversion of pyruvate to acetolactate;
ii) conversion of acetolactate to 2,3-dihydroxyisovalerate;
iii) conversion of 2,3-dihydroxyisovalerate to α-ketoisovalerate;
iv) conversion of isobutyraldehyde to α-ketoisovalerate; And
v) conversion of isobutyraldehyde to isobutanol. 174. The recombinant yeast cell of claim 174, wherein said recombinant yeast cell is substantially free of pyruvate decarboxylase activity. 175. The recombinant yeast cell of claim 175 comprising a deletion of the pdc1, pdc5, and pdc6 genes. 176. The recombinant yeast cell of claim 176, which is substantially free of enzymes having NAD dependent glycerol-3-phosphate dehydrogenase activity. 181. The recombinant yeast cell of claim 177 comprising a deletion of gpd2. 179. The recombinant yeast cell of claim 178, wherein both the enzyme of iii) and the enzyme of v) are encoded by a heterologous polynucleotide integrated into a chromosome. 179. The recombinant yeast cell of claim 179, wherein the isobutanol biosynthetic pathway comprises at least one heterologous polynucleotide encoding an enzyme for conversion of each substrate to a product. 182. The recombinant yeast cell of claim 180, further comprising a plasmid having at least 80% identity with each coding region of pYZ090 or a plasmid having at least 80% identity with each coding region of pLH468. 181. The recombinant yeast cell of claim 180, comprising a plasmid of SEQ ID NO: 1 or a plasmid of SEQ ID NO: 2, or both. (a) fermentable carbon substrate;
(b) a catalyst capable of esterifying free fatty acids with alcohols and fatty acid alkyl esters and optionally hydrolyzing glycerides with free fatty acids;
(c) alcohols;
(d) free fatty acids; And
(e) A fermentation composition comprising a fatty alcohol ester produced in situ from esterification of an alcohol with free fatty acid using a catalyst. 184. The composition of claim 183, further comprising an oil comprising glycerides. 185. The composition of claim 184, wherein the oil, free fatty acid, and fermentable carbon substrate are derived from biomass. 185. The composition of claim 185, wherein the oil and the fermentable carbon substrate are from the same biomass source or from different biomass sources. 185. The composition of claim 185, wherein the biomass source of oil is soybean or corn oil and the biomass source of fermentable carbon substrate is corn. 184. The composition of claim 183, wherein (d) the free fatty acid is corn oil fatty acid. 185. The composition of claim 183, wherein (d) the free fatty acid is produced from hydrolysis of at least some of the glycerides in the oil using a catalyst. 184. The composition of claim 183, further comprising at least one of diglycerides and monoglycerides resulting from partial hydrolysis of some of the glycerides in the oil using a catalyst. 184. The composition of claim 183, further comprising glycerol. 184. The composition of claim 183, further comprising an insoluble solid derived from a biomass source of fermentable carbon substrate. 192. The composition of claim 192, containing less than about 25 wt% of insoluble solids. 184. The composition of claim 183, further comprising a saccharifying enzyme capable of converting starch into fermentable sugars, wherein the alcohol is butanol. A composition comprising butanol produced by the method of claims 1, 12, 33, 54, 84, and 109. 197. The composition of claim 195, used as fuel additive, feedstock chemical, reagent, solvent, preservative, or food grade extractant. A method as claimed in claim 1, 12, 33, 54, 84, and 109 as a fuel additive, feedstock chemical, reagent, solvent, preservative, or food grade extractant. Use of Butanol.

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