KR20150036133A - Processes and systems for the production of fermentation products - Google Patents

Abstract

The present invention relates to processes and systems for the production of fermentation products such as alcohols. The present invention also provides a method for separating feed stream components for improved biomass processing and productivity.

Description

TECHNICAL FIELD [0001] The present invention relates to a process for producing a fermentation product,

This application is related to US Provisional Patent Application No. 61 / 674,607, filed July 23, 2012; U.S. Patent Application No. 61 / 699,976, filed September 12, 2012; U. S. Patent Application No. 61 / 712,385, filed October 11, 2012; U.S. Patent Application No. 13 / 828,353, filed March 14, 2013; And U.S. Patent Application No. 13 / 836,115, filed March 15, 2013, the entire contents of each of which are incorporated herein by reference.

Sequence listings related to the present application are filed in electronic form via EFS-Web, the disclosure of which is hereby incorporated by reference in its entirety.

The present invention relates to processes and systems for producing fermentation products such as alcohols. The present invention also provides a process for separating feed stream components for improved biomass process productivity.

Alcohols have various industrial and scientific applications such as fuels, reagents, and solvents. For example, butanol can be used as a fuel additive, as a feedstock chemical in the plastics industry, and as an important industrial use with various applications, including as a food-grade extractant in the food and flavor industries It is a chemical. Thus, there is a great need for an efficient and environmentally friendly production process involving alcohols such as butanol and also the use of biomass as a feedstock for, for example, fermentation processes and processes.

The production of alcohol by fermentation is one such environmentally friendly method of production. Some microorganisms that produce alcohol in high yields also have a low toxicity threshold so that the alcohol needs to be removed from the fermenter in which it is being produced. One method, in situ product removal (ISPR), can be used to remove the alcohol from the fermentor where the alcohol is produced, thereby allowing the microorganism to produce the alcohol in high yield. An example of an ISPR that has been described in the art is liquid-liquid extraction (see, for example, U.S. Patent Application Publication No. 2009/0305370). Liquid-liquid extraction, technically and economically available, is the contact between the extractant and the fermentation broth for efficient mass transfer; Phase separation of the extractant from the fermentation broth; Efficient recovery and recycling of the extractant; And minimal degradation and / or contamination of the extractant over long-term operation.

If the aqueous stream introduced into the fermenter contains undissolved solids from the feedstock, the undissolved solids may interfere with liquid-liquid extraction and the extraction process may not be technically and economically feasible, Which can lead to an increase in capital and operating costs. The presence of undissolved solids during extraction fermentation reduces the mass transfer coefficient, retarding phase separation and / or causing accumulation of oil from undissolved solids in the extractant, resulting in reduced extraction efficiency over time / Or the extractant is trapped in a solid and ultimately removed as Dried Distiller Grains with Solubles (DDGS), thus increasing the loss of the extractant and / or increasing the yield of the extractant from the fermentation broth It is possible to delay the dropout of the droplet and / or to produce a lower fermenter volumetric efficiency. Thus, solid removal provides an efficient means for producing and recovering alcohol from the fermentation process.

In addition to solids removal, the removal of oil from the feedstock can also provide commercial benefits as well as beneficial effects in the production of alcohol. For example, some oils, such as corn oil and soybean oil, can be used as feedstocks for biodiesel, thus providing additional revenue streams for alcohol producers. In addition, the removal of oil can also reduce the energy consumption of the production plant due to more efficient fermentation, fewer deposits due to removal of oil, increased fermenter volumetric efficiency, and energy requirements, such as drying distillers grains Which can lead to a reduction in the energy required to achieve the desired result.

There is a continuing need to develop more efficient processes and systems for producing alcohols such as ethanol and butanol. The present invention provides a process and system for producing alcohol comprising processes and systems for meeting this need and for controlling the amount of undissolved solids and / or oils in the fermentation process by separating the feed stream components prior to fermentation do.

The present invention relates to processes and systems for separating feed stream components and for controlling the amount of undissolved solids and / or oil in the fermenter feed stream during the production of fermentation products. The discrete components provide a mechanism for increasing biomass process productivity, including improving the alcohol fermentation co-product profile. (1) a water stream comprising a fermentable carbon source, (2) a feed stream comprising an oil, and (3) a feed stream comprising an undissolved solid. , These components can be recombined in a controlled manner or removed from the system for other uses. This provides a mechanism for developing co-product compositions for different market needs such as animal feed markets that require better protein or better fat feed.

The present invention also relates to a process and system for removing oil from a fermenter feed stream in the production of fermentation products. In some embodiments, undissolved solids and oil may be removed from the fermenter feed stream.

The present invention provides a method for producing a feedstock slurry comprising: providing a feedstock slurry comprising a fermentable carbon source, an undissolved solid, and an oil; Separating some of the undissolved solids and oil from the feedstock slurry to form an aqueous solution comprising a fermentable carbon source, a wet cake comprising the solids and an oil stream; And adding an aqueous solution to a fermentation broth comprising a microorganism to produce a product alcohol. In some embodiments, the method further comprises recovering the oil stream. In some embodiments, the method further comprises washing the wet cake to provide an aqueous stream comprising carbohydrates. In some embodiments, the method further comprises adding an aqueous stream to the fermentation broth. In some embodiments, the aqueous solution contains up to about 5% by weight of undissolved solids. In some embodiments, the oil is corn oil and comprises at least one triglyceride, fatty acid, diglyceride, monoglyceride, and phospholipid. In some embodiments, the method further comprises producing a wet cake comprising triglycerides, free fatty acids, diglycerides, monoglycerides, and phospholipids by combining a portion of the wet cake with a portion of the oil. In some embodiments, the method further comprises combining the aqueous solution with a portion of the wet cake to produce a mixture of aqueous solution and wet cake, and adding the mixture to a fermentation broth. In some embodiments, separating the feedstock slurry is a single step process. In some embodiments, the undissolved solids and oil are separated from the feedstock slurry by decanter bowl centrifugation, 3-phase centrifugation, disk stack centrifugation, filtration centrifugation, decanter centrifugation Filtration using a screen, screen separation, grating, porous grating, flotation, hydrotreating, centrifugal separation, filtration, decanter centrifugation, filtration, vacuum filtration, beltfilter, Such as a hydrocyclone, a filter press, a screw press, a gravity settler, a fluid separator, or a combination thereof. In some embodiments, one or more adjustment parameters of the separation device are adjusted to improve the separation of the feedstock slurry. In some embodiments, one or more of the control parameters are selected from the group consisting of differential speed, bowl speed, flow rate, impeller position, weir position, scroll pitch, residence time, and discharge volume ). In some embodiments, the product alcohol is selected from ethanol, propanol, butanol, pentanol, hexanol, and fusel alcohol. In some embodiments, the microorganism comprises a butanol biosynthetic pathway. In some embodiments, the butanol biosynthetic pathway is a 1-butanol biosynthetic pathway, a 2-butanol biosynthetic pathway, or an isobutanol biosynthetic pathway. In some embodiments, real time measurements are used to monitor the separation of the feedstock slurry. In some embodiments, the separation may be performed using Fourier transform infrared spectroscopy, near-infrared spectroscopy, Raman spectroscopy, high pressure liquid chromatography, viscometer, densitometer, tensiometer, A droplet size analyzer, a particle analyzer, or a combination thereof.

The present invention also provides a feedstock slurry comprising a fermentable carbon source and an undissolved solid; Producing a wet cake comprising an aqueous solution and a solid comprising a fermentable carbon source by separating at least a portion of the undissolved solids from the feedstock slurry; Contacting the wet cake with a liquid to form a wet cake mixture; And separating at least a portion of the undissolved solids from the wet cake mixture to produce a second wet cake comprising a second aqueous solution and a solid comprising a fermentable carbon source. In some embodiments, the liquid is selected from fresh water, a backset, a cook water, a process water, a lutter water, an evaporation water, or a combination thereof. In some embodiments, the contacting step and the separating step can be repeated.

The present invention provides a method for producing a feedstock slurry comprising: providing a feedstock slurry comprising a fermentable carbon source, an undissolved solid, and an oil; An oil stream comprising an aerobic fermentable carbon source by separating at least some of the oil and undissolved solids from the feed slurry; And a wet cake comprising a solid; And contacting the wet cake with a liquid to form a wet cake mixture; And a second aqueous solution comprising a carbon source fermentable by separating at least a portion of the undissolved solids and oil from the wet cake mixture; a second oil stream; And a second wet cake comprising a solid. In some embodiments, separating the feedstock slurry is a single step process. In some embodiments, the undissolved solids and oil are separated from the feedstock slurry by decanter bowl centrifugation, 3-phase centrifugation, disk stack centrifugation, filtration centrifugation, decanter centrifugation, filtration, vacuum filtration, Is separated by pressure filtration, membrane filtration, crossflow filtration, screen filtration, screen separation, grating, porous grating, suspension, hydrocyclone, filter press, screw press, gravity settler, vortex separator, . In some embodiments, one or more adjustment parameters of the separation device are adjusted to improve the separation of the feedstock slurry. In some embodiments, the at least one adjustment parameter is selected from differential speed, bowl speed, flow rate, impeller position, wear position, scroll pitch, residence time, and discharge volume. In some embodiments, real time measurements are used to monitor the separation of the feedstock slurry. In some embodiments, the separation is monitored by Fourier transform infrared spectroscopy, near infrared spectroscopy, Raman spectroscopy, high pressure liquid chromatography, viscometer, densitometer, tensiometer, droplet size analyzer, particle analyzer, or a combination thereof.

The present invention provides a method for producing a feedstock slurry comprising: providing a feedstock slurry comprising a fermentable carbon source, an undissolved solid, and an oil; By separating the feedstock slurry, a first aqueous solution comprising (i) a fermentable carbon source, (ii) a first wet cake comprising a solid, and (iii) a stream comprising the oil, a solid, and a fermentable carbon source To form an aqueous stream; And adding a first aqueous solution to a fermentation broth comprising a microorganism to produce a fermentation product. In some embodiments, the method comprises separating a stream comprising oil, a solid, and an aqueous stream comprising a fermentable carbon source to form a second aqueous solution comprising (i) a fermentable carbon source, (ii) 2 wet cake, and (iii) forming an oil stream. In some embodiments, the first and second aqueous solutions may be combined before adding to the fermentation broth. In some embodiments, the second aqueous solution may further comprise an oil. In some embodiments, the oil of the second aqueous solution or a portion thereof may be treated to produce an extractant. In some embodiments, the oil may be chemically or enzymatically treated. In some embodiments, the separation may be carried out in the following manner: decanter bowl centrifugation, 3-phase centrifugation, disk stack centrifugation, filtration centrifugation, decanter centrifugation, filtration, vacuum filtration, belt filtration, Screen separation, grating, porous grating, floating, hydrocyclone, filter press, screw press, gravity settler, vortex separator, or a combination thereof.

The present invention provides a method for producing a feedstock slurry comprising: providing a feedstock slurry comprising a fermentable carbon source, an undissolved solid, and an oil; Separating the feedstock slurry to form a first aqueous stream comprising (i) a first aqueous solution comprising a fermentable carbon source and a solid, (ii) a first wet cake comprising a solid, and (iii) a first oil stream; And a second aqueous solution comprising an oil layer comprising solids and a fermentable carbon source by applying the oil to the first aqueous solution. In some embodiments, the oil layer comprising solids comprises (i) a second oil stream, (ii) a second wet cake comprising a solid, and (iii) a third aqueous solution comprising a fermentable carbon source, And can be separated. In some embodiments, the second aqueous solution and the third aqueous solution may be added to a fermentation broth comprising a microorganism to produce a fermentation product. In some embodiments, the second aqueous solution and the third aqueous solution may further comprise an oil. In some embodiments, the extractant may be produced by combining the second aqueous solution and the third aqueous solution and treating the oil of the second aqueous solution and the third aqueous solution or a portion thereof. In some embodiments, the oil may be chemically or enzymatically treated. In some embodiments, the separation may be carried out in the following manner: decanter bowl centrifugation, 3-phase centrifugation, disk stack centrifugation, filtration centrifugation, decanter centrifugation, filtration, vacuum filtration, belt filtration, Screen separation, grating, porous grating, sub-organic, hydrocyclone, filter press, screw press, gravity settler, vortex separator, or a combination thereof.

The invention also relates to a process for producing a feedstock slurry, comprising at least one liquefaction unit configured to liquefy the feedstock to produce a feedstock slurry, wherein the liquefaction unit comprises an inlet for receiving the feedstock and an outlet for discharging the feedstock slurry Wherein the feedstock slurry comprises a fermentable carbon source, an oil, and an undissolved solid; And a wet cake comprising a fermentable carbon source, an oil stream, and a wet cake comprising a portion of the undissolved solids by removing oil and undissolved solids from the feed slurry, The centrifuge includes an inlet for receiving the feed slurry, a first outlet for discharging the aqueous solution, a second outlet for discharging the wet cake, and a third outlet for discharging the oil) / RTI > In some embodiments, the system includes one or more mixing devices; And at least one cleaning system configured to recover a fermentable carbon source from the wet cake, the at least one cleaning device comprising at least one separation device. In some embodiments, the separation device may be selected from the group consisting of decanter bowl centrifugation, 3-phase centrifugation, disk stack centrifugation, filtration centrifugation, decanter centrifugation, filtration, vacuum filtration, belt filtration, Is selected from filtration, filtration using screens, screen separation, gratings, porous gratings, suspended solids, hydrocyclones, filter presses, screw presses, gravity settlers, fluid separators, or combinations thereof. In some embodiments, the system further comprises at least one fermenter configured to ferment an aqueous solution to produce a product alcohol, the fermenter having an inlet for receiving an aqueous solution and / or a wet cake; And an outlet for discharging the fermentation broth comprising the product alcohol. In some embodiments, the system further comprises an on-line measurement device. In some embodiments, the on-line measurement device may be a particle size analyzer, a Fourier transform infrared spectroscope, a near infrared ray spectrometer, a Raman spectrometer, a high pressure liquid chromatography, a viscometer, a densitometer, a tensiometer, a droplet size analyzer, Or a combination thereof.

The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate the present invention and, together with the description, further serve to explain the principles of the invention and to enable a person skilled in the relevant art to make and use the invention .
Figure 1 schematically illustrates an exemplary process and system of the present invention in which the undissolved solids are removed by separation after liquefaction and prior to fermentation.
Figure 2 schematically depicts an exemplary alternative process and system of the present invention wherein the feedstock is pulverized.
Figure 3 schematically depicts another exemplary alternative process and system of the present invention wherein undissolved solids and oil are removed by separation.
Figure 4 schematically depicts another exemplary alternative process and system of the present invention wherein the wet cake is applied to one or more wash cycles.
Figure 5 schematically illustrates another exemplary alternative process and system of the present invention wherein the undissolved solids and oil are removed by separation and the wet cake is applied to one or more wash cycles.
Figure 6 schematically depicts another exemplary alternative process and system of the present invention wherein the aqueous solution and the wet cake are combined and processed for fermentation.
Figure 7 schematically illustrates another exemplary alternative process and system of the present invention wherein the aqueous solution is saccharified prior to fermentation.
Figure 8 schematically illustrates another exemplary alternative process and system of the present invention wherein the feedstock slurry is saccharified prior to separation.
Figures 9A-9D schematically illustrate exemplary alternative processes and systems of the present invention in which additional separation equipment is used to remove undissolved solids and oils.
Figure 10 schematically illustrates an exemplary fermentation process using on-line, in-line, at-line, and / or real-time measurements for monitoring the fermentation process.
Figure 11 schematically illustrates an exemplary fermentation process of the present invention including downstream processing.
12 is a graph showing the results of the measurement of the concentration of i- BuOH ( i- BuOH) in a dispersion of oleyl alcohol droplets flowing upward from an aqueous solution of liquefied corn starch through a bubble column when the oleyl alcohol is dispersed using a nozzle having an inner diameter of 2.03 mm The total volumetric mass transfer coefficient, k _L a, for delivering the unconverted corn mash solids.
13 is a graph showing the relationship between the amount of i- BuOH and the total amount of i- BuOH transferred to a dispersion of oleyl alcohol droplets flowing upward from an aqueous solution of liquefied corn starch through a bubble column when oleyl alcohol is dispersed using a nozzle having an inner diameter of 0.76 mm. The effect of the presence of undissolved maize solids on the volume mass transfer coefficient, k _L a, is shown.
Figure 14 shows the position of the liquid-liquid interface in the fermentation sample tube as a function of (gravity) settling time. The phase separated data are shown for run times: 5.3, 29.3, 53.3, and 70.3 hr. The sample data is from extractable-fermentation, where solids were removed from the mash feed and oleic alcohol (OA) was the solvent.
Figure 15 shows the position of the liquid-liquid interface of the final fermentation broth as a function of settling time (gravity). The data is from extractable-fermentation, where solids were removed from the mash feed and oleyl alcohol (OA) was the solvent.
Figure 16 shows the concentration of glucose in aqueous phase of the slurry as a function of time for batch 1 and batch 2.
Figure 17 shows the concentration of glucose in the aqueous phase of the slurry as a function of time for batch 3 and batch 4.
Figure 18 shows the effects of enzymatic loading and +/- high temperature steps that were applied sometime during liquefaction during starch conversion.
Figures 19a-e show the effect of a three phase centrifuge condition on the separation of the feedstock slurry.

Unless otherwise defined, 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 application, including the subject definition, will control. Also, unless the context otherwise requires, the singular terms shall include the plural and the plural terms shall include the singular. All publications, patents, and other references mentioned herein are incorporated by reference in their entirety for all purposes.

In order to further define the present invention, the following terms and definitions are provided herein.

As used herein, the terms "comprises," "including," "including," "including," "having," "having," "containing," or "containing" It will be understood that a variation includes a group of integers or integers as described, but does not exclude any other integer or group of integers. For example, a composition, mixture, process, method, article, or apparatus that comprises a list of components is not necessarily limited to these components, but may be any component, mixture, process, method, article, Other ingredients that are not unique may also be included. Also, unless expressly stated otherwise, "or" refers to inclusive or exclusive and not exclusive or. For example, condition A or B is satisfied by either: A is true (or exists), B is false (or does not exist), A is false (or does not exist) Truth (or exists), and both A and B are true (or exist).

Also, it is intended that the singular forms " a " and "an" preceding the components or parts of the invention are non-limiting in numerous instances, i. Accordingly, one ("a" or "an") should be interpreted as including one or at least one, and the singular word form of an element or part also includes a plurality, except where the number clearly implies a singular .

As used herein, the term " invention "or" invention "is a non-limiting term and is intended to refer to any single embodiment of the specific invention, as well as all possible implementations described in this application.

As used herein, the term "about" for modifying the amount of an ingredient or reagent of the present invention used, for example, through representative measurements and liquid handling procedures actually used to prepare the concentrate or solution; Through unintentional errors in these processes; Refers to a change in the numerical amount that may occur through the manufacture, source, or difference in purity of the ingredients used to make the composition or perform the method. The term " about "also includes a different amount due to the different equilibrium conditions for the composition resulting from the particular initial mixture. Regardless of whether the term "about" is modified or not, the claims include equivalents to amounts. In one embodiment, the term "about" means within 10% of the reported value, alternatively within 5% of the reported value.

As used herein, the term "biomass" is intended to encompass corn, sugarcane (or stem), wheat, cellulosic or lignocellulosic materials, and cellulose, hemicellulose, lignin, starch, oligosaccharides, disaccharides, and / Refers to natural products containing hydrolyzable polysaccharides that provide fermentable sugars, including certain saccharides and starches from natural sources, such as, for example, sugars, and mixtures thereof. Biomass may also include additional components such as proteins and / or lipids. The biomass may originate from a single source or the biomass may comprise a mixture derived from one or more sources. For example, the biomass may comprise a mixture of corn cob and corn stover, or a mixture of grass and leaves. Biomass includes biological energy crops, agricultural residues, municipal solid wastes, industrial solid wastes, sludge from paper manufacturing, yard waste, and wood and forest wastes (eg, by forests) , But is not limited thereto. Examples of biomass are corn residues such as corn kernels, corncobs, corn husks, cornstalks, weeds, wheat, rye, straw, spelled, triticale, barley, barley straw, oats, hay, Rice, rice straw, switchgrass, potato, sweet potato, cassava, Jerusalem artichoke, sugarcane remnants, sorghum, sugar cane, beetroot, beef cattle, soybean, palm, coconut, Vegetable, fruit, flower, animal manure, and mixtures thereof obtained by pulverizing sunflower flowers, sorghum, eucalyptus, wheat, abolished paper, grains, trees, branches, roots, leaves, wood chips, sawdust, shrubs and bushes But is not limited thereto. The mash, juice, molasses, or hydrolyzate may be removed from the biomass by any method known in the art for processing biomass for fermentation purposes such as grinding, processing (e.g., enzymatic, chemical), and / . The treated biomass may comprise fermentable sugars and / or water. The cellulosic and / or lignocellulosic biomass may be processed to yield a hydrolyzate containing the fermentable sugars by any method known to those skilled in the art. For example, low ammonia pretreatment is described in U. S. Patent Publication No. 2007/0031918 Al, which is incorporated herein by reference in its entirety. The enzymatic saccharification of cellulosic and / or lignocellulosic biomass typically produces hydrolysates containing saccharides including glucose, xylose, and arabinose, using enzymatic mixtures for the hydrolysis of cellulose and hemicellulose . Suitable glycosylating enzymes for cellulosic and / or lignocellulosic biomass are discussed in Lynd et al. (Microbiol. Mol. Biol. Rev. 66: 506-577, 2002).

As used herein, "fermentable carbon source" or "fermentable carbon substrate" refers to a carbon source that can be metabolized by a microorganism. Suitable fermentable carbon sources include monosaccharides such as glucose or fructose; Disaccharides such as lactose or sucrose; oligosaccharide; Polysaccharides such as starch or cellulose; One carbon substrate; And mixtures thereof.

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

As used herein, "feedstock" refers to a feedstock in a fermentation process. The feed may comprise a fermentable carbon source and may include undissolved solids and / or oils. If applicable, the feed may comprise a fermentable carbon source either before or after the fermentable carbon source is liberated from the starch or obtained from the hydrolysis of the complex saccharide by further processing, such as by liquefaction, saccharification, or other processes . The feedstock may comprise or be derived from a biomass. Suitable feedstocks include, but are not limited to, rye, wheat, corn, corn mash, sorghum, stem mash, barley, cellulosic material, lignocellulosic material, or mixtures thereof. When referring to "feedstock oil ", it will be appreciated that the term includes oils derived from the feedstock provided.

As used herein, the term "fermented broth" includes water, fermentable carbon sources (e.g. sugars, starches), dissolved solids, fermentation products which optionally produce microorganisms, optionally fermentation products are optionally insoluble solid, and the fermentation products are produced by the metabolism of a fermentable carbon source by the microorganism being refers to a mixture of the other components of the material held in the fermentation product, the fermentation to form water, and carbon dioxide (CO _2). As sometimes used herein, the terms "fermentation medium" and "fermented mixture" may be used synonymously with "fermentation broth".

As used herein, a "fermenter" or "fermentation vessel" is intended to encompass a container, unit, or container made from a fermentable carbon source of fermentation product (eg, a product alcohol such as ethanol or butanol) Tanks. The fermenter may also refer to a vessel, unit, or tank in which the growth of the microorganism takes place. In some instances, both microbial growth and fermentation can occur in a fermenter. The term "fermenter" may be used herein synonymously with "fermentation vessel ".

As used herein, "glycation vessel" refers to a vessel, device, or tank in which saccharification (i.e., hydrolysis of oligosaccharide to monosaccharide) is performed. When fermentation and saccharification occur at the same time, the saccharification vessel and the fermenter may be the same vessel.

As used herein, "saccharifying enzyme" refers to one or more enzymes capable of hydrolysing polysaccharides and / or oligosaccharides, for example alpha-1, 4-glucoside bonds, or starch of glycogen. The saccharifying enzyme may also comprise an enzyme capable of hydrolyzing the cellulosic or lignocellulosic material as well.

As used herein, "liquefier vessel" refers to a vessel, unit, or tank in which liquefaction is performed. Liquefaction is a process in which starch is hydrolyzed, for example, by an enzymatic process to obtain oligosaccharides. In embodiments where the feedstock is corn, the oligosaccharide is hydrolyzed from the corn starch component during liquefaction.

As used herein, "sugar" refers to oligosaccharides, disaccharides, monosaccharides, and / or mixtures thereof. The term "saccharide" may also include carbohydrates such as starch, dextran, glycogen, cellulose, pentanoic acid, and also sugars.

As used herein, "undissolved solids" refers to non-fermentable portions of feedstocks that are not soluble in the liquid or aqueous phase, such as embryos, fibers, and gluten. The non-fermentable portion of the feedstock remains as a solid and contains a portion of the feedstock that is capable of absorbing liquid from the fermentation broth. As sometimes used herein, the term "undissolved solid" may be used synonymously with "solid" or "suspended solid ".

As used herein, "extractant" refers to the solvent used to extract the fermentation product. As sometimes used herein, the term "extractant" may be used in the same language as "solvent ".

As used herein, the term " removal of products in a reaction system "(ISPR) refers to the control of product concentration in a biological process as the product is produced by selectively removing the product from biological processes such as fermentation.

As used herein, "product alcohol" refers to any alcohol that can be produced by a microorganism in a fermentation process that utilizes biomass as a fermentable carbon source. The product alcohols include, but are not limited to, C ₁ to C ₈ alkyl alcohols. In some embodiments, the product alcohol is a C ₂ to C ₈ alkyl alcohol. In another embodiment, the product alcohol is a C ₂ to C ₅ alkyl alcohol. C ₁ to C ₈ alkyl alcohols include, but are not limited to, methanol, ethanol, propanol, butanol, pentanol, hexanol, and isomers thereof. Similarly, C ₂ to C ₈ alkyl alcohols include, but are not limited to, ethanol, propanol, butanol, pentanol, hexanol, and isomers thereof. The term "alcohol" may also be used herein with reference to a product alcohol.

As used herein, "butanol ", individually or as a mixture thereof, includes butanol isomers such as 1-butanol (1-BuOH), 2-butanol (2-BuOH), tert- Butanol (iBuOH, i-BuOH, or i-BUOH).

As used herein, "propanol" refers to propanol isomers: isopropanol or 1-propanol, either individually or as a mixture thereof.

As used herein, "pentanol ", individually or as a mixture thereof, is selected from the group consisting of pentanol isomers: 1-pentanol, 3-methyl-1-butanol, 2-methyl-1-butanol, 2,2- , 3-pentanol, 2-pentanol, 3-methyl-2-butanol, or 2-methyl-2-butanol.

As used herein, "effective potency" refers to the total amount of a particular fermentation product (e.g., product alcohol) produced by fermentation. In some embodiments where the fermentation product is a product alcohol, an effective reversal refers to an alcohol equivalent of an alcohol ester produced by alcohol esterification per liter of fermentation medium.

As used herein, "water-immiscible" or "insoluble" means a chemical component, such as an extractant or solvent, which can not be mixed with an aqueous solution, such as a fermentation broth, .

As used herein, "aqueous phase" refers to an aqueous phase of at least two-phase mixture obtained by contacting a fermentation broth with an extractant, for example, a water-immiscible organic extractant. In one embodiment of the process described herein, including fermentative extraction, the term "fermentation broth" may hereinafter refer to an aqueous phase in two phase fermentation extraction.

As used herein, "aqueous phase titer" refers to a fermentation concentrate in aqueous phase (eg, product alcohol).

As used herein, "organic phase" refers to a non-aqueous phase of at least two-phase mixture obtained by contacting a fermentation broth with an extractant, for example, a water-immiscible organic extractant.

As used herein in connection with a process stream, a "portion" refers to any component or components of a stream, including all components of the stream, as well as any fractional portion of the stream that holds the composition of the stream comprising the entire stream .

As used herein, "by-product" or "co-product" refers to the product produced during the production of another product. In some cases, the term "co-product" may be used synonymously with the term "by-product ". The co-product includes, for example, feedstock slurry, wet cake, and oil recovered from DDGS. The co-product may also include oils, wet cakes and modifications of DDGS for the purpose of enhancing value and / or for producing other products such as biodiesel from oils.

A "distiller co-product" as used herein refers to a by-product from a product alcohol production process that can be separated prior to fermentation or during fermentation. The distiller co-product comprises a non-fermentable product that remains after the product alcohol has been removed from the fermented mash and from the solid isolated from the mash. As used herein, distiller co-products can be used in a variety of animal feed and non-animal feed products. Examples of distiller co-products are: fatty acids from oil solubilisation, lipids from evaporation of thin stillages, syrups, distiller grains, distiller grains and solubles, solids from the mesh before fermentation, and fermentation But are not limited to, solids, biodiesel, and acylglycerides from whole distillation wastewater.

As used herein, "animal feed distiller co-product" refers to a distiller co-product suitable for use in animal feed or as animal feed. Examples of animal feed distiller co-products are fatty acids from oil hydrolysis, lipids from evaporation of dilute distillery liquor, syrups, distiller grains, distiller grains and solids, solids from the mesh before fermentation, But are not limited to, solids.

As used herein, "distiller grain" or "DG" refers to a non-fermentable product that remains after the product alcohol has been removed from the fermented mash. Dried distiller grains are known as "distiller-dried grains" or "DDG". Non-dried distiller grains are known as "wet distiller grains" or "WDG ".

As used herein, "distiller grains and solubles" or "DGS" refers to a non-fermentable product that remains after the product alcohol has been removed from the fermented mash with the solubles. Dried distiller grains and soluble matter are known as "distiller dried grains and soluble matter" or "DDGS". Non-dried distiller grains and soluble matter are known as "wet retort grains and sweeteners" or "WDGS ".

As used herein, "dry distiller grains with solubles (DDGS)" refers to co-products or by-products from the fermentation of the feedstock or biomass (e.g., fermentation of the cereal or grain mixture producing the product alcohol) . In some embodiments, DDGS may also represent an animal feed produced from the process of producing product alcohols.

As used herein, "lipid" refers to any of a heterogeneous group of fatty and fatty substances, including fatty acids, triglycerides, waxes and steroids, which are water-insoluble and soluble in non-polar solvents. Examples of lipids include monoglycerides, diglycerides, triglycerides and phospholipids.

"Lipids from evaporation ", as used herein in connection with process streams, refers to lipid byproducts produced by evaporation and centrifugation of dilute distillery waste liquid after fermentation in a product alcohol production process.

"Syrup" or "concentrated distiller solubles" (CDS) as used herein in connection with process streams refers to byproducts produced by evaporation of dilute distillery waste after fermentation in a product alcohol production process.

As used herein, "process stream" refers to any by-product or co-product formed by the fermentation product production process. Examples of process streams include, but are not limited to, COFA, lipids from evaporation, syrup, DG, DDG, WDG, DGS, DDGS, and WDGS. Another example of a process stream is a solid (e. G., A solid removed from the maize mesh before fermentation) that has been removed from the mash prior to fermentation in the fermentation product production process (e. G., By centrifugation). These solids can be referred to as "wet cake" if they are not dried, and can be referred to as "dry cake" if they are dried. Another example of a process stream is a solid (e.g., by centrifugation) that has been removed from the entire distillation waste liquid after fermentation in the fermentation product production process. These solids can be referred to as "WS wet cake" if they are not dried and can be referred to as "WS dry cake" As sometimes used herein, the term "process stream" may be used synonymously with "stream ".

The present invention provides processes and methods for producing fermentation products such as product alcohols using fermentation. Other fermentation products that may be produced using the processes and methods described herein include acids such as propanediol, butanediol, acetone, lactic acid, acetic acid, butyric acid, and propionic acid; Hydrogen, methane, and carbon dioxide; amino acid; Biotin, vitamin B ₂ (riboflavin), vitamin B ₁₂ (e.g., cobalamin), ascorbic acid (e. G., Vitamin C), vitamin E (for example, a- tocopherol), and Vitamin K (e. G. , Menaquinone); Antibiotics such as erythromycin, penicillin, streptomycin, and tetracycline; And other products such as citric acid, invertase, sorbitol, pectinase, and xylitol.

As an example of the processes and methods provided herein, the feedstock may be liquefied to produce a feedstock slurry comprising a fermentable carbon source (e. G., Soluble sugars) and undissolved solids. In some instances, the terms "feedstock slurry" and "mesh" may be used interchangeably. In some embodiments, the feedstock slurry includes soluble sugars, undissolved solids, and oil. When the feedstock slurry is fed directly to the fermenter, the undissolved solids and / or oil may interfere with efficient removal and recovery of fermentation products such as product alcohol. For example, in the case of extracting the product alcohol from a fermentation broth using liquid-liquid extraction, the presence of the undissolved solid prevents the mass transfer rate of the product alcohol to the extractant by interfering with the contact between the extractant and the fermentation broth ≪ / RTI > Disturb phase separation of the extractant and the fermentation broth by producing an emulsion in the fermenter; Delaying the disengagement of the extractant from the fermentation broth; At least a portion of the extractant and product alcohol is "trapped" in the solid, thereby reducing the efficiency of recovery and recycling of the extractant; Shortening the life cycle of the extractant due to oil contamination; But it is possible to cause system inefficiency including, but not limited to, reducing the volume efficiency of the fermenter due to the presence of solids that occupy volume in the fermenter. These effects can result in higher capital and operating costs. In addition, extractants that are "trapped " in undissolved solids used to produce Distillers Dried Grains with Solubles (DDGS) with solubles may impair DDGS values and functionality for sale as animal feed . Thus, in order to avoid and / or minimize these problems, at least some of the undissolved solids may be removed from the feedstock slurry prior to feeding the feedstock slurry to the fermenter. Extraction activity and efficiency of product alcohol production is determined by performing extraction in a fermentation broth containing an aqueous solution in which the undissolved solids are removed as compared to the extraction performed in a fermentation broth containing an aqueous solution in which undissolved solids have not been removed And the like.

The process and system of the present invention will be described with reference to the drawings. In some embodiments, for example, as shown in Figure 1, the system includes a liquefaction 10 configured to liquefy the feedstock to produce a feedstock slurry.

For example, the feedstock 12 can be introduced into the inlet of the liquefaction 10. Feedstock 12 can be fermentable, such as, but not limited to, barley, oats, rye, sorghum, wheat, lime, spelled, spatula, sugarcane, corn, It may be any suitable biomaterial containing a carbon source. Water can also be introduced into the liquefaction 10.

The process of liquefying the feedstock 12 comprises hydrolyzing the feedstock 12 to generate a soluble sugar. Any known liquefaction process utilized in industry, including, but not limited to, an acid process, an enzymatic process, or an acid-enzyme process, may be used. These processes may be used alone or in combination. In some embodiments, an enzymatic process can be utilized and an appropriate enzyme 14, e.g., alpha-amylase, is introduced into the inlet of liquefaction 10. Examples of alpha-amylases that can be used in the processes and systems of the present invention are described in U.S. Patent Nos. 7,541,026; U.S. Patent Application Publication No. 2009/0209026; U.S. Patent Application Publication No. 2009/0238923; U.S. Patent Application Publication No. 2009/0252828; U.S. Patent Application Publication No. 2009/0314286; U.S. Patent Application Publication No. 2010/02278970; U.S. Patent Application Publication No. 2010/0048446; And U.S. Patent Application Publication No. 2010/0021587, the entire contents of each of which are incorporated herein by reference.

In some embodiments, the enzyme for liquefaction and / or glycation may be produced by a microorganism (e. G., Microorganism 32). Examples of microorganisms that produce such enzymes are described in U.S. Patent Nos. 7,498,159; U.S. Patent Application Publication No. 2012/0003701; U.S. Patent Application Publication No. 2012/0129229; PCT International Publication No. WO 2010/096562; And PCT International Publication No. WO 2011/153516, the entire contents of each of which are incorporated herein by reference.

The process of liquefying the feedstock 12 produces a feedstock slurry 16 comprising a fermentable carbon source and undissolved solids. In some embodiments, the feedstock slurry 16 may comprise a fermentable carbon source, oil, and undissolved solids. The undissolved solids are the non-fermentable portion of the feedstock 12. In some embodiments, the feedstock 12 may be anhydrous ground and corn, such as untreated corn flakes, and the undissolved solids may include bacteria, fibers, and gluten. In some embodiments, the feedstock 12 is a corn or corn grain and the feedstock slurry 16 is a corn mash slurry. The feedstock slurry 16 may be discharged from the outlet of the liquefier 10 and may be carried out in the separation 20.

In some embodiments, nutrients such as amino acids, nitrogen, minerals, trace elements, and / or vitamins may be added to the feed slurry 16 or fermentation 30. For example, the following: biotin, pantothenate, folic acid, niacin, aminobenzoic acid, pyridoxine, riboflavin, thiamine, vitamin A, vitamin C, vitamin D, vitamin E, vitamin K, inositol, potassium ), Boric acid, calcium chloride, chromium, copper (for example, copper sulfate), iodide (for example, potassium iodide) At least one of manganese (e.g., manganese sulfate), molybdenum, calcium chloride, phosphorus, potassium, sodium chloride, vanadium, zinc (e.g., zinc sulfate), yeast extract, soybean peptone, It can be added to the fermentation (30). Examples of amino acids include essential amino acids such as histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine and also amino acids such as alanine, arginine, aspartic acid, asparagine, cysteine, glutamic acid, glutamine, glycine, And other amino acids such as, for example, < RTI ID = 0.0 > lysine, < / RTI >

Separation 20 through the inlet can be configured to remove undissolved solids from the feedstock slurry 16. Separation 20 can be configured to remove oil or remove both oil and undissolved solids. Separation 20 can be any device capable of separating solids and liquids. For example, the separation 20 can be any conventional method used in the industry, including, for example, a decanter bowl centrifuge, a three-phase centrifuge, a disk stack centrifuge, a filtration centrifuge, or a decanter centrifuge. . ≪ / RTI > In some embodiments, the separation may be accomplished by any of a variety of methods, including filtration, vacuum filtration, belt filtration, membrane filtration, cross flow filtration, pressure filtration, screen filtration, screen separation, grates or gratings, porous gratings, Presses, gravity settlers, fluidized bed separators, or any method or separation device that can be used to separate solids and liquids.

Separating the feedstock slurry 16 performed in separation 20 to form a liquid phase, an aqueous stream, or an aqueous solution 22 (also known as a dilute mesh) and a solid phase, solid stream, or wet cake 24 can do. The aqueous solution 22 may comprise, for example, sugars in the form of oligosaccharides, and water. In some embodiments, the aqueous solution 22 may comprise at least about 10 weight percent oligosaccharides, at least about 20 weight percent oligosaccharides, or at least about 30 weight percent oligosaccharides. In some embodiments, the aqueous solution 22 may be discharged from an outlet located near the top of the separation 20. In some embodiments, the aqueous solution 22 may have a viscosity of about 20 centipoise (cP). In some embodiments, the aqueous solution 22 may comprise less than about 20 g / L of monomeric glucose, less than about 10 g / L of monomeric glucose, or less than about 5 g / L of monomeric glucose. A suitable methodology for determining the amount of monomeric glucose is well known in the art, such as high performance liquid chromatography (HPLC).

The wet cake 24 may be discharged from the separation 20. In some embodiments, the wet cake 24 may be discharged from an outlet located near the bottom of the separation 20. The wetting (24) may include undissolved solids. In some embodiments, wet cake 24 may also include sugar and a portion of water. The wet cake 24 can be washed with additional water using the separator 20 once the aqueous solution 22 is discharged from the separation 20. In some embodiments, the wet cake 24 may be washed with additional water using an additional separator. Washing the wet cake 24 will recover sugar or sugar sources (e.g., oligosaccharides) present in the wet cake and the recovered sugar and water will be recycled to the liquefaction 10. After washing, the wet cake 24 may be formed using any suitable known process to form DDGS. The formation of DDGS from the wet cake 24 has several advantages. Since undissolved solids do not apply to the conditions of the fermenter, for example, they are not added to the undissolved fermenter, the undissolved solids do not come into contact with the microorganisms present in the fermenter, and fermentation products such as product alcohol or extractants Are not trapped in undissolved solids. This effect is advantageous because the DDGS may not contain microorganisms or other components of the fermentation broth (e.g., product alcohol, extractant), thus providing advantages for subsequent processing and use of DDGS, such as animal feed do.

In some embodiments, the undissolved solids are separated from the feedstock slurry to form two product streams, for example, an aqueous solution of an oligosaccharide containing a lower concentration of solids as compared to a feedstock slurry, and a feedstock slurry A wet cake containing a higher concentration of solids can be formed. In addition, a third stream containing oil may be produced. As such, multiple product streams can be produced using different separation techniques or a combination thereof. As an example, the feedstock slurry 16 may be separated using a three-phase centrifuge. The three-phase centrifuge allows three-phase separation to produce two liquid phases (e.g., an aqueous stream and an oil stream) and a solid stream (e.g., a solid or wet cake) , Flottweg Tricanter ( ^R) , manufactured by Flottweg AG, Vilnius, Germany). The two liquid phases can be separated, for example, by decanting through a two-outlet system from the bowl to prevent cross-contamination, and the solid stream can be removed via a separate discharge system.

In some embodiments where corn is used as the feedstock 12, solids and corn oil may be simultaneously removed from the feedstock slurry 16 (e.g., liquefied corn mash) using a three-phase centrifuge . Solids are undissolved solids remaining after hydrolysis of the starch to soluble oligosaccharides during liquefaction, and corn oil is free oil released from the embryo during milling and / or liquefaction. In some embodiments, the three-phase centrifuge may have one feed stream and three discharge streams. The feed stream may consist of a liquefied corn mesh produced during liquefaction. The mesh comprises an aqueous solution of liquefied starch (e.g., oligosaccharides); An undissolved solid of a non-starch component from corn; And corn oil consisting of glycerides and free fatty acids. The three outlet streams from the three-phase centrifuge are wet cake (i.e., wet cake 24) containing undissolved solids from the mesh; A concentrated concentrate stream containing liquefied starch from the mesh; And a light concentrate stream containing corn oil from the mesh. In some embodiments, the light concentrate stream (i.e., oil 26) may be carried in a storage tank or any container suitable for oil storage. In some embodiments, the oil may be sold as a co-product and used in processes such as conversion to other co-products or conversion of corn oil to corn oil fatty acid. In some embodiments, a thick concentrate stream (i.e., aqueous solution 22) may be used for fermentation. In some embodiments, the wet cake is capable of recovering soluble starch in the liquid phase of the cake by washing with process recycle water, such as evaporator condensate and / or a backset as described herein.

In some embodiments, the wet cake 24 is a composition formed from the feedstock slurry 16 and comprises at least about 50% by weight of undissolved solids present in the feedstock slurry, at least about 55% At least about 60 weight percent of undissolved solids present in the feedstock slurry, at least about 65 weight percent undissolved solids present in the feedstock slurry, at least about 70 weight percent of the undissolved solids present in the feedstock slurry, % Of undissolved solids present in the feedstock slurry, at least about 75% undissolved solids present in the feedstock slurry, at least about 80% undissolved solids present in the feedstock slurry, at least about 85% By weight of undissolved solids, at least about 90% by weight of undissolved solids present in the feedstock slurry, At least about 95 weight percent undissolved solids present in the feedstock slurry, or at least about 99 weight percent undissolved solids present in the feedstock slurry.

In some embodiments, the aqueous solution 22 is formed from the feedstock slurry 16 and contains less than about 50 weight percent undissolved solids present in the feedstock slurry, less than about 45 weight percent dissolution present in the feedstock slurry Not more than about 40% by weight of undissolved solids present in the feedstock slurry, not more than about 35% by weight of undissolved solids present in the feedstock slurry, not more than about 30% by weight of the solids present in the feedstock slurry An undissolved solid, no more than about 25 weight percent undissolved solids present in the feedstock slurry, no more than about 20 weight percent undissolved solids present in the feedstock slurry, no more than about 15 weight percent present in the feedstock slurry Of undissolved solids, less than about 10 wt.% Of undissolved solids present in the feedstock slurry, about 5 percent of the solids present in the feedstock slurry % Of undissolved solids, or about 1 wt% of undissolved solids present in the feedstock slurry.

The fermentation 30, which is adapted to ferment the aqueous solution 22 to produce a fermentation product, such as product alcohol, has an inlet for receiving the aqueous solution 22. The fermentation 30 may include a fermentation broth. In some embodiments, microbes 32 selected from the group of bacteria, cyanobacteria, filamentous fungi, and yeast may be introduced into fermentation 30 to be included in the fermented broth. In some embodiments, the microorganism 32 may be a bacterium such as Escherichia coli . In some embodiments, the microorganism (32) is Saccharomyces cerevisiae cerevisiae . In some embodiments, the microorganism 32 consumes sugar in the aqueous solution 22 to produce butanol. In some embodiments, the microorganism 32 may be a recombinant microorganism.

In some embodiments, the microorganisms 32 may be engineered to include biosynthetic pathways. In some embodiments, the biosynthetic pathway may be a butanol biosynthetic pathway. In some embodiments, the butanol biosynthetic pathway may be a 1-butanol biosynthetic pathway, a 2-butanol biosynthetic pathway, or an isobutanol biosynthetic pathway. In some embodiments, the biosynthetic pathway converts pyruvate to a fermentation product. In some embodiments, the biosynthetic pathway converts pyruvate and also amino acids to fermentation products. In some embodiments, the biosynthetic pathway comprises at least one heterologous polynucleotide encoding a polypeptide that catalyzes the conversion of the substrate to a product of a biosynthetic pathway. In some embodiments, product conversion of each substrate of the biosynthetic pathway is catalyzed by a polypeptide encoded by a heterologous polynucleotide. Examples of the production of product alcohols by microorganisms including biosynthetic pathways are described, for example, in U.S. Patent No. 7,851,188 and U.S. Patent Application Publication No. 2007/0092957; 2007/0259410; 2007/0292927; 2008/0182308; 2008/0274525; 2009/0155870; 2009/0305363; And 2009/0305370, the entire contents of each of which are incorporated herein by reference.

In some embodiments, the microorganism 32 can also be immobilized, such as by absorption, covalent bonding, cross-linking, entrapment, and encapsulation. Methods for encapsulating cells are known in the art, such as in U.S. Patent Application Publication No. US2006 / 066116, the entire contents of which are incorporated herein by reference.

In some embodiments of the processes and systems described herein, in situ product removal (ISPR) can be used to remove fermentation products such as product alcohols (e.g., butanol) from the fermentation broth . In some embodiments, the ISPR may be performed in fermentation 30 as the fermentation product is produced by the microorganism, or may be performed outside fermentation 30, for example, by liquid-liquid extraction . Methods for producing and recovering product alcohols from fermentation broth using extractable fermentation are disclosed in U.S. Patent Application Publication No. 2009/0305370; U.S. Patent Application Publication No. 2010/0221802; U.S. Patent Application Publication No. 2011/0097773; U.S. Patent Application Publication No. 2011/0312044; U.S. Patent Application Publication No. 2011/0312043; And U.S. Patent Application Publication No. 2012/0156738, the entire contents of each of which are incorporated herein by reference.

In some embodiments, the fermentation 30 may have an inlet for receiving the extractant 34. In some embodiments, the extractant 34 may be added to the fermentation broth outside the fermentation 30. In some embodiments, the extractant 34 may be applied to an external extractor or to an external extraction loop. Alternative means of applying the extracting agent 34 to the outside of the fermentation 30 or the fermentation 30 are indicated by dotted lines. In some embodiments, the extractant 34 can be an incompatible organic daily. In some embodiments, the extractant 34 may be water-immiscible organic daily. In some embodiments, the extractant 34 may be an organic extractant selected from the group consisting of saturated, monounsaturated, highly unsaturated compounds, and mixtures thereof. In some embodiments, the extraction agent 34 is C ₁₂ to C ₂₂ fatty alcohols, C ₁₂ to C ₂₂ fatty acids, C ₁₂ to C ₂₂ fatty acid esters, C ₁₂ to C ₂₂ fat aldehydes, C ₁₂ to C ₂₂ fatty amides , And mixtures thereof. In some embodiments, the extractant 34 is selected from the group consisting of 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, Lt; / RTI > In some embodiments, the extraction agent 34 is C ₁₂ to C ₂₂ fatty alcohols, C ₁₂ to C ₂₂ fatty acids, C ₁₂ to C ₂₂ fatty acid esters, C ₁₂ to C ₂₂ fat aldehydes, C ₁₂ to C ₂₂ fatty amides , And a mixture thereof; And may include a C ₇ to C ₁₁ fatty alcohols, C ₇ to C ₁₁ fatty acid, C ₇ to C ₁₁ fatty acid esters, C ₇ to C ₁₁ aldehydes fat, extract and of the second selected mixtures thereof. In some embodiments, the extractant 34 may be a carboxylic acid. In some embodiments, the extractant 34 may be a corn oil fatty acid (COFA) or a soybean oil fatty acid (SOFA). In some embodiments, the extractant 34 is selected from the group consisting of oleic alcohol, behenyl alcohol, cetyl alcohol, lauryl alcohol, myristyl alcohol, stearyl alcohol, oleic acid, lauric acid, myristic acid, stearic acid, Organic such as methyl oleate, 1-nonanol, 1-decanol, 2-undecanol, 1-nonanal, 1-undecanol, ungecanol, lauric aldehyde, 20-methyl undecanol, May be an extracting agent. For processes and systems as described herein and shown in the drawings, the extractant may be added to a fermenter or an external extractor.

In some embodiments, the extractant may be selected on the basis of specific properties. For example, the extractant may have a high K _d . K _d is the partition coefficient of the fermentation product (e.g., product alcohol) between the extraction agent phase (e.g., organic phase) and the aqueous phase. In some embodiments, the extractant may be highly selective. For example, selectivity refers to the relative amount of product alcohol to the water occupied by the extract.

In some embodiments, the extractant may be biocompatible. In some embodiments, biocompatibility refers to a measure of the ability of a microorganism to utilize a carbon source capable of fermentation in the presence of an extractant. In some embodiments, the extractant may be a mixture of biocompatible and non-biocompatible extractants. In some embodiments, the non-biocompatible extractant refers to an extractant that interferes with the ability of the microorganism to utilize a fermentable carbon source. For example, in the presence of a non-biocompatible extracting agent, the microorganism does not utilize the fermentable carbon source at a utilization of greater than about 50% of the utilization rate in the absence of the extractant. In some embodiments, in the presence of a non-biocompatible extractant, the microorganism does not utilize the fermentable carbon source at a utilization rate of greater than about 25% of the utilization rate in the absence of the extractant. Examples of mixtures of biocompatible and non-biocompatible extractants include oleanolic and nonanol, oleyl alcohol and 1-undecanol, oleyl alcohol and 2-undecanol, oleyl alcohol and 1-nonanal, oleyl But are not limited to, alcohol and decanol, and oleyl alcohol and dodecanol. Additional examples of biocompatible and non-biocompatible extractants are described in U.S. Patent Publication No. 2011/0097773; The entire contents of which are incorporated herein by reference.

Extractant 34 is contacted with a fermentation broth formation stream 36 containing a biphasic mixture (i.e., an aqueous phase and an organic phase). If the fermentation product is a product alcohol, the product alcohol present in the fermentation broth is transferred to the extractant 34, which forms the extractant rich in product alcohol (e.g., organic phase). In some embodiments, the stream 36 may be discharged through the outlet at the fermentation 30. The product alcohol may be separated from the extractant in stream 36 using conventional techniques. The feed stream may be fed to fermentation (30). Fermentation 30 may be any suitable fermenter known in the art.

In some embodiments, when the extractant 34 is not added to the fermentation broth, the stream 36 contains fermentation broth and product alcohol. Stream 36 or a portion thereof containing product alcohol and fermentation broth may be discharged from fermentation 30 and further processed to recover product alcohol. In some embodiments, the fermentation broth may be recirculated to the fermentation (30).

In some embodiments, simultaneous saccharification and fermentation (SSF) may occur in fermentation (30). Any known saccharification process used in the industry, including, but not limited to, acid processing, enzymatic processing, or acid-enzyme processing may be used. In some embodiments, an enzyme 38 such as glucoamylase may be introduced to hydrolyze the feed slurry 16 or sugars (e.g., oligosaccharides) in the aqueous solution 22 to form a monosaccharide. Examples of glucoamylases that can be used in the processes and systems of the present invention are described in U.S. Patent No. 7,413,887; U.S. Patent No. 7,723,079; U.S. Patent Application Publication No. 2009/0275080; U.S. Patent Application Publication No. 2010/0267114; U.S. Patent Application Publication No. 2011/0014681; U.S. Patent Publication No. 2011/0020899, the entire contents of each of which are incorporated herein by reference. In some embodiments, the glucoamylase can be expressed by a recombinant microorganism that also produces a fermentation product (e. G., Product alcohol).

In some embodiments, enzymes such as glucoamylase may be added to the liquefaction. When an enzyme such as glucoamylase is added to the liquefaction, the viscosity of the feed slurry or liquefied mesh can be reduced and the separation efficiency can be improved. In some embodiments, any enzyme capable of reducing the viscosity of the feedstock slurry can be used (e.g., Viscozyme ^® , Sigma-Aldrich, St. Louis, Mo.). The viscosity of the feedstock can be measured by any method known in the art, including the method described in Example 22.

In some embodiments, stream 35 may be discharged from the outlet at fermentation 30. The drained stream 35 may comprise microorganisms 32 such as yeast. The microorganisms 32 can be separated from the stream 35, for example, by centrifugation (not shown). The microorganism 32 can then be recycled to the fermentation 30, which increases the product alcohol production efficiency by increasing the production rate of the product alcohol over time.

When a portion of the stream 35 exits the fermentation 30, the stream 35 contains less than about 50 weight percent undissolved solids present in the feedstock slurry, less than about 45 weight percent of the undissolved solids present in the feedstock slurry Up to about 40% by weight of undissolved solids present in the feedstock slurry, up to about 35% by weight of undissolved solids present in the feedstock slurry, up to about 30% by weight of the undissolved solids present in the feedstock slurry Of undissolved solids, up to about 25% undissolved solids present in the feedstock slurry, up to about 20% undissolved solids present in the feedstock slurry, about 15% by weight of the undissolved solids present in the feedstock slurry, Of undissolved solids, up to about 10% undissolved solids present in the feedstock slurry, up to about 5% undissolved solids present in the feedstock slurry, About 1 present in the slurry fee may include a non-dissolved solid.% Or less.

In some embodiments, for example, as shown in FIG. 2, the process and system of the present invention can include a mill 40 configured to dry-mill the feedstock 12. The feedstock 12 may be fed to the crusher 40 via an inlet. The pulverizer 40 may crush or grind the feedstock 12. In some embodiments, the feedstock 12 may not be classified. In some embodiments, the feedstock 12 may be unclassified corn flakes. Crusher 40 may be any suitable known crusher, such as a hammer crusher. The anhydrous pulverized feedstock 44 is discharged from the pulverizer 40 through the outlet and fed to the liquefaction 10. The rest of FIG. 2 is similar to FIG. 1 and will not be described in detail again. In other embodiments, the feedstock may be classified and / or wet pulverized as known in the industry as an alternative to being not classified and / or anhydrous.

Wet milling is a multi-step process in which biomass is separated into several components, such as bacteria, peel fibers, starch, and gluten, to capture the value separately from each co-product. When corn is used as the feedstock, the process produces several co-products: starch, gluten feed, gluten meal, and corn oil stream. These streams can be combined and processed to produce customized products for the feed industry. As an example of a wet grinding process, the feedstock (e.g., corn) may be run with a steeping tank, which may be, for example, at a temperature of 120 to 130 DEG F (about 50 to 55 DEG C) And immersed in sodium dioxide solution for about 30 to 50 hours. The nourishment released into the water can be collected and evaporated to produce condensed and fermented extracts (or stiff fluids). Bacteria can be removed from the immersed feedstock and further processed to recover oil and bacterial diets. After removing the germs, the remainder of the feedstock can be processed to remove bran and produce starch and gluten slurries. The slurry can be further processed to separate starch and gluten proteins, which can be dried to form a gluten meal. The starch stream may be further processed through fermentation to produce fermentation products (e. G., Product alcohols) or may be utilized in the food, paper or textile industries. For example, a starch stream can be used to create a sweetener. Gluten meal and gluten feed streams, including both protein, fat and fiber, can be used in cows, beef cattle, poultry, pigs, livestock, horses, aquaculture, and household pet feed. The gluten diet can also be used as a carrier for added micro-nutrients. Gluten diet also includes, for example, methionine and xanthophylls which can be used as pigment ingredients in poultry feed (for example, pigments provide yolk with yellow pigmentation). Condensed and fermented extracts containing proteins, growth factors, B vitamins and minerals can be used as high energy liquid feed ingredients. The condensed extract may also be used as a pellet binder. This process provides a purified starch stream, which is costly and involves separating the biomass into its non-starch stream, which may not be necessary for fermentation production.

The classification removes fibers and germs, which produces sorted corn having high starch (endosperm) content, including most of the lipids (e.g., oil) present in the whole corn of the ground. In some embodiments, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60% At least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95% or more oil can be removed from the bacteria by sorting. In some embodiments, the fractionation may be carried out at least about 0.5%, at least about 1%, at least about 2%, at least about 3%, at least about 4%, or at least about 5% of the undissolved solids content in the feedstock .

Anhydrous fractionation does not separate bacteria from the fibers, which is therefore cheaper than wet milling. Advantages of the classification include, for example, improved yield of product, increased volume in the fermenter (i.e., space), smaller column diameter, lower enzyme load and increased efficiency for glycation, increased oil removal, Less cleaning shutdown, increased protein levels in the DDGS, reduced drying time for DDGS, and reduced energy consumption. However, the classification does not remove the entire fiber or bacteria, nor does it remove all of the solids. Moreover, some of the starch is lost during the classification.

Anhydrous milling can also be used for feedstock processing. The feedstock can be milled, for example, using a hammer mill to produce a diet, which can then be mixed with water to form a slurry. Such a slurry can be liquefied by adding an enzyme such as amylase to hydrolyze the starch with sugar to form a mesh. The mash can be heated ("cooked") to inactivate the enzyme and then cool it for fermentation. The cooled mash, microorganisms, and enzymes, such as glucoamylase, may be added to the fermentation to produce a fermentation product (e. G., Product alcohol). After fermentation, the fermentation broth can be carried out by distillation to recover the fermentation product. If the undissolved solids are not removed, the bottom stream of the distillation column will contain unfermented solids (e.g., distiller grain solids), dissolved solids, and water that can be collected for further processing) Of the total distillation waste. For example, the entire distillation waste liquid can be separated into a solid (for example, a wet cake) and a dilute distillation waste liquid. Separation may be accomplished by a number of means including, but not limited to, centrifugation, filtration, screen separation, hydrocyclones, or any other means or separation device for separating liquid from solids. Dilute distillation waste can be carried out with condensed distiller solubles (CDS) or evaporation to form syrups. Dilute distillate wastes may include soluble nutrients, small grain solids (or particulates), and microorganisms. Solids (eg wet cake) can be combined with syrup and then dried to form DDS. Syrups include proteins, fats and fibers as well as vitamins and minerals such as phosphorus and potassium; It can be added to animal feed for its nutritional value and palatability. DDGS includes proteins, fats and fibers; Providing a source of bypassing protein. DDGS can be used in dairy cattle, beef cattle, poultry, pigs, livestock, horses, aquaculture and domestic pet feed.

In some embodiments, for example, as shown in FIG. 3, the process and system of the present invention may include draining the oil 26 from the outlet of the separation 20. Figure 3 is similar to Figure 1 except for the oil stream 26 exiting the separation 20 and will not be described in detail again.

The feedstock slurry 16 performed in separation 20 comprises a first liquid phase or an aqueous solution 22 containing a fermentation sugar, a second liquid phase containing oil 26, and an undissolved solid Lt; RTI ID = 0.0 > 24 < / RTI > Any suitable separation device, such as a 3-phase centrifuge, may also be used to discharge aqueous solution 22 (or aqueous stream), wet cake 24 (or solid stream), and oil 26 (or oil stream) Can be used. In some embodiments, the feedstock 12 is corn and the oil 26 is corn oil (e. G., Glass corn oil). As used herein, the term " free corn oil " means corn oil without corn bacteria. In some embodiments, the oil 26 may be treated in a storage tank or any vessel suitable for oil storage. In some embodiments, if the feedstock 12 is corn, some of the oil from the feedstock, such as corn oil, remains in the wet cake 24.

In some embodiments, the fermentation broth in the fermentation 30 includes a reduced amount of corn oil when the oil 26 is removed through the separation 20 from the feedstock 12. In some embodiments, the fermentation broth has less than about 25% by weight of the undissolved solids, wherein the fermentation broth has less than about 15% by weight of undissolved solids and the fermentation broth comprises less than about 10% by weight of undissolved solids Of the undissolved solids, the fermentation broth having less than or equal to about 5 weight percent of the undissolved solids, the fermentation broth having less than or equal to about 1 weight percent of the undissolved solids, and the fermentation broth having less than or equal to about 0.5 weight percent of the undissolved solids .

In some implementations, the process and system of FIG. 2 may be modified to include the discharge of an oil stream from the separation 20 as discussed herein in connection with the process and system of FIG.

As shown in FIG. 4, if the oil is not discharged separately, it can be removed with the wet cake 24. In some embodiments, when the wet cake 24 is separated through the separator 20, a portion of the oil from the feedstock, such as corn oil if the feedstock 12 is corn, do. Wet cake 24 may be treated with mix 60 and combined with water or other solvent to form wet cake mixture 65. [ In some embodiments, the water can be fresh water, reflux, cooking water, process water, Luther water, evaporative water, or any source of water available in a fermentation processing facility, or any combination thereof. The wet cake mixture 65 may be performed with a separation 70 that produces a fermentable sugar recovered from the wet cake 24 and a wash concentrate 75 comprising the wet cake 74. The wash concentrate 75 may be recycled to the liquefaction 10. The remainder of FIG. 4 is similar to FIG. 1 and will not be described in detail again.

In some embodiments, the separation 70 may be performed using a variety of techniques, including decanter bowl centrifugation, 3-phase centrifugation, disk stack centrifugation, filtration centrifugation, decanter centrifugation, filtration, vacuum filtration, belt filtration, membrane filtration, Separating solids and liquids, including pressurized filtration, screen filtration, screen separation, grating, porous grating, suspension, hydrocyclone, filter press, screw press, gravity settler, And may be any separating device that can be used.

In some embodiments, the wet cake may be treated in one or more cleaning cycles or cleaning systems. For example, the wet cake 74 can be further processed by performing the wet cake 74 with a second cleaning system. In some embodiments, the wet cake 74 may be performed with a second mix 60 'forming a wet cake mixture 65'. The wet cake mixture 65 'may be carried out with a second concentrate 70' to produce a wash concentrate 75 'and a wet cake 74'. The wash concentrate 75 'can be recycled to the liquefier 10 and / or the wash concentrate 75' can be combined with the wash concentrate 75 and the combined wash concentrate can be recycled to the liquefier 10 have. The wet cake 74 'may be combined with the wet cake 74 for further processing as described herein. In some embodiments, the separation 70 'may be performed by any suitable method, such as decanter bowl centrifugation, 3-phase centrifugation, disk stack centrifugation, filtration centrifugation, decanter centrifugation, filtration, vacuum filtration, Separation of solids and liquids, including filtration, pressure filtration, screen filtration, screen separation, grating, porous grating, suspension, hydrocyclone, filter press, screw press, gravity settler, And may be any separating device that can do so. In some embodiments, the wet cake may be treated with one, two, three, four, five or more wash cycles or a wash system.

The wet cake 74 may be combined with the solubles and then dried to form DDGS via any suitable known process. Formation of DDGS from the wet cake 74 has several advantages. For example, since the undissolved solids are not added to the fermenter, the undissolved solids are not subjected to the conditions of the fermenter, so that the undissolved solids do not come into contact with the microorganisms present in the fermenter, Other components such as products or extractants are not trapped in undissolved solids. This effect provides advantages for the use of DDGS as an animal feed and for subsequent processing, for example, since DDGS does not contain microorganisms or other components of fermented broth (e.g., product alcohol, extractants) do.

As shown in Fig. 4, the oil is not discharged separately from the wet cake, rather oil is included as part of the wet cake and ultimately in the DDGS. When corn is used as feedstock, the corn oil contains triglycerides, diglycerides, monoglycerides, fatty acids, and phospholipids, which provide a source of metabolizable energy for the animal. The presence of the wet cake and ultimately the oil in the DDGS can provide the desired animal feed, for example animal feed of high fat content.

In some embodiments, the oil may be separated from DDGS and converted to an ISPR extractant for subsequent use in the same or a different fermentation process. Methods for deriving an extractant from biomass are described in U.S. Patent Application Publication No. 2001/0312043, U.S. Patent Application Publication No. 2011/0312044, U.S. Patent Application Publication No. 2012/0156738, and PCT International Publication No. WO 2011/159998 Lt; / RTI > The entire contents of each are incorporated herein by reference. The oil may be separated from DDGS using any suitable known process, including, for example, a solvent extraction process. In one embodiment of the invention, DDGS is loaded into an extraction vessel and washed with a solvent such as hexane to remove oil. Other solvents that may be used include, for example, isobutanol, isohexane, ethanol, petroleum distillates such as petroleum ether, or mixtures thereof. After oil extraction, the DDGS can be treated to remove any remaining solvent. For example, DDGS can be heated to evaporate any remaining solvent using any method known in the art. After removal of the solvent, DDGS may be subjected to a drying process to remove any residues. Processed DDGS can be used as animal feed supplements such as cows, beef cattle, poultry, pigs, livestock, horses, aquaculture, and household pets.

After extraction from DDGS, the resulting oil and solvent mixture can be collected to separate the oil from the solvent. In one embodiment, the solvent can be evaporated, collected and recycled by processing the oil / solvent mixture by evaporation. The recovered oil may be converted to an ISPR extractant for subsequent use for use in the same or a different fermentation process.

 Removing the oil component of the feedstock is advantageous to produce because the oil present in the fermenter can be hydrolyzed to fatty acids and glycerin. The amount of water available for accumulating glycerin in water and recirculating throughout the fermentation system can be reduced. Thus, removing the oil component of the feedstock increases the production efficiency by increasing the amount of water that can be recirculated through the system. In addition, removal of the oil can lead to energy savings in the production plant due to more efficient fermentation, less attachment due to the removal of oil, increased efficiency of the fermentor volume, and energy requirements, such as drying the distiller grains The amount of energy required to achieve the desired performance can be reduced.

As shown in FIG. 5, the oil may be removed at various points during the process described herein. The feedstock slurry 16 is fed to a first liquid phase or aqueous solution 22 (or aqueous stream), a second liquid comprising an oil 26 (or oil stream), for example, using a three phase centrifuge, Phase, and a solid or wet cake 24 (or solid stream). The wet cake 24 may be further processed to recover fermentable sugars and oils. The wet cake 24 may be combined with water or other solvent to effect the mix 60 and form the wet cake mixture 65. In some embodiments, water can be countercurrent, cooked, processed, lutter water, water recovered from evaporation, or any source of water available in the fermentation process facility, or a combination thereof. The wet cake mixture 65 is conducted with a separation 70 (e.g., a three phase centrifuge) to produce a washing concentrate 75 comprising fermentable sugars, oil 76, and wet cake 74 can do. The wash concentrate 75 may be recycled to the liquefaction 10. Oil 76 and oil 26 may be combined and further processed for the manufacture of various consumer products. In some embodiments, the oil (e. G., Oil 26, oil 76) may be further processed to produce an extractant. For example, the oil may be chemically or enzymatically treated to generate an extractant. In some embodiments where the oil is corn oil, the corn oil may be chemically or enzymatically treated to generate a fatty acid (e.g., corn oil fatty acid) that can be used as an extractant. In some embodiments where the oil is enzymatically treated, the enzymatic reaction may be post-conversion treatment (e.g., heat) to deactivate the enzyme. In some embodiments, the oil may be an enzymatically utilized enzyme that is enzymatically treated, such as an esterase, a lipase, a phospholipase, a lysophospholipase, or a combination thereof. In some embodiments, the oil may be chemically treated with ammonium hydroxide, anhydrous ammonia, ammonium acetate, hydrogen peroxide, toluene, glacial acetic acid, or combinations thereof. Methods for deriving an extractant from biomass are described in U.S. Patent Application Publication No. 2011/0312043 and U.S. Patent Application Publication No. 2011/0312044, the entire contents of each of which are incorporated herein by reference. In some embodiments where the oil is corn oil, the feedstock slurry may contain at least about 1%, at least about 2%, at least about 3%, at least about 4% corn oil, or at least about 5% corn oil . ≪ / RTI > The remainder of FIG. 5 is similar to FIG. 1 and will not be described in detail again.

As described herein, the wet cake may be processed into one or more wash cycles or wash systems. In some embodiments, the wet cake 74 may be performed with a second mixture 60 'forming a wet cake mixture 65'. The wet cake mixture 65 'may be performed with a second separation 70' to produce a wash concentrate 75 ', oil 76' and a wet cake 74 '. The wash concentrate 75 'can be recycled to the liquefier 10 and / or the wash concentrate 75' can be combined with the wash concentrate 75 and the combined wash concentrate can be recycled to the liquefier 10 have. The wet cake 74 'may be combined with the wet cake 74 for further processing as described herein. Oil 76, oil 76 'and oil 26 can be combined and further processed for the manufacture of various consumer products. In some embodiments, an oil (e. G., Oil 26, oil 76, oil 76 ') may be further processed to produce an extractant as described herein. Methods for deriving an extractant from biomass are described in U.S. Patent Application Publication No. 2011/0312043 and U.S. Patent Application Publication No. 2011/0312044, each of which is incorporated herein by reference in its entirety.

As shown in FIG. 6, the aqueous solution 22 and the wet cake 24 may be combined, cooled and fermented (30). The feedstock slurry 16 is fed into a first liquid phase or aqueous solution 22 using a three-phase centrifuge, a second liquid phase comprising oil 26 and a solid phase or wet cake (24). ≪ / RTI > In some embodiments, the oil 26 (or oil stream) may be carried in a storage tank or any vessel suitable for oil storage. The aqueous solution 22 (or aqueous stream) and wet cake 24 (solid stream) may be subjected to mixing 80 and the remainder of the resulting aqueous / wet cake mixture 82 may be re-slurried. Mixture 82 may be performed with cooling 90 to produce a cooled mixture 92 that may be performed in fermentation 30. In some embodiments, when oil 26 is removed through separation 20 from feedstock 12, mixtures 82 and 92 contain a reduced amount of corn oil. The remainder of FIG. 6 is similar to FIG. 1 and will not be described in detail again.

In some embodiments, for example, as shown in FIGS. 7 and 8, saccharification may occur in a separate saccharification system 50 located between the separation 20 and the fermentation 30 (FIG. 7). Figures 7 and 8 are similar to Figure 1 except that the inclusion of a separate saccharide 50 and fermentation 30 does not receive the enzyme 38. In some embodiments, the enzyme 38 may also be added to the fermentation 30.

Any known saccharification process used in the industry may be used, including, but not limited to, acid processing, enzymatic processing, or acid-enzymatic processing. The saccharide 50 can be carried out in any suitable saccharification vessel. In some embodiments, an enzyme 38 such as glucoamylase may be introduced to hydrolyze the sugar in the feed slurry 16 or the sugar (e.g., oligosaccharides) in the aqueous solution 22 to form a monosaccharide. For example, in FIG. 7, the oligosaccharide present in the aqueous solution 22 discharged from the separation 20 and conducted through the inlet to the saccharification 50 is hydrolyzed to the monosaccharide. The fermentation 30 is performed by discharging the aqueous solution 52 containing the monosaccharide from the saccharification solution 50 through the outlet. Alternatively, as shown in FIG. 8, the oligosaccharide present in the feedstock slurry 16 discharged from the liquefaction 10 and conducted through the inlet to the saccharification 50 is hydrolyzed to the monosaccharide. The feedstock slurry 54 containing the monosaccharide is discharged from the saccharification 50 through the outlet and is carried out as a separation 20.

In some embodiments, the system and process of Figures 1-6 may be modified to include a separate saccharification system as discussed herein in connection with the systems and processes of Figures 7 and 8.

In some embodiments, for example, as shown in FIGS. 9A-9D, the system and process of the present invention may comprise a series of two or more separation devices. 9A-9D are similar to FIG. 1, except adding a separation system, and will not be described in detail again.

The aqueous solution 22 discharged from the separation 20 can be carried out as a separation 20 '. The separation 20 'may be the same as the separation 20 or may be different from the separation 20 and may operate in the same manner. The separation 20 'removes undissolved solids and oil that have not been separated from the aqueous solution 22 to form (i) a reduced amount of undissolved solids and oils similar to the aqueous solution 22, (Ii) a wet cake 24 'similar to the wet cake 24, and (iii) an oil 26' similar to the oil 26. The wet cake 24 ' Thereafter, the aqueous solution 22 'may be introduced into the fermentation 30. In some embodiments, there may be one or more additional separation devices after separation 20 '.

In some embodiments, the separation 20 'can be accomplished by, for example, decanter bowl centrifugation, 3-phase centrifugation, disk stack centrifugation, filtration centrifugation, decanter centrifugation, filtration, vacuum filtration, Including solid filtration, membrane filtration, cross flow filtration, pressure filtration, screen filtration, screen separation, grating, porous grating, suspension, hydrocyclone, filter press, screw press, gravity settler, vortex separator, And any separating device capable of separating the liquid.

In some embodiments, the systems and processes of FIGS. 1-9 may be modified to include additional separation devices for removing undissolved solids as discussed herein in connection with the systems and processes described herein .

In some embodiments, stream 35 may be discharged from the outlet at fermentation 30. The absence or minimization of undissolved solids exiting the fermentation 30 via stream 35 has several additional advantages. For example, the need for units and operation in downstream processes can reduce or eliminate, for example, beer columns or distillation columns, thereby increasing production efficiency. In addition, some or all of the total distillation waste liquid centrifuge can be removed as a result of less undissolved solids in the exiting stream of the fermenter.

Referring to FIG. 9B, the aqueous solution 22 discharged from the separation 20 can be carried out as a separation 20 '. The separation 20 'may be the same as the separation 20 or may be different from the separation 20. The separation 20 'may operate in a manner that may include a separate additive 28 such as an extractant or coagulant. Separation additive 28 may help to remove oil or solids. The separation 20 'removes oil and undissolved solids from the aqueous solution 22 to remove (i) a reduced amount of undissolved solids and oils similar to the aqueous solution 22, (Ii) an integrated stream of the oil 26 and the wet cake 24, and may also produce a stream 23 comprising the separation additive 28, . Stream 23 can be run with separation 20 ", producing a stream comprising separation additive 28 ', and wet cake 24'. Separation 20 " (20 ') or may be different from separation (20) and separation (20'). The aqueous solution 22 'may be introduced into the fermentation 30.

In some embodiments, the separation 20, separation 20 ', and separation 20 "may be accomplished, for example, by decanter bowl centrifugation, 3-phase centrifugation, disk stack centrifugation, Screen centrifugal separation, filtration, vacuum filtration, belt filter, membrane filtration, cross flow filtration, pressure filtration, screen filtration, screen separation, grating, porous grating, floating, hydrocyclone, filter press, screw press, , A fluidized bed separator, a fluidized bed separator, or a combination thereof.

Referring to FIG. 9C, the feedstock slurry 16 may be discharged from the liquefaction 10 and carried out by the separation 20. The feedstock slurry 16 is separated to form a stream: (i) an aqueous solution 22; (ii) a wet cake 24, and (iii) an aqueous stream comprising oil, solids, and a fermentable carbon source. In some embodiments, the solids in stream 25 may be light solids. In some embodiments, the light solids may be less dense than water but more dense solids than the oil. In some embodiments, light solids can be coated on oil to produce solids that are less dense than water. In some embodiments, the solids may have lipophilic and / or hydrophilic properties. In some embodiments, the solids of stream 25 may include one or more of the following: bacteria, fibers, starch, and gluten. In some embodiments, the solids of stream 25 may comprise particulates. In some embodiments, the solids of stream 25 include bacteria, gluten, and fibers. In some embodiments, an aqueous solution 22 comprising a fermentable carbon source can be performed in fermentation 30 to produce a fermentation product as described herein. In some embodiments, the wet cake 24 may be further processed, for example, as described herein, to form DDGS.

The stream 25 discharged from the separation 20 can be carried out as a separation 20 '. The separation 20 'may be the same as the separation 20 or may be different from the separation 20. In some embodiments, the separation 20 and separation 20 'may be performed using, for example, decanter bowl centrifugation, 3-phase centrifugation, disk stack centrifugation, filtration centrifugation, decanter centrifugation, Such as filtration, belt filtration, membrane filtration, crossflow filtration, pressure filtration, screen filtration, screen separation, grating, porous grating, suspension, hydrocyclone, filter press, screw press, gravity settler, Solids comprising the combination and any separating device capable of separating the liquid. In some embodiments, the separation may be a single step process.

Stream 25 can be separated by separation 20 'to generate streams: (i) aqueous solution 22', (ii) wet cake 24 ', and (iii) oil 26. In some embodiments, the aqueous solution 22 'may be combined with the aqueous solution 22, and the combined aqueous solution may be performed in the fermentation 30. In some embodiments, the amount of solids in aqueous solution 22 'is reduced compared to the amount of solids in aqueous solution 22. In some embodiments, the aqueous solution 22 'may comprise oil, and the oil in the aqueous solution 22' may be further processed to produce an extractant. For example, the aqueous solution 22 'may be chemically or enzymatically treated to generate an extractant. In some embodiments, the aqueous solution 22 'can be generated chemically or enzymatically to produce a fatty acid (e.g., corn oil fatty acid) that can be used as an extractant. In some embodiments in which the aqueous solution 22 'is enzymatically treated, the enzyme reaction can be treated (e. G., Heated) after conversion to deactivate the enzyme. Conversion of the oil in the aqueous solution 22 'having a relatively small stream volume to a fatty acid can reduce the capital cost of producing the extractant through enzymatic or chemical conversion. Methods for deriving an extractant from biomass are described in U.S. Patent Application Publication No. 2011/0312043 and U.S. Patent Application Publication No. 2011/0312044, the entire contents of each of which are incorporated herein by reference.

In some embodiments, the wet cake 24 'may be combined with the wet cake 24 and the combined wet cake may be further processed as described herein. In some embodiments, the wet cake 24' And such an oil-rich wet cake can be combined with the wet cake 24 to produce a wet cake having an increased fat content (e.g., increased triglyceride content) that can provide a metabolisable energy source for animal feed can do. In some embodiments, such wet cake with increased fat content may be combined with syrup to produce high triglycerides, high protein, low carbohydrate DDGS. In some embodiments, the wet cake 24 'comprising the oil may be further processed, for example, as described herein to produce DDGS.

In some embodiments, the oil 26 may be carried in a storage tank or any vessel suitable for oil storage. In some embodiments, the oil 26 or a portion thereof may be combined with the feedstock slurry 16 (dashed line, Figure 9c). Applying oil to the feedstock slurry can enhance the removal of solids through the stream 25 by increasing the amount of trapped solids.

In some embodiments, the oil 26 may be further processed to produce an extractant as described herein. Conversion of the oil 26, which has a relatively small stream volume and may have a reduced flow rate relative to the feedstock slurry 16, reduces energy costs as well as energy costs that generate the extractant through enzymatic or chemical conversion . In some embodiments, the flow rate of the oil 26 may be between about 1% and about 10% of the flow rate of the feedstock slurry 16. Methods for deriving an extractant from biomass are described in U.S. Patent Publication No. 2011/0312043 and U.S. Patent Application Publication No. 2011/0312044, the entire contents of each of which are incorporated herein by reference.

In some implementations, stream 25 may be generated by adjusting one or more parameters of the separation device. For example, stream 25 can be generated by adjusting the weir (or deep weir) of a centrifuge such as a decanter centrifuge or a 3-phase centrifuge.

As described herein, solids can interfere with liquid-liquid extraction, and thus, the use of extraction methods may not be technically or economically feasible. During the extraction process, a rag layer can be formed at the interface between the aqueous phase and the organic phase, and a lag layer composed of solids (e.g., light solids) can accumulate and possibly interfere with phase separation. In order to alleviate the formation of the lag layer, the removal of solids through stream 25 prior to fermentation can eliminate the formation of a lag layer, thereby enhancing the recovery of the fermentation product and downstream processing of the fermentation broth .

In another embodiment for alleviating the formation of the lag layer, the oil may be added to the aqueous solution as a means for selectively trapping the solid forming the lag layer. Referring to Fig. 9D, the feedstock slurry 16 may be discharged from the liquefaction 10 and carried out by the separation 20. The feedstock slurry 16 may be separated to produce a stream: (i) the aqueous solution 22, (ii) the wet cake 24, and (iii) the oil 26. In some embodiments, the wet cake 24 can be further processed, for example, as described herein to form DDGS. In some embodiments, the oil is added to the aqueous solution 22 and the resultant mixture is conducted to the vessel 80 wherein the mixture is precipitated or separated to form (i) an oil layer comprising the solid 86 and (ii) (22 '). In some embodiments, the aqueous solution 22 'may be performed in fermentation (30) to produce a fermentation product as described herein. In some embodiments, the amount of solids in aqueous solution 22 'is reduced compared to the amount of solids in aqueous solution 22. In some embodiments, the solid rich oil layer 86 may be removed (e.g., removed from the mixture) and filtered to remove solids from the oil layer. In some embodiments, the solid rich oil layer 86 is performed with separation 20 'and is separated into a stream: (i) an aqueous solution 22' ', (ii) a wet cake 24' The day of the week applied to the aqueous solution 22 may be separated from the feedstock slurry 16, the oil 26 ', and / or the external oil source, in some embodiments. Oil 26 may be an oil.

In some embodiments, the wet cake 24 'may be combined with the wet cake 24, and the combined wet cake may be further processed as described herein. In some embodiments, the wet cake 24 'may comprise an oil, and such an oil-rich wet cake may be combined with the wet cake 24 to produce a wet cake having an increased fat content, Can be further processed as described above. In some embodiments, the wet cake 24 'comprising oil may be further processed as described herein.

In some embodiments, the aqueous solution 22 "can be combined with the aqueous solution 22 ', and the combined aqueous solution can be treated with fermentation 30. The amount of solids in this combined aqueous solution is reduced relative to the aqueous solution 22 In some embodiments, the aqueous solution 22 'and the aqueous solution 22 "may comprise an oil, which oil may be further processed (e. G. An extracting agent can be generated. For example, the aqueous solution 22 'and the aqueous solution 22 "can be combined and treated chemically or enzymatically (dotted line in Figure 9d) to generate an extractant as described herein. Methods for deriving the agent are described in U.S. Patent Application Publication No. 2011/0312043 and U.S. Patent Application Publication No. 2011/0312044, the entire contents of each of which are incorporated herein by reference.

In some embodiments, the separation 20 and separation 20 'can be performed using a variety of techniques, including decanter bowl centrifugation, 3-phase centrifugation, disk stack centrifugation, filtration centrifugation, decanter centrifugation, filtration, vacuum filtration, Including solid filtration, membrane filtration, cross flow filtration, pressure filtration, screen filtration, screen separation, grating, porous grating, suspension, hydrocyclone, filter press, screw press, gravity settler, vortex separator, And any separating device capable of separating the liquid. In some embodiments, the separation may be a single step process. In some embodiments, the vessel 80 can be a static mixer, a mixer-settler, a decanter, a gravity settler, and combinations thereof. In some embodiments, the process can be maintained at a temperature that minimizes contamination (e.g., 70-110 ° C).

In some implementations, for example, as shown in FIG. 10, the system and process of the present invention may be implemented on-line, in-line, at-line and / Lt; / RTI > Fig. 10 is similar to Fig. 5, except adding a measurement device for on-line, in-line, at-line and / or real-time measurement and will not be described again in detail.

The process described herein may be used to determine the concentration of various streams (e.g., feed slurry, aqueous feed, oil stream, wet cake, wet cake mixture, wash concentrate, etc.) generated during fermentation and other physical properties - Integrated fermentation process using line, in-line, at-line, and / or real-time measurement. These measurements can be used, for example, in a feed-back loop to adjust and control the conditions of the fermenter, liquefaction unit, separation unit, and mixing unit and / or fermentation conditions. In some embodiments, the concentration of the fermentation product and / or other metabolites and substrate in the fermented broth is measured using any suitable measuring device for on-line, in-line, at-line, and / can do. In some embodiments, the measuring device may be one or more of the following: Fourier transform infrared spectroscope (FTIR), near-infrared spectroscope (NIR), Raman spectroscope, high pressure liquid chromatography (HPLC), viscometer, densitometer, tensiometer, droplet size analyzer, particle analyzer, pH meter, dissolved oxygen (DO) probe. In some embodiments, the off-gas evacuated from the fermenter can be analyzed, for example, by an in-line mass spectrometer. Measuring the off-gas evacuated from the fermenter can be used as a means to identify the species present in the fermentation reaction. The concentrations of fermentation products and other metabolites and substrates can also be measured using the techniques and apparatus described herein.

In some embodiments, the measured input values can be sent to the regulator and / or conditioning system and the conditions (temperature, pH, nutrients, enzyme and / or substrate concentration), liquefaction unit, separation unit, The concentration or concentration profile of various streams can be maintained. By using this conditioning system, process parameters can be maintained in a manner that enhances overall plant productivity and economic objectives. In some embodiments, real-time regulation of fermentation can be achieved by varying the concentration of components (e.g., biomass, sugar, enzymes, nutrients, microorganisms, etc.) in the fermenter, liquefaction unit, separation unit and mixing unit. In some embodiments, automated systems can be used to adjust flow rates, solids and oil removal, and sugar and starch recovery to and from separation and mixing conditions, separation and mixing operations.

During the fermentation process, the unit of operation can be performed at sub-optimal levels over time. In order to maintain overall plant productivity, it may be necessary to adjust flow rates, mixing rates, equipment settings, etc. for operations such as liquefaction and separation. The processes and systems described herein may be used for on-line, in-line monitoring of the concentration and other physical properties of the fermentation stream, such as the feedstock slurry 16, the aqueous solution 22, the oil 26 and the wet cake 24, , At-line, and / or real-time measurements. Such measurements can be used in a feed-back loop to adjust and condition the conditions of, for example, fermentation, separation of the feedstock slurry, and washing cycle performance. The on-line, in-line, By using real-time measurements, and / or by performing immediate feedback and adjustment of process conditions, an overall improved fermentation process can be created. For example, the amount of fermentable carbon source (e.g., starch, sugar), oil, and solids can be measured in feed slurry 16, aqueous solution 22, and streams 65 and 75, Or can be monitored using NIR. By monitoring these parameters, it is possible to improve the production of the feedstock slurry 16 by adjusting the enzyme concentration and residence time for liquefaction and saccharification, and by adjusting the separation and mixing conditions, for example, The amount of fermentable carbon source, oil, and / or solids in streams 65 and 75 can be increased.

As another example, washing the wet cake 24 allows the sugar, starch, and oil in the wet cake to be recovered, thereby minimizing the yield loss of such fermentable carbon source and oil. Starch, and oil content of the streams 24, 65, 74, and 75 can be adjusted to improve sugar, starch, and oil recovery by adjusting the cleaning performance. For example, real-time measurements of sugar, starch and oil content of these streams can be performed with FTIR and NIR, and these measurements allow for immediate feedback and adjustment of mixing (60) and separation (20, 70) conditions . 65, 74, and 75) by adjusting the differential speed, the feed speed, the bowl speed, the scroll differential speed, the impeller position, the ware position, the scroll pitch, the residence time, Starch and oil content can be modified. The moisture, sugar, starch, and oil content of the streams 24, 65, 74, and 75 can be modified by adjusting the mixing (60) conditions, such as pump speed or stirrer speed. It is also possible to modify the moisture, sugar, starch, and oil content of the streams 24, 65, and 74 by also adjusting the wash rate (e.g., the ratio between water and the wet cake). FTIR measurements and droplet imaging can be used to monitor the water content in the oil stream 26, 76. It is also possible to monitor the color and turbidity of the oil streams 26 and 76 to assess the quality of the oil. This real-time measurement allows adjustment for the separation 20, 70 conditions, Less water).

In another embodiment of the processes and systems described herein, the moisture content of the wet cake 24 can be monitored using real-time measurements. Real-time measurements of the moisture content of the wet cake can be performed with NIR, which allows for immediate feedback and adjustment of separation (20, 70) conditions. By reducing the water content of the wet cake, less energy is required to dry the wet cake, and thus total energy utilization can be increased for the production process. Also, the low water content of the wet cake can produce improved starch recovery.

As another example of a process control strategy using real-time measurements, the solids in the aqueous solution 22 and the oil 26, 76 were analyzed using a process particle analyzer (JM Canty, Inc., Buffalo, NY), a focused beam reflectometry ^®) it can be measured using a technique, or particle observation and particle size analysis, such as measurement (PVM ^®) technology (Mettler- Toledo, LLC, Columbus OH). By monitoring the solids in real time, process steps can be adjusted to enhance solids removal, thereby minimizing the amount of solids in the aqueous and oil streams, maximizing the recovery of solids, and enhancing the overall fermentation process including downstream processes .

The processes and systems described in Figures 1 to 10 include removing undissolved solids and / or oil from the feedstock slurry 16 and, as a result, improving process productivity and cost effectiveness. Improved productivity may include increased efficiency of fermentation product production and / or increased extraction activity for processes and systems that do not remove undissolved solids and / or oils prior to fermentation.

An exemplary fermentation process of the present invention including a downstream process is described in FIG. In Fig. 11, some processes and streams have been identified using the same naming and naming as used in Figs. 1-10 and represent processes and streams similar or similar to those described in Figs.

The feedstock 12 is processed and undissolved solids and / or oils are separated (100) as described herein with reference to FIGS. 1-10. In summary, the feedstock 12 can be liquefied to produce a feedstock slurry that contains undissolved solids, a fermentable carbon source, and oil. For example, the ground grains and one or more enzymes may be combined to produce a feedstock slurry. This feedstock slurry may be flashed with flash steam to produce heating (or cooking), liquefaction, and / or "cooked" feedstock slurries or feedstocks. In some embodiments, the feedstock slurry may be heated to at least about 100 < 0 > C. In some embodiments, the feedstock slurry can be heated for about 30 minutes. In some embodiments, the feedstock slurry may be treated with raw starch hydrolysis (also known as cold cooking or chilled hydrolysis). In the raw starch hydrolysis process, the heating (or cooking) step is omitted, omitting this step reduces energy consumption and vapor loading (e.g., water consumption). In some embodiments, liquefaction and / or saccharification can be performed at fermentation temperatures (e.g., from about 30 ° C to about 55 ° C). In some embodiments, liquefaction and / or glycation is performed using a raw starch enzyme or a low temperature hydrolytic enzyme such as Stargen ^™ (Genencor International, Palo Alto, CA) and BPX ^™ (Novozymes, Franklinton, NC) . In some embodiments, liquefaction and / or saccharification may be performed at a temperature of less than about 50 < 0 > C.

The feedstock slurry is then applied to the separation to remove the undissolved solids and the wet cake 24, the oil 26, and an aqueous solution 22 containing a fermentable carbon source, e. G., Dissolved fermentable sugar, (Or concentrate). Separation is carried out using decanter bowl centrifuge, 3-phase centrifuge, disk stack centrifuge, filtration centrifuge, decanter centrifuge, filtration, vacuum filtration, belt filtration, membrane filtration, cross flow filtration, But not limited to, filtration, screen separation, grating, porous grating, suspension, hydrocyclone, filter press, screw press, gravity settler, vortex separator, or combinations thereof. This separation step may comprise dissolving an undissolved solid of at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% Can be removed from the feedstock slurry. In some embodiments, the aqueous solution 22 may comprise at least about 0.5%, at least about 1%, or at least about 2% undissolved solids.

The wet cake 24 may be re-slurried or rinsed with water and applied to the separation to remove additional fermentable sugars and a washed wet cake (e.g., 74, 74 'as described in Figures 1 to 10) Can be generated. In some embodiments, about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, or about 15% fermentable sugars can be recovered from the washed wet cake. The washing process may be repeated a number of times, for example 1, 2, 3, 4, 5 or more times. The water used to re-slurry or wash the wet cake may be recycled water generated during the fermentation process (e.g., backwash, cooked water, process water, Luther water, evaporated water). In some embodiments, the wet cake may be re-slurried or beer-washed. The washing concentrate produced by the washing / separating process (for example, 75, 75 'as shown in FIGS. 4 and 5) may be returned to the mixing step to form a slurry with ground grains or used in the liquefaction process . In some embodiments, the washing concentrate may be heated or cooled prior to the mixing step.

The aqueous solution 22 may be further processed as described herein. For example, the aqueous solution 22 can be heated by steam or process-to-process heat exchange. The saccharifying enzyme may be added to the aqueous solution 22 and the dissolved fermentable sugar of the aqueous solution 22 may partially or completely be saccharified. The glycated aqueous solution 22 may be cooled by a number of means such as process-to-process exchange, exchange with cooling water, or exchange with quench water.

An aqueous solution 22 and a microorganism 32 may be added to the fermentation 30 wherein the fermentable sugar is metabolized by the microorganism 32 to produce a stream 105 containing the fermentation product (e.g., product alcohol) . In some embodiments, the microorganism 32 may be a recombinant microorganism capable of producing a product alcohol such as 1-butanol, 2-butanol or isobutanol. In some embodiments, the ammonia and recycle stream may also be added to the fermentation (30). In some embodiments, the process may include at least one fermenter, at least two fermentors, at least three fermentors, at least four fermenters, at least five fermenters, or more. In some embodiments, the carbon dioxide generated during fermentation may be vented to a scrubber to reduce air emissions (e. G., Alcohol air emissions) and increase product yield.

The stream 105 containing the product alcohol may be conducted into the via column 120 to produce an alcohol rich stream 122 and a lower stream 125. The alcohol rich stream 122 may be sent to the alcohol recovery 160 to recover the product alcohol. The product alcohols may be purified using methods known in the art including, but not limited to, distillation, adsorption (e.g., using a resin), separation using molecular sieves, dialysis evaporation, gas stripping, Enriched stream 122. In this way, The bottoms stream 125 containing dilute distillation waste having most of the solids removed prior to fermentation can be concentrated by evaporation through evaporation 130 to form syrup 135. The syrup 135 may be combined with the wet cake (24, 74, 74 'as described herein) in the mixer 140 and the combined stream 145 of the wet cake and syrup may then be dried in the dryer 150 So that DDGS can be generated.

In some embodiments, stream 105 may be degassed. In some embodiments, the stream 105 can be heated before degassing, for example, by process-to-process exchange using a heated mesh. In some embodiments, the vapor may be vented to a condenser and then vented to a scrubber. The degassed stream 105 can be further heated, for example, by process-to-process heat exchange with other streams in the distillation and / or alcohol recovery zone.

11, aqueous solution 22, microorganism 32, and extractant may be added to fermentation 30 to produce a two-phase stream. In some embodiments, the extractant may be added to fermentation 30 via a recirculated loop. In some embodiments, the extractant may be externally directed against the downstream or fermentation 30 of the fermentation 30. A stream comprising the fermentation broth and the fermentation product (e.g., product alcohol) may be performed with an external extractor to produce a stream comprising the product alcohol and the bottom stream. In some embodiments, a stream comprising the product alcohol may be run with alcohol recovery 160 for recovery of the product alcohol. In some embodiments, the bottoms stream can be treated with a separation apparatus to separate the bottoms stream into dilute distillation waste and extractants. In some embodiments, the recovered extractant may be recycled to extract the product alcohol. A dilute distillery waste liquid having most of the solids removed before fermentation can be concentrated by evaporation (130) to form a syrup. The syrup is combined with the wet cake (24, 74, 74 'as described herein) in the mixer 140 and the combined stream 145 of wet cake and syrup is then dried in the dryer 150 to produce DDGS can do.

In some embodiments, aqueous solution 22, microbes 32, and extractant may be added to fermentation 30 to form a single liquid phase stream. In some embodiments, a two-phase stream or a single liquid phase stream can be recovered from fermentation 30 in a batch mode or continuously recovered from fermentation 30. In some embodiments, the extractant may be externally directed downstream or fermentation of the fermentation 30 to form a single liquid phase stream. In some embodiments, the extractant may be added to an external extractor to form a single liquid phase stream.

Exemplary processes for alcohol recovery are described herein, and additional methods for recovering product alcohol from fermentation broth are disclosed in U.S. Patent Application Publication No. 2009/0305370; U.S. Patent Application Publication No. 2010/0221802; U.S. Patent Application Publication No. 2011/0097773; U.S. Patent Application Publication No. 2011/0312044; U.S. Patent Application Publication No. 2011/0312043; U.S. Patent Application Publication No. 2012/0035398; U.S. Patent Application Publication No. 2012/0156738; PCT International Publication No. WO 2011/159998; And PCT International Publication No. WO 2012/030374, the entire contents of each of which are incorporated herein by reference. For example, the product alcohol can be recovered from the fermentation broth using vacuum evaporation. Preheated vials (e.g., aqueous stream 22) and solvents (e. G., Extractants) may be fed to the preflash column, which may be a reattachment of the via column in a conventional anhydrous polishing fuel ethanol plant. Such a column may operate below atmospheric pressure and be driven by vapor from the evaporator train or feed cooking stage. The overhead of the preflash column can be condensed by heat exchange using some combination of cooling water and process-to-process heat exchange involving heat exchange with the preflash column feed. The liquid condensate can be directed to the alcohol / water decanter.

The lower portion of the preflash column may proceed to a solvent decanter. The bottom of the preflash column is capable of substantially stripping the product alcohol. The decanter may be a still well, a centrifuge, or a hydrocyclone. Water can separate from the solvent phase in these decanters and generate water. The aqueous phase containing the suspended and dissolved solids may be centrifuged to produce wet cake and dilute distillate waste. The wet cake can be combined with other streams and dried to produce DDGS, which can be dried and sold separately from other streams that produce DDGS, or sold as a wet cake. The aqueous phase may be divided to provide a partially countercurrent backwash to re-slurry the wet cake described herein. This division also provides a dilute distillation waste that can be pumped to an evaporator for further processing.

The organic phase produced in the solvent decanter may be an ester of an alcohol. The solvent can be hydrolyzed to regenerate the reactive solvent and recover additional alcohol. Alternatively, the organic phase may be filtered and sold as product. Hydrolysis can be thermally driven and can be uniformly catalyzed or heterogeneously catalyzed. The heat introduction for this process may be a flame heater, heating oil, electric heating introduction, or high pressure steam. The water applied to proceed with the hydrolysis may be from a recycled water stream, fresh water or steam.

The cooled and hydrolyzed solvent can be pumped into a solvent column below atmospheric pressure and the product alcohol can be substantially stripped using steam in the column. The steam may be steam from the evaporator, which may be steam from the flash stage of the mash process, or it may be steam from a boiler (see, for example, U.S. Patent Application Publication No. 2009/0171129; The entire contents of which are incorporated herein by reference. A rectifier column from a conventional anhydrous pulverized ethanol plant may be suitable as a solvent column. The rectifier column may be modified to act as a solvent column. The lower portion of the solvent column may be cooled, for example, by cooling water or process-to-process heat exchange. The cooled bottom can be obliquely filtered to remove residual water, which can be recycled to other stages of the process or recycled to liquefaction.

The solvent column overhead can be cooled by exchange with cooling water or by process-to-process heat exchange and the condensate can be directed to an evacuated alcohol / water decanter that can be shared with preflush column overhead . Other mixed water and product alcohol streams may be added to the decanter containing the scrubber bottom and condensate from the degassing step. The exhaust containing carbon dioxide can be directed to the scrubber. The aqueous layer of this decanter can also be fed into a solvent column or it can strip product alcohol in a small dedicated distillation column. The aqueous layer can be preheated by pre-flash column overhead, solvent column overhead, or process-to-process exchange with the solvent column bottom. This dedicated column can be deformed from a side stripper of a conventional anhydrous polished fuel ethanol process.

The organic layer of the alcohol / water decanter can be pumped into the alcohol column. Such a column may be a super-atmospheric column and may be driven by vapor condensation in the reboiler. The feed to the column can be heated by process-to-process heat exchange to reduce the energy requirement for operating the column. Such a process-to-process heat exchanger may comprise a partial condenser of the pre-flash column, a partial condenser of the solvent column, a product of the hydrolyzer, steam from the evaporator, or a lower portion of the alcohol column. The condensate of the alcohol column vapor can be cooled and returned to the alcohol / water decanter. The lower portion of the alcohol column may be cooled by process-to-process heat exchange involving an exchange with an alcohol column feed, further cooled with cooling water, filtered and sold as product alcohol.

The dilute distillation waste liquid generated from the bottom of the preflash column as described herein can be directed to a multiple effect evaporator (see, for example, U.S. Patent Application Publication No. US 2011/0315541; The entire contents of which are incorporated herein by reference. Such an evaporator may have two, three or more stages. The evaporator can have four body configurations due to two effects similar to the conventional design of a fuel ethanol plant, which can have three bodies by three effects, or it can have a different structure. Dilute distillation wastewater can be supplied from any of these effects. At least one of the first effect bodies may be heated with steam from a super-atmospheric alcohol column. Steam can be taken from the lowest pressure effect to provide steam-like heat to sub-atmospheric preflash columns and solvent columns. The syrup from the evaporator can be added to the still grain dryer.

Carbon dioxide emissions from fermentors, deaerator, alcohol / water decanter, and other sources can be directed to the water scrubber. The water supplied to the top of such a scrubber may be fresh water or recycled water. The recycled water can be treated (e. G., Biologically digested) to remove volatile organic compounds and can be quenched. The bottom of the scrubber can be sent to an alcohol / water decanter, solvent column, or used with other recycled water to re-slurry the wet cake described herein. The concentrate from the evaporator can be treated with anaerobic biological digestion or other processes to purify water prior to recycling to re-slurry the wet cake.

The oil can be separated from the process stream at any of several points. For example, a centrifuge may be operated to produce an oil stream after filtration of the cooked mesh, or the preflash column aqueous centrifuge may be operated to produce an oil stream. The intermediate concentrated syrup or the final syrup can be centrifuged to produce an oil stream.

In another embodiment, the multi-phase material may leave the bottom of the preflash column and be processed in a separation system as described herein. The concentrated solids can be redispersed in the aqueous stream and this combined stream can be used to re-pulp and pump the washed low starch solids from the liquefied mesh.

In another exemplary process for product alcohol recovery, an extractant may be used to remove the product alcohol from the fermentation broth during fermentation to maintain the product alcohol in the fermentation broth below a certain concentration. In some embodiments, removal of the product alcohol may be accomplished by esterifying the product with a carboxylic acid in the presence of a catalyst to produce an alcohol ester. A description of processes and systems for the extraction of alcohol by the formation of alcohol esters can be found in U.S. Patent No. 2012/0156738, the entire contents of which are incorporated herein by reference. For example, the oil separated from the feedstock slurry can be hydrolyzed with a catalyst such as an esterase (e.g., lipase) that converts the triglyceride in the oil to a fatty acid such as a carboxylic acid. These fatty acids can be used as extractants for the recovery of the product alcohol. In some embodiments, hydrolysis of the oil can occur in the fermenter by adding the catalyst to the fermenter. In some embodiments, the hydrolysis of the oil can occur in a separate vessel, and fatty acids can be added to the fermenter. For example, the feedstock slurry may be carried in a vessel or tank, and an esterase such as a lipase may be added to the vessel, which converts the oil present in the feedstock slurry to a fatty acid. Feedstock slurries containing fatty acids can be carried out with a fermenter.

The product alcohol produced by fermentation can react with fatty acids to produce alcohol esters. In some embodiments, these alcohol esters can be extracted from fermentation broth. For example, fermentation broth containing alcohol esters may be transferred to a separation device such as a 3-phase centrifuge to separate the fermentation broth into three streams: undissolved solids (including microorganisms), aqueous streams, and alcohol esters Organic < / RTI > stream. In some embodiments, the fermentation broth can be separated into two streams: an undissolved solid (including microorganisms), and a biphasic mixture comprising an aqueous phase and an organic phase. This separation of the fermentation broth can occur continuously during fermentation, for example, by removing a portion of the fermentation broth for separation, or in the batch mode, for example, the entire contents of the fermenter can be removed for separation .

In some embodiments, the two-phase mixture can be separated into an ester-containing organic phase and an aqueous phase, and this separation can be accomplished by various methods including siphoning, aeration, gradient filtration, centrifugation, gravity settling, , Hydrocyclones, and the like, which are known in the art. The alcohol ester-containing organic phase may be further processed to recover the product alcohol. For example, the alcohol ester-containing organic phase can be transferred to a vessel, where the alcohol ester can hydrolyze to form product alcohols and fatty acids, and this mixture of product alcohols and fatty acids is removed by distillation It can be processed to separate product alcohols and fatty acids.

In some embodiments, the fatty acid may be recycled to a fermenter or extractor column. In some embodiments, the aqueous stream and undissolved solids can be recycled to the fermenter.

In some embodiments, the extraction of the product alcohol may occur at the downstream stream of the fermenter or externally relative to the fermenter. In some embodiments, the fermentation system may include an external extraction system, including, for example, a mixing device and a separation system. The fermentation broth can be carried out with a mixing device, which can be added to the mixing device and combined with the fermentation broth to produce a two-phase mixture. The two-phase mixture may be carried out in a separation system, wherein the separation of the two-phase mixture produces an alcohol-containing organic phase and an aqueous phase. In some embodiments, the aqueous phase or a portion thereof may be returned to the fermenter. In some embodiments, the alcohol-containing organic phase may be carried out with an extractant column. Bioprocessing productivity in these embodiments is substantially improved by separating the biomass feed stream components after liquefaction but prior to fermentation. In particular, reducing the amount of undissolved solids and / or oil increases the efficiency of the external alcohol extraction system.

In some embodiments, the oil may be separated from the feedstock or feedstock slurry and stored in an oil storage vessel. For example, the oil may be separated from the feedstock or feedstock slurry using any suitable means for separation, including 3-phase centrifugation or mechanical extraction. In order to enhance the removal of oil from the feedstock or feedstock slurry, an oil extraction aid such as a surfactant, an anti-emulsifier, or a flocculant may be used in addition to the enzyme. Examples of oil extraction aids include non-polymeric, liquid surfactants; Talc powder; Fine talc powder; Enzymes, for example, Pectinex ^® Ultra SP-L, Celluclast ^{®, ®} L and Viscozyme (Sigma-Aldrich, St. Louis, MO), and NZ 33095 (Novozymes, Franklinton, NC ); Salt (NaOH); And calcium carbonate. Another means for enhancing oil removal may be pH control, such as raising or lowering the pH. Additional benefits for oil removal include increased oil yield, improved oil quality, reduced system attachment, and reduced downtime. In addition, oil removal can also produce a scrubber, high quality oil.

The remaining feedstock or feedstock slurry may then be further treated to remove any residual oil. For example, the feedstock or feedstock slurry can be run as a vessel or tank after oil separation and a catalyst such as an esterase (e.g., lipase) can be added to the vessel to convert the oil present in the feedstock or feedstock slurry into fatty acid . ≪ / RTI > Removal of the oil from the feedstock or feedstock slurry not only enhances the enzyme efficiency, but also reduces the amount of enzyme required for the process described herein. The feedstock or feedstock slurry can then be run with a fermenter and the microbe can also be added to the fermenter to produce the product oil. In some embodiments, the catalyst may be deactivated by, for example, heating. In some embodiments, the deactivation may be performed in a separate vessel, for example, a deactivating vessel. The deactivated feedstock or feedstock slurry can be carried out with a fermenter and the microorganism can also be added to the fermenter to produce the product alcohol. Removal of the oil from the feedstock or feedstock slurry by converting the oil to a fatty acid can result in energy savings in the production plant due to more efficient fermentation, less attachment due to removal of the oil, and energy requirements, The energy required to dry distiller grains can be reduced. After fermentation, the fermentation broth containing the product alcohol may be carried out in an external vessel, for example, an external extractor or an external extraction loop, for recovering the product alcohol. Removal of the oil as provided herein may be advantageous if the embodiment of the present invention separates the oil from the feedstock slurry so that the stable emulsion is less prone to occur with the aid of the feed stream separation process and the recovered entrapped bio- (See, for example, U.S. Patent No. 7,601,858; U.S. Patent No. 8,192,627), as well as the need to add a quantum solvent to destroy the emulsions formed together. In some embodiments of the present invention, the emulsion can be formed, but is easily destroyed by mechanical processing or other conventional means.

When recovering the product alcohol using an extractant, removing the oil prior to the fermentation process can reduce the amount of oil taken up by the extractant and thereby expand the effectiveness of the extractant to recover the product alcohol. The oil taken by the extractant can reduce the K _d as well as the selectivity of the extractant and can increase the operating cost of the production process again. Since the extractant can be recycled in the production process, each fermentation cycle exposes the extractant to more oil taken by the extractant and can significantly reduce the selectivity and K _d of the extractant over time. The processes and systems described herein provide a means to maintain the K _d and selectivity of the extractant by removing oil from the feedstock or feedstock slurry and / or converting the oil to a fatty acid by the addition of a catalyst.

The processes and systems described herein can increase extraction activity and / or efficiency in producing fermentation products (e.g., product alcohols) as a result of removal of undissolved solids. For example, extraction fermentation without the presence of undissolved solids can be advantageously used in the production of extractables as a result of the increased mass transfer rate of the product alcohol from the fermentation broth to the extractant, better phase separation, It is possible to induce a low hold-up. Also, for example, an extractant droplet that is stagnant in the fermentation broth during fermentation will dissociate more rapidly and more completely from the fermentation broth, thereby creating less glass extractant in the fermentation broth. In addition, the lower stagnation of the extractant may reduce the amount of extractant lost in the process. Additional advantages of solids removal include, for example, removal of the stirrer in downstream stream processing equipment and fermentors such as centrifuges and via columns, resulting in a reduction of capital costs and energy use; Increased fermenter volume resulting in increased fermenter productivity; Increased production efficiency of the extractant, increased recovery and regeneration, reduced flow rate of the extractant to lower operating cost, and reduced extraction agent hold-up resulting in the potential to use continuous fermentation or mini-fermentors, . In some embodiments, the volume of fermenter available for fermentation will be increased by at least about 5%, at least about 10% or more.

Examples of increased extraction efficiencies include, for example, stabilization of partition coefficients, enhanced phase separation, enhanced mass transfer coefficients, operation at low titer, increased process stream reproducibility, increased fermentation volume efficiency, Increased feed load load, increased product alcohol tolerance of microorganisms, water regeneration, energy savings, increased regeneration of extractants, and microbial regeneration. For example, since the oil in the fermentation broth can be reduced by removing solids from the feedstock slurry prior to fermentation, the extractant is exposed to less oil that can be combined with the extractant and lowers the partitioning factor of the extractant . Thus, the reduction of oil in the fermentation broth produces a more stable fractionation factor over many fermentation cycles. In some embodiments, the partition coefficient may be reduced to less than about 10%, less than about 5%, or less than about 1% over 10 or more fermentation cycles. As another example of increased extraction efficiency, higher mass transfer rates (e.g., in the form of higher mass transfer coefficients) may result in increased efficiency of product alcohol production. In some embodiments, the mass transfer coefficient may increase by at least 2-fold, at least 3-fold, at least 4-fold, or at least 5-fold.

Increasing the phase separation between the fermentation broth and the extractant reduces the tendency of emulsion formation and increases the efficiency of product alcohol production. For example, in the absence of an emulsion, phase separation can occur more rapidly and can be more perfect. In some embodiments, the phase separation may occur if no previously recognized phase separation is observed. In some embodiments, phase separation may occur within 24 hours. In some embodiments, the phase separation is at least about 2X (2x) faster, at least about 5X (5X) faster, or at least about 10X (x 10) times faster than phase separation in which the solid is not removed or the emulsion formed. ) Can occur quickly.

As described herein, nutrients such as nitrogen, minerals, trace elements, and / or vitamins can be added to the feedstock slurry or fermenter. These nutrients as well as the naturally occurring nutrients in the feedstock may be soluble in the oil and thus the presence of oil in the feedstock or feedstock slurry may reduce the concentration of these nutrients in the fermented broth. Removal of oil from the feedstock or feedstock slurry can minimize the loss of nutrients. In addition, the presence of solids in the fermented broth may also result in a reduction in the concentration of nutrients. Removal of solids and / or oils from the feedstock or feedstock slurry can minimize the loss of nutrients.

In the processes and systems described herein, the presence of oil in a fermentation process can affect the partitioning factor of the extractant over a number of fermentation processes. Removing the oil from the feedstock or feedstock slurry can reduce the diversity of the partitioning factor of the extractant over a number of fermentation processes, thereby improving the extensibility of the processes and systems described herein. Scalability refers to the ability to modify (e.g., expand or condense) a process or system to accommodate manufacturing requirements (e.g., operating volume) without defects in functionality, for example.

In addition, removal of solids from the feedstock or feedstock slurry can also have an effect on scalability. For example, when using an external extractor, reduced solids (e.g., reduced total suspended solids, TSS) can produce enhanced performance and good scalability. Reduced solids increase the mass transfer rate of the product alcohol between the aqueous phase and the organic phase (e.g., fermentation broth and extractant). The solid particles cover the surface of the extraction droplet which effectively reduces the area for mass transfer. Solid particles also inhibit phase separation by increasing the tendency towards viscosity and emulsification. Removal of solids from the feedstock or feedstock slurry provides improved extensibility and reliability of the external extractor because the presence of solids can interfere with the operation of the external extractor.

Thus, removal of solids and / or oils may enhance the extensibility of the unit operations of the processes and systems described herein. For example, retention of solids and / or oil may enhance unit operation, such as but not limited to extractor performance, distillation column performance, heat exchanger performance, and / or evaporator performance.

As an example of improved unit operation, referring to FIG. 11, stream 105 may be conducted to via column 120 to produce stream 122 and stream 125 that are rich in alcohol. The bottoms stream 125 containing dilute distillation waste liquid, having most of the solids removed prior to fermentation, can be concentrated through evaporation 130. By removing the solids before fermentation, less solids can be sent to the evaporator to produce a lower feed rate for the evaporator. Lower feed rates require less energy and therefore lower costs due to less energy requirements.

In some embodiments, there may be a need to remove water from the recovered oil from the feedstock or feedstock slurry. In some embodiments, the water may be removed by gravity separation, coalescing seperator, centrifugation (e.g. decanter), adsorption or absorption, distillation, heating, vacuum dehydration, and / or air stripping ). ≪ / RTI > Examples of adsorption media include, but are not limited to, activated alumina, bentonite clay, calcium chloride, calcium sulfate, cellulose, magnesium sulfate, molecular sieve, polymer and / or silica gel. In some embodiments, the adsorption medium may be continuously agitated with oil, or the oil may flow through a packed bed having an adsorption medium.

Sometimes it may be necessary to clean and / or sterilize equipment used to produce a fermentation product, such as product alcohol. Examples of equipment include, but are not limited to, fermenters, liquefier vessels, saccharification vessels, support tanks, storage tanks, heat exchangers, pipelines, equipment connections, nozzles, fittings, and valves. Cleaning and sterilization not only reduces or eliminates microbial contamination, but also minimizes accumulation of residues (eg, carbohydrates, sugars) in the equipment. Increasing residues in the equipment can provide a source of nutrients for unwanted microorganisms, which can lead to the proliferation of these microorganisms. There are a number of methods used to clean and / or sterilize fermentation equipment, including in situ cleaning (CIP) and in situ sterilization (SIP). CIP and SIP can be performed manually or using an automated system. Adequate as well as efficient CIP or SIP can minimize manufacturing plant compliance by, for example, minimizing the need for disruption due to microbial contamination.

In the processes and systems described herein, for example, removal of solids and oils from the feedstock or feedstock slurry prior to fermentation can enhance the efficiency of CIP and SIP. The lack of solids in the fermentation equipment may require less cleaning water, less cleaning solution, and less time to perform CIP and SIP. Thus, removal of solids can increase the efficiency of CIP and SIP processes and reduce costs. In addition, the caustic formulation used in CIP (e.g., sodium hydroxide) can react with triglycerides in oil-forming soaps (i.e., saponification), which may have an effect on the efficiency of CIP. Thus, the removal of oil can reduce the formation of soap during CIP and improve the efficiency of CIP.

During the fermentative process, the adherence can affect the productivity and efficiency of the production process. In general, the adherence refers to the adherence of foreign matter or particles, for example, the adherence of a substance on the surface of a heat exchanger and a distillation column reboiler. This adherence of the material on the surface of the heat exchanger can interfere with the heat transfer and reduce the ability of the heat exchanger to operate. For example, material attachment can interfere with fluid flow through the exchanger causing an increase in flow resistance. In addition, attachment of materials on the surface of a heat exchanger or other equipment may require additional cleaning. These additional cleaning requirements may require plant shutdowns that can result in reduced plant productivity. In the processes and systems described herein, for example, removal of oil and / or solids from a feedstock or feedstock slurry prior to fermentation can minimize the attachment of the material and may reduce the adhesion of the material on the surface of equipment such as a heat exchanger The speed can be lowered. Thus, solid and oil removal can reduce the rate of attachment of the heat exchanger and minimize the impact of adhesion on heat transfer and operating capability.

In another example of an embodiment of the process and system of the present invention, the material discharged from the fermenter is processed in a separation system comprising a device such as a centrifuge, a settler, a hydrocyclone, and the like, The microbes are recovered in a concentrated form that can be regenerated for re-use with the following re-conditioning. The ability to regenerate microorganisms can increase the overall rate of fermentation product production, such as product alcohol production, and can degrade overall drop-off requirements and / or degrade aqueous drop-in requirements, resulting in more healthy microbes and higher production rates . Such a separation system may also produce an aqueous stream containing an organic stream comprising fermentation products (e.g., product alcohols) and other by-products resulting from fermentation, and only trace amounts of incompatible organic matter. This aqueous stream can be used before or after stripping the product alcohol content for washing the solids separated from the feedstock slurry. This has the advantage of avoiding that it may be a long belt-driven delivery system for the transfer of these solids from the liquefaction zone to the grain drying and syrup blending zone. Moreover, the entire distillation waste liquid produced after stripping the product alcohol will need to be separated into dilute distillate waste and wet cake fractions using existing or new separation equipment. The dilute distillate wastewater can partially form backflow that can be combined with the number of cooks to produce a new batch of fermentable mesh. Another advantage of this embodiment is that any remaining fermentable sugar retained in the solids separated from the feedstock slurry can be partially captured and recovered through this backwash. Alternatively, the microorganisms contained in the solid stream can be redispersed in the aqueous stream and this combined stream distils any product alcohol content remaining from the fermentation. If the microorganism is not viable, the non-viable microorganism can be further separated for use as a nutrient in the propagation process, for example.

In some embodiments of the processes and systems described herein, the by-product (or co-product) of the fermentation process can be further processed, for example, the undissolved solids can be processed to generate DDGS. Other by-products, such as fatty acid esters, which may have an inhibitory effect on microorganisms, may be recovered from the fermentation broth and / or the by-product stream to increase the yield of product alcohol. Recovery of fatty acid esters or other lipids can be accomplished using a solvent to extract the fatty acid esters from the by-product stream. In some embodiments, several byproduct streams can be combined and fatty acid esters can be recovered from the combined stream.

In embodiments of solvent extraction of lipids (e.g., fatty acid esters), solids can be separated from the total distillation waste liquid ("isolated solids") because such streams can contain large amounts of fatty acid esters . This separated solid can then be fed to the extractor and washed with solvent. In some embodiments, the separated solids can be washed at least two times. After washing, the resulting mixture of lipid and solvent, known as miscella, may be collected for the separation of the lipids extracted from the solvent. For example, the resulting mixture of lipid and solvent can be carried out with a separator or extractor for further processing. During the extraction process, the solvent not only extracts lipids into the solution but also collects particulates ("fine water"). These microfilters are generally undesirable impurities in the genus Micas, and in one embodiment, may be carried out with a separator that separates or scrubs fine material from the micellar, which is discharged from a separator or extractor called micelles.

In order to separate lipids and solvents contained in the micelles, it can be applied to a distillation step called micelles. At this stage, the micelles, e. G., Micelles, can be processed through an evaporator heated to a temperature sufficiently high 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, for example, in a condenser and recycled for future use. Separation of the solvent from the micelle produces a crude lipid source which can be further processed to separate water, fatty acid esters (e.g., fatty acid isobutyl esters), fatty acids, and triglycerides. A solvent system extraction system for recovering triglyceride is described in U.S. Patent Publication No. 2010/0092603, the entire contents of which are incorporated herein by reference.

After extracting the lipids, the solids can be carried from the extractant and treated with a stripping device (e.g., solvent remover) to remove residual solvent. Removal of residual solvent may be important to process economics. In some embodiments, the solids can be conveyed to a solvent remover in a vacuum-tight environment to preserve and collect solvents that can be temporarily evaporated from the solids. As the solids are fed to the solvent remover, the solids can be heated to evaporate and remove the residual solvent. In order to heat the solids, the solvent remover may include a mechanism for dispensing the solids over one or more trays, the solids may be heated either directly, such as through direct contact with heated air or steam, Or indirectly, such as by heating. To facilitate the transfer of solids from one tray to another, the trays carrying the solids may include openings through which solids pass from one tray to the next. From the solvent remover, the solids can optionally be conveyed to a mixer, where the solids are mixed with other by-products before being conveyed to the dryer. In some embodiments, the solids are conducted with a solvent remover and the solids are contacted with the vapor. In some embodiments, the flow of vapor and solids in the solvent remover may be countercurrent. In some embodiments, the vapors exiting the solvent remover can be condensed, optionally mixed with the micelles, and then fed to a decanter to form a water-rich phase. Such a water-rich phase exiting the decanter can be fed into the distillation column, where the solvent is removed from the water-rich stream. In some embodiments, the solvent-depleted water-rich stream can be discharged to the bottom of the distillation column and recycled to the fermentation process, for example, to use it to process the feedstock. In some embodiments, the overhead and bottom products of the distillation column can be recycled to the fermentation process. For example, a lipid-rich bottom can be added to the feed of the hydrolyzate. The overhead can be fed, for example, to a decanter that can be condensed and form a solvent-rich stream and a water-rich phase. A solvent rich stream exiting the decanter is optionally used as the solvent feed to the extractor and a water-rich phase exiting the decanter is fed into the stripping column to strip the solvent from the water.

In some embodiments, the by-product or co-product can be derived from the feedstock slurry used in the fermentation process. As described herein, if corn is used as the feedstock, the corn oil may be separated from the feedstock slurry prior to fermentation. The benefits of removing corn oil prior to the fermentation process include recovering more corn oil compared to corn oil removal at the end of the fermentation process (e.g., from syrup), high quality and / Thus, recovering more expensive oil, generating corn oil as co-product, and converting corn oil to other products.

For example, corn oil can be selected from the group consisting of triglycerides, diglycerides, monoglycerides, fatty acids, phytosterols, vitamin E, carotenoids (e.g., beta -carotene, beta -cryptotaxanthin, Because it contains antioxidants such as tocopherol, it can be added to the other co-product at different concentrations or rates to create the ability to change the amount of these components in the resulting co-product. In this way, it is possible to control the fat content of the resulting co-product to obtain a low fat, high protein animal feed which can, for example, be better suited to the needs of the cow compared to the high fat product. In another embodiment where high fat animal feed may be desirable, corn oil can be used as a component of animal feed because its high triglyceride content can provide a source of metabolizable energy. In addition, natural antioxidants in corn oil not only provide a source of vitamin E but also reduce the development of odors.

The corn oil separated from the feedstock may be further processed to produce refined corn oil or edible oil for consumer use. For example, crude corn oil is further processed to degumming the gum to remove phosphatide, alkali refining to neutralize the free fatty acids, decolorizing to remove hues and trace elements, removing the wax (Eg, corn oil, 5 ^th Edition, Corn Refiners Association, Washington, DC, 2006). The term " corn oil " Purified corn oil can be used, for example, by food manufacturers to produce food products. Free fatty acids removed by alkali refining can be used as soap raw materials and the wax recovered from the winter preparation step can be used in animal feed.

Corn oil can be used in the manufacture of resins, plastics, polymers, lubricants, paints, varnishes, printing inks, soaps, and fabrics; It may also be used in the pharmaceutical industry as a component of drug formulations. Corn oil can also be used as a feedstock for biodiesel or renewable diesel.

In some embodiments, an oil such as corn oil may be used as a feedstock for the production of an extractant for extractive fermentation. For example, the oil derived from the biomass may be converted to an extractant available for removal of the product alcohol, such as butanol, from the fermentation broth. The glycerides in the oil can be chemically modified with reaction products, such as fatty acids, fatty alcohols, fatty amides, fatty acid alkyl esters, fatty acid glycol esters, and hydroxylated triglycerides, or mixtures thereof, which can be used as fermentation product extractants ≪ / RTI > or enzymatically. When corn oil is used as an example, corn oil triglyceride can react with bases such as ammonia hydroxide or sodium hydroxide to obtain fatty amides, fatty acids, and glycerol. These fatty amides, fatty acids, or a mixture thereof can be used as an extracting agent. In some embodiments, vegetable oils such as corn oil may be hydrolyzed with an enzyme such as lipase to form fatty acids (e.g., corn oil fatty acid). Methods for deriving an extractant from biomass are described in U.S. Patent Application Publication No. 2011/0312043, U.S. Patent Application Publication No. 2011/0312044, and PCT International Publication No. WO 2011/159998. In some embodiments, the extractant may be used wholly or in part as a component of an animal feed, or it may be used as a feedstock for biodiesel or renewable diesel.

In some embodiments, corn oil may also be used as biodiesel or as a renewable diesel. In some embodiments, a combination of oils or oils may be used as the feedstock for biodiesel or renewable diesel. Examples of oils include canola, caster, corn, jojoba, carranza, mahayana, flaxseed, soybean, palm, peanut, rapeseed, rice, safflower, and sunflower oil. Biodiesel can be derived from the esterification or esterification of plant oils using alcohols such as methanol, ethanol and butanol. For example, biodiesel can be used as an acid-catalyzed, alkali-catalyzed, or enzyme-catalyzed ester conversion reaction or esterification reaction (e.g., ester conversion reaction of plant oil-derived triglycerides, An esterification reaction of an oil-derived free fatty acid). Inorganic acids such as sulfuric acid, hydrochloric acid and phosphoric acid; Organic acids such as toluene sulfonic acid and naphthalene sulfonic acid; Solid acids such as Amberlyst ^® sulfonated polystyrene resins; Or zeolites can be used as catalysts for acid-catalyzed ester conversion reactions or esterification reactions. A base such as potassium hydroxide, potassium methoxide, sodium hydroxide or sodium methoxide or calcium hydroxide can be used as a catalyst for the alkali-catalyzed ester conversion reaction or esterification reaction. In some embodiments, biodiesel can be prepared by an integrated process, such as acid-catalyzed esterification of free fatty acids and subsequent base-catalyzed ester conversion of triglycerides.

Enzymes such as lipase or esterase can be used to catalyze ester or etherification reactions. Lipases may be derived from bacteria or fungi, for example, Pseudomonas Monastir (Pseudomonas), Terre Mo My process (Thermomyces), Burkholderia (Burkholderia), Candida (Candida), and Lee jomu cor (Rhizomucor). In some embodiments, the lipase is Pseudomonas fluorescein sense (Pseudomonas fluorescens , Pseudomonas sp. cepacia ), Rizomukor Hey, Mie (Rhizomucor miehei ), Burkholderia Sefar Asia (Burkholderia cepacia), Terminus all my process called leakage labor (Thermomyces lanuginosa), or it may be derived from Candida not teuak urticae (Candida antarctica). In some embodiments, the enzyme can be immobilized on a soluble or insoluble support. Immobilization of the enzyme may be accomplished by 1) binding of the enzyme to a porous or non-porous carrier support via covalent support, physical adsorption, electrostatic binding or affinity binding; 2) cross-linking with difunctional or multifunctional reagents; 3) capture in gel matrix, polymer, emulsion, or some form of membrane; And 4) using any of a variety of techniques including any combination of these methods. In some embodiments, the lipase can passivate, for example, acrylic resin, silica, or beads (e.g., polymethacrylate beads). In some embodiments, the lipase may be soluble.

In some embodiments, the biodiesel described herein may comprise one or more of the following fatty acid alkyl esters (FAAEs): fatty acid methyl esters (FAME), fatty acid ethyl esters (FAEE), and fatty acid butyl esters (FABE). In some embodiments, the biodiesel described herein may comprise one or more of the following: pre-stearate, palmitate, stearate, oleate, linoleate, linolenate, arachidate, and behenate.

In some embodiments, the oil from the fermentation process may be recovered by evaporation to form a non-aqueous stream. Such non-aqueous streams may include fatty acid esters and fatty acids, which may be carried out with a hydrolyzate to recover product alcohols and fatty acids. In some embodiments, such a stream may be used as a feedstock for biodiesel production.

In some embodiments, the biodiesel described herein meets the specifications of the American Society for Testing and Materials (ASTM) D6751. In some embodiments, the biodiesel described herein meets the specification of European standard EN 14214.

In some embodiments, the reactor structure for producing biodiesel includes, for example, a batch-stirred tank reactor, a continuous-stirred tank reactor, a packed bed reactor, a fluid bed reactor, an expandable bed reactor, Membrane reactor.

In some embodiments, the composition comprises at least 2% biodiesel, at least 5% biodiesel, at least 10% biodiesel, at least 20% biodiesel, at least 30% biodiesel, at least 40% biodiesel, at least 50% 60% biodiesel, at least 70% biodiesel, at least 80% biodiesel, at least 90% biodiesel, or 100% biodiesel.

In some embodiments, the biodiesel described herein can be combined with a petroleum-based diesel fuel to form a biodiesel blend. In some embodiments, the biodiesel blend comprises at least 2% by volume biodiesel, at least 3% by volume biodiesel, at least 4% by volume biodiesel, at least 5% by volume biodiesel, at least 6% by volume biodiesel, at least 7% by volume biodiesel At least 8% by volume biodiesel, at least 9% by volume biodiesel, at least 10% by volume biodiesel, at least 11% by volume biodiesel, at least 12% by volume biodiesel, at least 13% by volume biodiesel, at least 14% by volume biodiesel, At least 15% by volume biodiesel, at least 16% by volume biodiesel, at least 17% by volume biodiesel, at least 18% by volume biodiesel, at least 19% by volume biodiesel, or at least 20% by volume biodiesel. In some embodiments, the biodiesel blend may comprise up to about 20% by volume biodiesel.

The by-product of the biodiesel product is glycerol. Glycerol may also be a by-product of the production of extractants from oils and by-products of the fermentation process. Feedstocks for biodiesel can be produced by reacting fatty acids such as COFA with glycerol. The reaction can be catalyzed by a solid acid catalyst such as an inorganic strong acid or Amberlyst ^TM polymeric catalyst and an ion exchange resin such as sulfuric acid. A high conversion rate can be obtained by recovering water from the reactants. The reaction product may include monoglyceride, diglyceride, and triglyceride as a ratio measured by the ratio of the reactants to the reactants. The glyceride mixture can be used in place of the triglyceride feeds commonly used to make biodiesel. In some embodiments, the glyceride can be used as a surfactant or as a feedstock for biodiesel.

In some embodiments, the solids can be separated from the feedstock slurry and can include triglycerides and fatty acids. Such solids can be used as an animal feed, recovered as an effluent from centrifugation or after drying. Solids may be particularly suitable as rats for ruminants (e.g. dairy cows) because of the high content of lysine and bypass or lumen nonsoluble protein available. For example, these solids can be particularly valuable in high protein, low fat feeds. In some embodiments, these solids can be used as bases, i.e., other by-products such as syrups can be added to the solid to form a product that can be used as an animal feed. In some embodiments, different amounts of different byproducts are added to the solid to provide the solid (such as cows, beef cattle, poultry, pigs, livestock, horses, aquaculture, and household pets) The properties of the product can be controlled.

In some embodiments where low fat animal feed is desired, the oil may be removed from the feedstock prior to fermentation. By removing the corn oil, the resulting DDGS can have low fat, high protein content. If the corn oil is not removed, the oil present in the wet cake may be oxidized by a drying process. This oxidation leads to a darkening effect and produces a darker color of DDGS. If the oil is removed from the feedstock prior to fermentation, the resulting DDGS may be brighter in color and this brightly colored DDGS may be desirable for some animal feed products.

Solid compositions separated from the whole distillation waste liquid may contain, for example, crude proteins, fatty acids, and fatty acid esters. In some embodiments, such compositions may be used in wet or dry form, for example, as animal feeds where high protein (e.g., high lysine), low fat, and high fiber content are desirable. In some embodiments, for example, fat can be added to the composition from another byproduct stream if a high fat, low fiber animal feed is desired. In some embodiments, such high fat, low fiber animal feed can be used in pigs or poultry. In some embodiments, non-aqueous compositions of CDS can include, for example, proteins, fatty acids, and fatty acid esters, and also other dissolved and suspended solids, such as salts and carbohydrates. Such a CDS composition can be used, for example, as an animal feed in a wet or dry state, wherein high protein, low fat, high inorganic salt feed ingredients are preferred. In some embodiments, such a composition may be used as a component of a cow allocation.

In some embodiments, one or more streams generated by the production of the product alcohol through a fermentation process can be combined to produce a composition comprising at least about 90% fatty acid that can be used as a fuel source, such as biodiesel.

Various streams generated by the production of the product alcohol through a fermentation process can be combined in a number of ways to generate multiple co-product compositions. For example, if the crude corn from the mash is used to extract fatty acids to be used as extractants and to extract lipids with an evaporator, then the remaining streams are combined and processed to produce crude proteins, crude fat, trichlcercides, To produce a co-product composition comprising a fatty acid ester. In another example, when an oil, such as corn oil, is removed from the feedstock slurry, the oil may be added to the distiller grains to produce, for example, animal feed products.

In some embodiments, the compositions of the processes and systems described herein comprise at least about 20-35 wt% crude protein, at least about 1-20 wt% crude fat, at least about 0-5 wt% triglyceride, at least about 4-10 wt% % Fatty acids, and at least about 2 to 6 wt% fatty acid esters. In some embodiments, the composition may comprise about 25 wt% crude protein, about 10 wt% crude fat, about 0.5 wt% triglyceride, about 6 wt% fatty acid, and about 4 wt% fatty acid ester. In some embodiments, the composition comprises at least about 25 to 31 weight percent crude protein, at least about 6 to 10 weight percent crude fat, at least about 4 to 8 weight percent triglyceride, at least about 0 to 2 weight percent fatty acid, and at least about 1 To 3% by weight fatty acid esters. In some embodiments, the composition may comprise about 28 wt% crude protein, about 8 wt% crude fat, about 6 wt% triglyceride, about 0.7 wt% fatty acid, and about 1 wt% fatty acid ester. In some embodiments, the fatty acid ester may be a fatty acid methyl ester, a fatty acid ethyl ester, a fatty acid butyl ester, or a fatty acid isobutyl ester.

In some embodiments, the total distillation effluent extracted from the feedstock slurry and the solids separated from the oil may be combined and the resulting composition may contain crude protein, crude fat, triglyceride, fatty acid, fatty acid ester, lysine, neutral detergent fiber (NDF) , And acid detergent fiber (ADF). In some embodiments, the composition comprises at least about 26-34 wt% crude protein, at least about 15-25 wt% crude fat, at least about 12-20 wt% triglyceride, at least about 1-2 wt% fatty acid, 4 wt% fatty acid ester, at least about 1 to 2 wt% lysine, at least about 11 to 23 wt% NDF, and at least about 5 to 11 wt% ADF. In some embodiments, the composition comprises about 29 weight percent crude protein, about 21 weight percent crude fat, about 16 weight percent triglyceride, about 1 weight percent fatty acid, about 3 weight percent fatty acid ester, about 1 weight percent lysine, about 17 weight % NDF, and about 8 wt% ADF. In some embodiments, the fatty acid ester may be a fatty acid methyl ester, a fatty acid ethyl ester, a fatty acid butyl ester, or a fatty acid isobutyl ester. The high fat, triglyceride, and lysine content and low fiber content of such compositions may be desirable as feed for pigs and poultry.

As described herein, various streams generated by the production of product alcohols through a fermentation process can be combined in various ways to produce a composition comprising crude protein, crude fat, triglycerides, fatty acids, and fatty acid esters. 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 a cow animal. Compositions comprising at least about 6% crude fat and at least about 26% crude protein can be used as animal feed products for livestock husbandry use but contain at least about 1% crude fat and at least about 27% crude protein in an animal feed for winter It can be used as a product. A composition comprising at least about 13% crude fat and at least about 27% crude protein can be used as an animal feed product for poultry. 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. It is possible to use various streams for specific animal species such as livestock, ruminants, cows, dairy animals, pigs, goats, sheep, poultry, horses, aquaculture or domestic pets such as dogs, cats, Can be combined as a method for customizing feed products.

DDGS generated by the process of the present invention can be modified by adding one or more of the following to produce customized and expensive feed products: proteins, fats, fibers, ash, lipids, amino acids, vitamins and minerals. Amino acids include, for example, essential amino acids such as histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan and valine and also other amine acids such as alanine, arginine, aspartic acid, asparagine , Cysteine, glutamic acid, glutamine, glycine, hydroxylysine, hydroxyproline, ornithine, proline, serine, and tyrosine. Minerals include, for example, calcium, chloride, cobalt, copper, fluoride, iodine, iron, magnesium, manganese, phosphorus, potassium, selenium, sodium, sulfur and zinc. Vitamins include, for example, vitamins A, C, D, E, K, and B (thiamine, riboflavin, niacin, pantothenic acid, biotin, vitamin B6, vitamin B12 and folate).

As described herein, the processes and systems of the present invention provide a number of advantages that can improve the production of product alcohols such as butanol. For example, improvements in mass transfer enable operation at lower aqueous additions, resulting in "healthier" microorganisms. Better phase separation can also lead to improved fermenter volumetric efficiency as well as the possibility of processing less reactor contents via via columns, distillation columns, and the like. In addition, there is less solvent loss through solids and the possibility of cell recirculation exists. The process and system of the present invention can also provide advanced DDGS.

The processes and systems described herein also provide for the removal of the oil prior to fermentation, which in turn allows controlled addition of the oil for fermentation. Moreover, removal of the oil before fermentation may allow flexibility in the amount of oil present in the DDGS. That is, the oil is added to DDGS in different amounts to produce DDGS with different fat contents depending on the nutritional needs of particular animal species.

The processes and systems described herein can be verified using computer modeling such as Aspen modeling (see, e.g., U.S. Patent No. 7,666,282). For example, commercially available modeling software Aspen Plus ^® (Aspen Technology, Inc., Burlington, Mass.) Is available from the American Institute of Chemical Engineers, Inc., New York, NY , And DIPPR (Design Institute for Physical Property Research) to develop an Aspen model for integrated alcohol fermentation, refining and water management processes. Such process modeling can perform many basic engineering calculations, such as mass and energy balances, vapor / liquid equilibrium, and reaction rate calculations. In order to generate the Aspen model, the information input may include, for example, experimental data, the water content and composition of the feedstock, the temperature for mash cooking and flashing, the saccharification conditions (e.g., enzyme feed, starch conversion, temperature, , A fermentation condition (e.g., microbial feed, glucose conversion, temperature, pressure), a degassing condition, a solvent column, a preflash column, a condenser, an evaporator, a centrifuge and the like.

Recombinant  microbe

While not intending to be bound by theory, the process described herein is useful with respect to any alcohol-producing microorganism, particularly a recombinant microorganism that produces an alcohol at a lower than its resistance level.

Alcohol-producing microorganisms are known in the art. For example, methanotrophs (e.g., Methylosinus fermentative oxidation of methane by trichosporium) generates methanol, a yeast strain CEN.PK113-7D (CBS 8340, the Centraal Buro voor Schimmelculture; van Dijken, et al, Enzyme Microb Techno 26:... 706-714, 2000) produces ethanol. Recombinant microorganisms that produce alcohol are also known in the art (see, for example, Ohta, et al., Appl. Environ. Microbiol. 57: 893-900, 1991; Underwood, et al., Appl. Microbiol. 68: 1071-1081, 2002; Shen and Liao, Metab. Eng. 10: 312-320, 2008; Hahnai, et al., Appl. Environ Microbiol. 73: 7814-7818, 2007; Science, 267: 240-351, 1992; Zhang, et al., Science 267: 240-351, 243, 1995; U.S. Patent Application Publication No. 2007/0031918 A1; U.S. Patent No. 7,223,575; U.S. Patent No. 7,741,119; U.S. Patent No. 7,851,188; U.S. Patent Application Publication No. 2009/0203099 A1; U.S. Patent Application Publication No. 2009 / 0246846 A1, and PCT Application Publication No. WO 2010/075241, all of which are incorporated herein by reference).

In addition, microorganisms can be modified using recombinant techniques to generate recombinant microorganisms capable of producing product alcohols such as ethanol and butanol. Microorganisms that can be transformed with recombinant volume to produce the product alcohol from the biosynthetic pathway in Clostridium (Clostridium), jimo Pseudomonas (Zymomonas), Escherichia (Escherichia), Salmonella (Salmonella), Serratia marcescens (Serratia) , Er Winiah (Erwinia), keulrep when Ella (Klebsiella), Shh Gela (Shigella), also co kusu (Rhodococcus), Pseudomonas (Pseudomonas), Bacillus (Bacillus), Lactobacillus bacteria (Lactobacillus), Enterobacter nose kusu (Enterococcus), But are not limited to, Alcaligenes , Kl ebsiella , Paenibacillus , Arthrobacter , Corynebacterium , Brevibacterium , (Schizosaccharomyces), Cluj Vero My process (Kluyveromyces), Yarrow subtotal (Yarrowia), blood teeth (Pichia), Candida (Candida), Hanse Cronulla (Han enula s), Issa teuchen Escherichia (Issatchenkia), also Includes members of the Saccharomyces family. In some embodiments, the recombinant microorganism is selected from the group consisting of Escherichia Escherichia E. coli , Lactobacillus plantarum , Kluy veromyces ( Kluy veromyces lactis) lactis), Cluj Vero My Marcia Seth Augustine (Kluyveromyces marxianus) and in Saccharomyces (Saccharomyces Mai Jia access Celebi cerevisiae ). In some embodiments, the recombinant microorganism is yeast. In some embodiments, the recombinant microorganism is selected from the group consisting of Saccharomyces , Zygosaccharomyces , Schizosaccharomyces , Dekkera , Torulopsis , a positive yeast-Mai access (Brettanomyces), and crab tree selected from Candida (Candida) of some species. Gargoyle-positive yeast species include Saccharomyces cerevisiae, Saccharomyces cerevisiae, Schizosaccharomyces pombe , Saccharomyces bacillus , Saccharomyces spp. bayanus ), Saccharomyces < RTI ID = 0.0 > mikitae , < / RTI > Saccharomyces paradoxus , Zygosaccharomyces rouxii , Candida glabrata , and the like.

Saccharomyces cerevisiae is well known in the art and is commercially available from the American Type Culture Collection (Rockville, MD), Centraalbureau voor Schimmelcultures (CBS) Feral Biodiversity Center, LeSaffre, Gert Strand AB, Ferm Solutions, North American Bioproducts, Martrex, and Lallemand. ≪ / RTI > including but not limited to < / RTI > Saccharomyces cerevisiae includes BY4741, CEN.PK 113-7D, Ethanol Red ^占 yeast, Ferm Pro ^TM Yeast, Bio-Ferm ^® XR yeast, Gert Strand Prestige Batch Turbo alcohol yeast, Gert Strand Pot Distillers yeast, Gert Strand Distillers Turbo yeast, FerMax ^TM Green yeast, FerMax ^TM Gold yeast, Thermosacc ^® yeast, BG-1, PE-2, CAT-1, CBS7959, CBS7960, and CBS7961.

In some embodiments, the microorganism may be passivated or encapsulated. For example, the microorganism can be produced by using alginate, calcium alginate, or polyacrylamide gel, or on a variety of high surface area support matrices such as diatomaceous earth, celite, diatomaceous earth, silica gel, plastic, Lt; RTI ID = 0.0 > encapsulation < / RTI > In some embodiments, ISPR can be used with immobilized or encapsulated microorganisms. This combination can enhance productivity such as specific volume productivity, metabolic rate, yield of product alcohol, and tolerance to product alcohol. In addition, passivation and encapsulation can minimize the effects of process conditions such as shear on the microbial bed.

The production of butanol using fermentation and also microorganisms that produce butanol is described, for example, in U.S. Patent No. 7,851,188 and U.S. Patent Publication No. 2007/0092957; 2007/0259410; 2007/0292927; 2008/0182308; 2008/0274525; 2009/0155870; 2009/0305363; And 2009/0305370, the entire contents of each of which are incorporated herein by reference. In some embodiments, the microorganism may be engineered to include biosynthetic pathways. In some embodiments, the biosynthetic pathway is a engineered butanol biosynthetic pathway. In some embodiments, the biosynthetic pathway converts pyruvate to a fermentation product. In some embodiments, the biosynthetic pathway converts pyruvate and also amino acids to fermentation products. In some embodiments, at least one, at least two, at least three, or at least four polypeptides that catalyze substrate conversion of the pathway are encoded by heterologous polynucleotides in the microorganism. In some embodiments, all polypeptides that catalyze substrate conversion of the pathway are encoded by heterologous polynucleotides in the microorganism. In some embodiments, the polypeptide and / or substrate catalyzing the conversion of the substrate to product conversion of acetolactate to 2,3-dihydroxyisovalerate can be achieved by converting the product of isobutyraldehyde to isobutanol, May use reduced nicotinamide adenine dinucleotide (NADH) as a cofactor.

Biosynthetic pathway

Biosynthetic pathways for producing isobutanol which may be used include those described in U.S. Patent No. 7,851,188, which is incorporated herein by reference. In one embodiment, the isobutanol biosynthetic pathway comprises product conversion of the following substrates:

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

b) the conversion of acetolactate to 2,3-dihydroxyisovalerate, which can be catalysed, for example, by acetohydroxy acid reductoisomerase;

c) conversion of 2,3-dihydroxyisovvalerate, which can be catalyzed, for example, by acetohydroxy acid dehydratase to alpha -ketoisovvalerate;

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

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

In another embodiment, the isobutanol biosynthetic pathway comprises product conversion of the following substrates:

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

b) the conversion of acetolactate to 2,3-dihydroxyisovalerate, which can be catalysed, for example, by ketol-acid reductoisomerase;

c) conversion of 2,3-dihydroxyisovvalerate to alpha -ketoisovalerate, which can be catalysed, for example, by dihydroxy acid dehydratase;

d)? -ketoisovalerate, which can be catalyzed, for example, by transaminase or valine dehydrogenase;

e) conversion of valine to isobutylamine, for example, which can be catalyzed by valine decarboxylase;

f) the conversion of isobutylamine to isobutyraldehyde, which can be catalyzed, for example, by omega transaminase; And

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

In another embodiment, the isobutanol biosynthetic pathway comprises product conversion of the following substrates:

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

b) the conversion of acetolactate to 2,3-dihydroxyisovalerate, which can be catalysed, for example, by acetohydroxy acid reductoisomerase;

c) conversion of 2,3-dihydroxyisovvalerate to alpha -ketoisovalerate, which can be catalysed, for example, by acetohydroxy acid dehydratase;

d) the conversion of alpha -ketoisovalerate to isobutyryl-CoA, which can be catalyzed, for example, by branched chain ketoacid dehydrogenases;

e) conversion of isobutyryl-CoA to isobutyraldehyde, which can be catalyzed, for example, by acylating an aldehyde dehydrogenase; And

f) the conversion of isobutyraldehyde to isobutanol, which can be catalysed, for example, by branched alcohol dehydrogenases.

Biosynthetic pathways for producing 1-butanol which may be used are described in U.S. Patent Application Publication No. 2008/0182308, which is incorporated herein by reference. In one embodiment, the 1-butanol biosynthetic pathway involves product conversion of the following substrates:

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

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

c) conversion of 3-hydroxybutyryl-CoA to crotyl-CoA, which can be catalyzed, for example, by crotonazene;

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

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

f) Conversion of butyraldehyde to 1-butanol, which can be catalyzed by butanol dehydrogenase.

Biosynthetic pathways for producing 2-butanol that may be used include those described in U.S. Patent Application Publication No. 2007/0259410 and U.S. Patent Application Publication No. 2009/0155870, incorporated herein by reference. In one embodiment, the 2-butanol biosynthetic pathway involves product conversion of the following substrates:

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

b) the conversion of alpha-acetolactate to acetonine, which can be catalyzed, for example, by acetolactate decarboxylase;

c) conversion of acetone to 3-amino-2-butanol, which can be catalysed, for example, by acetoninaminase;

d) conversion of 3-amino-2-butanol to 3-amino-2-butanol phosphate, which can be catalyzed, for example, by amino butanol kinase;

e) conversion of 3-amino-2-butanol phosphate to 2-butanol, which can be catalyzed, for example, by aminobutanol phosphate phosphorylase; And

f) Conversion of 2-butanone, which can be catalysed, for example, by butanol dehydrogenase to 2-butanol.

In another embodiment, the 2-butanol biosynthetic pathway comprises product conversion of the following substrates:

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

b) the conversion of alpha-acetolactate to acetone, which can be catalyzed, for example, by acetolactate decarboxylase;

c) conversion of acetone to 2,3-butanediol, which can be catalyzed, for example, by butanediol dehydrogenase;

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

e) Conversion to 2-butanone 2-butanol, which can be catalysed, for example, by butanol dehydrogenase.

Biosynthetic pathways for producing 2-butanone that may be used are described and included in U.S. Patent Application Publication No. 2007/0259410 and U.S. Patent Application Publication No. 2009/0155870, which are incorporated herein by reference. In one embodiment, the 2-butanone biosynthetic pathway involves product conversion of the following substrates:

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

b) the conversion of alpha-acetolactate to acetone, which can be catalyzed, for example, by acetolactate decarboxylase;

c) conversion of acetone to 3-amino-2-butanol, which can be catalysed, for example, by acetoninaminase;

d) conversion of 3-amino-2-butanol to 3-amino-2-butanol phosphate, which can be catalyzed, for example, by amino butanol kinase; And

e) Conversion of 3-amino-2-butanol phosphate to 2-butanone, which can be catalyzed, for example, by aminobutanol phosphate phosphorylase.

In another embodiment, the 2-butanone biosynthetic pathway involves product conversion of the following substrates:

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

b) the conversion of alpha-acetolactate to acetone, which can be catalyzed, for example, by acetolactate decarboxylase;

c) conversion of acetone to 2,3-butanediol, which can be catalyzed, for example, by butanediol dehydrogenase; And

d) Conversion of 2,3-butanediol to 2-butanone, which can, for example, be catalyzed by diol dehydratase.

The terms "acetohydroxy acid synthase", "acetolactate synthase" and "acetolactate synthase" (abbreviated as "ALS") are used interchangeably herein and include acetolactate and (Or polypeptides) having an enzyme activity catalyzing the conversion to CO ₂ . An example of acetolactate synthase is known from EC No. 2.2.1.6 (Enzyme Nomenclature 1992, Academic Press, San Diego). These unmodified enzymes are called Bacillus subtilis subtilis) (each, Gene Bank (GenBank) Number: CAB 15618 (SEQ ID NO: 1), Z99122 (SEQ ID NO: 2), NCBI (National Center for Biological Information (National Center for Biotechnology Information)) amino acid sequence, NCBI nucleotide in the sequence), when keulrep Ella peune feather California (Klebsiella pneumoniae) (Gene Bank ID: AAA25079 (SEQ ID NO: 3), M73842 (SEQ ID NO: 4)), and Lactobacillus nose kusu lactis (Lactococcus lactis) (Gene Bank ID: AAA25161 (SEQ ID NO: 5), L16975 (SEQ ID NO: No. 6)). ≪ / RTI >

The term "ketol-acid reductoisomerase"("KARI"),"acetohydroxy acid isomeruryldextyrase "," acetohydroxy acid reductoisomerase " ) -Acetolactate with 2,3-dihydroxyisovalerate. The term " polypeptide " refers to a polypeptide (or polypeptides) having an enzymatic activity that catalyzes the reaction of acetolactate with 2,3-dihydroxyisovalerate. Examples of the KARI enzyme EC number EC 1.1.1.86: can be divided into (see Enzyme Nomenclature 1992, Academic Press, San Diego), Escherichia coli (Escherichia coli) (Gene bank number: NP_418222 (SEQ ID NO: 7), NC_000913 (SEQ ID NO: 8), to), my process three Levy Jia as Saccharomyces (Saccharomyces cerevisiae) (Gene bank number: NP_013459 (SEQ ID NO: 9), NC_001144 (SEQ ID NO: 10)), a meta Noko kusu grains arm rudiseu (Methanococcus maripaludis) (Gene bank number: CAF30210 (SEQ ID NO: 11), BX957220 (SEQ ID NO: 12)), and Bacillus subtilis (Bacillus subtilis) (Gene Bank Number: CAB14789 (SEQ ID NO: 13), No. Z99118 (SEQ ID NO: No. 14)), which are commercially available. KARI is an aerostrict cocktail ( Anaerostipes Kacc variants "K9G9" and "K9D3" (SEQ ID NOs: 15 and 16, respectively). The ketol-acid reductoisomerase (KARI) enzyme is described in U.S. Patent Application Publication Nos. 2008/0261230, 2009/0163376, and 2010/0197519, and PCT Application Publication No. WO / 2011/041415 , Which is incorporated herein by reference. Examples of KARI's described herein include Lactococcus lactis , Vibrio cholera ), Pseudomonas aeruginosa aeruginosa) PAO1, and Pseudomonas fluorescein sense (Pseudomonas fluorescens PF5 mutants. In some embodiments, KARI utilizes NADH. In some embodiments, KARI utilizes reduced nicotinamide adenine dinucleotide phosphate (NADPH).

The terms "acetohydroxy acid dehydratase" and "dihydroxy acid dehydratase"("DHAD") refer to the conversion of 2,3-dihydroxyisobalurate to a- Refers to polypeptides (or polypeptides) having catalytic enzyme activity. An example of an acetohydroxy acid dehydratase is known under EC number 4.2.1.9. These enzymes include Escherichia coli (Gene Bank No .: YP_026248 (SEQ ID NO: 17), NC000913 (SEQ ID NO: 18)), Saccharomyces cerevisiae (Gene Bank No .: NP_012550 : 19), NC 001142 (SEQ ID NO: 20), M. maripaludis (Gene Bank number: CAF29874 (SEQ ID NO: 21), BX957219 (SEQ ID NO: 22) But not limited to, B. subtilis (Gene Bank No .: CAB14105 (SEQ ID NO: 23), Z99115 (SEQ ID NO: 24)), L. Iactis , and N. crassa U.S. Patent Application Publication No. 2010/0081154 and U.S. Patent No. 7,851,188, which are incorporated herein by reference, disclose that dehydrohalogenation from Streptococcus mutans , Describes DHAD containing Roxithan dehydratase (DHAD).

The term "side chain α-keto acid decarboxylase", "α-keto acid decarboxylase", "α-ketoisovalerate decarboxylase" or "2-ketoisovalerate decarboxylase" KIVD ") refers to polypeptides (or polypeptides) having enzymatic activity catalyzing the conversion of alpha -ketoisovalerate to isobutyraldehyde and CO ₂ . Quot; side chain a-keto acid decarboxylase "is known as EC No. 4.1.1.72 and is referred to as Lactococcus lactis) (Gene bank number: AAS49166 (SEQ ID NO: 25), AY548760 (SEQ ID NO: 26); CAG34226 (SEQ ID NO: 27), AJ746364 (SEQ ID NO: 28), Salmonella typhimurium (Salmonella typhimurium) (Gene bank number: NP_461346 (SEQ ID NO: 29), NC_003197 (SEQ ID NO: 30)), Clostridium acetonitrile unit Tilikum (Clostridium acetobutylicum) (Gene bank number: NP_149189 (SEQ ID NO: 31), NC_001988 ( SEQ ID NO: 32)), M. < RTI ID = 0.0 > M. caseolyticus (SEQ ID NO: 33), and EL. (SEQ ID NO: 34). ≪ / RTI >

The term "branched alcohol dehydrogenase"("ADH") refers to polypeptides (or polypeptides) having an enzymatic activity catalyzing the conversion of isobutyraldehyde to isobutanol. Examples of branched alcohol dehydrogenases are known under EC No. 1.1.1.265, but may also be classified under other alcohol dehydrogenases (specifically EC 1.1.1.1 or 1.1.1.2). The alcohol dehydrogenase may be NADPH-dependent or NADH-dependent. This enzyme is the My process three Levy Jia in Saccharomyces (Saccharomyces cerevisiae) (Gene bank number: NP_010656 (SEQ ID NO: 35), NC_001136 (SEQ ID NO: 36), NP_014051 (SEQ ID NO: 37), NC_001145 (SEQ ID NO: 38)), Escherichia coli (Escherichia coli) (Gene bank number: NP_417484 (SEQ ID NO: 39), NC_000913 (SEQ ID NO: 40)), seed. One including, whereby; tikum butyl acetoacetate (C. acetobutylticum) (44): : 43), NC_003030 ( SEQ ID NO: Gene bank number: NP_349892 (SEQ ID NO:: 41), NC_003030 (SEQ ID NO: NP_349891 (SEQ ID NO: 42)) And are available from a number of sources, including but not limited to. U.S. Patent Application Publication No. 2009/0269823 describes an alcohol dehydrogenase (ADH) from Achromobacter xylosoxidans , SadB. Alcohol dehydrogenase is also known to be involved in the production of endogenous ADH and Beijerinkia < RTI ID = 0.0 > indica ) ADH (described in U.S. Patent Application Publication No. 2011/0269199, incorporated herein by reference).

The term "butanol dehydrogenase" refers to polypeptides (polypeptides) having an enzymatic activity of catalyzing the conversion of isobutyraldehyde to isobutanol or the conversion of 2-butanone and 2-butanol. Butanol dehydrogenase is a subset of a broad family of alcohol dehydrogenases. Butanol dehydrogenase is NAD- or NADP-dependent. NAD-dependent enzymes are known as EC 1.1.1.1 and include, for example, Rhodococcus ruber (Gene Bank No .: CAD36475, AJ491307). NADP-dependent enzymes are known as EC 1.1.1.2 and include, for example, Pyrococcus furiosus (Gene Bank number: AAC25556, AF013169). In addition, butanol dehydrogenase is available from Escherichia coli (Gene Bank No: NP 417484, NC_000913) and cyclohexanol dehydrogenase is available from Acinetobacter sp . ) (Gene Bank No .: AAG10026, AF282240). The term "butanol dehydrogenase" also refers to an enzyme that catalyzes the conversion of butyraldehyde to 1-butanol using NADH or NADPH as cofactor. Butanol dehydrogenase is, for example, C. acetobutylicum (Gene Bank No .: NP_149325, NC_001988; note: the enzyme has both aldehyde and alcohol dehydrogenase activity); NP_349891, NC_003030; And NP_349892, NC_003030) and Escherichia coli (Gene Bank No .: NP_417-484, NC_000913).

The term "side chain keto acid dehydrogenase" is typically NAD ⁺ isobutyryl -CoA (isobutyryl of, α- Kane toy Nassau multiple rate using the (nicotinamide adenine di-nucleotide) as an electron acceptor-coenzyme (Or polypeptides) having an enzyme activity that catalyzes the conversion of A to A). An example of a branched chain keto acid dehydrogenase is known as EC No. 1.2.4.4. These side chain keto acid dehydrogenases are composed of four subunits and the sequence from all subunits is Bacillus subtilis (Gene Bank number: CAB14336 (SEQ ID NO: 45), Z99116 (SEQ ID NO: 46); CAB14335 (SEQ ID NO: 47), Z99116 (SEQ ID NO: 48), CAB14334 (SEQ ID NO: 49), Z99116 (SEQ ID NO: 55), M57613 (SEQ ID NO: 56), AAA65617 (SEQ ID NO: 57), Pseudomonas putida (Gene Bank No .: AAA65614 ), M57613 (SEQ ID NO: 58), and AAA65618 (SEQ ID NO: 59), M57613 (SEQ ID NO: 60)).

The term "acylated aldehyde dehydrogenase" refers to a polypeptide (or polypeptide) having an enzymatic activity that catalyzes the conversion of isobutyryl-CoA to isobutyraldehyde, typically using NADH or NADPH as the electron donor ). Examples of acylated aldehyde dehydrogenases are known as EC numbers 1.2.1.10 and 1.2.1.57. These enzymes include Clostridium < RTI ID = 0.0 > beijerinckii) (Gene Bank ID: AAD31841 (SEQ ID NO: 61), AF157306 (SEQ ID NO: 62)), Clostridium acetonitrile unit Tilikum (Clostridium acetobutylicum) (Gene bank number: NP_149325 (SEQ ID NO: 63), NC_001988 (SEQ ID NO: 64); NP_149199 (SEQ ID NO: 65), NC_001988 (SEQ ID NO: 66), c), footage Pseudomonas (Pseudomonas putida ) (Gene Bank No .: AAA89106 (SEQ ID NO: 67), U13232 (SEQ ID NO: 68)), and Thermus stearothermophilus but are not limited to, a variety of sources including, but not limited to, thermophilus (Gene Bank No .: YP_145486 (SEQ ID NO: 69), NC_006461 (SEQ ID NO: 70)

The term "transaminase" refers to polypeptides (or polypeptides) having an enzymatic activity catalyzing the conversion of alpha -ketoisovalerate to L-valine using alanine or glutamate as an amine donor. Examples of transaminases are known as EC numbers 2.6.1.42 and 2.6.1.66. Such enzymes are available from multiple sources. Examples of sources for alanine-dependent enzymes include Escherichia coli (Gene Bank No .: YP_ 026231 (SEQ ID NO: 71), NC_000913 (SEQ ID NO: 72)) and Bacillus licheniformis ) (Gene Bank No .: YP_093743 (SEQ ID NO: 73), NC_006322 (SEQ ID NO: 74)). Examples of sources for glutamate-dependent enzymes include Escherichia coli (Gene Bank No .: YP_026247 (SEQ ID NO: 75), NC_000913 (SEQ ID NO: 76)), Saccharomyces cerevisiae (SEQ ID NO: 77), NC_001142 (SEQ ID NO: 78) and Methanobacterium thermoautotrophicum (Gene Bank No .: NP_276546 (SEQ ID NO: 79), NC_000916 : 80)), but is not limited thereto.

The term "valine dehydrogenase" typically encompasses the enzymatic activity of catalyzing the conversion of alpha -ketoisobalrate to L-valine using NAD (P) H as the electron donor and ammonia as the amine donor (Or < / RTI > polypeptides). Examples of the valine dehydrogenase are known to the EC number 1.4.1.8 and 1.4.1.9, and these enzymes are Streptomyces nose Elie Le Colo (Streptomyces a) coelicolor) (Gene bank number: NP_628270 (SEQ ID NO: 81), NC_003888 (SEQ ID NO: 82)) and Bacillus subtilis (Gene Bank Number: CAB14339 (SEQ ID NO:: 83), Z99116 (84 SEQ ID NO) But are not limited to, a number of sources.

The term "valynecarboxylase" refers to polypeptides (or polypeptides) having an enzymatic activity catalyzing the conversion of L-valine to isobutylamine and CO ₂ . An example of valindeca fucilase is known as EC number 4.1.1.14. Such enzymes include, for example, Streptomyces irregularities Defining Keyence (Streptomyces viridifaciens) (Gene bank number: AAN10242 (SEQ ID NO: 85) is found in Streptomyces, such as (SEQ ID NO: 86)), AY116644.

The term "omega transaminase" refers to polypeptides (or polypeptides) having an enzymatic activity catalyzing the conversion of isobutylamine to isobutyraldehyde, using the appropriate amino acid as the amine donor. An example of an omega transaminase is known as EC number 2.6.1.18 and is derived from Alcaligenes denitrificans (AAP92672 (SEQ ID NO: 87), AY330220 (SEQ ID NO: 88)), Ralstonia eutropha) (Gene Bank ID: YP_294474 (SEQ ID NO: 89), NC_007347 (SEQ ID NO: 90)), and guilloche Nella ohneyi den sheath (Shewanella oneidensis) (Gene bank number: NP_719046 (SEQ ID NO: 91), NC_004347 (SEQ ID NO: 92)), and Pseudomonas footage is (Pseudomonas but are not limited to, putida (Gene Bank number: AAN66223 (SEQ ID NO: 93), AEO16776 (SEQ ID NO: 94)).

The term "acetyl-CoA acetyltransferase" refers to polypeptides (or polypeptides) having an enzymatic activity catalyzing the conversion of two molecules of acetyl-CoA to acetoacetyl-CoA and coenzyme A (CoA). An example of an acetyl-CoA acetyltransferase is an acetyl-CoA acetyltransferase with substrate preference (positive reaction) for short chain acyl-CoA and acetyl-CoA, and an enzyme with a broader substrate range (EC 2.3.1.16) It will also be functional, but EC 2.3.1.9 [Enzyme Nomenclature 1992, Academic Press, San Diego]. Acetyl-CoA acetyltransferase may be obtained from a number of sources such as, for example, Escherichia coli (Gene Bank No: NP_416728, NC_000913; NCBI (National Center for Biotechnology Information) amino acid sequence, NCBI nucleotide sequence), Clostridium aceto (Gene Bank Number: NP_015297, NC_001148), Bacillus subtilis (Gene Bank Number: NP_390297, NC_000964), and Saccharomyces cerevisiae (Gene Bank No .: NP_349476.1, NC_003030; NP_149242, Lt; / RTI >

The term "3-hydroxybutyryl-CoA dehydrogenase" refers to polypeptides (or polypeptides) having an enzymatic activity that catalyze the conversion of acetoacetyl-CoA to 3-hydroxybutyryl-CoA. An example of a 3-hydroxybutyryl-CoA dehydrogenase is NADH-dependence ((S) -3-hydroxybutyryl-CoA having substrate preference for Lt; / RTI > Examples can be categorized as EC 1.1.1.35 and EC 1.1.1.30, respectively. In addition, 3-hydroxybutyryl-CoA dehydrogenase is NADPH-dependent and has a substrate preference for (S) -3-hydroxybutyryl-CoA or (R) -3-hydroxybutyryl- And are classified as EC 1.1.1.157 and EC 1.1.1.36, respectively. 3-hydroxy-butyryl -CoA dehydrogenase includes a plurality of sources, for example, Clostridium acetonitrile unit Tilikum (Clostridium acetobutylicum ) (Gene Bank No: NP_349314, NC_003030), Bacillus subtilis (Gene Bank No .: AAB09614, U29084), Ralstonia eutropha ) (Gene Bank No .: YP_294481, NC_007347), and Alcaligenes eutrophus ) (Gene Bank No .: AAA21973, J04987).

The term "crotonase" refers to polypeptides (or polypeptides) having an enzymatic activity catalyzing the conversion of 3-hydroxybutyryl-CoA to crotyl-CoA and H ₂ O. Examples of crotonazines may have substrate preferences for (S) -3-hydroxybutyryl-CoA or (R) -3-hydroxybutyryl-CoA and are described in EC 4.2.1.17 and EC 4.2.1.55 Can be classified. The crotonazate may be obtained from a number of sources such as, for example, Escherichia coli (Gene Bank No .: NP_415911, NC_000913), Clostridium acetobutylicum (Gene Bank No .: NP_349318, NC_003030), Bacillus subtilis No. CAB13705, Z99113), and Aeromonas caviae) (Gene bank number is available from BAA21816, D88825).

The term "butyryl-CoA dehydrogenase" refers to polypeptides (or polypeptides) having an enzymatic activity catalyzing the conversion of crotyl-CoA to butyryl-CoA. Examples of butyryl-CoA dehydrogenases are NADH-dependent, NADPH-dependent, or flavin-dependent, and can be classified as EC 1.3.1.44, EC 1.3.1.38, and EC 1.3.99.2, respectively. Butyryl -CoA dehydrogenase includes a plurality of sources, for example, Clostridium acetonitrile unit Tilikum (Clostridium acetobutylicum ) (Gene Bank No: NP_347102, NC_003030), Euglena gracilis) (Gene bank number: Q5EU90, AY741582), Streptomyces call Linus (Streptomyces collinus) (Gene bank number: AAA92890, U37135), and Streptomyces nose Elie Le Colo (Streptomyces coelicolor (Gene Bank number: CAA22721, AL939127).

The term "butyraldehyde dehydrogenase" refers to polypeptides (or polypeptides) having an enzymatic activity that catalyzes the conversion of butyryl-CoA to butyraldehyde using NADH or NADPH as an assistant . Unit having a preference for NADH butyraldehyde dehydrogenase are known as EC 1.2.1.57, and, for example, Clostridium bay jerin key (Clostridium beijerinckii ) (Gene Bank No .: AAD31841, AF157306) and Clostridium Acetobutylicum (Gene Bank No .: NP.sub .-- 149325, NC.sub .-- 001988).

The term "isobutyryl-CoA mutease" refers to polypeptides (or polypeptides) having an enzymatic activity catalyzing the conversion of butyryl-CoA to isobutyryl-CoA. This enzyme uses coenzyme B ₁₂ as a cofactor. An example of an isobutyryl-CoA mutease is known as EC number 5.4.99.13. The enzyme is called Streptomyces cinnamonensis cinnamonensis) (Gene bank number: AAC08713 (SEQ ID NO: 95), U67612 (SEQ ID NO: 96); CAB59633 (SEQ ID NO: 97), AJ246005 (SEQ ID NO: 98)), Streptomyces nose Eli coke (Streptomyces coelicolor) (Gene Bank Number: CAB70645 (SEQ ID NO: 99), AL939123 (SEQ ID NO: 100); CAB92663 (SEQ ID NO: 101), AL939121 (SEQ ID NO: 102)), and Streptomyces Abbe reumi subtilis (Streptomyces but are not limited to , avermitilis (SEQ ID NO: 103), NC_003155 (SEQ ID NO: 104), NP_824637 (SEQ ID NO: 105), NC_003155 It is found in myces.

The term "acetolactate decarboxylase" refers to polypeptides (or polypeptides) having an enzymatic activity catalyzing the conversion of alpha-acetolactate to acetone. Examples of acetolactate decarboxylases are known as EC 4.1.1.5 and include, for example, Bacillus subtilis (Gene Bank No .: AAA22223, L04470), Klebsiella terrigena (Gene Bank No .: AAA25054, L04507) and Klebsiella pneumoniae ) (Gene Bank No .: AAU43774, AY722056).

The term "acetoinaminase" or "acetoin transaminase" refers to polypeptides (or polypeptides) having an enzymatic activity catalyzing the conversion of acetone to 3-amino-2-butanol. Acetone aminase may utilize co-factor pyridoxal 5'-phosphate or NADH or NADPH. The resulting product may have (R) or (S) stereochemistry at the 3-position. Pyridoxal phosphate-dependent enzymes can use amino acids such as alanine or glutamate as amino donors. NADH- and NADPH-dependent enzymes can use ammonia as a secondary substrate. Suitable examples of NADH-dependent acetylinaminases also known as aminoalcohol dehydrogenases are described by Ito et al. (U.S. Patent No. 6,432,688). Examples of pyridoxal-dependent acetylinaminases include the amine: pyruvate aminotransferase described by Shin and Kim et al. (J. Org. Chem. 67: 2848-2853, 2002) Amine: pyruvate transaminase).

The term "acetoin kinase" refers to polypeptides (or polypeptides) having an enzymatic activity catalyzing the conversion of acetone to phosphoacetone. Acetone kinase can use ATP (adenosine triphosphate) or phosphoenolpyruvate as a phosphate donor in the reaction. For example, an enzyme that catalyzes a similar reaction in a similar substrate dihydroxyacetone includes an enzyme known as EC 2.7.1.29 (Garcia-Alles, et al., Biochemistry 43: 13037-13046, 2004 ).

The term "acetoinphosphate aminase" refers to polypeptides (or polypeptides) having an enzymatic activity catalyzing the conversion of phosphoacetone to 3-amino-2-butanol O-phosphate. Acetoin phosphate aminase may use the coenzyme pyridoxal 5'-phosphate, NADH, or NADPH. The resulting product may have (R) or (S) stereochemistry at the 3-position. Pyridoxal phosphate-dependent enzymes can use amino acids such as alanine or glutamate. NADH-dependent and NADPH-dependent enzymes can use ammonia as a secondary substrate. Although no enzyme has been reported to catalyze the reaction in phosphoacetone, there is a pyridoxal phosphate-dependent enzyme proposed to perform homologation in a similar substrate serinol phosphate (see Yasuta, et al. Appl. Environ. Microbiol. 67: 4999-5009, 2001).

The term "amino butanol phosphate phospholipase " also referred to as" amino alcohol O-phosphate lyase "refers to a polypeptide having an enzymatic activity that catalyzes the conversion of 3-amino-2-butanol O- (Or polypeptides). Aminobutanol phosphate phospho-lyase can utilize the coenzyme pyridoxal 5'-phosphate. Enzymes catalyzing a similar reaction in a similar substrate 1-amino-2-propanol phosphate have been reported (Jones, et al., Biochem J. 134: 167-182, 1973). U.S. Patent Application Publication No. 2007/0259410 discloses an organism Erwinia carotovora . < / RTI >

The term "aminobutanol kinase" refers to polypeptides (or polypeptides) having an enzymatic activity catalyzing the conversion of 3-amino-2-butanol to 3-amino-2-butanol O-phosphate. Aminobutanol kinase can utilize ATP as a phosphate donor. Although enzymes catalyzing this reaction have not been reported in 3-amino-2-butanol, enzymes catalyzing homologous reactions in similar substrates ethanolamine and 1-amino-2-propanol have been reported (Jones , et al., supra). U.S. Patent Application Publication No. 2009/0155870 describes an amino alcohol kinase of Erwinia carotovora subsp. Atroseptica in Example 14.

The term "butanediol dehydrogenase " also known as" acetoyl reductase "refers to polypeptides (or polypeptides) having an enzymatic activity catalyzing the conversion of acetone to 2,3-butanediol. Butanedialdehydrogenase is a subset of a broad family of alcohol dehydrogenases. The butanediol dehydrogenase enzyme may have specificity for the production of (R) - or (S) - stereochemistry at the time of alcohol production. (S) -specific butanediol dehydrogenase is known as EC 1.1.1.76 and is described, for example, in Klebsiella pneumoniae ) (Gene Bank number: BBA13085, D86412). (R) - specific butanediol dehydrogenase are known as EC 1.1.1.4 and, for example, Bacillus cereus (Bacillus cereus) (Gene bank number NP 830481, NC_004722; available from AAP07682, AE017000), and Lactococcus lactis kusu nose (Lactococcus lactis) (Gene bank number AAK04995, AE006323).

The term "butanediol dehydratase " also known as" dial dehydratase "or" propanediol dehydratase "refers to an enzyme having the enzymatic activity of catalyzing the conversion of 2,3-butanediol to 2- Refers to polypeptides (or polypeptides). The butanediol dehydratase can utilize coenzyme adenosyl cobalamin (also known as coenzyme Bw or vitamin B12, although vitamin B12 can also refer to other forms of cobalamin, not coenzyme B12). Adenosyl cobalamin-dependent enzymes are known as EC 4.2.1.28, and include, for example, Klebsiella oxytoca) (Gene bank number: AA08099 (alpha subunit), D45071; BAA08100 (beta subunit), D45071; and BBA08101 (gamma subunit), D45071 (all three subunits are also noted a need for an activity), and keurep when Ella Klebsiella pneumonia ) (available from Gene Bank Number: AAC98384 (alpha subunit), AF102064; Gene Bank Number: AAC98385 (beta subunit), AF102064, Gene Bank Number: AAC98386 (Gamma Subunit), AF102064. Other suitable Diallo to Hydra hydratase is Salmonella typhimurium (Salmonella typhimurium ) (Gene Bank Number: AAB84102 (Giant Subunit), AF026270; Gene Bank Number: AAB84103 (intermediate subunit), AF026270; Gene Bank Number: AAB84104 (small subunit), AF026270); And Lactobacillus < RTI ID = 0.0 > collinoides ) (Gene Bank Number: CAC82541 (giant subunit), AJ297723; Gene Bank Number: CAC82542 (intermediate subunit); AJ297723; Gene Bank Number: CAD01091 (small subunit), AJ297723); And enzymes from Lactobacillus brevis (in particular strains CNRZ 734 and CNRZ 735, see Speranza, et al., J. Agric. Food Chem. 45: 3476-3480, 1997), and from nucleotide sequences encoding the corresponding enzymes But are not limited to, the available B12-dependent dialdehyde hydratase. Methods for the isolation of didehydratase genes are well known in the art (see, for example, U.S. Patent No. 5,686,276).

The term " pyruvate decarboxylase "refers to polypeptides (or polypeptides) having enzymatic activity catalyzing decarboxylation of pyruvic acid to acetaldehyde and carbon dioxide. Pyruvate dehydrogenase is known as EC number 4.1.1.1. These enzymes are in my process to the three Levy Jia Saccharomyces (Saccharomyces cerevisiae) (Gene bank number: CAA97575 (SEQ ID NO: 107), CAA97705 (SEQ ID NO: 109), CAA97091: It is found in a number of yeast containing (SEQ ID NO: 111)).

It will be appreciated that a microorganism comprising an isobutanol biosynthetic pathway as provided herein may comprise one or more additional modifications. U.S. Patent Application Publication No. 2009/0305363 (incorporated herein by reference) discloses a method for the production of acetolactate synthetase from cytoplasm and the production of acetolactate from pyruvate by treating the yeast for substantial elimination of skin bait decarboxylase activity ≪ / RTI > In some embodiments, the microorganism is a variant for reducing glycerol-3-phosphate dehydrogenase activity and / or as described in U.S. Patent Application Publication No. 2009/0305363 (incorporated herein by reference), pyruvate decarboxylase Destruction of at least one gene encoding a polypeptide having activity, or destruction of at least one gene encoding a regulatory element that controls pyruvate decarboxylase gene expression, and / or the disruption of at least one gene encoding a polypeptide having activity (US Patent Publication No. 2010/0120105 The Entner-Doudoroff Pathway as described in U.S. Patent Application Serial No. 10/1992, the disclosure of which is incorporated herein by reference, or a modification that provides increased carbon efflux through equivalent balance reduction. Other variations include the incorporation of at least one polynucleotide encoding a polypeptide that catalyzes a step in a pyruvate-utilizing biosynthetic pathway. Other variations include deletion, mutation, and / or substitution of at least one endogenous polynucleotide encoding a polypeptide having acetolactate reductase activity. In some embodiments, the polypeptide having acetolactate reductase activity is YMR226C (SEQ ID NO: 127, 128) of Saccharomyces cerevisiae or a homologue thereof. Further modifications include deletions, mutations and / or substitutions in endogenous polynucleotides encoding polypeptides with aldehyde dehydrogenase and / or aldehyde oxadase activity. In some embodiments, the polypeptide having aldehyde dehydrogenase activity is ALD6 from Saccharomyces cerevisiae or analogs thereof. Genetic variants that have the effect of reducing yeast-producing host cell pdc- inhibiting glucose are described in U.S. Patent Application Publication No. 2011/0124060, incorporated herein by reference. In some embodiments, the deleted or down-regulated pyruvate decarboxylase is selected from the group consisting of PDC1 , PDC5 , PDC6 , and combinations thereof. In some embodiments, the pyruvate decarboxylase is selected from the enzymes of Table 1. In some embodiments, the microorganism may contain deletion or down-regulation of a polynucleotide encoding a polypeptide that catalyzes the conversion of glyceraldehyde-3-phosphate to glycerate 1,3, bisphosphate. In some embodiments, the enzyme that catalyzes the reaction is glyceraldehyde-3-phosphate dehydrogenase.

[Table 1]

In some embodiments, any particular nucleic acid molecule or polypeptide is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence or polypeptide sequence described herein. Can be the same. As is known in the art, the term "homology percentage" is the relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences. In the art, "homology" also refers to the degree of sequence relatedness between a polypeptide or polynucleotide sequence, as it may be the case, as determined by the correspondence between the strings of such sequences. Homology and similarity are described in Computational Molecular Biology (Lesk, AM, Ed.) Oxford University: NY (1988); Biocomputing: Informatics and Genome Projects (Smith, DW, Ed.) Academic: NY (1993); Computer Analysis of Sequence Data , Part I ( Griffin, AM, and Griffin, HG, Eds.) Humania: NJ (1994); Sequence Analysis in Molecular Biology (von Heinje, G., Ed.) Academic (1987); and Sequence Analysis Primer (Gribskov, M. and Devereux, J., Eds.) Stockton: NY (1991).

Methods for measuring homology are designed to provide the best match between tested sequences. Methods of measuring homogeneity and similarity are encrypted in commercially available computer programs. Sequence alignments and percent homogeneity calculations can be performed using the MegAlign ^TM program of the LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wis.), Madison, Wis. Multiple alignments of sequences are described in Clustal V (Higgins and Sharp, CABIOS. 5: 151-153, 1989; Higgins, et al., Comput. Appl. Biosci. 8: 189-191, ) Containing a number of various algorithms including the clusteral V method of alignment corresponding to the alignment method identified in the MegAlign ^TM program of the LASERGENE bioinformatics computing suite (manufacturer: DNASTAR Inc.) Can be carried out using a star sorting method. For multiple alignments, the default value corresponds to GAP PENALTY = 10 and GAP LENGTH PENALTY = 10. The default parameters for calculation of percent identity and pairwise alignment of protein sequences using the clustering method are KTUPLE = 1, GAP PENALTY = 3, WINDOW = 5, and DIAGONALS SAVED = 5. For nucleic acids, these parameters are KTUPLE = 2, GAP PENALTY = 5, WINDOW = 4 and DIAGONALS SAVED = 4. It is possible to obtain homogeneity percentages by aligning the sequences using a Cluster V program and then observing the sequence distance table in the same program. Clustal W methods of alignment are also available and are available from Clustal W (Higgins and Sharp, CABIOS. 5: 151-153, 1989; Higgins, et al., Comput. Appl. Biosci. 8: 189-191, 1992) and corresponds to the alignment method revealed in the MegAlign ^TM v6.1 program of the LASERGENE bioinformatics calculation set (DNASTAR Inc.). (GAP PENALTY = 10, GAP LENGTH PENALTY = 0.2, Delay Divergen Seqs (%) = 30, DNA transfer weight = 0.5, Weight Matrix = Gonnet Series, DNA Weight Matrix = IUB). After aligning the sequence with the cluster W program, it is possible to observe the sequence distance table in the same program to obtain the percent identity.

Standard recombinant DNA and molecular cloning techniques are well known in the art and are described in Sambrook et al. (Sambrook, J., Fritsch, EF and Maniatis, T. (Molecular Cloning: A Laboratory Manual; Cold Spring Harbor (Ausubel, et al., Current Protocols in Molecular Biology, pub. By Greene Publishing Assoc, and Wiley-Interscience, 1987) Examples of methods for constructing a microorganism including a butanol biosynthetic pathway are described in, for example, U.S. Patent No. 7,851,188 and U.S. Patent Application Publication 2007/0092957; 2007/0259410; 2007 / 0292927, 2008/0182308, 2008/0274525, 2009/0155870, 2009/0305363 and 2009/0305370, the entire contents of each being incorporated herein by reference.

In addition, while various embodiments of the invention have been described herein, it should be understood that they have been provided by way of example only, and not limitation. It will be apparent to those skilled in the relevant art that various changes in form and details may be made therein 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 implementations, but should be defined only in accordance with the following claims and their equivalents.

All publications, patents, and patent applications mentioned in this specification are indicative of the level of skill of those skilled in the art to which this invention pertains and each individual publication, patent or patent application is herein incorporated by reference in specificity and in its Quot; is < / RTI > incorporated herein by reference to the same extent.

Example

The following non-limiting examples will further illustrate the present invention. It should be understood that the following examples include corn as the feedstock, but other biomass feedstocks can be used for the feedstock without departing from the invention.

As used herein, the abbreviations used have the following meanings: "g" means gram, "kg" means kilogram, "lbm" means pound mass, "gpm" means gallon per minute , "Gal" means gallon, and "MMGPY" means million gallons per year. ML / min " means milliliters per liter, "mL / min" means milliliters per minute, "&Quot; um "means micrometer," nm "means nanometer, and "" w / v "means weight / volume," wt% "means weight percent," OD "means optical density," OD ₆₀₀ "means optical density at wavelength of 600 nM , "dcw" means the weight of anhydrous cells, "rpm" means the number of revolutions per minute, "C" means Celsius, "C / min" means Celsius temperature per minute, "slpm" Standard liter, "ppm" means one million, "cP" means centipoise, "ID" means inner diameter, and "GC" means gas chromatograph.

Example  One

Effect of undissolved solids on mass transfer rate

Fermentation broth derived from corn mash, approximately half-way through simultaneous saccharification and fermentation (SSF) fermentation (i.e., about 50% conversion of the oligosaccharide) to simulate the liquid composition for the SSF batch, The effect of undissolved solids on the mass transfer rate of i- BuOH from the aqueous phase simulating the composition of < RTI ID = 0.0 >

Approximately 100 kg of liquefied corn mash was prepared in three equivalent batches using a 30 liter glass, jacketed resin kettle. The kettle was set using mechanical stirring, temperature control, and pH control. The protocol used for the three batches was as follows: (a) mixing the ground corn with tap water (30 wt% corn on anhydrous basis), (b) heating the slurry to 55 & Adjusting the pH of the slurry to 5.8 using NaOH or H ₂ SO ₄ , (d) adding alpha-amylase (0.02 wt% on anhydrous maize), (e) heating the slurry to 85 ° C, adjusting the pH to 5.8, (g) maintaining the slurry at 85 ° C for 2 hours while maintaining the pH at 5.8, and (h) cooling the slurry to 25 ° C.

Pioneer 3335 compound corn, whole kernel yellow corn (Pioneer Hi-Bred International, Inc., Johnston, Iowa) was used and ground in a hammer-grinder using a 1 mm screen. The moisture content of the ground corn was 12 wt%, and the content of starch in the ground corn was 71.4 wt% based on anhydrous corn. Alpha-amylase enzyme Liquozyme ^® SC DS: It was (manufactured by Novozymes North Carolina Franklin t material). The total amount of ingredients used for the three batches combined was as follows: 33.9 kg of ground corn (12% moisture), 65.4 kg of tap water, and 0.006 kg of Liquozyme ^® SC DS. A total of 0.297 kg of NaOH (17% by weight) was added to adjust the pH. No H ₂ SO ₄ was needed. The total amount of liquefied corn mash recovered from the three 30L batches was 99.4 kg.

The solids were removed from the mesh by centrifugation in a large floor centrifuge containing 6 1L bottles. The mesh (73.4 kg) was centrifuged at 8000 rpm for 20 minutes at 25 ° C to yield 44.4 kg of concentrate and 26.9 kg of wet cake. The concentrate contained <1 wt.% Of the suspended solids and the wet cake contained approximately 18 wt.% Of the suspended solids. This suggests that the original liquefied mesh contained approximately 7 wt% suspended solids. This is consistent with the starch content and corn loadings of corn used assuming most of the starch was liquefied. When all of the starch was liquefied, 44.4 kg of the concentrate directly recovered from the centrifuge could contain approximately 23 wt% dissolved oligosaccharides (liquefied starch). About 0.6 kg of i- BuOH was added to 33.4 kg of concentrate to maintain it. 36.0 kg of the obtained concentrate containing 1.6 wt% of i- BuOH was used as the raw material solution. The concentrate simulates the liquid phase composition at the beginning of the SSF. Thus, a portion thereof was diluted with an equal amount of H ₂ O on a mass basis to produce a concentrate that simulates SSF at about 50% conversion. More i- BuOH was added to make the final concentration of i- BuOH in the diluted concentrate 3.0 wt% (about 30 g / L).

The diluted concentrate was prepared as follows: 18 kg of a concentrate stock solution containing 1.6% by weight of i - BuOH was mixed with 18 kg of tap water and 0.82 kg of i- BuOH was added. A resulting solution of 36.8 kg of the diluted concentrate consisted of approximately 11% by weight of oligosaccharide and approximately 30 g / L of i- BuOH. This solution simulates a liquid phase of corn silk fermentation (SSF) at a conversion of approximately 50% of the oligosaccharide and an aqueous titre of 30 g / L i- BuOH.

The mass transfer test was carried out using this solution as an aqueous phase to simulate the mass transfer performance in the broth derived from the liquefied corn mash after most of the undissolved solids had been removed. The purpose of the mass transfer test is to determine the total volume mass transfer coefficient ( i. E.) For transferring the i- BuOH from the simulated broth derived from the liquefied corn mash to the dispersion of solvent (extractant) droplets rising through the simulated broth It was to determine the effects of non-dissolved solids on the k _L a). The correlation between the main design of the operating parameters and k _L a can be used to expand the mass transfer operation. Examples of parameters that must be kept as constant as possible in order to generate a correlation of k _L a from small data useful for magnification include an image that measures droplet size (e.g., nozzle diameter, velocity of the image dispersed through the nozzle) And the design parameters.

From a solution of oligosaccharide (derived from a liquefied corn mash), using a 6-inch diameter, 7 foot long glass, jacketed column, both with and without a suspended mesh solid, i- BuOH the rising through the simulated broth of oleyl alcohol (OA) k _L a for movement in the dispersion of the droplets was measured. i- BuOH was added to the aqueous phase to obtain an initial concentration of i- BuOH of approximately 30 g / L. Certain amount of aqueous phase containing about 11% of oligosaccharides and i -BuOH of about 30g / L of the weight (typically about 35kg) for sikyeotgo filled in the column, heated with warm H ₂ O to the column through the jacket at 30 ℃ by flowing Respectively. There was no aqueous phase flow in or out of the column during the test.

Fresh oleic alcohol (80/85% grade, Cognis Corporation, Cincinnati, Ohio) was sparged through a single nozzle into the bottom of the column to produce a dispersion of extractant droplets that flowed through the aqueous phase. After reaching the top of the aqueous phase, the extractant droplets formed a separate organic phase, which was subsequently exchanged from the top of the column and recovered into the receiver. Typically, 3 to 5 grams of oleyl alcohol has flowed through the column for a single test.

Samples of the aqueous phase were collected from a number of water columns throughout the test and a composite sample of the total "rich" oleyl alcohol collected from the oil animals was collected at the end of the test. All samples were analyzed for i-BuOH using HP-6890 GC (Agilent Technologies, Inc., Santa Clara, Calif.). The concentration profile for i- BuOH in aqueous phase (i.e., i- BuOH concentration versus time) was used to calculate k _L a for a given set of operating conditions. The mass balance for i- BuOH was checked using the final composite sample of total "rich" oleyl alcohol collected during the test.

The effect on k _L a was observed by varying the nozzle size and nozzle speed (mean velocity of oleo alcohol through the feed nozzle). A series of tests were performed using an aqueous solution of oligosaccharide (diluted concentrate obtained from liquefied corn mash). A similar series of tests were performed using the same aqueous solution of oligosaccharide after the mesh solids were again applied to simulate liquefied corn mashes (including undissolved solids) in mid-SSF. Under some operating conditions (e.g., high oleic alcohol flow rates), poor phase separation was obtained at the top of the prepared column to make it difficult to obtain representative composite samples of the total "rich" oleyl alcohol collected during the test . It is also noted that under some operating conditions, the aqueous phase samples contained significant amounts of organic phase. Using specific sample processing and fabrication techniques, a sample of aqueous phase that was as representative of the aqueous phase in the column as possible at the time the sample was collected was obtained.

The aqueous phase in the column was "mixed well" for all practical purposes because the concentration of i- BuOH did not change much along the length of the column at any given point in time. Assuming that the solvent droplet phase is also well mixed, the total mass transfer of i-BuOH from the aqueous phase to the solvent phase in the column can be calculated by the following equation:

(1)

(One)

In the above equation (1)

r _B is the total mass of i- BuOH transferred from the aqueous phase to the solvent, expressed in grams i- BuOH / liter aqueous phase / hr or g / L / hr per unit time per aqueous phase unit.

k _L a is the total volumetric mass transfer coefficient, expressed in hr ^-1 , which describes the mass transfer of i- BuOH from the aqueous phase to the solvent phase.

C _{B, broth,} the average concentration of the i- BuOH on g i -BuOH / liter aqueous phase or g / L shown in, the simulated over the test broth (aqueous).

C _{B , and the solvent} is the average concentration of i - BuOH in the solvent phase over the entire test, expressed in grams i- BuOH / liter solvent phase or g / L.

K _B is the average equilibrium partition coefficient for i - BuOH between the solvent and the aqueous phase, expressed in grams i- BuOH / liter solvent phase / (gram i- BuOH / Liter aqueous phase)

The parameters r _B , C _{B, Broth} , and C _{B, solvents} were calculated for each test from the concentration data obtained from aqueous and solvent phase samples. The parameter K _B was measured independently by mixing the liquefied corn mash, oleyl alcohol, and aqueous concentrates from i- BuOH and vigorously mixing the system until the two liquid phases reached equilibrium. The concentration of i- BuOH was determined in two phases to determine K _B. After determining r _B , C _{B , Broth} , C _{B , solvent} , and K _B for a given test, K _L a could be calculated by rearranging equation (1)

(2)

(2)

The mass transfer test was performed using two different sized nozzles at a nozzle speed ranging from 5 ft / s to 21 ft / s using diluted concentrate (solids removed) as the aqueous phase. Three tests were performed using a nozzle with an ID of 0.76 mm and three tests were conducted using a nozzle with an ID of 2.03 mm. All tests were carried out at 30 ° C in a 6 inch diameter column as described above using oleyl alcohol as solvent. The equilibrium partition coefficient of i- BuOH between the oleyl alcohol and the diluted concentrate obtained from the liquefied corn mash by removing the solids was determined to be approximately 5. [ The results of the mass transfer test using the diluted concentrate (solid removed) are shown in Table 2.

[Table 2]

A portion of the wet cake (initially obtained by removing solids from the liquefied corn mash) was added to the dilute concentrate to simulate the fermentation broth from the corn mash (containing undissolved solids) that was half liquefied through the SSF Was synthesized. Some water is also added to dilute the liquid phase held in the wet cake, since this liquid has the same composition as the concentrated concentrate. The diluted top layer (17.8 kg), 13.0 kg wet cake (containing ~ 18 wt% insoluble mesh solids), 5.0 kg of H ₂ O, and 0.83 kg of i- BuOH were mixed together to give a weight of approximately 6.3 wt % Of undissolved solids, and a liquid phase consisting of approximately 13 wt% liquefied starch and approximately 2.4 wt% i- BuOH (balanced with H ₂ O). This slurry simulates the composition of the fermentation broth in half-way through the SSF of corn for i- BuOH at approximately 30% corn load, because the levels of undissolved solids and oligosaccharides found in this type of broth Is about 6 to 8 wt% and 10 to 12 wt%, respectively.

Mass transfer tests were performed using two different sized nozzles at a nozzle speed ranging from 5 ft / s to 22 ft / s using a slurry of diluted concentrate and undissolved mesh solid as the aqueous phase. Three trials were performed using a nozzle with an ID of 0.76 mm and three trials were conducted using a nozzle with an ID of 2.03 mm. All tests were carried out at 30 [deg.] C in a 6 inch diameter column as described above using oleyl alcohol as solvent. The results of the mass transfer test using a slurry of diluted concentrate and undissolved mesh solids are shown in Table 3.

[Table 3]

12 is not dissolved in the total volume of the mass transfer coefficient, k _L a of the upflow oleyl transfer to the dispersion of the alcohol droplets through a bubble column of the i-BuOH from an aqueous solution of liquefied corn starch (i.e., oligosaccharides) Indicating the effect of the presence of non-corn mash solids. The oleyl alcohol was fed to the column via an ID nozzle of 2.03 mm. The ratio of k _L a of the k _L a large solid system is not removed on the solid is removed, the system was found to be a 2 to 5 according to the nozzle speed when the 2.03 mm nozzle.

13 is a full-volume mass-transfer coefficient for the liquefied corn starch passed the i-BuOH to the dispersion of oleyl alcohol droplets that from the flow upward through the bubble column of the i-BuOH in water (i.e., oligosaccharides), k _L a Of undissolved corn silicate solids. The oleyl alcohol was fed to the column via a 0.76 mm ID nozzle. The ratio of k _L a of the k _L a large solid system is not removed on the solid is removed, the system was found to be a 2 to 4 according to the nozzle speed of the 0.76 mm nozzle.

Example  2

Aqueous phase and Solid phase  The effect of removal of undissolved solids on phase separation between

This example illustrates an improvement between an aqueous solution of oligosaccharides originating from a liquefied corn mash and a solvent phase in which undissolved solids have been removed as compared to an oligosaccharide aqueous solution originating from the same solvent as the liquefied corn mash in which the undissolved solids have not been removed Lt; / RTI &gt; phase separation. Both systems contained i- BuOH. Proper separation of the solvent phase from the aqueous phase is important as liquid-liquid extraction is a valuable separation method for conducting ISPR.

Approximately 900 g of liquefied corn mash was prepared in a 1 liter glass, jacketed resin kettle. The kettle was set for mechanical stirring, temperature control, and pH control. The following protocol was used: milled corn was mixed with tap water (26 wt% corn on anhydrous basis) and the slurry heated to 55 ° C with stirring and the pH adjusted to 5.8 with NaOH or H ₂ SO ₄ , Added with alpha-amylase (0.02 wt% based on anhydrous corn), heated continuously to 85 ° C, held at 85 ° C for 2 hours while adjusting the pH to 5.8, and cooled to 25 ° C.

Using Pioneer 3335 hybrid corn, whole grain yellow corn (Pioneer Hi-Bred International, Inc., Johnston, Iowa), it was ground in a hammer-grinder using a 1 mm screen. The moisture content of the ground corn was 12 wt%, and the starch content of the ground corn was 71.4 wt% on anhydrous corn. Alpha-amylase enzyme was Liquozyme ^® SC DS from Novozymes (North Carolina Franklin t material). The total amount of ingredients used was 265.9 g ground maize (12% moisture), 634.3 g tap water, and 0.056 g Liquozyme ^® SC DS. The total amount of recovered liquefied corn mash was 883.5 g.

A portion of the liquefied corn mash was used directly without removing undissolved solids to prepare an aqueous phase for phase separation tests involving solids. Some of the liquefied corn mashes were centrifuged to remove most of the undissolved solids and an aqueous phase was prepared for phase separation tests involving the absence of solids.

The solids were removed from the mesh by centrifugation in a large bottom centrifuge from the mesh. The mash (583.5 g) was centrifuged at 5000 rpm for 20 minutes at 35 DEG C to obtain 394.4 g of concentrate and 189.0 g of wet cake. The concentrate contained approximately 0.5% by weight of suspended solids, and the wet cake was determined to contain approximately 20% by weight of suspended solids. This suggests that the original liquefied mesh contained approximately 7 wt% suspended solids. This is consistent with the corn loading and the starch content of corn used, and it is estimated that most of the starch was liquefied. When all of the starch was liquefied, the concentrate directly recovered from the centrifuge could contain approximately 20 wt% of dissolved oligosaccharides (liquefied starch) based on the absence of solids.

The purpose of the phase separation test is to determine the effect of undissolved solids on the degree of phase separation between the liquid phase and the solvent phase, which simulates the broth originating from the liquefied corn mash. In all the tests, the aqueous liquid phase contained about 20% by weight dissolved oligosaccharide, and the organic phase contained oleyl alcohol (OA). Also, i-BuOH was added to all the tests to obtain approximately 25 g / L in the aqueous phase when the phases were equilibrated. Two shaking tests were performed. The aqueous phase (containing solids) for the first test was prepared by mixing 60.0 g of liquefied corn mash with 3.5 g of i- BuOH. The aqueous phase for the second test (solids removed) was prepared by mixing 60.0 g of the concentrate obtained by removing solids from the liquefied corn mash with 3.5 g of i- BuOH. Oleyl alcohol (15.0 g, 80/85% grade, manufactured by Cognis Corporation, Cincinnati, OH) was added to each of the shaking test bottles. The oleyl alcohol formed liquid phase separations on top of the aqueous phase in both the bottles and the mass ratio of the phases was obtained such that the aqueous phase / solvent phase was about 1/4. Both bottles were shaken vigorously for 2 minutes to bring the aqueous and organic phases intimately in contact so that i- BuOH reached equilibrium between the two phases. The bottles were allowed to settle for 1 hour. Various time a picture (0, 15, 30, and 60 minutes) on the one originating from the aqueous phase, the solvent phase containing the oleyl alcohol, and a solvent containing i- BuOH derived from the liquefied corn mash aqueous choelyoung The effect of undissolved solids on phase separation in systems containing phases was observed. The 0 o'clock (0) corresponds to the time immediately after the 2 min shaking period is completed.

The degree of separation between the organic (solvent) phase and the aqueous phase as a function of time for the system with solids (liquefied corn mash) and solids removed (liquid concentrate from the liquefied corn mash) Both appeared almost identical. The organic phase was slightly darker and turbid, and the interface was almost indistinguishable (with a solid "rag" layer around the interface) with solids. However, in the case of extractive fermentation in which the solvent is continuously operated, the composition on top of the organic phase is of interest in the case of a sub-process of extractive fermentation in which the next step is distillation.

Since the microorganisms will be thermally deactivated in the distillation column, it may be advantageous to minimize the amount of microorganisms at the top of the organic phase. The undissolved solvent on the top of the organic phase can clog the distillation column, contaminate the reboiler, cause poor phase separation of the solvent / water decanter located at the bottom of the column, or any combination of the aforementioned concerns It may be advantageous to minimize the amount of undissolved solvent at the top of the organic phase. It may be advantageous to minimize the amount of supernatant at the top of the organic phase. The constant is water present in a separate aqueous phase. The additional amount of aqueous phase will increase loading and energy requirements in the distillation column. A 10 milliliter (10 mL) sample was removed from the top of the organic layer from the "solids depleted" and "solids not removed" bottles and both samples were centrifuged to remove " &Quot; solids removed "bottle. The results indicate that at the end of both shaking tests the organic phase contained some undesirable phase (s) (both organic phases are turbid). However, the results also show that the top layer from the phase separation test, which includes the concentrate from which the solid is removed, is removed so that it essentially does not contain undissolved solids. On the other hand, undissolved solids are clearly observed at the bottom of a 10 mL sample collected from the top of the organic phase of the test containing the mesh. It was estimated that 3% of the samples were collected from the top of the organic layer wash mesh solid. If the rich solvent phase present in the fermentor of the extraction fermentation process contained 3% undissolved solids, one or more of the following problems could have occurred: a significant amount of microbial loss, contamination of the solvent column boiler, Clogging of the column. The results also indicate that the top layer from the phase separation test containing the concentrate contained very little water. Table 4 shows estimates of the relative amounts of the phases dispersed throughout the top "organic" layer in both shaking test bottles after 60 minutes of settling time.

[Table 4]

This example demonstrates that removing most of the undissolved solids from the liquefied corn mash produces improved phase separation after contacting the liquid obtained from the mesh, the aqueous phase with a solvent such as oleyl alcohol . This example shows that the solids will contain substantially no dissolved solids if they are first removed before contacting the liquid portion of the mesh containing the organic solvent. This demonstrates the advantage of minimizing the undissolved solids content of the mesh in the upper ("organic") layer of the phase separation for extraction fermentation.

The sample was also allowed to settle for several days after the completion of the sample preparation before repeating the phase separation shaking test described in this example. The samples containing solids were composed of liquefied corn mash, i-BuOH, and oleyl alcohol, and the solid-removed samples contained most of the undissolved solids from liquefied corn mash, i-BuOH, and oleyl alcohol &Lt; / RTI &gt; The liquefied mesh contained approximately 7 wt% suspended solids, and the concentrate from the mesh contained approximately 0.5 wt% suspended solids. When all of the corn starch in the ground corn was liquefied, the liquid phase in the liquefied mesh and the concentrate produced from the mesh contained solids up to approximately 20 wt% dissolved oligosaccharides (liquefied starch) on a non-containing basis. Both samples contained an amount of oleyl alcohol to obtain a ratio of about 1/4 of the phase: solvent phase / aqueous phase mass. In addition, i-BuOH was added to all the tests to obtain approximately 25 g / L in the aqueous phase when the phases were equilibrium.

The purpose of the phase separation test is to measure the effect of undissolved solids at the degree of phase separation after placing the multiphase mixture for several days at room temperature and to simulate potential changes in the characteristics of the system throughout the extraction fermentation. Two shaking tests were performed. Both bottles were vigorously shaken for two minutes to bring the aqueous and organic phases intimately in contact. The bottle was left for one hour. Photographs were taken at various times (0, 2, 5, 10, 20, and 60 minutes) and aged for several days to observe the effect of solubilized solids upon phase separation in the system. 0 Time (0) corresponds to the time immediately after placing the bottle on the bench.

The phase separation began to occur two minutes after the solid was removed from the sample. Nearly complete phase separation appeared to occur in the sample when removed after only 5 to 10 minutes, based on the fact that the organic phase accounts for approximately 25% of the total volume of the two phase mixture. Complete separation can be expected to occur when the organic phase occupies approximately 20% of the total volume since then, corresponding to the initial ratio of phases. No apparent phase separation occurred even after 1 hour in the sample without solid removal.

The composition on top of both samples was also compared. The composition on the top affects the process downstream of the extractive fermentation, the next step being distillation. Since the microorganisms will be thermally deactivated in the distillation column, it is advantageous to minimize the amount of microorganisms on top of the organic phase. Because the solids can block the distillation column, contaminate the reboiler, cause poor phase separation in the solvent / water decanter located at the bottom of the column, or cause any combination of the above mentioned meshes, Another ingredient to minimize is the amount of undissolved solids. In addition, the additional amount of the aqueous phase will increase loading and energy requirements in the subsequent distillation column, so the other ingredient to minimize at the top of the organic phase is the amount of water that is water present as a separate aqueous phase.

A 10 milliliter (10 mL) sample was removed from the top of the organic layer from the " solids containing "and" solids removed "bottles, and both samples were centrifuged to obtain" The organic phase compositions in the "solids removed" bottle were compared and compared. The composition of the sample collected from the top of the "solid containing" sample confirms that there is essentially no phase separation occurring in these samples within 60 minutes. Specifically, the ratio of the solvent phase to the total aqueous phase (aqueous liquid + suspended solid) in the sample collected from the top of the shaking test bottle containing "solids " is approximately 1/4 w / w, &Lt; / RTI &gt; Also, the amount of undissolved solids in the sample collected from the top of the shaking test bottle containing "solids" is almost the same as that found in the liquefied corn mash, which means that the solids precipitated in the shaking test bottle Is not necessarily present. On the other hand, the upper layer from the phase separation test, which included a solid-depleted concentrate ("solids removed"), essentially did not contain undissolved solids. The results also indicate that the top layer from the phase separation test containing the concentrate contained very little water. This indicates that the ratio of solvent phase to aqueous phase in the sample bottle is approximately 1/1 w / w, indicating that the solvent (oleyl alcohol) was concentrated in the organic phase in the test in which the solid was removed. Table 5 shows an estimate of the relative amount of phase dispersed throughout the upper "organic" layer in both shaking test bottles after a settling time of 60 minutes.

[Table 5]

This example demonstrates that removing the undissolved solids from the liquefied corn mash containing i- BuOH, contacting it with the solvent phase, allowing it to settle for several days, and then re-mixing the phase, &Lt; / RTI &gt; produces improved phase separation compared to samples that were not removed from the liquefied mesh. Indeed, this example shows that essentially no phase separation occurs even after 60 minutes in a sample in which undissolved solids have not been removed. This example shows that the top phase obtained after phase separation is substantially free of undissolved solids when the solids are first removed prior to contacting the liquid portion of the mesh with the organic solvent. This is important because two of the most important species that must be minimized in the upper ("organic") layer of phase separation for extraction fermentation are the levels of microorganisms and levels of undissolved solids from the mash. The previous example showed that removing the solids from the liquefied corn mash resulted in improved phase separation immediately after contacting the aqueous phase with the solvent phase. This allows the extraction fermentation to be available at an early stage in fermentation. This example also shows that removing the solids from the liquefied corn mash results in improved phase separation in the aged sample containing the aqueous phase (solids-free oligosaccharide solution) in contact with the solvent phase. This may also make the extract fermentation available later in the fermentation.

Example  3

ISPR  Effect of removal of undissolved solids on loss of extraction solvent-disk stack centrifuge

This example demonstrates the ability to reduce solvent loss through DDGS produced by an extractive fermentation process by removing undissolved solids from pre-fermented corn silages using a semi-continuous disc-stack centrifuge.

Approximately 216 kg of liquefied corn mash was produced in a jacketed stainless steel reactor. The reactor was set to mechanical stirring, temperature control, and pH control. The protocol is as follows was used: heating the comminuted corn water (anhydrous basis with 25% by weight of corn) and mixed, and the slurry with 55 ℃ with stirring at 400rpm, and using NaOH or H ₂ SO ₄ pH Adjusted to 5.8, alpha-amylase (0.02% by weight on anhydrous corn) was added, heating was continued at 85 DEG C, the pH was adjusted to 5.8, the pH was maintained at 5.8 and kept at 85 DEG C for 30 minutes, Heated to 121 캜 using live steam injection and maintained at 121 캜 for 30 minutes to simulate a jet cooker, cooled to 85 캜, adjusted to pH 5.8, loaded with alpha-amylase (0.02% by weight on anhydrous corn) of a second filler is added and maintained at 85 DEG C for 60 minutes while maintaining the pH at 5.8 to complete liquefaction. Thereafter, the mesh was cooled to 60 DEG C and transferred to a centrifuge feed tank.

Pioneer 3335 hybrid corn, whole grain yellow corn (manufactured by Pioneer Hi-Bred International, Inc., Johnston, Iowa) was crushed in a hammer-grinder using a 1 mm screen. The moisture content of the ground corn was 12 wt%, and the starch content of the ground corn was 71.4 wt% on anhydrous corn. Alpha-amylase enzyme was Liquozyme ^® SC DS from Novozymes (North Carolina Franklin materials). The amount of ingredients used 61.8kg of crushed corn (12% moisture), 147.3kg of water, the alpha 1-amylases Liquozyme solution of 0.0109kg in the water for the filling of 1kg ^® SC DS, of a second alpha- amylase filler was a different solution of Liquozyme ^® SC DS of 0.0109kg in the water of 1kg for (after cooking step). About 5 kg of H ₂ O was added to the batch via the steam concentrate during the cooking step. A total of 0.25 kg of NaOH (12.5 wt%) and 0.12 kg of H ₂ SO ₄ (12.5 wt%) were added throughout the run to adjust the pH. The total amount of recovered liquefied corn mash was 216 kg.

The final liquefied corn mash slurry composition was estimated to be approximately 7 wt% undissolved solids and 93 wt% liquid. The liquid phase contained about 19% by weight (190 g / L) of liquefied starch (soluble oligosaccharides). The rheology of the mesh is important in relation to its ability to separate the slurry into its constituents. The liquid phase in the mesh was measured to be a Newtonian fluid having a viscosity of about 5.5 cP at 30 ° C. The mesh slurry was measured to be a shear thinning fluid having a bulk viscosity of about 10 to 70 cP at 85 DEG C, depending on the shear rate.

The liquefied mesh (209 kg, 190 L) was centrifuged using a disk-stack split-bowl centrifuge (Alfa Laval Inc., Richmond, Va.). The centrifuge operated in a semi-batch mode using continuous feed, continuous concentrate outlet, and batch drainage of the wet cake. The liquefied corn mash was continuously fed at a rate of 1 L / min, the clarified concentrate was continuously removed, and the wet cake was periodically drained every 4 minutes. To determine the proper discharge interval for solids from the disk stack, a sample of the mesh to be fed into the disk stack was centrifuged in a high speed lap centrifuge. The mesh (48.5 g) was spun at room temperature to 11,000 rpm (room temperature to about 21,000 g for about 10 minutes). The clarified concentrate (36.1 g) and 12.4 g of pellets (wet cake) were recovered. The clarified concentrate contained about 0.3% by weight of undissolved solids and the pellets (wet cake) contained about 27% by weight of undissolved solids. Based on this data, a 4 minute discharge interval was selected for operation of the disk stack centrifuge.

The disk stack centrifuge was operated at about 60 ° C at 9000 rpm (6100 g) using a liquefied corn meal feed rate of 1 L / min. The mesh (209 kg) was separated into 155 kg of a clear, concentrated concentrate and 55 kg of wet cake. The split defined by (amount of concentrate) / (quantity of supplied mesh) achieved by the semi-continuous disk stack was similar to the fraction achieved in the batch centrifuge. (155 kg / 209 kg) = 74% for a disk stack half-batch centrifuge operating at 6100 g, a feed rate of 1 L / min, and a discharge interval of 4 minutes, and a lap batch The fraction (36.1 g / 48.5 g) for the centrifuge was 74%.

45 mL of the clarified centrifuge sample recovered from the disk stack centrifuge was spun in a lap centrifuge at 21,000 g for 10 minutes to assess the level of suspended solids in the concentrate. About 0.15 to 0.3 g of undissolved solid was recovered from 45 mL of the concentrate. This corresponds to 0.3 to 0.7% by weight of undissolved solids in the concentrate which is about 10 times less in the undissolved solids from the mesh fed to the centrifuge. ISPR Extraction Solvent Loss via DDGS is a process whereby the level of undissolved solids present in the extract fermentation is reduced to an order of magnitude using some solid / liquid separator or combination of devices, To remove suspended solids from the pre-fermented corn mash. Minimizing solvent loss through DDGS is an important factor in the quality and economics of DDGS for the extraction fermentation process.

Example  4

ISPR  Effect of removal of undissolved solids on loss of extraction solvent-bottle rotation test

This example demonstrates that the ability to reduce solvent losses through DDGS has been generated in the fermentation process by removing undissolved solids from the corn mash prior to fermentation using a centrifuge.

Laboratory-scale bottle rotation tests were performed using liquefied corn mash. This test simulates the operating conditions of a typical decanter centrifuge used to remove undissolved solids from the entire distillation waste liquid in a conventional ethanol plant. In a typical ethanol plant, a decanter centrifuge typically operates at a relative centrifugal force (RCF) of about 3000 g and a total distillery waste retention time of about 30 seconds. These centrifuges typically remove about 90% of suspended solids in a total distillation wastewater containing about 5% to 6% of suspended solids (after the beer column), resulting in a suspension containing about 0.5% of the suspended solids A light distillation waste liquid is produced.

Liquefied corn mashes were prepared according to the protocol described in Example 2. About 10 mL of the mesh was placed in a centrifuge tube. The sample was centrifuged for a total of 1 minute at an RCF of about 3000 g (rotor speed of 4400 rpm). Due to the acceleration and deceleration of the centrifuge the sample was consumed at 3000 g for about 30 to 40 seconds and at a rate of less than 3000 g for a total of 20 to 30 seconds. The sample temperature was about 60 ° C.

A mesh (10 mL) containing about 7 wt% of the suspended solids was separated into about 6.25 mL of the clarified concentrate and 3.75 mL of the wet cake (pellet at the bottom of the centrifuge tube). The fraction divided by (amount of concentrate) / (amount of original mesh filled) achieved by the bottle rotation test was about 62%. The clear concentrate contained about 0.5 wt.% Suspended solids, which was determined to be a 10-fold decrease in suspended solids compared to the level of suspended solids in the original mesh. The clear pellets were also measured to contain about 18% by weight suspended solids.

Table 6 summarizes the suspended (undissolved) solids mass balance for the bottle rotation test under conditions indicative of the operation of a decanter centrifuge to convert the entire distillation waste liquid to a light distillation waste liquid in a commercial ethanol process. All values provided in Table 6 are approximate.

[Table 6]

The concentrate was also measured to contain about 190 g / L of dissolved oligosaccharide (liquefied starch). This is because most of the ground corn starch is liquefied (i. E., Hydrolyzed to soluble oligosaccharides) during the liquefaction process based on the corn load (about 26% by weight on anhydrous maize) used and the starch content of the corn used, (About 71.4% by weight starch on anhydrous maize) of liquefied mash. Hydrolysis of most of the corn starch ground at 26% anhydrous maize load resulted in about 7 wt% suspended (undissolved) solids in the liquefied corn mache filled in the centrifuge used in the bottle rotation test Will be.

The fact that the clarified concentrate contained only about 0.5% by weight of undissolved solids indicates that the conditions used in the bottle rotation test resulted in a 10-fold or more reduction in the undissolved solids from the filled mash . It is well assumed that the same solids removal performance could be achieved by a pre-fermentation continuous decanter centrifuge, the loss of ISPR extraction solvent in DDGS could be reduced to approximately one order of magnitude. Minimizing solvent loss through DDGS is an important factor in DDGS quality and economics for the extraction fermentation process.

Example  5

Removal of corn oil by removing undissolved solids

This example demonstrates the ability to remove and recover corn oil from corn mash by removing undissolved solids before fermentation. The efficacy of the extraction solvent can be improved if corn oil is removed through the removal of undissolved solids. In addition, removal of corn oil through removal of undissolved solids can minimize any reduction in the solvent partition coefficient, creating a potentially improved extraction fermentation process.

Approximately 1000 g of liquefied corn mash was prepared in a 1 L glass, jacketed resin kettle. The kettle was set up using mechanical stirring, temperature control, and pH control. The following protocol was used: milled corn was mixed with tap water (26 wt% corn on anhydrous basis) and the slurry heated to 55 ° C with stirring and the pH was adjusted to 5.8 using NaOH or H ₂ SO ₄ (0.02 wt% on anhydrous corn), heated continuously to 85 ° C, adjusted to pH 5.8, held at 85 ° C for 2 hours while maintaining the pH at 5.8, maintained at 25 ° C Lt; / RTI &gt;

Pioneer 3335 hybrid corn, whole grain yellow corn (Pioneer Hi-Bred International, Inc., Johnston, Iowa) was used and milled in a hammer-grinder using a 1 mm screen. The moisture content of the ground corn was about 11.7 wt%, and the starch content of the ground corn was about 71.4 wt% on anhydrous corn. Alpha-amylase enzyme Liquozyme ^® SC DS: It was (manufactured by Novozymes North Carolina Franklin t material). 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 Liquozyme ^® SC DS. Water (4.3 g) was added to dilute the enzyme, and a total of 2.3 g of 20% NaOH solution was added to adjust the pH. Approximately 952 g of the mesh was recovered.

The liquefied corn mash was centrifuged at 5000 rpm (7260 g) for 30 minutes at 40 &lt; 0 &gt; C to remove undissolved solids from the aqueous solution of oligosaccharide. Removal of the solids by centrifugation also resulted in the removal of glass corn oil as a separate organic liquid on top of the aqueous phase. Approximately 1.5 g of corn oil was recovered from the organic layer suspended on top of the aqueous phase. It was determined by hexane extraction that the ground corn used to produce the liquefied mesh contained about 3.5 wt.% Corn oil on anhydrous maize. This corresponds to about 9 grams of corn oil supplied to the liquefaction process using ground corn.

After recovering the corn oil from the liquefied mesh, an aqueous solution of oligosaccharide was decanted away from the wet cake. About 617 g of the liquefied starch solution was recovered leaving about 334 g of wet cake. The wet cake contained most of the undissolved solids that were present in the liquefied mesh. The liquefied starch solution contained about 0.2 wt% undissolved solids. The wet cake contained about 21% by weight of undissolved solids. The wet cake was washed with 1000 g of tap water to remove oligosaccharide still present 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 mesh to recover washed solids. Centrifugation of the wash slurry to remove washed solids also resulted in the removal of some additional free corn oil that had to remain with the original wet cake produced from the liquefied mesh. This additional corn oil was observed as a separate, pale, organic liquid layer on top of the aqueous phase of the centrifuged wash mixture. Approximately 1 g of additional corn oil was recovered from the wash process.

The wet solid was washed twice more each time with 1000 g of tap water to remove essentially all liquefied starch. No visible additional corn oil was removed from the second and third water wash of the mesh solid. The final washed solids were dried in a vacuum oven at 80 &lt; 0 &gt; C overnight and about 20 inches of Hg vacuum. As a dry solid, the amount of corn oil that was still remaining in the embryo was probably determined by hexane extraction. A relatively anhydrous solid sample (3.60 g, approximately 2 wt% moisture) contained 0.22 g of corn oil. The results correspond to a dry solid of 0.0624 g of corn oil / g. This was the case for washed solids, meaning that no residual oligosaccharides were present in the wet solid. The liquefied corn mash was centrifuged to separate a layer of the aqueous corn oil and oligosaccharide solution from the wet cake and then determined that about 334 grams of wet cake remained, containing about 21 weight percent undissolved solids. This corresponds to a wet cake containing about 70.1 g of undissolved solids. In a dry solid of 0.0624 g of corn oil / g, the solids in the wet cake should contain about 4.4 g of corn oil.

In summary, approximately 1.5 g of free corn oil was recovered by centrifuging the liquefied mesh. In addition, 1 g of glassy corn oil was recovered by centrifugation of the first (water) wash slurry generated to wash the original wet cake produced from the mesh. Finally, the washed solid was determined to still contain about 4.4 g of corn oil. The liquefied corn was also measured to contain about 9 grams of corn oil. Thus, a total of 6.9 g of corn oil was recovered from the following process steps: liquefaction, removal of solids from the liquefied mesh, washing of the solids from the mesh, and final washing of the solids. As a result, approximately 76% of the total corn oil in the corn fed to the liquefaction was recovered during the liquefaction and solid removal processes described herein.

Example  6

Extraction fermentation using concentrate and mesh as a party

This example illustrates an extractive fermentation carried out using corn mash and corn mash concentrate as a source. The corn mash concentrate was produced by removing undissolved solids from the corn mash before fermentation. Four extracted fermentations were performed twice using liquefied corn mash (solid not removed) as a source and liquefied mesh concentrate obtained by removing most of the undissolved solids from the liquefied corn mash (an aqueous solution of oligosaccharide ). &Lt; / RTI &gt; The product ( i- BuOH) formed from the broth was extracted by adding two oleyl alcohol (OA), one using solids and one using the removed solids. A mixture of corn oil fatty acid (COFA) was added to the other two fermentations, one using solids and one using solids removed, to extract the product formed from the broth. COFA was prepared by hydrolyzing corn oil. The purpose of these fermentations is to test the effect of removing solids during phase separation between solvent and broth and to determine the amount of residual solvent trapped in undissolved solids recovered from solids-free or non-solids fermentation broth Respectively.

Liquefied corn Mash  Produce

Approximately 31 kg of liquefied corn mash was produced in a 30 L jacketed glass resin kettle. The reactor was equipped with mechanical stirring, temperature control, and pH adjustment. The protocol used was as follows: milled corn was mixed with tap water (40 wt.% Corn on anhydrous basis) and the slurry heated to 55 ° C with stirring at 250 rpm and the pH was adjusted to pH 5 using NaOH or H ₂ SO ₄ , Adjusted to 5.8, added with a dilute aqueous solution of alpha-amylase (0.16% by weight on anhydrous corn), maintained at 55 DEG C for 60 minutes, heated to 95 DEG C, adjusted to pH 5.8, Lt; / RTI &gt; for 5 min. The mesh was transferred into a sterile centrifuge bottle to prevent contamination.

The corn used was whole-grain yellow corn (Pioneer Hi-Bred International of Johnston, Iowa), which was ground using a 1 mm screen in a pilot-scale hammer-grinder. The moisture content of the ground corn was about 12 wt%, and the starch content of the ground corn was about 71.4 wt% on anhydrous corn. Using the Alpha-amylase enzyme Spezyme ^® Fred-L: It was ^(® Manufacturer of Palo Alto, Genencor). The amount of ingredients used was: a solution of alpha-amylase consisting of 14.1 kg of ground corn (12% moisture), 16.9 kg of tap water, 19.5 g of Spezyme ^® Fred-L in 2.0 kg of water. Alpha-amylase was sterile filtered. A total of 0.21 kg of NaOH (17% by weight) was adjusted throughout the run.

The liquefied corn mash was estimated to contain approximately 28 wt.% (About 280 g / L) of liquefied starch based on the corn load used, the starch content of maize, and all starches were estimated to be hydrolyzed during liquefaction . The mash was prepared using higher concentrations of oligosaccharides than required for fermentation, allowing dilution when the nutrients, inoculum, glucoamylase, and base were added to the initial fermentation broth. After dilution by addition of nutrients, inoculum, glucoamylase, and base, the predicted total initial soluble sugar in the mash (solid not removed) was about 250 g / L.

Approximately 13.9 kg of the liquefied mesh was centrifuged using a bottle centrifuge containing six 1 L bottles. The centrifuge was operated at room temperature for 20 minutes at 5000 rpm (7260 RCF). The mesh was separated into about 5.5 kg of the clarified concentrate and about 8.4 kg of wet cake (pellet on the bottom of the centrifuge bottle). (5.5 kg / 13.9 kg) = 40% was defined as the amount of water (concentrated amount) / (amount of supplied mesh).

The solids were not removed from the filled mashes at the 2010Y034 and 2010Y036 fermentations described below. Concentrates filled in fermentations 2010Y033 and 2010Y035 (also described below) were produced by centrifuging most of the suspended solids from the mesh according to the protocol.

Common methods for growing fermented seed flasks

Strains ( pdcl , pdc5 , and pdc6 deleted) to Saccharomyces cerevisiae processed to produce isobutanol from a carbohydrate source were fed in a seed flask from a frozen culture at 0.55-1.1 g / L dcw ( OD ₆₀₀ 1.3-2.6 - Thermo Helios a manufacturer: Thermo Fisher Scientific Inc., Walton, Mass.). The strain Saccharomyces cerevisiae is described in U.S. Patent Application Publication No. 2012/0164302 incorporated herein by reference. The cultures were grown in a thermostat rotating at 300 rpm at 26 &lt; 0 &gt; C. The frozen cultures were pre-stored at -80 &lt; 0 &gt; C. The composition of the first seed flask culture was as follows:

3.0 g / L dextrose

3.0 g / L of ethanol,

Complete amino acid (Kaiser) drop-out of 3.7 g / L ForMedium ^TM synthesis:

         HIS member, URA member (DSCKl62CK)

Difco yeast nitrogen base (No. 291920) without 6.7 g / L amino acid.

Twelve milliliters (12 mL) from the first seed flask culture was transferred to a 2 L flask and grown at 30 C in a thermostat rotating at 300 rpm. The second seed flask has 220 mL of the following medium:

30.0 g / L dextrose

5.0 g / L of ethanol,

A complete amino acid of 3.7 g / L ForMedium ^TM synthesis (manufacturer: Kaiser) Drop-out:

    HIS member, URA member (DSCKl62CK)

6.7 g / L of Difco yeast nitrogen base without amino acid (No. 291920)

Titration to pH 5.5 to 6.0 with 0.2 M MES buffer.

The cultures were incubated with 0.55-1.1 g / L dcw (OD ₆₀₀ 1.3-2.6). An additive of 30 mL of solution containing 200 g / L of peptone and 100 g / L of yeast extract was added to the cell concentrate. Thereafter, 300 mL of sterilized 0.2 [mu] M filter was added and 90 to 95% of oleic alcohol (Cognis Corporation, Cincinnati, OH) was added to the flask. Cultures were continuously grown to> 4 g / L dcw (OD ₆₀₀ > 10) before harvest and added to the fermenter.

Fermentation manufacture

Initial fermenter manufacturing

A 2L glass-jacketed fermenter (Sartorius AG, Gottingen, Germany) was charged with a liquefied mesh with or without solid (concentrate). The pH probe (Hamilton Easyferm Plus K8, part no. 238627, manufactured by Hamilton Bonaduz AG, Bonaduz, Switzerland) was calibrated using the Sartorius DCU-3 Control Tower Calibration menu. 0 was corrected at pH = 7. The period was corrected at pH = 4. Thereafter, the probe was placed into the fermenter through the stainless steel head plate. A dissolved oxygen probe (pO ₂ probe) was also placed in the fermenter through the head plate. The tubing used to deliver the nutrients, seed culture, extraction solvent, and base was attached to the head plate and the ends were foil treated. The entire fermenter was placed in an autoclave (Steris Corporation, Mentor, OH) and sterilized with a liquid cycle for 30 minutes.

The fermenter was removed from the autoclave and placed in a load cell. A jacket water supply and return line was connected to each of the house water and rinse drain lines. The condenser cooling water inside and outside the line was connected to a 6-L recirculation temperature bath operating at 7 ° C. The exhaust line carrying the gas from the fermenter was connected to a delivery line connected to a thermal mass spectrometer (Prima dB, manufactured by Thermo Fisher Scientific Inc., Walton, Mass.). The spreader line was connected to the gas supply line. The conduits for delivering nutrients, extraction solvents, seed cultures, and bases were connected to the drainage line via a pump or closed by clamping. The autoclaved material, 0.9% w / v NaCl, was drained before adding the liquefied mesh.

After lipase treatment - liquefaction

The fermenter temperature was set at 55 占 폚 instead of 30 占 폚 after the liquefaction cycle was completed. The pH was adjusted manually at pH = 5.8 by adding lumps of acid or base if necessary. The lipase enzyme stock solution was added to a fermenter to give a final lipase concentration of 10 ppm. A fermenter at 55 ℃, 300 rpm, and 0.3 slpm N ₂ overlaid (overlay)> was maintained for 6 hours. After completion of the lipase treatment, the temperature of the fermenter was set to 30 占 폚.

Nutrient addition before inoculation

7.0 mL / L (volume after inoculation) of ethanol (200 proof, anhydrous) was added just before inoculation. Immediately prior to inoculation, thiamine was added to a final concentration of 20 mg / L. 100 mg / L of nicotinic acid was added immediately before inoculation.

Inoculation of fermenter

The fermentor pO ₂ probe was calibrated to zero while N _{2 was added} to the fermenter. Fermenters were corrected for a period between the pO ₂ probe with sterile air spraying at 300 rpm. The fermenter was inoculated after the second seed flask had> 4 g / L dcw. The shake flask was removed from the fermenter / shaker for 5 minutes to allow phase separation of the oleanolic and aqueous phases. The aqueous phase (55 mL) was transferred to a sterile, inoculated bottle. The inoculum was pumped into the fermenter through a peristaltic pump.

After inoculation Oleyl alcohol  Or corn oil fatty acid addition

1 L / L (volume after inoculation) oleic alcohol or corn oil fatty acid was added immediately after inoculation.

Fermenter operating conditions

The fermentor was operated at 30 &lt; 0 &gt; C during the entire growth and production phase. The pH was allowed to decrease from a pH of 5.7 to 5.9, without any acid, to a regulated setpoint of 5.2. The pH was adjusted using ammonium hydroxide at pH = 5.2 for the remainder of the growth and production steps. Sterile air was added via a sparger to the fermenter at 0.3 slpm for the remainder of the growth and production steps. pO ₂ was set to 3.0% controlled by a Sartorius DCU-3 Control Box PID control loop using only stirring control while setting the stirrer to a minimum of 300 rpm and setting it to 2000 rpm to a maximum. Glucose was fed through continuous saccharification and fermentation of liquefied corn mash by adding? -Amylase (glucoamylase). Glucose was maintained at an excess (1 to 50 g / L) as much as the starch was available for glycation.

sample  A

Experimental Verifier 2010Y033 included: seed flask growth method, initial fermenter preparation method using solid maize mesh, lipase treatment after liquefaction, inoculation method All nutrient addition, fermenter inoculation method, fermenter operating condition method and analysis method . The corn oil fatty acid was added in a single batch between 0.1 and 1.0 hour after inoculation.

sample  B

Experimental Verifier 2010Y034 included: seed flask growth method, initial fermenter manufacturing method using corn silicate containing solids, lipase treatment after liquefaction, inoculation method addition of whole nutrients, fermenter inoculation method, fermenter operating condition method, and analysis method all. Corn oil fatty acid was added to a single batch between 0.1 and 1.0 hour after inoculation.

sample  C

Experimental verifier 2010Y035 included: seed flask growth method, initial fermenter manufacturing method using solid maize mesh, inoculation method, all nutrient addition, fermenter inoculation method, fermenter operating condition method, and analysis method. Oleyl alcohol was added to a single batch between 0.1 and 1.0 hour after inoculation.

sample  D

Experimental verifier 2010Y036 included: seed flask growth method, initial fermenter manufacturing method using corn silicate containing solids, inoculation method, all nutrient addition, fermenter inoculation method, fermenter operating condition method, and analysis method. Oleyl alcohol was added to a single batch between 0.1 and 1.0 hour after inoculation. The results for Samples A to D are shown in Table 7.

[Table 7]

Example  7

Fermenter for improvement of volumetric efficiency of fermenter From the feed  Removal effect of undissolved solids

This example demonstrates the effect of removing undissolved solids from the pre-fermentation mesh for the volume efficiency of the fermenter. The undissolved solids in the maize mesh occupy at least 5% of the mesh volume depending on the corn load and starch content. Removing the pre-fermentation solids can increase batching productivity by allowing at least 5% of the sugar to be filled with the fermenter.

Based on the corn loading used, it was found that approximately 28 wt% (280 g / L) of liquefied starch (based on 40% anhydrous corn), corn starch content (based on an anhydrous maize of 71.4%), It was assumed that the liquefied corn mash produced in Example 5 was hydrolyzed to soluble oligosaccharides during liquefaction. The mash was prepared with oligosaccharides at concentrations higher than the desired concentrations in the fermenter to dilute the nutrients, inoculum, glucoamylase, and base when added to the initial fermentation broth. The mesh was diluted to approximately 10% after adding these ingredients. Thus, at the onset of fermentation 2010Y034 and 2010Y036, the predicted concentration of liquefied starch in the mash (including solids) was about 250 g / L. The actual glucose equivalents charged at the 2010Y034 and 2010Y036 fermentations were determined to be 239 g / kg and 241 g / kg, respectively. In comparison, the glucose equivalents charged at the 2010 Y033 and 2010 Y035 fermentations were determined to be 257 g / kg and 263 g / kg, respectively. Note that the feed for these fermentations was a concentrate (a mash with most solids removed). Approximately 1.2 liters of sugar (mash or concentrate) was charged to each fermentation. Thus, based on these data, the fermenter was filled with concentrates (2010Y033 and 2010Y035) that were approximately 8.3% or more sugar compared to mash (2010Y033 and 2010Y036). These results demonstrate that the removal of undissolved solids from the corn silage prior to fermentation can result in a significant increase in the sugars charged per unit volume. This implies that when solids are present, they occupy valuable fermenter volumes. When the solids are removed from the feed, more sugars may be applied ("tailed") to the fermenter due to the absence of undissolved solids. This example demonstrates that the volume efficiency of the fermenter can be significantly improved by removing undissolved solids from the mesh before fermentation.

Example  8

Extraction solvent Brods  Effect of elimination of undissolved solids on phase separation between extractive fermentation

This example demonstrates improved separation between the solvent phase and Broth phase during and after the extraction fermentation process by removing undissolved solids from the corn mash prior to fermentation. One using the liquefied corn mash as a sugar source (no solids are removed) and one concentrate (an aqueous solution of oligosaccharides) generated by removing most of the undissolved solids from the liquefied corn mash , And performed in parallel. Oleyl alcohol (OA) was added to both fermentations to extract the product ( i- BuOH) formed from the broth. The fermentation broth mentioned in this example in which the solids were not removed from the feed (using a corn mash) was 2010Y036 as described in Example 6. The fermented broth mentioned in this example was the 2010 Y035 as described in Example 6, in which the solids were removed from the feed (using the concentrate produced from the corn mash). The oleyl alcohol was the extraction solvent used in both fermentation. The speed and degree of phase separation were measured and compared for the entire fermentation and for the final fermentation broth. Proper phase separation in an extractive fermentation process can lead to minimal loss of microorganisms and also minimal solvent losses and also lower capital and operating costs of the wake process.

During fermentation, the phase between the solvent and broth phase  detach

Approximately 10 mL of sample was withdrawn from each fermenter at 5.3, 29.3, 53.3, and 70.3 hours, and phase separation was performed on samples from solid fermentation (2010Y035) and solid fermentation (2010Y036) Respectively. Phase separation was observed and samples were compared for all samples from all run times by setting the sample for about 2 hours and tracking the position of the liquid-liquid interface. Essentially, phase separation was not observed for any of the samples taken from the fermentation where solid was not removed. Phase separation was observed for all samples from the fermentation removed from the liquefied corn mash prior to solid fermentation. The separation began to occur within about 10 to 15 minutes after the solid was removed from the run for all fermentation times, and was continuously enhanced over a period of 2 hours. Phase separation began to occur in the sample taken from the fermentation of the concentrate (solid removed) after about 7 minutes of precipitation time and 5.3 hours after fermentation. The phase separation began to occur in the sample taken 53.5 hours after the fermentation of the concentrate (solid was removed) after about 17 minutes of precipitation time.

Figure 13 is a plot of the position of the liquid-liquid interface in the fermentation sample tube as a function of (gravity) settling time. The data are for a sample from which the concentrate was fed as a source (solids removed from corn mash) and the oleic alcohol was taken from an extractive fermentation (conducted in Example 6, 2010Y035), which is an ISPR extraction solvent. The phase separated data in the plot are for samples taken at run times of 5.3, 29.3, 53.3, and 70.3 hours from fermentation 2010Y035. The interfacial position is recorded as a percentage of the total brot height in the sample tube. For example, the interfacial position in the sample taken from the 2010Y035 fermentation (concentrate / oleyl alcohol) at 5.3 hours of run time increased to 3.5 mL after 120 minutes of sedimentation time from the bottom of the sample tube (not separated). There was about 10 mL total broth in this particular sample tube. Thus, the interfacial position for such a sample is reported as 35% in FIG. Similarly, the interfacial position in the sample taken from the 2010Y035 fermentation (concentrate / oleyl alcohol) at 53.3 h run time increased to about 3.9 mL after the 125 min precipitation time from the bottom of the sample tube (not separated). About 10 mL of total broth was present in this particular sample tube. Therefore, the interface position for this sample is reported as 39% in FIG.

After complete fermentation, Brods  Phase separation between phases

After a running time of 70 hours, the fermentation was stopped and two broths from oleyl alcohol-extracted fermentation were transferred to separate 2 L glass graduated cylinders. Solvent and broth phases were observed and compared. For the broth that had not been removed before the solid fermentation (Run 2010Y036), most phase separation was not observed after about 3 hours. Phase separation was observed for the broth in which the solids were removed from the liquefied corn mash before fermentation (performance 2010Y035). Separation began to occur after a settling time of about 15 minutes and continued to improve over a period of 3 hours. After 15 minutes, the liquid-liquid interface was set at a level that was about 10% of the total height of the two phase mixture. This indicates that the aqueous phase is first separated from the dispersion and begins to accumulate at the bottom of the mixture. As time progresses, more aqueous phase accumulates at the bottom of the mixture, causing the position of the interface to rise. After a settling time of about 3 hours, the interface increased to a level of about 40% of the total height of the two phase mixtures. This resulted in an almost complete phase separation based on the amount of the initially charged concentrate and oleyl alcohol for fermentation after about 3 hours (gravity) settling time for the final two-phase broth in which solids were removed . Approximately the same volume of initial concentrate and solvent was charged to both fermentations. Approximately 1.2 L of liquefied corn mash and approximately 1.1 L of oleyl alcohol were charged to fermentation 2010Y036. Approximately 1.2 L of concentrate produced from the same batch of mesh, and approximately 1.1 L of oleyl alcohol were charged to fermentation 2010Y035. After considering the fact that approximately 100 g / kg of the initial sugar in the aqueous phase has been consumed and about 75% of the i-BuOH produced was in the solvent phase, the final relative amounts of aqueous and organic phases It can be estimated that this can be about 1: 1. Figure 14 is a plot of the liquid-liquid interfacial location as a function of (gravity) settling time for the final two phase broth from extract fermentation with solids removed (2010Y035). The interfacial position is reported as a percentage of the total broth height in a 2L graduated cylinder used to observe the phase separation of the final broth. From the 2010Y035 fermentation, the interfacial position of the final broth increased to a level of about 40% of the total height of the two phase mixtures after a settling time of 175 minutes from the bottom of the graduated cylinder (not detached). Thus, almost complete separation of the two phases in the final broth occurred after a settling time of 3 hours. An interfacial position of approximately 50% may correspond to complete separation.

10 mL of sample was taken from the top of the organic phase of the final broth (which had settled for about 3 hours) from the solid-free fermentation. The sample was rotated with a high-speed centrifuge to allow the broth to settle for 3 hours and the amount of aqueous phase present in the organic phase was measured. The results showed that about 90% of the top layer of the final broth was in the solvent phase. About 10% of the top layer of the final broth was an aqueous phase containing a relatively small amount of undissolved solids. Some solids were located at the bottom of the aqueous phase (more dense than the aqueous phase) and also a small amount of solid was accumulated at the liquid-liquid interface.

10 mL of sample was also taken from the bottom of the final broth (which was settled after about 3 hours) from the solids-free fermentation. The sample was rotated by a high-speed centrifuge to allow the broth to settle for 3 hours and the amount of organic phase present in the aqueous phase was measured. After the broth was settled for 3 hours, it was determined that the organic phase was essentially not present in the bottom (aqueous) phase of the final broth from the solid-free fermentation. The results demonstrate that nearly complete phase separation has occurred for the final broth from solids-free fermentation. Almost no phase separation was evident for the final broth from the solid-free fermentation. The data show that the removal of solids from liquefied corn mashes prior to extraction fermentation can result in significant improvements in phase separation during fermentation and later in phase separation, resulting in loss of microorganisms, undissolved solids, It implies that it does not exist.

After the broth was settled for about 3 hours, 10 mL of the sample from the non-solid fermentation was taken out from the top of the final broth. The sample was rotated in a high-speed laboratory centrifuge to measure the relative amounts of solvent and aqueous phase at the top of the final broth. The broth contained all solids from the liquefied corn mash solids. About half of the sample was an aqueous phase and about half was organic phase. The aqueous phase contained significantly more undissolved solids (from the liquefied mesh) as compared to the sample of the top layer from the solids-free broth. The amounts of aqueous and solvent phases in the sample are approximately the same, indicating that phase separation did not necessarily occur in the final broth where solids were not removed (even after a 3 hour precipitation time). This data implies little or no phase separation which tends to occur during and after fermentation if the solids are not removed from the liquefied corn mash prior to extraction fermentation. This could lead to significant loss of microorganisms, undissolved solids, and water to the wake process.

Example  9

ISPR  Effect of elimination of undissolved solids on loss of extraction solvent-extracted fermentation

This example demonstrates that the ability to reduce solvent is lost to DDGS at the end of the extractive fermentation process by removing undissolved solids from the pre-fermentation maize mesh. Example 6 is a liquefied mesh concentrate (2010Y036) obtained by using liquefied corn mash (2010Y036 - solid not removed) as a source and one removing most of the undissolved solids from the liquefied corn mash - &lt; / RTI &gt; aqueous solution of oligosaccharide). Oleic alcohol (OA) was added to both fermentations to extract the product isobutanol ( i- BuOH) from broth when it was formed. The amount of residual solvent trapped in the undissolved solids recovered from the final fermentation broth was measured and compared.

After the fermentations 2010Y035 and 2010Y036 described in Example 6 were completed, the broth was collected and used to perform the phase separation test. Subsequently, undissolved solids (micro-water from corn silks not removed prior to fermentation) were recovered from each broth and analyzed for total extractable oil. The oil recovered from each lot of solids was analyzed for oleyl alcohol and corn oil. For both brochures we have followed the following protocol:

The broth was centrifuged to separate the organic, aqueous, and solid phases.

The organic and aqueous phases were removed by obliquely filtering from the solids coming off the wet cake at the bottom of the centrifuge tube.

The wet cake was thoroughly washed with water to remove essentially all dissolved solids retained in the cake, such as residual sugars, glucose, salts, enzymes, and the like.

The washed wet cake was dried in a vacuum oven overnight (house-vacuum at 80 ° C) to essentially remove all of the water in the cake.

A portion of the anhydrous solid was completely contacted with hexane in a Soxhlet extractor to remove the oil from the solid.

The oil recovered from the solid was analyzed by GC to determine the relative amounts of oleyl alcohol and corn oil present in the recovered oil from the solid.

The particle size distribution (PSD) was measured for solids recovered from both fermentation broths.

Data for recovery of undissolved solids from both fermentation broths and hexane extraction are shown in Table 8. The data indicate that approximately the same amount of oil was absorbed by the solid (per unit weight of solids) in both fermentations.

[Table 8]

Example  10

By washing the wet cake with water, liquefied corn From mesh  With removal of solid Occur Recovery of soluble starch from wet cake - Two-step process

This example demonstrates the recovery of soluble starch from a wet cake by washing the wet cake twice with water, where the cake was generated by centrifuging the liquefied mesh. The liquefied corn mash was fed to a continuous decanter centrifuge to produce a concentrate stream (C-1) and a wet cake (WC-1). The concentrate was an aqueous solution which contained relatively no solids of soluble starch, and the wet cake was concentrated in the solid as compared to the feed mash. A portion of the wet cake was mixed with hot water to form a slurry (S-1). The slurry was back-pumped through a decanter centrifuge to produce a wash water concentrate (C-2) and a washed wet cake (WC-2). C-2 was a dilute aqueous solution containing relatively no solids of soluble starch. The concentration of soluble starch at C-2 was less than the concentration of soluble starch in the concentrate resulting from the separation of the mash. The liquid phase retained in the WC-2 was more diluted than in the wet cake produced from the separation of the mash than in the starch. The washed wet cake (WC-2) was mixed with hot water to form a slurry (S-2). The ratio of the amount of soluble starch in the filled water to the filled wet cake was the same in both wash steps. The second wash slurry was pumped back through a decanter centrifuge to form a second wash water concentrate (C-3) and a second washed wet cake (WC-3). C-3 was a dilute aqueous solution containing relatively no solids of soluble starch. Since the concentration of soluble starch in C-3 was less than the concentration of soluble starch in the concentrate produced in the first washing step (C-2), the liquid phase retained in WC-3 (second washed wet cake) -2 (the first washed wet cake) than in the starch. The total soluble starch in the two wash concentrates (C-2 and C-3) is a starch that can be recovered and recycled back to liquefaction. The soluble starch in the liquid retained in the final washed wet cake is much less than that in the wet cake produced in the original separation of the mesh.

Liquefied corn Mash  productivity

Approximately 1000gal of liquefied corn mash was produced in a continuous aeration system with a hammer mill, slurry mixer, slurry tank, and liquefied tank. Crushed corn, water, and alpha-amylase were continuously fed. The reactor was equipped with mechanical stirring, temperature control, and pH adjustment with ammonia or sulfuric acid. The reaction conditions were as follows:

· Hammer crusher body size: 7/64 "

· Feed rate to slurry mixer

    - Ground corn: 560 lbm / hr (14.1 wt% moisture)

    - Number of processes: 16.6 lbm / min (200 F)

- Alpha-amylase: 61 g / hr (Genecor: Spezyme ^® ALPHA)

· Slurry tank conditions:

     - Temperature: 185 ° F (85 ° C)

     - pH: 5.8

     - Retention time: 0.5 hour

    - anhydrous maize load: 31 wt%

    - Enzyme load: 0.028 wt% (based on anhydrous corn)

· Liquefaction tank conditions:

    - Temperature: 185 ° F (85 ° C)

    - pH: 5.8

    - Retention time: about 3 hours

    - No additional enzyme added.

The production rate of the liquefied corn mash was about 3 gpm. The starch content of the ground corn was determined to be about 70% by weight on anhydrous maize. The total solids (TS) of the liquefied mesh was about 31 wt% and the total suspended solids (TSS) was about 7 wt%. The liquid phase contained about 23 to 24% by weight of liquefied starch (soluble oligosaccharide) as determined by HPLC.

The liquefied mesh was centrifuged in a continuous decanter centrifuge under the following conditions:

Bowl speed: 5000 rpm (about 3600g's)

· Differential speed: 15 rpm

· Weir Diameter: 185 mm (wear plate removed)

Feed rate: varies from 5 to 20 gpm.

Approximately 850 g of concentrate and approximately 1400 lbm of wet cake were produced by centrifuging the mesh. The total solids in the wet cake were determined to be about 46.3% (suspended + dissolved) by moisture balance. It was assumed that the total suspended solids in the wet cake were about 28% by weight, with the liquid phase knowing that it contained about 23% by weight of soluble starch. The wet cake was estimated to contain approximately 12% of the soluble starch present in the liquefied mash prior to centrifuge operation.

Recovery of soluble starch from wet cake by washing the solids with water - first  wash

Approximately 707 lbm of the wet cake recovered from the separation of the liquefied mesh was mixed with 165 grams of hot (91 DEG C) water and 300 grams of stainless steel vessel. The resulting slurry was mixed for about 30 minutes. The slurry was continuously fed into a decanter centrifuge to remove washed solids from the slurry. The centrifuge used to separate the wash slurry was the same as that used to remove solids from the liquefied mesh and was rinsed with fresh water before feeding the slurry. The centrifuge was run under the following conditions to remove solids from the wash slurry:

Bowl speed: 5000 rpm (about 3600g's)

· Differential speed: 5 rpm

· Wear diameter: 185 mm (wear plate removed)

· Feed rate: 5gpm.

Approximately 600 lbm of washed wet cake was produced with a centrifuge, but only 400 lbm was recovered due to material loss. The total solids in the wet cake were determined to be about 36.7% (suspended + dissolved) by moisture balance. The total soluble starch (sum of glucose, DP2, DP3, and DP4 +) in the liquid phase and wash water concentration of the slurry (obtained from the slurry) was determined to be about 6.7 wt% and 6.9 wt% respectively by HPLC. DP2 refers to a dextrose polymer containing two glucose units (glucose dimer). DP3 refers to a dextrose polymer containing three glucose units (glucose trimer). DP4 + refers to a dextrose polymer containing four or more glucose units (glucose tetramer and more). This proved that a well-mixed dilute washing step was achieved. Thus, the concentration of soluble starch in the liquid phase retained in the washed wet cake should be about 6.8% by weight (by mass balance) for the dilute wash concerned. Based on the total solid and dissolved oligosaccharide data, it was estimated that the total suspended solids in the washed wet cake were about 32% by weight. When all 600 lbm of the cake produced by centrifugation had been washed, the washed wet cake was estimated to contain approximately 2.6% of the soluble starch present in the original liquefied mesh. This represents about 78% reduction in soluble starch in the wet cake washed compared to the pre-wash mesh wet cake. If the wet cake produced from the separation of the liquefied mesh is not cleaned, about 12% of the total starch in the mesh may be lost as soluble (liquefied) starch. When the wet cake produced from the separation of the mashes was washed with water under the proven conditions of this example, 2.6% of the total starch from the mash could be lost as soluble (liquefied) starch.

Approximately 400 lbm of the washed wet cake recovered from the first re-slurry wash of the liquefied mesh wet cake was mixed with 110 grams of hot (90 占 폚) water and 300 grams of stainless steel vessel. The resulting slurry was mixed for about 30 minutes. The slurry was continuously fed into a decanter centrifuge to remove washed solids from the slurry. The centrifuge used to separate the second wash slurry was the same as that used in the first wash, which was rinsed with fresh water before feeding the second wash slurry. The centrifuge was operated under the following conditions to remove solids from the wash slurry:

Bowl speed: 5000 rpm (about 3600g's)

· Differential speed: 5 rpm

· Wear diameter: 185 mm (remove the wear plate)

· Feeding speed: 4gpm.

Approximately 322 lbm of the cleaned wet cake was produced with a centrifuge. The total solids in the wet cake from the second wash were determined to be about 37.4% (suspended + dissolved) by moisture balance. The total soluble starch (sum of glucose, DP2, DP3, and DP4 +) in the liquid phase and wash water concentration of the slurry (obtained from the slurry) was measured by HPLC to be about 1.6% by weight and 1.6% by weight respectively. This proved that a well-mixed dilute wash step was achieved in the second wash. Thus, the concentration of soluble starch in the liquid phase retained in the washed wet cake should be about 1.6% by weight (by mass balance) for this dilute wash. Based on the total solid and dissolved oligosaccharide data, it was estimated that the total suspended solids in the washed wet cake was about 36 wt%. If all 600 lbm of the cake produced in the first washing step had been washed, the washed wet cake was estimated to contain approximately 0.5% of the soluble starch present in the original liquefied mesh. This represents a total reduction in soluble starch in the double-washed wet cake compared to about 96% wet wet cake before washing. When the wet cake produced from the separation of the liquefied mesh was not cleaned, about 12% of the total starch in the mesh could be lost as soluble (liquefied) starch. If the wet cake produced from the separation of the mashes was washed twice with water in the proven conditions of this example, 0.5% of the total starch from the mash could be lost as soluble (liquefied) starch.

Example  11

Effect of high temperature step during liquefaction on conversion of starch in corn solids to soluble (liquefied) starch

This example demonstrates that operation liquefaction using a high temperature (or "cooking") step at some time in the middle of the reaction can produce a higher conversion of the starch in corn solids into soluble (liquefied) starch. The proven " cooking "step in this example is to raise the liquefaction temperature at some point after liquefaction has started, to maintain it at a higher temperature for some period, and then to drop the temperature back to its original value to complete the liquefaction .

A. Procedure for measuring unhydrolyzed starch remaining in solid after liquefaction

A liquefied corn mesh was prepared in a single run according to the protocol of Example 1 (no intermediate high temperature steps). The liquefied corn mash was prepared in another run under the same conditions as in the first run except for the addition of intermediate high temperature stages in another run. The mesh from both runs was post-processed according to the following steps. This was centrifuged to separate an aqueous solution of liquefied starch from undissolved solids. The aqueous solution of the liquefied starch was removed by oblique filtration to recover the wet cake. The wet cake contained most of the undissolved solids from the mesh, but the solids were still wet with the liquefied starch solution. The wet cake was thoroughly washed with water and the subsequent slurry was centrifuged to separate the aqueous layer from the undissolved solid. The cake was washed five times total with water sufficient to approximately remove all of the soluble starch retained in the original wet cake recovered from liquefaction. As a result, the liquid phase was maintained in a final washed wet cake consisting essentially of water containing no soluble starch.

The final washed wet cake was re-slurried in water and added with both large excess of alpha-amylase and glucoamylase. The temperature and pH were adjusted while mixing the slurry for at least 24 hours to convert essentially all of the unhydrolyzed starch remaining in the solubilized alpha-amylase to soluble oligosaccharides. The soluble oligosaccharide generated from the residual starch (which was not hydrolyzed during liquefaction at the desired conditions) was subsequently converted to glucose by the existing glucoamylase. Glucose concentration was monitored with HPLC over time to ensure that all oligosaccharide from the residual starch was converted to glucose and glucose concentration did not increase with time.

B. Liquefied corn Mash  produce

Two batches of liquefied corn mashes (approximately 1 L each) were prepared at 85 ° C using Liquozyme ^® SC DS (alpha-amylase, Novozymes, Franklin, North Carolina). Both batches were run at 85 ° C for a little longer than 2 hours. However, the "cooking" period was added in the middle of the second batch ("Batch 2"). The temperature profile for batch 2 was about 30 minutes at 85 캜, the temperature was raised from 85 캜 to 101 캜, held at 101 캜 for about 30 minutes, dropped to 85 캜, and liquefied for another 120 minutes . The ground corn used in both batches was the same as in Example 1. A corn load of 26 wt.% (Based on anhydrous maize) was used in both batches. The total amount of enzyme used in both runs corresponded to 0.08 wt% (on anhydrous corn). The pH was adjusted at 5.8 during both liquefaction runs. Liquefaction was carried out in a glass, jacketed resin kettle. The cakes were set up with mechanical stirring, temperature control, and pH control.

A liquefied corn mash for batch 1 was prepared according to the following protocol:

Alpha-amylase was diluted with tap water (0.418 g enzyme in 20.802 g water)

704.5 g of tap water was charged into the kettle.

I turned on the shaker.

198 g of the first filler of ground corn was prepared.

Heat with shaking at 55 ° C.

The pH was adjusted to 5.8 using H ₂ SO ₄ or NaOH.

7.111 g of the first charge of the alpha-amylase solution were prepared.

Gt; 85 C. &lt; / RTI &gt;

Gt; 85 C &lt; / RTI &gt; for 30 minutes.

3.501 g of a second packing of alpha-amylase solution was prepared.

97.5 g of a second packing of ground corn was prepared.

Gt; 85 C &lt; / RTI &gt; for another 100 minutes.

After the liquefaction was completed, it was cooled to 60 캜.

The reactor was emptied and 998.5 g of liquefied mesh was recovered.

A liquefied corn mash for Batch 2 was prepared according to the following protocol:

Alpha-amylase was diluted in tap water (0.3366 g of enzyme in 16.642 g of water)

562.6 g of tap water was charged to the kettle.

I turned on the shaker.

237.5 g of ground corn were charged.

Heat with shaking at 55 ° C.

The pH was adjusted to 5.8 using dilute H ₂ SO ₄ or NaOH.

The first charge of the alpha-amylase solution, 4.25 g, was prepared.

Gt; 85 C. &lt; / RTI &gt;

Gt; 85 C &lt; / RTI &gt; for 30 minutes.

Gt; 101 C. &lt; / RTI &gt;

Gt; 101 C &lt; / RTI &gt; for 30 minutes.

The temperature of the mesh was dropped again to 85 ° C.

The pH was adjusted to 5.8 using diluted H ₂ SO ₄ or NaOH.

A second charge of alpha-amylase solution, 4.2439 g, was prepared.

Gt; 85 C &lt; / RTI &gt; for another 120 minutes.

After the liquefaction was completed, it was cooled to 60 캜.

C. liquefied From mesh  Removal of undissolved solids and washing of wet cake with water to remove soluble starch

Most of the solids were removed from both batches of the liquefied mesh by centrifugation at 5000 rpm for 20 minutes at room temperature in a large bottom centrifuge. Centrifugation of 500 g of mesh from batch 1 gave 334.1 g of concentrate and 165.9 g of wet cake. Centrifugation of 872 g of mesh from batch 2 yielded 654.7 g of concentrate and 217 g of wet cake. The wet cake recovered from each batch of liquefied mesh was washed five times with tap water to essentially remove all soluble starch retained in the cake. Washing was carried out in the same bottle used to centrifuge the original mesh to prevent transfer of the cake between containers. For each washing step, the cake was mixed with water and the resulting wash slurry was centrifuged (5000 rpm for 20 minutes) at room temperature. This was done for all five wash steps performed in the wet cake recovered from both batches of the mash. Approximately 165 g of water was used in each of the five washes of the wet cake from Batch 1 to produce a total of 828.7 g of water used to wash the wet cake from Batch 1. Approximately 500 g of water was used in each of the five washes of the wet cake from Batch 2 to produce a total of 2500 g of water used to wash the wet cake from Batch 2. [ The total washing concentrate recovered from all five washes of the wet cake from batch 1 was 893.1 g. The total washing concentrate recovered from all five water washes of the wet cake from batch 2 was 2566.3 g. The final washed wet cake recovered from batch 1 was 101.5 g and the final washed wet cake recovered from batch 2 was 151.0 g. The final washed wet cake obtained from each batch had essentially no soluble starch; The liquid held in each cake was mainly water. The total solids (TS) of the wet cake was measured using a wet balance. The total solids of the wet cake from batch 1 were 21.63 wt% and the TS for wet cake from batch 2 was 23.66 wt%.

D. Liquefaction of the washed wet cake to determine the level of hydrolyzed starch remaining in the undissolved solids after liquefaction / Glycation

The level of hydrolyzed starch remaining in the solids present in both washed wet cake was measured by reslurrying the cake in water and adding excess alpha-amylase and excess glucoamylase. Alpha-amylase converts the remaining unhydrolyzed starch in the solid into a soluble oligosaccharide that is soluble in the aqueous phase of the slurry. Glucoamylase subsequently converts soluble oligosaccharides produced by alpha-amylase to glucose. The reaction is carried out for at least 24 hours at 55 ° C (the maximum recommended temperature for glucoamylase) to ensure that all the remaining starch in the solid is converted to soluble oligosaccharides and ensures that all soluble oligosaccharides are converted to glucose. The remaining unhydrolyzed starch, which was in the solid and was not hydrolyzed during liquefaction, can be calculated from the amount of glucose produced by the process.

Alpha using the following protocol-amylase and glucoamylase enzymes are each Liquozyme ^® SC and DS Spirizyme Fuel ^® (Novozymes, Franklin, North Carolina). The vessel used to treat the washed wet cake was a 250 mL jacketed glass resin kettle equipped with mechanical stirring, temperature control, and pH adjustment. The amount of Liquozyme ^® used corresponds to an enzyme load of 0.08% by weight on "anhydrous corn basis". The amount of Sprizyme ^® used corresponds to an enzyme load of 0.2 wt.% On a "anhydrous maize basis. &Quot; The criteria are defined as the amount of ground corn required to provide the amount of undissolved solids held in the washed cake and it is assumed that all of the starch is hydrolyzed to soluble oligosaccharides. The undissolved solids retained in the washed cake are considered to be non-fermentable portions, most of which are starch free of corn. These enzyme loads are at least four times higher than those required to complete liquefaction and saccharification at a 26% corn load. The enzyme has been used in extreme amounts to ensure complete hydrolysis of the residual starch in the solid and to ensure complete conversion of the oligosaccharide to glucose.

The level of unhydrolyzed starch in the solid present in the washed wet cake from one batch of batches was determined according to the following protocol:

Alpha-amylase was diluted in tap water (0.1297 g of enzyme in 10.3607 g of water)

Glucoamylase was diluted in tap water (0.3212 g in 15.6054 g water)

132 g of tap water was charged to the kettle.

I turned on the shaker.

68 g of the washed wet cake produced from liquefied batch 1 was filled (TS = 21.63 wt%).

Heat with shaking at 55 ° C.

The pH was adjusted to 5.5 using dilute H ₂ SO ₄ or NaOH.

Alpha-amylase solution, 3.4992 g.

· 5.319 g of glucoamylase solution was charged.

The slurry for glucose was periodically sampled by performing at 55 [deg.] C for 24 hours while adjusting the pH to 5.5.

The level of unhydrolyzed starch in the solid present in the washed wet cake from batch 2 was determined according to the following protocol.

Alpha-amylase was diluted in tap water (11.709 g of 0.2384 g of enzyme in water).

Glucoamylase was diluted in tap water (0.3509 g in 17.5538 g water).

154.3 g of tap water was charged to the kettle.

I turned on the shaker.

70.7 g of the washed wet cake produced from liquefied batch 1 was filled (TS = 23.66 wt%).

Heat with shaking at 55 ° C.

The pH was adjusted to 5.5 using dilute H ₂ SO ₄ or NaOH.

Alpha-amylase solution, 2.393 g.

· 5.8314 g of glucoamylase solution was charged.

The slurry for glucose was periodically sampled by performing at 55 [deg.] C for 24 hours while adjusting the pH to 5.5.

Washed  The liquefaction / On saccharification  Comparison of results for

As described above, the washed wet cake from Batches 1 and 2 is re-slurried in water and both very high amounts of alpha-amylase and glucoamylase are added to the slurry to hydrolyze any remaining starch in the solid, Glucose. Figure 15 shows the concentration of glucose in the aqueous phase of the slurry as a function of time.

Glucose concentration increased with time and peaked at approximately 24 hours for both of the reactors. A slight decrease in glucose between 24 and 48 hours may be due to microbial contamination; The maximum level of glucose reached in each system was used to calculate the level of residual unhydrolyzed starch in the solids of the washed wet cake. The maximum level of glucose reached (in the presence of alpha-amylase and glucoamylase) by reacting the washed wet cake obtained from batch liquor was 4.48 g / L. In comparison, the washed wet cake obtained from batch liquefaction was reacted (in the presence of alpha-amylase and glucoamylase) to a maximum level of 2. 4 g / L of glucose reached.

The level of residual unhydrolyzed starch present in the undissolved solids in the liquefied mesh (which was not hydrolyzed during liquefaction) is based on the glucose data obtained from the washed wet cake obtained from the corresponding batch of mesh Respectively.

Liquefied Batch 1: The remaining unhydrolyzed starch in the solid corresponds to 2.1% of the total starch in the corn fed to the liquefaction. This implies that 2.1% of the corn starch was not hydrolyzed during the batch liquefaction condition. No intermediate high temperature ("cooking") step occurred during liquefaction batch 1.

Liquefied Batch 2: The remaining unhydrolyzed starch in the solid corresponds to 1.1% of the total starch in the corn fed to the liquefaction. This implies that 1.1% of the corn starch was not hydrolyzed during the batch liquefaction condition. The high temperature ("cooking") step did not occur during liquefaction batch 2.

This example demonstrates that the addition of the high temperature "cooking" step at some time during liquefaction could produce higher starch conversion. This will produce very little residual hydrolyzed starch remaining in the undissolved solids in the liquefied corn mash and will cause little starch loss in the process where undissolved solids are removed from the mesh before liquefaction.

Example  12

Effect of high temperature stage during liquefaction in converting cornstarch starch into soluble (liquefied) starch

Two batches of the liquefied corn mash ^® Liquozyme SC DS in a 85 ℃ (batch 3 batch and 4) was prepared by using (alpha-amylase, manufactured by Novozymes North Carolina Franklin material). Both batches were run at 85 캜 for slightly over 2 hours. However, the "cooking" period was added in the middle of batch 4. The temperature profile for batch 4 was about 30 minutes at 85 캜 and the temperature was raised from 85 캜 to 121 캜, held at 121 캜 for about 30 minutes, cooled to 85 캜, liquefied for another 90 minutes Respectively. The ground corn used for both batches was the same as in Example 1. Corn loading of 26 wt% (based on anhydrous corn) was used for both batches. The total amount of enzyme used in both runs corresponded to 0.04 wt% (on anhydrous corn). The pH was adjusted at 5.8 during both liquefaction runs. Liquefaction for batch 3 was carried out in a 1 L glass, jacketed resin kettle and liquefaction for batch 4 was carried out in a 200 L stainless steel fermenter. Both reactors were equipped with mechanical stirring, temperature control, and pH control.

The experimental conditions for this example were similar to those described for Example 9 with the following differences.

For the production of a liquefied corn mash for batch 3: 0.211 g of alpha-amylase was diluted in 10.403 g tap water. The first charge of the alpha-amylase solution was 3.556 g. The second charge of the alpha-amylase solution was 1.755 g and the reaction was allowed to continue at 85 [deg.] C for another 90 minutes.

For the production of a liquefied corn mash for batch 4: 22 g of alpha-amylase was diluted in 2 kg of tap water, 147.9 kg of tap water was charged to the fermenter, and 61.8 kg of ground corn was charged. The first charge of the alpha-amylase solution was 1.0 kg and the reaction was heated to 85 DEG C and held at 85 DEG C for 30 minutes, then the reaction was heated to 121 DEG C and held at 121 DEG C for 30 minutes. The second charge of the alpha-amylase solution was 1 kg and the reaction was allowed to continue for another 90 minutes at 85 ° C.

Liquefied From mesh  Removal of undissolved solids and washing of wet cake with water to remove soluble starch

Most of the solids were removed from both batches of the liquefied mesh by centrifugation at 5000 rpm at room temperature in a large bottom centrifuge for 15 minutes. Centrifugation of 500.1 g of mesh from batch 3 yielded 337.2 g of concentrate and 162.9 g of wet cake. Centrifugation of 509.7 g of mesh from batch 4 yielded 346.3 g of concentrate and 163.4 g of wet cake. The wet cake recovered from each batch of liquefied mesh was washed five times with tap water to essentially remove all soluble starch retained in the cake. Washing was performed in the same bottle used to centrifuge the original mesh to prevent the cake from migrating between containers. For each wash step, the cake was mixed with water and the resulting wash slurry was centrifuged at room temperature (5000 rpm for 15 minutes). This was done for all five wash steps performed in the wet cake recovered from both batches of the mash. Approximately 164 g of water was used in each of the five washes of the wet cake from batch 3 to produce a total of 819.8 g of water used to wash the wet cake from batch 3. Approximately 400 g of water was used in each of the five washes of the wet cake from Batch 4 to produce a total of 2000 g of water used to clean the wet cake from Batch 4. The total washing concentrate recovered from all five water washes of the wet cake from batch 3 was 879.5 g. The total wash concentrate recovered from all five washes of the wet cake from batch 4 was 2048.8 g. The final washed wet cake recovered from batch 3 was 103.2 g and the final washed wet cake recovered from batch 4 was 114.6 g. The final washed wet cake obtained from each batch had essentially no soluble starch; The liquid held in each cake was mainly water. The total solids (TS) of the wet cake was measured using a moisture balance. The total solids of the wet cake from batch 3 were 21.88 wt.% And the TS for wet cake from batch 4 was 18.1 wt.%.

The experimental conditions for this example were similar to those described for Example 9 with the following differences:

For liquefaction / saccharification of the washed wet cake for measuring the level of unhydrolyzed starch remaining in undissolved solids after liquefaction for batch 3: 68 g of the washed wet cake produced from the liquefaction of batch 3 is charged (TS = 21.88 wt%). 3.4984 g of alpha-amylase solution and 5.3042 g of glucoamylase were charged. The reaction was carried out at 55 ° C for 47 hours while the pH was adjusted at 5.5 and the slurry for glucose was periodically sampled.

For liquefaction / saccharification of the washed wet cake to determine the level of hydrolyzed starch remaining in undissolved solids after liquefaction for batch 4: 0.1663 g of alpha-amylase was diluted in 13.8139 g of tap water, 0.213 g of glucoamylase was diluted in 20.8002 g of tap water. 117.8 g of tap water was charged to the kettle. 82.24 g of the washed wet cake produced from the liquefaction of batch 4 was filled (TS = 18.1 wt%). 3.4952 g of alpha-amylase solution and 10.510 g of glucoamylase were charged. The reaction was carried out at 55 ° C for 50 hours while the pH was adjusted to 5.5 and the slurry for glucose was periodically sampled.

Washed  The liquefaction / On saccharification  Comparison of results for

As described above, the washed wet cake from batches 3 and 4 is re-slurried in water and very high amounts of alpha-amylase and glucoamylase are added to the slurry to hydrolyze any starch residing in the solid, Glucose. Figure 16 shows the concentration of glucose in the aqueous phase of the slurry as a function of time.

Glucose concentration increased with time and peaked at approximately 26 hours for the washed wet cake from batch 3. For the washed wet cake of Batch 4, the glucose concentration continued to increase slightly between 24 and 47 hours. The glucose concentration measured at 47 hours relative to the wet cake of Batch 4 is estimated to be approximately equal to the maximum value. The maximum level of glucose reached (in the presence of alpha-amylase and glucoamylase) by reacting the washed wet cake obtained from batch 3 liquefaction was 8.33 g / L. In comparison, the washed wet cake obtained from batch 4 liquefaction was reacted (in the presence of alpha-amylase and glucoamylase) to a maximum level of 4. 25 g / L of glucose reached.

The level of residual unhydrolyzed starch present in the undissolved solids in the liquefied mesh (which was not hydrolyzed during liquefaction) "hydrolyzes" the washed wet cake obtained from the corresponding batches of the mash In the presence of alpha-amylase and glucoamylase).

Liquefied Batch 3: The remaining unhydrolyzed starch in the solid corresponds to 3.8% of the total starch fed to the liquefaction. This implies that 3.8% of the starch in the corn was not hydrolyzed during batch 3 liquefaction conditions. No intermediate high temperature ("cooking") step occurred during liquefaction batch 3.

Liquefied Batch 4: The remaining unhydrolyzed starch in the solid corresponds to 2.2% of the total starch in the corn fed to the liquefaction. This implies that 2.2% of the corn starch was not hydrolyzed during batch 4 liquefaction conditions. The high temperature ("cooking") step occurred during liquefaction batch 4.

This example demonstrates that the addition of the high temperature "cooking" step at some time during liquefaction could produce higher starch conversion. This will produce very little residual hydrolyzed starch remaining in the undissolved solids in the liquefied corn mash and will cause little starch loss in the process where undissolved solids are removed from the mesh before liquefaction.

Example  Summary and comparison of 11 and 12

Liquefaction conditions can affect the conversion of starch in corn solids to soluble (liquefied) starch. Possible liquefaction conditions that can affect the conversion of starch in ground corn to soluble starch are temperature, enzyme (alpha-amylase) load, and +/- high temperature ("cooking") step occurs at some time during liquefaction . Examples 11 and 12 demonstrate that the implementation of the high temperature ("cooking") step at some time during liquefaction can result in higher conversion of the starch in corn solids into soluble (liquefied) starch. The liquefaction high temperature steps described in Examples 11 and 12 raise the liquefaction temperature at some point after the start of liquefaction and then maintain it at a higher temperature for some period and then drop the temperature back to its original value to complete liquefaction .

The liquefaction reactions compared in Example 11 were performed at different enzyme loading than the reactions compared in Example 12. [ This example demonstrates the effect of two major liquefaction conditions on starch conversion: (1) enzyme load, and (2) high temperature step applied at some time during liquefaction.

The conditions used to prepare the four batches of the liquefied corn mash described in Examples 11 and 12 are summarized below and in Table 9.

General conditions for all batches:

· Liquefaction temperature - 85 ℃

· Total time at liquefaction temperature - approximately 2 hours

Sieve size used to crush corn - 1 mm

PH - 5.8

· Anhydrous corn load - 26%

· Alpha-amylase - Liquozyme ^® SC DS (manufactured by North Carolina Franklin Nozymes of material).

[Table 9]

The temperature profiles for batches 2 and 4 are as follows (all values are approximate): 85 ° C for 30 minutes, 30 minutes high temperature step, 85 ° C for 90 minutes. One half of the enzyme was added before the initial 85 ° C period and half was added after the high temperature step for the final 85 ° C period.

Figure 17 shows that the effects of enzyme loading and +/- high temperature steps were applied at some time during liquefaction in starch conversion. The level of residual unhydrolyzed starch in the solid is starch that is not hydrolyzed during the desired liquefaction conditions. Figure 17 shows that the level of unhydrolyzed starch in the solid was reduced to almost one-half by applying a high temperature ("cooking") step at some point during liquefaction. This was demonstrated at two different enzyme loads. The data in Figure 17 also shows that with a double enzyme load, the hot stage produced almost half of the level of residual unhydrolyzed starch in the solid, regardless of whether it was applied during liquefaction. These examples demonstrate that the addition of a higher enzyme (alpha-amylase) load and / or a high temperature ("cooking") step at some time during the reaction is carried out in the unhydrolyzed solid remaining in the undissolved solids present in the liquefied corn mash Could lead to a significant reduction in starch and demonstrate that the loss of starch in the process can be reduced if the undissolved solids are removed from the mesh prior to liquefaction. Any residual starch in the solid after liquefaction will not have the potential to be hydrolyzed during fermentation in a process where the solid is removed prior to fermentation.

Example 13

After the enzyme decomposition at 85 ° C, Insoluble  Sieve separation

Embodiment the mash (301 grams) prepared according to the method described in Example 1, the adjustment using the drip of the NaOH solution was maintained at pH 5.8, if necessary, Liquozyme ^® of about 0.064 grams of alpha-amylase enzyme (manufactured by North Carolina Novozyme, Franklin, Tenn.) And maintained at 85 占 폚 for 5 hours. The product was refrigerated.

The refrigerated product was warmed to approximately 50 ° C and 48 g was poured into a filter assembly containing a 100 mesh sieve and connected to a storage vacuum source at -15 Hg to -20 Hg. The exposed surface area of the sieve plate was 44 cm ² and was sealed inside a plastic filter housing provided by Nalgene ^® (Thermo Fisher Scientific, Rochester, NY). The slurry was filtered to form 40.4 g of a yellow turbid filtrate in a wet cake and a receiver bottle on the sieve. Immediately after washing the wet cake with water in place, the vacuum source was paused while still drawing any liberated moisture through the final washed cake. Filtration was terminated when dripping from the bottom of the filter ceased. A further 28.5 g of wash filtrate was collected over the three stages of filtration of the final step, at least showing color and turbidity. 7.6 g of the final wet cake mass was air dried at 2.1 g over 24 hours at room temperature. 2.1 g was measured to contain 7.73% water after drying with a heat lamp. Vacuum filtration of this experiment produced a wet cake containing 25% total dry solids.

Samples of the filtrate were combined with oleic alcohol at room temperature and mixed vigorously to precipitate. The interface was restored within approximately 15 minutes, but a hazy rag layer remained.

1 g of 99.99% (trace metal based) iodine, 2 g of ReagentPlus ^® grade (> 99%) potassium iodide (both from Sigma-Aldrich, St. Louis, Mo.), and one drop of 17 g Lugol's solution (starch indicator) consisting of house deionized water was added to the sample of the filtrate and the control sample of cake-solids and water re-slurried in water was dried. The filtrate turned dark blue or purple, the solid slurry changed to a very dark blue color and the water became pale amber. Any color darker than amber indicates the presence of oligosaccharides longer than 12 units.

This experiment showed that most of the suspended solids can be separated from the prepared starch solution at a medium speed in a 100 mesh sieve as described above and the starch remains with the filter cake solids. This represents an incomplete washing of the cake where some of the hydrolyzed starch remains.

This experiment was repeated with a mesh of 156 grams in a 100 mesh sieve having a diameter of 63 mm. The maximum temperature was 102 ° C, the enzyme was Spezyme ^® , and the slurry was held at 85 ° C for 3 hours. The rate of sieving was measured to be less than 0.004 gals per minute per square foot of body area.

Example  14

After enzyme degradation at &lt; RTI ID = 0.0 &gt; 115 C & Insoluble  Sieve separation

House deionized water (200 grams) was charged into an open Parr Model 4635 1 liter pressure vessel (Moline, Ill.) And heated to a temperature of 85 degrees Celsius. The water was shaken with a magnetic stir bar. Anhydrous ground corn (90 g) prepared as described in Example 1 was added spoon-wise. The pH was raised to around 5.2 to 6.0 using a stock aqueous ammonia solution. Approximately 0.064 grams of Liquozyme ^® solution was added using a small graduated pipette. The lid of the pressure vessel was sealed and the vessel was pressurized to 50 psig using house nitrogen. The shaken mixture was heated to &lt; RTI ID = 0.0 &gt; 110 C &lt; / RTI &gt; within 6 minutes and maintained between 106 and 116 C for a total of 20 minutes. Reducing heating, relieving pressure, and opening the container. An additional 0.064 g of Liquozyme ^® was added and the temperature was maintained at 63 to 75 ° C for an additional 142 minutes.

A small amount of slurry was taken from a Parr vessel and gravity was blocked through a pile of 100, 140, and 170 mesh sieves. Slides were kept in 100 mesh sieve only.

A portion, about 40%, of the slurry was transferred while heating on top of a double screen assembly of 100 and 200 mesh dishes of 75 mm diameter. Some gravity filtration was performed. Vacuum between -15 and -20 inches of mercury was drawn into the filtrate receiver and steady filtration was established. The filtrate was yellow and cloudy but stably dispersed. The cake surface was exposed within 5 minutes. The cake was washed with deionized water spray for 2 to 3 minutes and repeated with a total of 5 doses, varying the receiver until the turbidity of the filtrate was constant. The sieve was tested and all the solids were present on a 100 mesh sieve and not on 200 mesh. The wet cake mass had a thickness of 5 mm. The wet cake mass was measured to be 18.9 g and the combined filtrate mass was 192 g.

The mass of the remaining slurry was transferred to a filter assembly at 65 DEG C to a 100 mesh sieve and filtered for 5 to 10 minutes. The cake was washed with a deionized water sprayer for 3-4 minutes until the turbidity of the filtrate became constant - the changes in the receiver were repeated with a total of 8 doses. The vacuum was maintained until no further drips were observed to fall off the underside of the filter. The wet cake had a thickness of 8 mm, a diameter of 75 mm, and a mass of 36.6 g. The combined filtrate was weighed to 261 g.

Three vials were tested for starch according to the method described above. One vial contained water and the other two contained a sample of the wet cake slurried in deionized water. All vials changed color to yellow-amber. This was interpreted to mean that the filter cake was washed so that the starch did not contain oligosaccharides. These solids were then rigorously analyzed using extended liquefaction and subsequent saccharification to confirm that, on a glucose basis, the wet cake contained less than 0.2% of the total starch present in the original corn.

The sample of the filtrate was combined with the oleyl alcohol in the vial, mixed vigorously and allowed to settle. A clear oil layer was obtained quickly and the interface was well defined to have fewer lag layers. This example demonstrates that in a process in which the corn mash is further liquefied at 85 캜 for more than 2 hours before being heated and filtered during 20 minutes of cooking under hydrothermal conditions of ~ 110 캜, the total filtrate is essentially fed to the grain Indicating that it contains all of the starch. Further, no significant interference between the oleyl alcohol and the impurities contained in the filtrate was observed.

This experiment was repeated using a mesh of 247 grams in an 80 mesh sieve having a diameter of 75 mm. The maximum cooking temperature was 115 ° C, the enzyme was Liquozyme ^®, and the slurry was held at 85 ° C for 3 hours. The sieving rate was measured and determined to be greater than 0.1 gal per minute per square foot of body area.

Example  15

This example shows the recovery of fatty acids, esters, and triglycerides from solids by removal of the solid from the distillation waste liquid and extraction with the desolvating agent. During fermentation, the solids are separated from the total distillation waste liquid and fed to the desolvation unit, where they are contacted with 1.1 ton / hr of steam. The flow rates for the whole distilled waste liquid wet cake (extractor feed), solvent, extractor micellar, and extractor discharge solids are shown in Table 10. The value in the table is short ton / hr.

[Table 10]

The solids exiting the desolvator are fed to the dryer. The vapors exiting the desolvator contain 0.55 ton / hr of hexane and 1.102 ton / hr of water. The stream is concentrated and fed to a decanter. The water-rich phase leaving the decanter contains about 360 ppm of hexane. The stream is fed to a distillation column where the hexane is removed from the water-rich stream. The hexane-enriched stream exiting the top of the distillation column is concentrated and fed to the decanter. The organic rich stream exiting the decanter feeds to the distillation column. The stream (11.02 ton / hr) is fed to the bottom of the distillation column. The composition of the upper and lower products of the column is shown in Table 11. The table value is ton / hr.

[Table 11]

Example  16

By-product recovery

This example shows the recovery of by-products from the mesh. The corn oil was separated from the mesh under the conditions described in Example 6, except that a 3-phase centrifuge (Flottweg Tricanter ^® Z23-4 bowl diameter, 230 mm, length to diameter ratio of 4: 1) :

· Bowl speed: 5000 rpm

· Differential speed: 10 rpm

· Feeding speed: 3gpm

· Phase separator disk: 138 mm

· Impeller setting: 144 mm.

Separated corn oil has 81% triglyceride, 6% free fatty acid, 4% diglyceride, and 5% total phospholipid and monoglyceride as measured by gas chromatography and thin layer chromatography (see, For example, U.S. Patent Application Publication No. 2012/0164302).

The solids separated from the mash under the conditions described above had a moisture content of 58% as measured by weight loss on drying and had 1.2% triglyceride and 0.27% free fatty acids as measured by gas chromatography (cf. &lt; RTI ID = For example, U.S. Patent Application Publication No. 2012/0164302).

The composition of the solid separated from the total distillation waste liquid, the oil extracted between the evaporator stages, the by-product extract and the condensed distiller solubles (CDS) in Table 14 are shown in Table 12 and the estimated (three-phase Separated from the centrifuge). The values in Table 11 were obtained from an Aspen Plus ^® model (Aspen Technology, Inc., Burlington, Mass.). The model assumes that the corn oil is not extracted from the mesh. The protein content on the anhydrous basis of cells, dissolved solids, and suspended solids is estimated to be approximately 50%, 22%, and 35.5%, respectively. The composition of the by-product extract is estimated to be 70.7% fatty acid and 29.3% fatty acid isobutyl ester on anhydrous basis.

[Table 12]

[Table 13]

[Table 14]

Example  17

Liquefied corn From mesh Removal of corn oil

This example describes the use of a three-phase centrifuge to remove corn oil from a liquefied corn mash. The whole corn grain typically contains about 3 to 6 wt% corn oil, most of which remain in the embryo. The corn oil is released from the embryo during dry milling and liquefaction. Subsequently, the corn mash contains glass corn oil.

Liquefied corn mashes were produced using, for example, a standard continuous liquefaction process as used in the process from anhydrous-ground corn to ethanol. The ground corn contained 4.16 wt% corn oil (based on anhydrous corn) and had a water content of 14.7 wt%. Crushed corn and water were fed into the slurry tanks at 10.2 lbm / min and 17.0 lbm / min, respectively, to yield a 32% by weight anhydrous maize load. The alpha-amylase was fed to the slurry tank at a rate corresponding to an enzyme load of about 0.025% by weight on anhydrous corn. The slurry and liquefaction tank were both run at 85 [deg.] C and pH 5.8. The total residence time at 85 캜 was about 2 hours. The mesh was produced at a rate of about 3 gpm and contained about 1.3 wt.% Corn oil on a wet basis. Some of the oil present as a glass oil and as a part were undissolved solids. This corresponds to the total corn oil content of the mesh being approximately 2.0 lbm of corn oil / corn bushel. The total solids (TS) in the mesh was 32 wt% and the total suspended solids (TSS) was 7.7 wt%.

The liquefied corn mash was supplied with approximately 3gpm speed three-phase centrifuge (Model Z23-4 / 441, Flottweg Tricanter ®, Flottweg AG Germany Freiburg Bill Nashville materials). The feed temperature was about 80 ° C. The mesh was separated into three streams: (1) corn oil, (2) an aqueous solution of oligosaccharides (liquefied starch), and (3) wet cake. The operating conditions of Tricanter ^® were as follows:

· Bowl speed: 5000 rpm

· G-Power: about 4000g's

· Differential speed: 10 rpm

· Impeller setting: approximately 145

· Phase separator disk: approx. 138 mm.

Table 15 summarizes the measured data (flow rate, density, solids content, and corn oil content) for the feed stream and the three outlet streams from Tricanter ^® .

[Table 15]

The corn oil removed from the mesh by Tricanter ^® was counted in 39% total corn oil in the mash feed. The removal rate of corn oil is about 0.8 lbm / equivalent to the bovine corn. The corn oil recovered from the liquefied corn mash contained about 85% by weight of glyceride.

In a process in which about 0.8 lb of corn oil / bovine corn was removed, the mesh flow rate could be reduced to 3.9 gal / min:

In a production plant with a total mesh flow of about 700 gpm for fermentation, the removed oil can produce about 0.55% of the total mesh flow. Assuming the production plant increases the throughput proportionally and uses additional volumes, the annual production could increase to 0.55%, which means that the 56 MMGPY plant could generate an additional 310,000 gals of ethanol.

Example  18

Liquefied corn From mesh  Removal of corn oil - adjustment of feed rate

In this embodiment, the liquefied corn mash was fed to a three-phase centrifuge at a feed rate of 1 gpm. Liquefied corn mashes were generated using, for example, a standard continuous liquefaction process used in a process from anhydrous-ground corn to ethanol. The ground corn contained 4.16 wt% corn oil (based on anhydrous corn) and the moisture content was 14.7 wt%. The ground corn and water were fed into the slurry tanks at 8.2 lbm / min and 19.0 lbm / min, respectively, to yield a dry corn load of approximately 26% by weight. Alpha-amylase was fed to the slurry tank at a rate of 50 g / hr, corresponding to an enzyme load of about 0.026% by weight on a dry corn basis. Both the slurry and the liquefaction tank were operated at 85 [deg.] C and pH 5.8. No jet cookers were used. The total residence time at 85 캜 was about 2 hours. The mesh was produced at a rate of about 3 gpm and packed into a 1500 gal tank for centrifugation. The mesh contained about 1.1% by weight corn oil on a wet basis. The glass oil and some of the oil present as part were present in undissolved solids. This corresponds to the total corn oil content of the mesh, which is approximately 2.0 lbm of corn oil / maize cores. The TS in the mesh was 25.6 wt% and the TSS was 5.3 wt%.

3-phase centrifuges for liquefied corn mash to feed tank: was supplied with a velocity of about 1gpm (model Z23-3, Flottweg Tricanter ^®, manufactured by Flottweg AG Germany Freiburg Bill fertilizing material). The feed temperature was about 80 ° C. The mesh was separated into three streams: (1) corn oil, (2) an aqueous solution of oligosaccharides (liquefied starch), and (3) wet cake. The operating conditions of Tricanter ^® were as follows:

· Bowl speed: 5000 rpm

· G-Power: about 4000g's

· Differential speed: 12 rpm

· Impeller setting: Approximately 156

· Phase separator disk: approx. 140 mm.

Table 16 summarizes the measured data (flow rate, density, solids content, and corn oil content) for the feed stream and the three outlet streams from Tricanter ^® . The quality of the corn oil mesh balance was 102% and the quality of the total solid mesh balance was 105%.

[Table 16]

The corn oil removed from the mesh by Tricanter ^® was calculated as 17% total corn oil in the mash feed. This corn oil removal rate is equivalent to about 0.34 lbm / bushel of maize. The corn oil recovered from the liquefied corn mash contained about 81.4 wt% glyceride and 8.3 wt% free fatty acid.

Example  19

Liquefied corn From mesh  Removal of corn oil - adjustment of feed rate

In this example, the liquefied corn mash was fed to a three-phase centrifuge at a feed rate of 10.1 gpm. Liquefied corn mashes, for example, were generated using a standard continuous liquefaction process as used in the process from anhydrous-ground corn to ethanol. The ground corn contained 4.16 wt% corn oil (based on anhydrous corn) and the moisture content was 14.7 wt%. The ground corn and water were fed into the slurry tanks at 8.2 lbm / min and 19.0 lbm / min, respectively, to yield a dry corn load of approximately 26% by weight. The alpha-amylase was fed to the slurry tank at a rate of 50 g / hr, corresponding to an enzyme load of about 0.026% by weight on a dry corn basis. Both the slurry and the liquefaction tank were operated at 85 [deg.] C and pH 5.8. No jet cookers were used. The total residence time at 85 캜 was about 2 hours. The mesh was produced at a rate of about 3 gpm and packed into a 1500 gal tank for centrifugation. The mesh contained about 1.1% by weight corn oil on a wet basis. The glass oil and some of the oil present as part were present in undissolved solids. This corresponds to the total corn oil content of the mesh, which is approximately 2.0 lbm of corn oil / maize cores. The TS in the mesh was 26.2 wt% and the TSS was 6.7 wt%.

The liquefied corn mash was fed from the feed tank to a 3-phase centrifuge (Model Z23-4 / 441, Flottweg Tricanter ^® Flottweg AG, Wiltshire, Germany) at a rate of about 10.1 gpm. The feed temperature was about 80 ° C. The mesh was separated into three streams: (1) corn oil, (2) an aqueous solution of oligosaccharides (liquefied starch), and (3) wet cake. The operating conditions of Tricanter ^® were as follows:

· Bowl speed: 5000 rpm

· G-Power: about 4000g's

· Differential speed: 20 rpm

· Impeller setting: approx. 148

· Phase separator disk: approx. 138 mm.

Table 17 summarizes the measured data (flow rate, density, solids content, and corn oil content) for the feed stream and the three outlet streams from Tricanter ^® . The quality of the lump balance of corn oil was 95%.

[Table 17]

The corn oil removed from the mesh by Tricanter ^® was calculated as 18% whole corn oil in the mesh feed. This corn oil removal rate was equivalent to about 0.36 lbm / bushel of maize. The corn oil recovered from the liquefied corn mash contained about 81.4 wt% glyceride and 8.3 wt% free fatty acid.

Example  20

Effect of liquefied corn mash pH on recovery of maize oil from mash

The liquefied corn mash was generated using a standard continuous liquefaction process typically used in a dry-milled corn versus ethanol process. The ground corn contained 4.6 wt% corn oil (based on anhydrous corn) and the moisture content was 12.5 wt%. The ground corn and water were fed to the slurry tank at a rate of 3 gpm at a rate of producing corn mash with a 25.9 wt% dry maize load in the slurry tank. The slurry tank was operated at 85 캜 for a residence time of 30 minutes. Subsequently, the slurry was heated to 105 DEG C in a jet cooker using the raw stream and held at that temperature for about 30 minutes. After draining the retained tube, the slurry was fed into a liquefaction tank operating at 85 DEG C with a residence time of 90 minutes. Alpha-amylase: it was fed continuously to the process in a whole corresponding to the enzyme of the enzyme load of the speed (Spezyme ^®, manufactured by ALPHA Genencor Palo Alto ^® materials) and 0.04% by weight, based on dry corn. 40% (40%) of total enzyme was added to the slurry tank and 60% was added to the liquefaction tank. Both the slurry and the liquefaction tank were run at pH 5.8. The mesh was produced at a rate of approximately 3 gpm and packed in a 1500 gal tank for centrifuge testing. The liquefied agate mesh contained about 1.12 wt% corn oil on a wet basis. This corresponds to a total corn oil content of approximately 2.2 lbm corn oil / bushel corn mesh. Some of the oil was present as glass oil; Some were still in undissolved solids. The ratio of glyceride to free fatty acid in the corn oil in the mesh was about 7.6 to 1. The total solids (TS) in the mesh was 25.9 wt% and the total suspended solids (TSS) was 4.7 wt%. The DE (dextrose equivalent) and pH of the final mesh were 15.9 and 5.75, respectively. The density of the mesh was 1.08 g / mL.

A three-phase centrifugal separator the liquefied mash: using (Model Z23-4 / 441, Flottweg Tricanter ®, manufactured by Flottweg AG of Germany bill Nashville Freiburg material) were separated in three different feed flow rate: 1.24gal / min , 5.1gal / min and 10gal / min. The feed temperature was about 80 ° C. The mesh was separated into three streams: (1) corn oil, (2) an aqueous solution of oligosaccharide (liquefied starch), and (3) wet cake. Bowl speed was kept constant at about 5000 rpm (about 4000 g's). Table 18 compares corn oil recovery as a function of mash feed rate to Tricanter ^® for a mesh pH of 5.8. The data presented in Table 18 indicate that there is an effect of the feed rate on Tricanter ^® in the recovery of corn oil at pH = 5.8.

[Table 18]

Corn oil recovery is based on total oil (both glass oil and solid oil) contained in the mesh. The mesh supplied to Tricanter ^® contained 1.1-1.2 wt% corn oil (including glass oil and oil in solid).

The data in Table 18 indicate that there is an effect of the mash feed rate on the corn oil recovery rate (under the conditions tested). Table 19 summarizes the amount of solids in the oil phase in an aqueous concentrate, the aqueous phase in an oil concentrate, and the oil concentrate for the three conditions tested.

[Table 19]

^* Measured using LuMiSizer ^® (manufactured by LUM GmbH, Berlin, Germany).

The data in Table 19 indicate that the recovered corn oil has very little transparency because it contains little aqueous phases and solids. The corn oil recovered from the liquefied corn mash contained about 85.1 wt% glyceride and 8.0 wt% free fat. Balances were solid, aqueous phase, and other extractable materials (e.g., phospholipids, sterols, etc.).

The pH of the remaining liquefied corn mash in the Tricanter ^® feed tank was reduced to about 3. Acid mesh three-phase centrifuge: using (model Z23-4 / 441, Flottweg Tricanter ^®, manufactured by German bill fertilization Flottweg AG of Freiburg material) two different feed flow rate: 1.3gal / min and 5gal / min . The feed temperature was about 80 ° C. The mesh was separated into three streams: (1) corn oil, (2) an aqueous solution of oligosaccharides (liquefied starch), and (3) wet cake. Bowl speed was kept constant at about 5000 rpm (about 4000 g's). Table 20 compares corn oil recovery as a function of feed rate with Tricanter ^® for a mesh pH of 3.0.

[Table 20]

After producing the mesh at pH = 5.8, the pH of the final mesh was lowered to 3 before feeding to the centrifuge. Corn oil recovery is based on the total oil contained in the mesh (both glass and solid oils). The mesh supplied to Tricanter ^® contained about 0.9% by weight of corn oil (including glass oil and oil in the solid). The total solid of the mesh fed to Tricanter ^® was 27.1 wt% and the total suspended solids of the mesh was 5.5 wt%.

Table 21 summarizes the amounts of solids in the oil phase, the aqueous phase in the oil concentrate, and the oil concentrate in an aqueous concentrate for the two conditions tested. The data in Table 21 show that the recovered corn oil is very transparent because it contains little aqueous phase and solids.

[Table 21]

Test A, (Table 18) compared to the results of Test D (Table 20) The results of comparing the results of Test B (Table 19) and Test E (Table 21) of mash pH in corn oil return with Tricanter ^® Effect. It is suggested that reducing the pH of the mesh before it is separated using the Tricanter ^® produces higher corn yields. These comparisons are shown in Table 22.

[Table 22]

The percentage shown in Table 22 is the recovery of corn oil based on total oil (both glass oil and solid oil) contained in the mesh. The composition of the corn oil in the mesh supplied to Tricanter ^® ranged from 0.9% to 1.2% by weight of corn oil (including glass oil and solid in oil) for all of the tests described in this example. Tricanter ^® was operated at 5000 rpm (~ 4000 G's), differential speed and impeller settings were 5 to 10 rpm and 145 mm respectively.

Example  21

corn From mesh Recovery of corn oil and solids

The liquefied corn mash was generated using a standard continuous liquefaction process used in a dry-milled corn-to-ethanol process with 30 to 31 wt% on a dry corn basis. Recirculating water consisting of cooking water and reflux was used, which increased the total solids (TS) to approximately 33% by weight. Alpha-amylase (Spezyme ^® RSL, Genencor ^® , Palo Alto, Calif.) Corresponds to approximately 0.02 wt% anhydrous corn-based enzyme load in a slurry tank (85 ° C, pH of approximately 5.8, residence time of 30 min) I went at a speed. Using a jet cooker, the temperature was raised to 105 to 110 DEG C with a 18 minute cooking time. The liquefaction tank was operated at 85 캜 using a pH of approximately 5.8. Spezyme ^® RSL (manufactured in Palo Alto, Genencor ^®) was added in addition to one equivalent to about 0.005% by weight of the dry maize base load liquefaction enzymes tank speed, total residence time of liquid in the tank was about 90 minutes. The side stream of the mesh was collected from the liquefaction tank and transferred to a mini-dilution tank where process condensate was added to achieve the desired dilution. The original mesh contained about 1.55 wt% corn oil on a wet basis. The glass oil and some of the oil present as a part were undissolved solids. This corresponds to the total corn content of the original mesh, which is approximately 3.0 lbm corn oil / boucle maize. The TS in the original mesh was 33.2 wt% and the total suspended solids (TSS) was 6.5 wt%. Dilution with process concentrate reduced TS to approximately 27 wt%, TSS to approximately 5.5 wt%, and oil content to approximately 1.3 wt% (wetting basis).

A three-phase centrifugal separator the liquefied corn mash from the feed tank: was fed at a rate of between 9 to 11gpm (model Z23-4 / 441, Flottweg Tricanter ^®, manufactured by Flottweg AG of Germany bill fertilizing material Freiburg). The feed temperature was about 85 ° C. The mesh was separated into three streams: (i) corn oil, (2) an aqueous solution of oligosaccharide (liquefied starch), and (3) wet cake. The operating conditions of the three-phase centrifuge were as follows:

· Bowl speed: 5000 rpm

· G-Force: Approximately 3200g

· Differential speed: 25 rpm

· Impeller settings: see table

· Phase separator disk: approx. 138 mm

Table 23 summarizes the conditions and characteristics of the three-phase centrifuge after separation. At both corn loadings of 33 wt% and 26 wt%, the stream was separated into a very transparent corn oil stream and a wet cake at 38-41 wt% total solids. The solid concentrate suspended in the heavy phase was strongly affected by the corn load. A sample of 33 wt% yielded approximately 3.5 to 4 wt% of concentrated TSS, while 26 wt% of TS produced approximately 1.7 to 2 wt% of less TSS concentrate.

[Table 23]

The results are also shown in Figs. 19A to 19E. 19A, at a low flow rate of approximately 4 gpm, the concentrate TSS was approximately 3.3% and the concentrate TSS increased to approximately 4.2 to 4.7% at a flow rate of 11.5 gpm.

Figure 19b shows the number of solids suspended as a function of flow rate. At low flow rates of approximately 4 gpm, approximately 60% of the suspended solids were recovered in the wet cake. By increasing the flow rate to about 11.5 gpm, the recovery was reduced to about 40 to 50%.

Figure 19c shows the total cake of the wet cake as a function of flow rate. At low flow rates of approximately 4 gpm, the total solids of the wet cake was approximately 41%. By increasing the flow rate to about 11.5 gpm, the total solids of the wet cake was reduced to about 39%.

Figure 19d shows the effect of the differential rpm on the overall wet cake solids. The wet cake solids were reduced to increased differential rpm.

Figure 19e shows the effect of flow rate on corn oil recovery. At low flow rates, the oil recovery was about 48%. If the flow rate is increased to approximately 11.5 gpm, the oil recovery is reduced to approximately 35%. The larger the flow rate, the less oil is separated from the feed stream.

Example  22

Rheological properties of corn mash

The viscosity of the maize mesh is measured using an AR-G2 rotary flow meter (TA Instruments, New Castle, Del.) With vane geometry. A slurry of ground corn and water is prepared, mixed and heated to 55 ° C in a resin cake. The pH is adjusted to 5.8 and the enzyme (e.g., alpha-amylase and / or glucoamylase) is added to the slurry. The slurry is heated to 65 DEG C at a rate of 2 DEG C / min. The sample is removed and transferred to a flow meter equipped with a narrow gap concentric cylinder geometry preheated to 65 ° C. Thereafter, the temperature ramp is performed by raising the temperature at 65 [deg.] C to 85 [deg.] C at a rate of 2 [deg.] C / min. The temperature ramp is performed at a fixed shear rate (e.g., 75-200 s ^-1 ). The viscosity is measured as a function of time.

While various embodiments of the present invention have been described above, it should be understood that they have been presented by way of example only, and not limitation. It will be apparent to one skilled in the relevant art that various changes in form and details may be made therein 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 implementations, but should be defined only in accordance with the following claims and their equivalents.

All publications, patents, and patent applications mentioned in this specification are indicative of the level of skill of those skilled in the art to which this invention pertains and are specifically and individually indicated to be incorporated by reference to the respective individual publication, patent or patent &Lt; / RTI &gt; are incorporated herein by reference.

                         SEQUENCE LISTING <110> Butamax Advanced Biofuels LLC   <120> Processes and Systems for the Fermentative Production of Alcohols <130> CL4723WOPCT1 <160> 128 <170> PatentIn version 3.5 <210> 1 <211> 570 <212> PRT <213> Bacillus subtilis <400> 1 Met Thr Lys Ala Thr Lys Glu Gln Lys Ser Leu Val Lys Asn Arg Gly 1 5 10 15 Ala Glu Leu Val Val Asp Cys Leu Val Glu Gln Gly Val Thr His Val             20 25 30 Phe Gly Ile Pro Gly Ala Lys Ile Asp Ala Val Phe Asp Ala Leu Gln         35 40 45 Asp Lys Gly Pro Glu Ile Ile Val Ala Arg His Glu Gln Asn Ala Ala     50 55 60 Phe Met Ala Gln Ala Val Gly Arg Leu Thr Gly Lys Pro Gly Val Val 65 70 75 80 Leu Val Thr Ser Gly Pro Gly Ala Ser Asn Leu Ala Thr Gly Leu Leu                 85 90 95 Thr Ala Asn Thr Glu Gly Asp Pro Val Val Ala Leu Ala Gly Asn Val             100 105 110 Ile Arg Ala Asp Arg Leu Lys Arg Thr His Gln Ser Leu Asp Asn Ala         115 120 125 Ala Leu Phe Gln Pro Ile Thr Lys Tyr Ser Val Glu Val Gln Asp Val     130 135 140 Lys Asn Ile Pro Glu Ala Val Thr Asn Ala Phe Arg Ile Ala Ser Ala 145 150 155 160 Gly Gln Ala Gly Ala Phe Val Ser Phe Pro Gln Asp Val Val Asn                 165 170 175 Glu Val Thr Asn Thr Lys Asn Val Arg Ala Val Ala Ala Pro Lys Leu             180 185 190 Gly Pro Ala Ala Asp Asp Ala Ile Ser Ala Ala Ila Ala Lys Ile Gln         195 200 205 Thr Ala Lys Leu Pro Val Val Leu Val Gly Met Lys Gly Gly Arg Pro     210 215 220 Glu Ala Ile Lys Ala Val Arg Lys Leu Leu Lys Lys Val Gln Leu Pro 225 230 235 240 Phe Val Glu Thr Tyr Gln Ala Gly Thr Leu Ser Arg Asp Leu Glu                 245 250 255 Asp Gln Tyr Phe Gly Arg Ile Gly Leu Phe Arg Asn Gln Pro Gly Asp             260 265 270 Leu Leu Leu Glu Gln Ala Asp Val Val Leu Thr Ile Gly Tyr Asp Pro         275 280 285 Ile Glu Tyr Asp Pro Lys Phe Trp Asn Ile Asn Gly Asp Arg Thr Ile     290 295 300 Ile His Leu Asp Glu Ile Ile Ala Asp Ile Asp His Ala Tyr Gln Pro 305 310 315 320 Asp Leu Glu Leu Ile Gly Asp Ile Pro Ser Thr Ile Asn His Ile Glu                 325 330 335 His Asp Ala Val Lys Val Glu Phe Ala Glu Arg Glu Gln Lys Ile Leu             340 345 350 Ser Asp Leu Lys Gln Tyr Met His Glu Gly Glu Gln Val Pro Ala Asp         355 360 365 Trp Lys Ser Asp Arg Ala His Pro Leu Glu Ile Val Lys Glu Leu Arg     370 375 380 Asn Ala Val Asp Asp His Val Thr Val Thr Cys Asp Ile Gly Ser His 385 390 395 400 Ala Ile Trp Met Ser Arg Tyr Phe Arg Ser Tyr Glu Pro Leu Thr Leu                 405 410 415 Met Ile Ser Asn Gly Met Gln Thr Leu Gly Val Ala Leu Pro Trp Ala             420 425 430 Ile Gly Ala Ser Leu Val Lys Pro Gly Glu Lys Val Val Ser Ser Ser         435 440 445 Gly Asp Gly Gly Phe Leu Phe Ser Ala Met Glu Leu Glu Thr Ala Val     450 455 460 Arg Leu Lys Ala Pro Ile Val His Ile Val Trp Asn Asp Ser Thr Tyr 465 470 475 480 Asp Met Val Ala Phe Gln Gln Leu Lys Lys Tyr Asn Arg Thr Ser Ala                 485 490 495 Val Asp Phe Gly Asn Ile Asp Ile Val Lys Tyr Ala Glu Ser Phe Gly             500 505 510 Ala Thr Gly Leu Arg Val Glu Ser Pro Asp Gln Leu Ala Asp Val Leu         515 520 525 Arg Gln Gly Met Asn Ala Glu Gly Pro Val Ile Ile Asp Val Pro Val     530 535 540 Asp Tyr Ser Asp Asn Ile Asn Leu Ala Ser Asp Lys Leu Pro Lys Glu 545 550 555 560 Phe Gly Glu Leu Met Lys Thr Lys Ala Leu                 565 570 <210> 2 <211> 1716 <212> DNA <213> Bacillus subtilis <400> 2 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> 3 <211> 559 <212> PRT <213> Klebsiella pneumoniae <400> 3 Met Asp Lys Gln Tyr Pro Val Arg Gln Trp Ala His Gly Ala Asp Leu 1 5 10 15 Val Val Ser Gln Leu Glu Ala Gln Gly Val Arg Gln Val Phe Gly Ile             20 25 30 Pro Gly Ala Lys Ile Asp Lys Val Phe Asp Ser Leu Leu Asp Ser Ser         35 40 45 Ile Arg Ile Ile Pro Val Arg His Glu Ala Asn Ala Ala Phe Met Ala     50 55 60 Ala Ala Val Gly Arg Ile Thr Gly Lys Ala Gly Val Ala Leu Val Thr 65 70 75 80 Ser Gly Pro Gly Cys Ser Asn Leu Ile Thr Gly Met Ala Thr Ala Asn                 85 90 95 Ser Glu Gly Asp Pro Val Val Ala Leu Gly Gly Ala Val Lys Arg Ala             100 105 110 Asp Lys Ala Lys Gln Val His Gln Ser Met Asp Thr Val Ala Met Phe         115 120 125 Ser Pro Val Thr Lys Tyr Ala Ile Glu Val Thr Ala Pro Asp Ala Leu     130 135 140 Ala Glu Val Val Ser Asn Ala Phe Arg Ala Ala Glu Gln Gly Arg Pro 145 150 155 160 Gly Ser Ala Phe Val Ser Leu Pro Gln Asp Val Val Asp Gly Pro Val                 165 170 175 Ser Gly Lys Val Leu Pro Ala Ser Gly Ala Pro Gln Met Gly Ala Ala             180 185 190 Pro Asp Asp Ala Ile Asp Gln Val Ala Lys Leu Ile Ala Gln Ala Lys         195 200 205 Asn Pro Ile Phe Leu Leu Gly Leu Met Ala Ser Gln Pro Glu Asn Ser     210 215 220 Lys Ala Leu Arg Arg Leu Leu Glu Thr Ser His Ile Pro Val Thr Ser 225 230 235 240 Thr Tyr Gln Ala Ala Gly Ala Val Asn Gln Asp Asn Phe Ser Arg Phe                 245 250 255 Ala Gly Arg Val Gly Leu Phe Asn Gln Ala Gly Asp Arg Leu Leu             260 265 270 Gln Leu Ala Asp Leu Val Ile Cys Ile Gly Tyr Ser Pro Val Glu Tyr         275 280 285 Glu Pro Ala Met Trp Asn Ser Gly Asn Ala Thr Leu Val His Ile Asp     290 295 300 Val Leu Pro Ala Tyr Glu Glu Arg Asn Tyr Thr Pro Asp Val Glu Leu 305 310 315 320 Val Gly Asp Ile Ala Gly Thr Leu Asn Lys Leu Ala Gln Asn Ile Asp                 325 330 335 His Arg Leu Val Leu Ser Pro Gln Ala Ala Glu Ile Leu Arg Asp Arg             340 345 350 Gln His Gln Arg Glu Leu Leu Asp Arg Arg Gly Ala Gln Leu Asn Gln         355 360 365 Phe Ala Leu His Pro Leu Arg Ile Val Arg Ala Met Gln Asp Ile Val     370 375 380 Asn Ser Asp Val Thr Leu Thr Val Asp Met Gly Ser Phe His Ile Trp 385 390 395 400 Ile Ala Arg Tyr Leu Tyr Thr Phe Arg Ala Arg Gln Val Met Ile Ser                 405 410 415 Asn Gly Gln Gln Thr Met Gly Val Ala Leu Pro Trp Ala Ile Gly Ala             420 425 430 Trp Leu Val Asn Pro Glu Arg Lys Val Val Ser Val Ser Gly Asp Gly         435 440 445 Gly Phe Leu Gln Ser Ser Met Glu Leu Glu Thr Ala Val Arg Leu Lys     450 455 460 Ala Asn Val Leu His Leu Ile Trp Val Asp Asn Gly Tyr Asn Met Val 465 470 475 480 Ala Ile Gln Glu Glu Lys Lys Tyr Gln Arg Leu Ser Gly Val Glu Phe                 485 490 495 Gly Pro Met Asp Phe Lys Ala Tyr Ala Glu Ser Phe Gly Ala Lys Gly             500 505 510 Phe Ala Val Glu Ser Ala Glu Ala Leu Glu Pro Thr Leu Arg Ala Ala         515 520 525 Met Asp Val Asp Gly Pro Ala Val Val Ala Ile Pro Val Asp Tyr Arg     530 535 540 Asp Asn Pro Leu Leu Met Gly Gln Leu His Leu Ser Gln Ile Leu 545 550 555 <210> 4 <211> 2055 <212> DNA <213> Klebsiella pneumoniae <400> 4 tcgaccacgg ggtgctgacc ttcggcgaaa ttcacaagct gatgatcgac ctgcccgccg 60 acagcgcgtt cctgcaggct aatctgcatc ccgataatct cgatgccgcc atccgttccg 120 tagaaagtta agggggtcac atggacaaac agtatccggt acgccagtgg gcgcacggcg 180 ccgatctcgt cgtcagtcag ctggaagctc agggagtacg ccaggtgttc ggcatccccg 240 gcgccaaaat cgacaaggtc tttgattcac tgctggattc ctccattcgc attattccgg 300 tacgccacga agccaacgcc gcatttatgg ccgccgccgt cggacgcatt accggcaaag 360 cgggcgtggc gctggtcacc tccggtccgg gctgttccaa cctgatcacc ggcatggcca 420 ccgcgaacag cgaaggcgac ccggtggtgg ccctgggcgg cgcggtaaaa cgcgccgata 480 aagcgaagca ggtccaccag agtatggata cggtggcgat gttcagcccg gtcaccaaat 540 acgccatcga ggtgacggcg ccggatgcgc tggcggaagt ggtctccaac gccttccgcg 600 ccgccgagca gggccggccg ggcagcgcgt tcgttagcct gccgcaggat gtggtcgatg 660 gcccggtcag cggcaaagtg ctgccggcca gcggggcccc gcagatgggc gccgcgccgg 720 atgatgccat cgaccaggtg gcgaagctta tcgcccaggc gaagaacccg atcttcctgc 780 tcggcctgat ggccagccag ccggaaaaca gcaaggcgct gcgccgtttg ctggagacca 840 gccatattcc agtcaccagc acctatcagg ccgccggagc ggtgaatcag gataacttct 900 ctcgcttcgc cggccgggtt gggctgttta acaaccaggc cggggaccgt ctgctgcagc 960 tcgccgacct ggtgatctgc atcggctaca gcccggtgga atacgaaccg gcgatgtgga 1020 acagcggcaa cgcgacgctg gtgcacatcg acgtgctgcc cgcctatgaa gagcgcaact 1080 acaccccgga tgtcgagctg gtgggcgata tcgccggcac tctcaacaag ctggcgcaaa 1140 atatcgatca tcggctggtg ctctccccgc aggcggcgga gatcctccgc gaccgccagc 1200 accagcgcga gctgctggac cgccgcggcg cgcagctcaa ccagtttgcc ctgcatcccc 1260 tgcgcatcgt tcgcgccatg caggatatcg tcaacagcga cgtcacgttg accgtggaca 1320 tgggcagctt ccatatctgg attgcccgct acctgtacac gttccgcgcc cgtcaggtga 1380 tgatctccaa cggccagcag accatgggcg tcgccctgcc ctgggctatc ggcgcctggc 1440 tggtcaatcc tgagcgcaaa gtggtctccg tctccggcga cggcggcttc ctgcagtcga 1500 gcatggagct ggagaccgcc gtccgcctga aagccaacgt gctgcatctt atctgggtcg 1560 ataacggcta caacatggtc gctatccagg aagagaaaaa atatcagcgc ctgtccggcg 1620 tcgagtttgg gccgatggat tttaaagcct atgccgaatc cttcggcgcg aaagggtttg 1680 ccgtggaaag cgccgaggcg ctggagccga ccctgcgcgc ggcgatggac gtcgacggcc 1740 cggcggtagt ggccatcccg gtggattatc gcgataaccc gctgctgatg ggccagctgc 1800 atctgagtca gattctgtaa gtcatcacaa taaggaaaga aaaatgaaaa aagtcgcact 1860 tgttaccggc gccggccagg ggattggtaa agctatcgcc cttcgtctgg tgaaggatgg 1920 atttgccgtg gccattgccg attataacga cgccaccgcc aaagcggtcg cctccgaaat 1980 caaccaggcc ggcggccgcg ccatggcggt gaaagtggat gtttctgacc gcgaccaggt 2040 atttgccgcc gtcga 2055 <210> 5 <211> 554 <212> PRT <213> Lactococcus lactis <400> 5 Met Ser Glu Lys Gln Phe Gly Ala Asn Leu Val Val Asp Ser Leu Ile 1 5 10 15 Asn His Lys Val Lys Tyr Val Phe Gly Ile Pro Gly Ala Lys Ile Asp             20 25 30 Arg Val Phe Asp Leu Leu Glu Asn Glu Glu Gly Pro Gln Met Val Val         35 40 45 Thr Arg His Glu Gln Gly Ala Ala Phe Met Ala Gln Ala Val Gly Arg     50 55 60 Leu Thr Gly Glu Pro Gly Val Val Val Val Thr Ser Gly Pro Gly Val 65 70 75 80 Ser Asn Leu Ala Thr Pro Leu Leu Thr Ala Thr Ser Glu Asp Ala                 85 90 95 Ile Leu Ala Ile Gly Gly Gln Val Lys Arg Ser Asp Arg Leu Lys Arg             100 105 110 Ala His Gln Ser Met Asp Asn Ala Gly Met Met Gln Ser Ala Thr Lys         115 120 125 Tyr Ser Ala Glu Val Leu Asp Pro Asn Thr Leu Ser Glu Ser Ile Ala     130 135 140 Asn Ala Tyr Arg Ile Ala Lys Ser Gly His Pro Gly Ala Thr Phe Leu 145 150 155 160 Ser Ile Pro Gln Asp Val Thr Asp Ala Glu Val Ser Ile Lys Ala Ile                 165 170 175 Gln Pro Leu Ser Asp Pro Lys Met Gly Asn Ala Ser Ile Asp Asp Ile             180 185 190 Asn Tyr Leu Ala Gln Ala Ile Lys Asn Ala Val Leu Pro Val Ile Leu         195 200 205 Val Gly Ala Gly Ala Ser Asp Ala Lys Val Ala Ser Ser Leu Arg Asn     210 215 220 Leu Leu Thr His Val Asn Ile Pro Val Val Glu Thr Phe Gln Gly Ala 225 230 235 240 Gly Val Ile Ser His Asp Leu Glu His Thr Phe Tyr Gly Arg Ile Gly                 245 250 255 Leu Phe Arg Asn Gln Pro Gly Asp Met Leu Leu Lys Arg Ser Asp Leu             260 265 270 Val Ile Ala Val Gly Tyr Asp Pro Ile Glu Tyr Glu Ala Arg Asn Trp         275 280 285 Asn Ala Glu Ile Asp Ser Ile Ile Ile Val Ile Asp Asn Ale Ile Ala     290 295 300 Glu Ile Asp Thr Tyr Gln Pro Glu Arg Glu Leu Ile Gly Asp Ile 305 310 315 320 Ala Ala Thr Leu Asp Asn Leu Leu Pro Ala Val Arg Gly Tyr Lys Ile                 325 330 335 Pro Lys Gly Thr Lys Asp Tyr Leu Asp Gly Leu His Glu Val Ala Glu             340 345 350 Gln His Glu Phe Asp Thr Glu Asn Thr Glu Glu Gly Arg Met His Pro         355 360 365 Leu Asp Leu Val Ser Thr Phe Gln Glu Ile Val Lys Asp Asp Glu Thr     370 375 380 Val Thr Val Asp Val Gly Ser Leu Tyr Ile Trp Met Ala Arg His Phe 385 390 395 400 Lys Ser Tyr Glu Pro Arg His Leu Leu Phe Ser Asn Gly Met Gln Thr                 405 410 415 Leu Gly Val Ala Leu Pro Trp Ala Ile Thr Ala Ala Leu Leu Arg Pro             420 425 430 Gly Lys Lys Val Tyr Ser His Ser Gly Asp Gly Gly Phe Leu Phe Thr         435 440 445 Gly Gln Glu Leu Glu Thr Ala Val Arg Leu Asn Leu Pro Ile Val Gln     450 455 460 Ile Ile Trp Asn Asp Gly His Tyr Asp Met Val Lys Phe Gln Glu Glu 465 470 475 480 Met Lys Tyr Gly Arg Ser Ala Ala Val Asp Phe Gly Tyr Val Asp Tyr                 485 490 495 Val Lys Tyr Ala Glu Ala Met Arg Ala Lys Gly Tyr Arg Ala His Ser             500 505 510 Lys Glu Glu Leu Ala Glu Ile Leu Lys Ser Ile Pro Asp Thr Thr Gly         515 520 525 Pro Val Val Ile Asp Val Pro Leu Asp Tyr Ser Asp Asn Ile Lys Leu     530 535 540 Ala Glu Lys Leu Leu Pro Glu Glu Phe Tyr 545 550 <210> 6 <211> 3220 <212> DNA <213> Lactococcus lactis <400> 6 tagatccgga aacaactgat tacctgagtt aacttagcag aaattgcaga agataacggt 60 aatttggatg aagcattaaa ttacctttat caaattccgg tgaatgatga aaattatatt 120 gctgctttaa tcaaaattgc tgacttatat caatttgaag ttgattttga aacagcaatt 180 tctaagttag aagaagcaag agaattatcg gattctcctc tgattacttt tgctttggct 240 gagtcctact ttgaacaagg tgattattca gctgccatta ccgaatatgc aaaactttca 300 gaacgaaaaa ttttacatga aacaaaaatt tctatttatc aaagaattgg tgactcttat 360 gcccaattag gtaattttga gaatgccata tcatttcttg aaaaatcact tgaatttgat 420 gaaaaaccgg aaaccttgta taaaattgct cttctttatg gagaaactca taatgaaaca 480 agagccattg ctaatttcaa acggttagaa aaaatggatg ttgaattttt gaactatgaa 540 ttagcctatg cccaaaccct agaagctaat caagaattta aagctgcact agaaatggca 600 aagaaaggga tgaaaaaaaa tcctaatgcc gttcctctct tacacttcgc ttcaaaaatt 660 tgtttcaaac ttaaggacaa agctgcagca gaacgttatc tcgtggatgc tttaaattta 720 ccagaattac atgacgaaac agtctttttg cttgctaatt tatacttcaa cgaagaagat 780 tttgaagctg tcattaatct tgaagagctt ttagaagatg aacatttatt agctaaatgg 840 ctttttgcag gagcacataa agctttggaa aatgattctg aagcggctgc tttgtatgaa 900 gaactcattc aaaccaatct gtcagagaat ccagagtttt tagaagacta tattgatttt 960 cttaaagaaa ttggtcaaat ttctaaaaca gaaccaatta ttgaacaata tttggaactt 1020 gttccagatg atgaaaatat gagaaattta ctgacagact taaaaaataa ttactgacaa 1080 agctgtcagt aattattttt attgtaagct agaaaattca aaaacttgcg tcaaaataat 1140 tgtaaaaggt tctattatct gataaaatga ttgtgaagta atccaagaga ttatgaaata 1200 tgaattagaa caaatagagg taaaataaaa aatgtctgag aaacaatttg gggcgaactt 1260 ggttgtcgat agtttgatta accataaagt gaagtatgta tttgggattc caggagcaaa 1320 aattgaccgg gtttttgatt tattagaaaa tgaagaaggc cctcaaatgg tcgtgactcg 1380 tcatgagcaa ggagctgctt tcatggctca agctgtcggt cgtttaactg gcgaacctgg 1440 tgtagtagtt gttacgagtg ggcctggtgt atcaaacctt gcgactccgc ttttgaccgc 1500 gacatcagaa ggtgatgcta ttttggctat cggtggacaa gttaaacgaa gtgaccgtct 1560 taaacgtgcg caccaatcaa tggataatgc tggaatgatg caatcagcaa caaaatattc 1620 agcagaagtt cttgacccta atacactttc tgaatcaatt gccaacgctt atcgtattgc 1680 aaaatcagga catccaggtg caactttctt atcaatcccc caagatgtaa cggatgccga 1740 agtatcaatc aaagccattc aaccactttc agaccctaaa atggggaatg cctctattga 1800 tgacattaat tatttagcac aaganattaa aaatgctgta ttgccagtaa ttttggttgg 1860 agctggtgct tcagatgcta aagtcgcttc atccttgcgt aatctattga ctcatgttaa 1920 tattcctgtc gttgaaacat tccaaggtgc aggggttatt tcacatgatt tagaacatac 1980 tttttatgga cgtatcggtc ttttccgcaa tcaaccaggc gatatgcttc tgaaacgttc 2040 tgaccttgtt attgctgttg gttatgaccc aattgaatat gaagctcgta actggaatgc 2100 agaaattgat agtcgaatta tcgttattga taatgccatt gctgaaattg atacttacta 2160 ccaaccagag cgtgaattaa ttggtgatat cgcagcaaca ttggataatc ttttaccagc 2220 tgttcgtggc tacaaaattc caaaaggaac aaaagattat ctcgatggcc ttcatgaagt 2280 tgctgagcaa cacgaatttg atactgaaaa tactgaagaa ggtagaatgc accctcttga 2340 tttggtcagc actttccaag aaatcgtcaa ggatgatgaa acagtaaccg ttgacgtagg 2400 ttcactctac atttggatgg cacgtcattt caaatcatac gaaccacgtc atctcctctt 2460 ctcaaacgga atgcaaacac tcggagttgc acttccttgg gcaattacag ccgcattgtt 2520 gcgcccaggt aaaaaagttt attcacactc tggtgatgga ggcttccttt tcacagggca 2580 agaattggaa acagctgtac gtttgaatct tccaatcgtt caaattatct ggaatgacgg 2640 ccattatgat atggttaaat tccaagaaga aatgaaatat ggtcgttcag cagccgttga 2700 ttttggctat gttgattacg taaaatatgc tgaagcaatg agagcaaaag gttaccgtgc 2760 acacagcaaa gaagaacttg ctgaaattct caaatcaatc ccagatacta ctggaccggt 2820 ggtaattgac gttcctttgg actattctga taacattaaa ttagcagaaa aattattgcc 2880 tgaagagttt tattgattac aatcaagcaa tttgtggcat aacaaaataa aagaagaagg 2940 ccttgaacac ctaagcgttc agggcctttt tttgtgaaat aaattagatg aaatttacaa 3000 tgagttttgt gaaactagct tctagtttgt gaaaaattgc ctataattgc cgaataaaaa 3060 tacccattta ccactccaag aggatgcttc aaattagcta aatacccgtt ttagaggatg 3120 cgtaaaaaca acaaaagagg atgagtatag aacgataaaa cttttttatg ataggttgag 3180 agaattgaat ataaaatata ataagtagaa ggcagcaatt 3220 <210> 7 <211> 491 <212> PRT <213> Escherichia coli <400> 7 Met Ala Asn Tyr Phe Asn Thr Leu Asn Leu Arg Gln Gln Leu Ala Gln 1 5 10 15 Leu Gly Lys Cys Arg Phe Met Gly Arg Asp Glu Phe Ala Asp Gly Ala             20 25 30 Ser Tyr Leu Gln Gly Lys Lys Val Val Ile Val Gly Cys Gly Ala Gln         35 40 45 Gly Leu Asn Gln Gly Leu Asn Met Arg Asp Ser Gly Leu Asp Ile Ser     50 55 60 Tyr Ala Leu Arg Lys Glu Ala Ile Ala Glu Lys Arg Ala Ser Trp Arg 65 70 75 80 Lys Ala Thr Glu Asn Gly Phe Lys Val Gly Thr Tyr Glu Glu Leu Ile                 85 90 95 Pro Gln Ala Asp Leu Val Ile Asn Leu Thr Pro Asp Lys Gln His Ser             100 105 110 Asp Val Val Arg Thr Val Gln Pro Leu Met Lys Asp Gly Ala Ala Leu         115 120 125 Gly Tyr Ser His Gly Phe Asn Ile Val Glu Val Gly Glu Gln Ile Arg     130 135 140 Lys Asp Ile Thr Val Val Met Ala Pro Lys Cys Pro Gly Thr Glu 145 150 155 160 Val Arg Glu Glu Tyr Lys Arg Gly Phe Gly Val Pro Thr Leu Ile Ala                 165 170 175 Val His Pro Glu Asn Asp Pro Lys Gly Glu Gly Met Ala Ile Ala Lys             180 185 190 Ala Trp Ala Ala Ala Thr Gly Gly His Arg Ala Gly Val Leu Glu Ser         195 200 205 Ser Phe Val Ala Glu Val Lys Ser Asp Leu Met Gly Glu Gln Thr Ile     210 215 220 Leu Cys Gly Met Leu Gln Ala Gly Ser Leu Leu Cys Phe Asp Lys Leu 225 230 235 240 Val Glu Glu Gly Thr Asp Pro Ala Tyr Ala Glu Lys Leu Ile Gln Phe                 245 250 255 Gly Trp Glu Thr Ile Thr Glu Ale Leu Lys Gln Gly Gly Ile Thr Leu             260 265 270 Met Met Asp Arg Leu Ser Asn Pro Ala Lys Leu Arg Ala Tyr Ala Leu         275 280 285 Ser Glu Gln Leu Lys Glu Ile Met Ala Pro Leu Phe Gln Lys His Met     290 295 300 Asp Asp Ile Ile Ser Gly Glu Phe Ser Ser Gly Met Met Ala Asp Trp 305 310 315 320 Ala Asn Asp Asp Lys Lys Leu Leu Thr Trp Arg Glu Glu Thr Gly Lys                 325 330 335 Thr Ala Phe Glu Thr Ala Pro Gln Tyr Glu Gly Lys Ile Gly Glu Gln             340 345 350 Glu Tyr Phe Asp Lys Gly Val Leu Met Ile Ala Met Val Lys Ala Gly         355 360 365 Val Glu Leu Ala Phe Glu Thr Met Val Asp Ser Gly Ile Ile Glu Glu     370 375 380 Ser Ala Tyr Tyr Glu Ser Leu His Glu Leu Pro Leu Ile Ala Asn Thr 385 390 395 400 Ile Ala Arg Lys Arg Leu Tyr Glu Met Asn Val Val Ile Ser Asp Thr                 405 410 415 Ala Glu Tyr Gly Asn Tyr Leu Phe Ser Tyr Ala Cys Val Pro Leu Leu             420 425 430 Lys Pro Phe Met Ala Glu Leu Gln Pro Gly Asp Leu Gly Lys Ala Ile         435 440 445 Pro Glu Gly Ala Val Asp Asn Gly Gln Leu Arg Asp Val Asn Glu Ala     450 455 460 Ile Arg Ser His Ala Ile Glu Gln Val Gly Lys Lys Leu Arg Gly Tyr 465 470 475 480 Met Thr Asp Met Lys Arg Ile Ala Val Ala Gly                 485 490 <210> 8 <211> 1476 <212> DNA <213> Escherichia coli <400> 8 atggctaact acttcaatac actgaatctg cgccagcagc tggcacagct gggcaaatgt 60 cgctttatgg gccgcgatga attcgccgat ggcgcgagct accttcaggg taaaaaagta 120 gtcatcgtcg gctgtggcgc acagggtctg aaccagggcc tgaacatgcg tgattctggt 180 ctcgatatct cctacgctct gcgtaaagaa gcgattgccg agaagcgcgc gtcctggcgt 240 aaagcgaccg aaaatggttt taaagtgggt acttacgaag aactgatccc acaggcggat 300 ctggtgatta acctgacgcc ggacaagcag cactctgatg tagtgcgcac cgtacagcca 360 ctgatgaaag acggcgcggc gctgggctac tcgcacggtt tcaacatcgt cgaagtgggc 420 gagcagatcc gtaaagatat caccgtagtg atggttgcgc cgaaatgccc aggcaccgaa 480 gtgcgtgaag agtacaaacg tgggttcggc gtaccgacgc tgattgccgt tcacccggaa 540 aacgatccga aaggcgaagg catggcgatt gccaaagcct gggcggctgc aaccggtggt 600 caccgtgcgg gtgtgctgga atcgtccttc gttgcggaag tgaaatctga cctgatgggc 660 gagcaaacca tcctgtgcgg tatgttgcag gctggctctc tgctgtgctt cgacaagctg 720 gtggaagaag gtaccgatcc agcatacgca gaaaaactga ttcagttcgg ttgggaaacc 780 atcaccgaag cactgaaaca gggcggcatc accctgatga tggaccgtct ctctaacccg 840 gcgaaactgc gtgcttatgc gctttctgaa cagctgaaag agatcatggc acccctgttc 900 cagaaacata tggacgacat catctccggc gaattctctt ccggtatgat ggcggactgg 960 gccaacgatg ataagaaact gctgacctgg cgtgaagaga ccggcaaaac cgcgtttgaa 1020 accgcgccgc agtatgaagg caaaatcggc gagcaggagt acttcgataa aggcgtactg 1080 atgattgcga tggtgaaagc gggcgttgaa ctggcgttcg aaaccatggt cgattccggc 1140 atcattgaag agtctgcata ttatgaatca ctgcacgagc tgccgctgat tgccaacacc 1200 atcgcccgta agcgtctgta cgaaatgaac gtggttatct ctgataccgc tgagtacggt 1260 aactatctgt tctcttacgc ttgtgtgccg ttgctgaaac cgtttatggc agagctgcaa 1320 ccgggcgacc tgggtaaagc tattccggaa ggcgcggtag ataacgggca actgcgtgat 1380 gtgaacgaag cgattcgcag ccatgcgatt gagcaggtag gtaagaaact gcgcggctat 1440 atgacagata tgaaacgtat tgctgttgcg ggttaa 1476 <210> 9 <211> 395 <212> PRT <213> Saccharomyces cerevisiae <400> 9 Met Leu Arg Thr Gln Ala Ala Arg Leu Ile Cys Asn Ser Arg Val Ile 1 5 10 15 Thr Ala Lys Arg Thr Phe Ala Leu Ala Thr Arg Ala Ala Ala Tyr Ser             20 25 30 Arg Pro Ala Ala Arg Phe Val Lys Pro Met Ile Thr Thr Arg Gly Leu         35 40 45 Lys Gln Ile Asn Phe Gly Gly Thr Val Glu Thr Val Tyr Glu Arg Ala     50 55 60 Asp Trp Pro Arg Glu Lys Leu Leu Asp Tyr Phe Lys Asn Asp Thr Phe 65 70 75 80 Ala Leu Ile Gly Tyr Gly Ser Gln Gly Tyr Gly Gln Gly Leu Asn Leu                 85 90 95 Arg Asp Asn Gly Leu Asn Val Ile Ile Gly Val Arg Lys Asp Gly Ala             100 105 110 Ser Trp Lys Ala Ala Ile Glu Asp Gly Trp Val Pro Gly Lys Asn Leu         115 120 125 Phe Thr Val Glu Asp Ala Ile Lys Arg Gly Ser Tyr Val Met Asn Leu     130 135 140 Leu Ser Asp Ala Ala Gln Ser Glu Thr Trp Pro Ala Ile Lys Pro Leu 145 150 155 160 Leu Thr Lys Gly Lys Thr Leu Tyr Phe Ser His Gly Phe Ser Pro Val                 165 170 175 Phe Lys Asp Leu Thr His Val Glu Pro Pro Lys Asp Leu Asp Val Ile             180 185 190 Leu Val Ala Pro Lys Gly Ser Gly Arg Thr Val Arg Ser Leu Phe Lys         195 200 205 Glu Gly Arg Gly Ile Asn Ser Ser Tyr Ala Val Trp Asn Asp Val Thr     210 215 220 Gly Lys Ala His Glu Lys Ala Gln Ala Leu Ala Val Ala Ile Gly Ser 225 230 235 240 Gly Tyr Val Tyr Gln Thr Thr Phe Glu Arg Glu Val Asn Ser Asp Leu                 245 250 255 Tyr Gly Glu Arg Gly Cys Leu Met Gly Gly Ile His Gly Met Phe Leu             260 265 270 Ala Gln Tyr Asp Val Leu Arg Glu Asn Gly His Ser Pro Ser Glu Ala         275 280 285 Phe Asn Glu Thr Val Glu Glu Ala Thr Gln Ser Leu Tyr Pro Leu Ile     290 295 300 Gly Lys Tyr Gly Met Asp Tyr Met Tyr Asp Ala Cys Ser Thr Thr Ala 305 310 315 320 Arg Arg Gly Ala Leu Asp Trp Tyr Pro Ile Phe Lys Asn Ala Leu Lys                 325 330 335 Pro Val Phe Gln Asp Leu Tyr Glu Ser Thr Lys Asn Gly Thr Glu Thr             340 345 350 Lys Arg Ser Leu Glu Phe Asn Ser Gln Pro Asp Tyr Arg Glu Lys Leu         355 360 365 Glu Lys Glu Leu Asp Thr Ile Arg Asn Met Glu Ile Trp Lys Val Gly     370 375 380 Lys Glu Val Arg Lys Leu Arg Pro Glu Asn Gln 385 390 395 <210> 10 <211> 1188 <212> DNA <213> Saccharomyces cerevisiae <400> 10 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> 11 <211> 330 <212> PRT <213> Methanococcus maripaludis <400> 11 Met Lys Val Phe Tyr Asp Ser Asp Phe Lys Leu Asp Ala Leu Lys Glu 1 5 10 15 Lys Thr Ile Ala Val Ile Gly Tyr Gly Ser Gln Gly Arg Ala Gln Ser             20 25 30 Leu Asn Met Lys Asp Ser Gly Leu Asn Val Val Gly Leu Arg Lys         35 40 45 Asn Gly Ala Ser Trp Asn Asn Ala Lys Ala Asp Gly His Asn Val Met     50 55 60 Thr Ile Glu Glu Ala Gla Lys Ala Asp Ile Ile His Ile Leu Ile 65 70 75 80 Pro Asp Glu Leu Gln Ala Glu Val Tyr Glu Ser Gln Ile Lys Pro Tyr                 85 90 95 Leu Lys Glu Gly Lys Thr Leu Ser Phe Ser His Gly Phe Asn Ile His             100 105 110 Tyr Gly Phe Ile Val Pro Pro Lys Gly Val Asn Val Val Leu Val Ala         115 120 125 Pro Lys Ser Pro Gly Lys Met Val Arg Arg Thr Tyr Glu Glu Gly Phe     130 135 140 Gly Val Pro Gly Leu Ile Cys Ile Glu Ile Asp Ala Thr Asn Asn Ala 145 150 155 160 Phe Asp Ile Val Ser Ala Met Ala Lys Gly Ile Gly Leu Ser Arg Ala                 165 170 175 Gly Val Ile Gln Thr Thr Phe Lys Glu Glu Thr Glu Thr Asp Leu Phe             180 185 190 Gly Glu Gln Ala Val Leu Cys Gly Gly Val Thr Glu Leu Ile Lys Ala         195 200 205 Gly Phe Glu Thr Leu Val Glu Ala Gly Tyr Ala Pro Glu Met Ala Tyr     210 215 220 Phe Glu Thr Cys His Glu Leu Lys Leu Ile Val Asp Leu Ile Tyr Gln 225 230 235 240 Lys Gly Phe Lys Asn Met Trp Asn Asp Val Ser Asn Thr Ala Glu Tyr                 245 250 255 Gly Gly Leu Thr Arg Arg Ser Ser Ile Val Thr Ala Asp Ser Lys Ala             260 265 270 Ala Met Lys Glu Ile Leu Arg Glu Ile Gln Asp Gly Arg Phe Thr Lys         275 280 285 Glu Phe Leu Leu Glu Lys Gln Val Ser Tyr Ala His Leu Lys Ser Met     290 295 300 Arg Arg Leu Glu Gly Asp Leu Gln Ile Glu Glu Val Gly Ala Lys Leu 305 310 315 320 Arg Lys Met Cys Gly Leu Glu Lys Glu Glu                 325 330 <210> 12 <211> 993 <212> DNA <213> Methanococcus maripaludis <400> 12 atgaaggtat tctatgactc agattttaaa ttagatgctt taaaagaaaa aacaattgca 60 gtaatcggtt atggaagtca aggtagggca cagtccttaa acatgaaaga cagcggatta 120 aacgttgttg ttggtttaag aaaaaacggt gcttcatgga acaacgctaa agcagacggt 180 cacaatgtaa tgaccattga agaagctgct gaaaaagcgg acatcatcca catcttaata 240 cctgatgaat tacaggcaga agtttatgaa agccagataa aaccatacct aaaagaagga 300 aaaacactaa gcttttcaca tggttttaac atccactatg gattcattgt tccaccaaaa 360 ggagttaacg tggttttagt tgctccaaaa tcacctggaa aaatggttag aagaacatac 420 gaagaaggtt tcggtgttcc aggtttaatc tgtattgaaa ttgatgcaac aaacaacgca 480 tttgatattg tttcagcaat ggcaaaagga atcggtttat caagagctgg agttatccag 540 acaactttca aagaagaaac agaaactgac cttttcggtg aacaagctgt tttatgcggt 600 ggagttaccg aattaatcaa ggcaggattt gaaacactcg ttgaagcagg atacgcacca 660 gaaatggcat actttgaaac ctgccacgaa ttgaaattaa tcgttgactt aatctaccaa 720 aaaggattca aaaacatgtg gaacgatgta agtaacactg cagaatacgg cggacttaca 780 agaagaagca gaatcgttac agctgattca aaagctgcaa tgaaagaaat cttaagagaa 840 atccaagatg gaagattcac aaaagaattc cttctcgaaa aacaggtaag ctatgctcat 900 ttaaaatcaa tgagaagact cgaaggagac ttacaaatcg aagaagtcgg cgcaaaatta 960 agaaaaatgt gcggtcttga aaaagaagaa taa 993 <210> 13 <211> 342 <212> PRT <213> Bacillus subtilis <400> 13 Met Val Lys Val Tyr Tyr Asn Gly Asp Ile Lys Glu Asn Val Leu Ala 1 5 10 15 Gly Lys Thr Val Ala Val Ile Gly Tyr Gly Ser Gln Gly His Ala His             20 25 30 Ala Leu Asn Leu Lys Glu Ser Gly Val Asp Val Ile Val Gly Val Arg         35 40 45 Gln Gly Lys Ser Phe Thr Gln Ala Gln Glu Asp Gly His Lys Val Phe     50 55 60 Ser Val Lys Glu Ala Ala Ala Gl Ala Glu Ile Ile Met Val Leu Leu 65 70 75 80 Pro Asp Glu Gln Gln Gln Lys Val Tyr Glu Ala Glu Ile Lys Asp Glu                 85 90 95 Leu Thr Ala Gly Lys Ser Leu Val Phe Ala His Gly Phe Asn Val His             100 105 110 Phe His Gln Ile Val Pro Pro Ala Asp Val Asp Val Phe Leu Val Ala         115 120 125 Pro Lys Gly Pro Gly His Leu Val Arg Arg Thr Tyr Glu Gln Gly Ala     130 135 140 Gly Val Pro Ala Leu Phe Ala Ile Tyr Gln Asp Val Thr Gly Glu Ala 145 150 155 160 Arg Asp Lys Ala Leu Ala Tyr Ala Lys Gly Ile Gly Gly Ala Arg Ala                 165 170 175 Gly Val Leu Glu Thr Thr Phe Lys Glu Glu Thr Glu Thr Asp Leu Phe             180 185 190 Gly Glu Gln Ala Val Leu Cys Gly Gly Leu Ser Ala Leu Val Lys Ala         195 200 205 Gly Phe Glu Thr Leu Thr Glu Ala Gly Tyr Gln Pro Glu Leu Ala Tyr     210 215 220 Phe Glu Cys Leu His Glu Leu Lys Leu Ile Val Asp Leu Met Tyr Glu 225 230 235 240 Glu Gly Leu Ala Gly Met Arg Tyr Ser Ile Ser Asp Thr Ala Gln Trp                 245 250 255 Gly Asp Phe Val Ser Gly Pro Arg Val Val Asp Ala Lys Val Lys Glu             260 265 270 Ser Met Lys Glu Val Leu Lys Asp Ile Gln Asn Gly Thr Phe Ala Lys         275 280 285 Glu Trp Ile Val Glu Asn Gln Val Asn Arg Pro Arg Phe Asn Ala Ile     290 295 300 Asn Ala Ser Glu Asn Glu His Gln Ile Glu Val Val Gly Arg Lys Leu 305 310 315 320 Arg Glu Met Met Pro Phe Val Lys Gln Gly Lys Lys Lys Glu Ala Val                 325 330 335 Val Ser Val Ala Gln Asn             340 <210> 14 <211> 1476 <212> DNA <213> Bacillus subtilis <400> 14 atggctaact acttcaatac actgaatctg cgccagcagc tggcacagct gggcaaatgt 60 cgctttatgg gccgcgatga attcgccgat ggcgcgagct accttcaggg taaaaaagta 120 gtcatcgtcg gctgtggcgc acagggtctg aaccagggcc tgaacatgcg tgattctggt 180 ctcgatatct cctacgctct gcgtaaagaa gcgattgccg agaagcgcgc gtcctggcgt 240 aaagcgaccg aaaatggttt taaagtgggt acttacgaag aactgatccc acaggcggat 300 ctggtgatta acctgacgcc ggacaagcag cactctgatg tagtgcgcac cgtacagcca 360 ctgatgaaag acggcgcggc gctgggctac tcgcacggtt tcaacatcgt cgaagtgggc 420 gagcagatcc gtaaagatat caccgtagtg atggttgcgc cgaaatgccc aggcaccgaa 480 gtgcgtgaag agtacaaacg tgggttcggc gtaccgacgc tgattgccgt tcacccggaa 540 aacgatccga aaggcgaagg catggcgatt gccaaagcct gggcggctgc aaccggtggt 600 caccgtgcgg gtgtgctgga atcgtccttc gttgcggaag tgaaatctga cctgatgggc 660 gagcaaacca tcctgtgcgg tatgttgcag gctggctctc tgctgtgctt cgacaagctg 720 gtggaagaag gtaccgatcc agcatacgca gaaaaactga ttcagttcgg ttgggaaacc 780 atcaccgaag cactgaaaca gggcggcatc accctgatga tggaccgtct ctctaacccg 840 gcgaaactgc gtgcttatgc gctttctgaa cagctgaaag agatcatggc acccctgttc 900 cagaaacata tggacgacat catctccggc gaattctctt ccggtatgat ggcggactgg 960 gccaacgatg ataagaaact gctgacctgg cgtgaagaga ccggcaaaac cgcgtttgaa 1020 accgcgccgc agtatgaagg caaaatcggc gagcaggagt acttcgataa aggcgtactg 1080 atgattgcga tggtgaaagc gggcgttgaa ctggcgttcg aaaccatggt cgattccggc 1140 atcattgaag agtctgcata ttatgaatca ctgcacgagc tgccgctgat tgccaacacc 1200 atcgcccgta agcgtctgta cgaaatgaac gtggttatct ctgataccgc tgagtacggt 1260 aactatctgt tctcttacgc ttgtgtgccg ttgctgaaac cgtttatggc agagctgcaa 1320 ccgggcgacc tgggtaaagc tattccggaa ggcgcggtag ataacgggca actgcgtgat 1380 gtgaacgaag cgattcgcag ccatgcgatt gagcaggtag gtaagaaact gcgcggctat 1440 atgacagata tgaaacgtat tgctgttgcg ggttaa 1476 <210> 15 <211> 343 <212> PRT <213> Anaerostipes caccae <400> 15 Met Glu Glu Cys Lys Met Ala Lys Ile Tyr Tyr Gln Glu Asp Cys Asn 1 5 10 15 Leu Ser Leu Leu Asp Gly Lys Thr Ile Ala Val Ile Gly Tyr Gly Ser             20 25 30 Gln Gly His Ala His Ala Leu Asn Ala Lys Glu Ser Gly Cys Asn Val         35 40 45 Ile Ile Gly Leu Tyr Glu Gly Ala Lys Glu Trp Lys Arg Ala Glu Glu     50 55 60 Gln Gly Phe Glu Val Tyr Thr Ala Ala Glu Ala Ala Lys Lys Ala Asp 65 70 75 80 Ile Ile Met Ile Leu Ile Asn Asp Glu Lys Gln Ala Thr Met Tyr Lys                 85 90 95 Asn Asp Ile Glu Pro Asn Leu Glu Ala Gly Asn Met Leu Met Phe Ala             100 105 110 His Gly Phe Asn Ile His Phe Gly Cys Ile Val Pro Pro Lys Asp Val         115 120 125 Asp Val Thr Met Ile Ala Pro Lys Gly Pro Gly His Thr Val Arg Ser     130 135 140 Glu Tyr Glu Glu Gly Lys Gly Val Pro Cys Leu Val Ala Val Glu Gln 145 150 155 160 Asp Ala Thr Gly Lys Ala Leu Asp Met Ala Leu Ala Tyr Ala Leu Ala                 165 170 175 Ile Gly Gly Ala Arg Ala Gly Val Leu Glu Thr Thr Phe Arg Thr Glu             180 185 190 Thr Glu Thr Asp Leu Phe Gly Glu Gln Ala Val Leu Cys Gly Gly Val         195 200 205 Cys Ala Leu Met Gln Ala Gly Phe Glu Thr Leu Val Glu Ala Gly Tyr     210 215 220 Asp Pro Arg Asn Ala Tyr Phe Glu Cys Ile His Glu Met Lys Leu Ile 225 230 235 240 Val Asp Leu Ile Tyr Gln Ser Gly Phe Ser Gly Met Arg Tyr Ser Ile                 245 250 255 Ser Asn Thr Ala Glu Tyr Gly Asp Tyr Ile Thr Gly Pro Lys Ile Ile             260 265 270 Thr Glu Asp Thr Lys Lys Ala Met Lys Lys Ile Leu Ser Asp Ile Gln         275 280 285 Asp Gly Thr Phe Ala Lys Asp Phe Leu Val Asp Met Ser Asp Ala Gly     290 295 300 Ser Gln Val His Phe Lys Ala Met Arg Lys Leu Ala Ser Glu His Pro 305 310 315 320 Ala Glu Val Val Gly Glu Glu Ile Arg Ser Leu Tyr Ser Trp Ser Asp                 325 330 335 Glu Asp Lys Leu Ile Asn Asn             340 <210> 16 <211> 343 <212> PRT <213> Anaerostipes caccae <400> 16 Met Glu Glu Cys Lys Met Ala Lys Ile Tyr Tyr Gln Glu Asp Cys Asn 1 5 10 15 Leu Ser Leu Leu Asp Gly Lys Thr Ile Ala Val Ile Gly Tyr Gly Ser             20 25 30 Gln Gly His Ala His Ala Leu Asn Ala Lys Glu Ser Gly Cys Asn Val         35 40 45 Ile Ile Gly Leu Tyr Glu Gly Ala Lys Asp Trp Lys Arg Ala Glu Glu     50 55 60 Gln Gly Phe Glu Val Tyr Thr Ala Ala Glu Ala Ala Lys Lys Ala Asp 65 70 75 80 Ile Ile Met Ile Leu Ile Asn Asp Glu Lys Gln Ala Thr Met Tyr Lys                 85 90 95 Asn Asp Ile Glu Pro Asn Leu Glu Ala Gly Asn Met Leu Met Phe Ala             100 105 110 His Gly Phe Asn Ile His Phe Gly Cys Ile Val Pro Pro Lys Asp Val         115 120 125 Asp Val Thr Met Ile Ala Pro Lys Gly Pro Gly His Thr Val Arg Ser     130 135 140 Glu Tyr Glu Glu Gly Lys Gly Val Pro Cys Leu Val Ala Val Glu Gln 145 150 155 160 Asp Ala Thr Gly Lys Ala Leu Asp Met Ala Leu Ala Tyr Ala Leu Ala                 165 170 175 Ile Gly Gly Ala Arg Ala Gly Val Leu Glu Thr Thr Phe Arg Thr Glu             180 185 190 Thr Glu Thr Asp Leu Phe Gly Glu Gln Ala Val Leu Cys Gly Gly Val         195 200 205 Cys Ala Leu Met Gln Ala Gly Phe Glu Thr Leu Val Glu Ala Gly Tyr     210 215 220 Asp Pro Arg Asn Ala Tyr Phe Glu Cys Ile His Glu Met Lys Leu Ile 225 230 235 240 Val Asp Leu Ile Tyr Gln Ser Gly Phe Ser Gly Met Arg Tyr Ser Ile                 245 250 255 Ser Asn Thr Ala Glu Tyr Gly Asp Tyr Ile Thr Gly Pro Lys Ile Ile             260 265 270 Thr Glu Asp Thr Lys Lys Ala Met Lys Lys Ile Leu Ser Asp Ile Gln         275 280 285 Asp Gly Thr Phe Ala Lys Asp Phe Leu Val Asp Met Ser Asp Ala Gly     290 295 300 Ser Gln Val His Phe Lys Ala Met Arg Lys Leu Ala Ser Glu His Pro 305 310 315 320 Ala Glu Val Val Gly Glu Glu Ile Arg Ser Leu Tyr Ser Trp Ser Asp                 325 330 335 Glu Asp Lys Leu Ile Asn Asn             340 <210> 17 <211> 616 <212> PRT <213> Escherichia coli <400> 17 Met Pro Lys Tyr Arg Ser Ala Thr Thr His Gly Arg Asn Met Ala 1 5 10 15 Gly Ala Arg Ala Leu Trp Arg Ala Thr Gly Met Thr Asp Ala Asp Phe             20 25 30 Gly Lys Pro Ile Ile Ala Val Val Asn Ser Phe Thr Gln Phe Val Pro         35 40 45 Gly His Val His Leu Arg Asp Leu Gly Lys Leu Val Ala Glu Gln Ile     50 55 60 Glu Ala Ala Gly Gly Val Ala Lys Glu Phe Asn Thr Ile Ala Val Asp 65 70 75 80 Asp Gly Ile Ala Met Gly His Gly Gly Met Leu Tyr Ser Leu Pro Ser                 85 90 95 Arg Glu Leu Ile Ala Asp Ser Val Glu Tyr Met Val Asn Ala His Cys             100 105 110 Ala Asp Ala Met Val Cys Ile Ser Asn Cys Asp Lys Ile Thr Pro Gly         115 120 125 Met Leu Met Ala Ser Leu Arg Leu Asn Ile Pro Val Ile Phe Val Ser     130 135 140 Gly Gly Pro Met Glu Ala Gly Lys Thr Lys Leu Ser Asp Gln Ile Ile 145 150 155 160 Lys Leu Asp Leu Val Asp Ala Met Ile Gln Gly Ala Asp Pro Lys Val                 165 170 175 Ser Asp Ser Gln Ser Asp Gln Val Glu Arg Ser Ala Cys Pro Thr Cys             180 185 190 Gly Ser Cys Ser Gly Met Phe Thr Ala Asn Ser Met Asn Cys Leu Thr         195 200 205 Glu Ala Leu Gly Leu Ser Gln Pro Gly Asn Gly Ser Leu Leu Ala Thr     210 215 220 His Ala Asp Arg Lys Gln Leu Phe Leu Asn Ala Gly Lys Arg Ile Val 225 230 235 240 Glu Leu Thr Lys Arg Tyr Tyr Glu Gln Asn Asp Glu Ser Ala Leu Pro                 245 250 255 Arg Asn Ile Ala Ser Lys Ala Ala Phe Glu Asn Ala Met Thr Leu Asp             260 265 270 Ile Ala Met Gly Gly Ser Thr Asn Thr Val Leu His Leu Leu Ala Ala         275 280 285 Ala Gln Glu Ala Glu Ile Asp Phe Thr Met Ser Asp Ile Asp Lys Leu     290 295 300 Ser Arg Lys Val Pro Gln Leu Cys Lys Val Ala Pro Ser Thr Gln Lys 305 310 315 320 Tyr His Met Glu Asp Val His Arg Ala Gly Gly Val Ile Gly Ile Leu                 325 330 335 Gly Glu Leu Asp Arg Ala Gly Leu Leu Asn Arg Asp Val Lys Asn Val             340 345 350 Leu Gly Leu Thr Leu Pro Gln Thr Leu Glu Gln Tyr Asp Val Met Leu         355 360 365 Thr Gln Asp Asp Ala Val Lys Asn Met Phe Arg Ala Gly Pro Ala Gly     370 375 380 Ile Arg Thr Thr Gln Ala Phe Ser Gln Asp Cys Arg Trp Asp Thr Leu 385 390 395 400 Asp Asp Arg Ala Asn Gly Cys Ile Arg Ser Leu Glu His Ala Tyr                 405 410 415 Ser Lys Asp Gly Gly Leu Ala Val Leu Tyr Gly Asn Phe Ala Glu Asn             420 425 430 Gly Cys Ile Val Lys Thr Ala Gly Val Asp Asp Ser Ile Leu Lys Phe         435 440 445 Thr Gly Pro Ala Lys Val Tyr Glu Ser Gln Asp Asp Ala Val Glu Ala     450 455 460 Ile Leu Gly Gly Lys Val Val Ala Gly Asp Val Val Ile Arg Tyr 465 470 475 480 Glu Gly Pro Lys Gly Gly Pro Gly Met Gln Glu Met Leu Tyr Pro Thr                 485 490 495 Ser Phe Leu Lys Ser Met Gly Leu Gly Lys Ala Cys Ala Leu Ile Thr             500 505 510 Asp Gly Arg Phe Ser Gly Gly Thr Ser Gly Leu Ser Ile Gly His Val         515 520 525 Ser Pro Glu Ala Ala Ser Gly Gly Ser Ile Gly Leu Ile Glu Asp Gly     530 535 540 Asp Leu Ile Ale Ile Asp Ile Pro Asn Arg Gly Ile Gln Leu Gln Val 545 550 555 560 Ser Asp Ala Glu Leu Ala Ala Arg Arg Glu Ala Gln Asp Ala Arg Gly                 565 570 575 Asp Lys Ala Trp Thr Pro Lys Asn Arg Glu Arg Gln Val Ser Phe Ala             580 585 590 Leu Arg Ala Tyr Ala Ser Leu Ala Thr Ser Ala Asp Lys Gly Ala Val         595 600 605 Arg Asp Lys Ser Lys Leu Gly Gly     610 615 <210> 18 <211> 1851 <212> DNA <213> Escherichia coli <400> 18 atgcctaagt accgttccgc caccaccact catggtcgta atatggcggg tgctcgtgcg 60 ctgtggcgcg ccaccggaat gaccgacgcc gatttcggta agccgattat cgcggttgtg 120 aactcgttca cccaatttgt accgggtcac gtccatctgc gcgatctcgg taaactggtc 180 gccgaacaaa ttgaagcggc tggcggcgtt gccaaagagt tcaacaccat tgcggtggat 240 gatgggattg ccatgggcca cggggggatg ctttattcac tgccatctcg cgaactgatc 300 gctgattccg ttgagtatat ggtcaacgcc cactgcgccg acgccatggt ctgcatctct 360 aactgcgaca aaatcacccc ggggatgctg atggcttccc tgcgcctgaa tattccggtg 420 atctttgttt ccggcggccc gatggaggcc gggaaaacca aactttccga tcagatcatc 480 aagctcgatc tggttgatgc gatgatccag ggcgcagacc cgaaagtatc tgactcccag 540 agcgatcagg ttgaacgttc cgcgtgtccg acctgcggtt cctgctccgg gatgtttacc 600 gctaactcaa tgaactgcct gaccgaagcg ctgggcctgt cgcagccggg caacggctcg 660 ctgctggcaa cccacgccga ccgtaagcag ctgttcctta atgctggtaa acgcattgtt 720 gaattgacca aacgttatta cgagcaaaac gacgaaagtg cactgccgcg taatatcgcc 780 agtaaggcgg cgtttgaaaa cgccatgacg ctggatatcg cgatgggtgg atcgactaac 840 accgtacttc acctgctggc ggcggcgcag gaagcggaaa tcgacttcac catgagtgat 900 atcgataagc tttcccgcaa ggttccacag ctgtgtaaag ttgcgccgag cacccagaaa 960 taccatatgg aagatgttca ccgtgctggt ggtgttatcg gtattctcgg cgaactggat 1020 cgcgcggggt tactgaaccg tgatgtgaaa aacgtacttg gcctgacgtt gccgcaaacg 1080 ctggaacaat acgacgttat gctgacccag gatgacgcgg taaaaaatat gttccgcgca 1140 ggtcctgcag gcattcgtac cacacaggca ttctcgcaag attgccgttg ggatacgctg 1200 gcgccgatc gcgccaatgg ctgtatccgc tcgctggaac acgcctacag caaagacggc 1260 ggcctggcgg tgctctacgg taactttgcg gaaaacggct gcatcgtgaa aacggcaggc 1320 gtcgatgaca gcatcctcaa attcaccggc ccggcgaaag tgtacgaaag ccaggacgat 1380 gcggtagaag cgattctcgg cggtaaagtt gtcgccggag atgtggtagt aattcgctat 1440 gaaggcccga aaggcggtcc ggggatgcag gaaatgctct acccaaccag cttcctgaaa 1500 tcaatgggtc tcggcaaagc ctgtgcgctg atcaccgacg gtcgtttctc tggtggcacc 1560 tctggtcttt ccatcggcca cgtctcaccg gaagcggcaa gcggcggcag cattggcctg 1620 attgaagatg gtgacctgat cgctatcgac atcccgaacc gtggcattca gttacaggta 1680 agcgatgccg aactggcggc gcgtcgtgaa gcgcaggacg ctcgaggtga caaagcctgg 1740 acgccgaaaa atcgtgaacg tcaggtctcc tttgccctgc gtgcttatgc cagcctggca 1800 accagcgccg acaaaggcgc ggtgcgcgat aaatcgaaac tggggggtta a 1851 <210> 19 <211> 585 <212> PRT <213> Saccharomyces cerevisiae <400> 19 Met Gly Leu Leu Thr Lys Val Ala Thr Ser Arg Gln Phe Ser Thr Thr 1 5 10 15 Arg Cys Val Ala Lys Lys Leu Asn Lys Tyr Ser Tyr Ile Ile Thr Glu             20 25 30 Pro Lys Gly Gln Gly Ala Ser Gln Ala Met Leu Tyr Ala Thr Gly Phe         35 40 45 Lys Lys Glu Asp Phe Lys Lys Pro Gln Val Gly Val Gly Ser Cys Trp     50 55 60 Trp Ser Gly Asn Pro Cys Asn Met His Leu Leu Asp Leu Asn Asn Arg 65 70 75 80 Cys Ser Gln Ser Ile Glu Lys Ala Gly Leu Lys Ala Met Gln Phe Asn                 85 90 95 Thr Ile Gly Val Ser Asp Gly Ile Ser Met Gly Thr Lys Gly Met Arg             100 105 110 Tyr Ser Leu Gln Ser Arg Glu Ile Ile Ala Asp Ser Phe Glu Thr Ile         115 120 125 Met Met Ala Gln His Tyr Asp Ala Asn Ile Ala Ile Pro Ser Cys Asp     130 135 140 Lys Asn Met Pro Gly Val Met Met Ala Met Gly Arg His His As Arg Pro 145 150 155 160 Ser Ile Met Val Tyr Gly Gly Thr Ile Leu Pro Gly His Pro Thr Cys                 165 170 175 Gly Ser Ser Lys Ile Ser Lys Asn Ile Asp Ile Val Ser Ala Phe Gln             180 185 190 Ser Tyr Gly Glu Tyr Ile Ser Lys Gln Phe Thr Glu Glu Glu Arg Glu         195 200 205 Asp Val Val Glu His Ala Cys Pro Gly Pro Gly Ser Cys Gly Gly Met     210 215 220 Tyr Thr Ala Asn Thr Met Ala Ser Ala Ala Glu Val Leu Gly Leu Thr 225 230 235 240 Ile Pro Asn Ser Ser Ser Phe Pro Ala Val Ser Lys Glu Lys Leu Ala                 245 250 255 Glu Cys Asp Asn Ile Gly Glu Tyr Ile Lys Lys Thr Met Glu Leu Gly             260 265 270 Ile Leu Pro Arg Asp Ile Leu Thr Lys Glu Ala Phe Glu Asn Ala Ile         275 280 285 Thr Val Val Ala Thr Gly Gly Ser Thr Asn Ala Val Leu His Leu     290 295 300 Val Ala Val Ala His Ser Ala Gly Val Lys Leu Ser Pro Asp Asp Phe 305 310 315 320 Gln Arg Ile Ser Asp Thr Thr Pro Leu Ile Gly Asp Phe Lys Pro Ser                 325 330 335 Gly Lys Tyr Val Met Ala Asp Leu Ile Asn Val Gly Gly Thr Gln Ser             340 345 350 Val Ile Lys Tyr Leu Tyr Glu Asn Asn Met Leu His Gly Asn Thr Met         355 360 365 Thr Val Thr Gly Asp Thr Leu Ala Glu Arg Ala Lys Lys Ala Pro Ser     370 375 380 Leu Pro Glu Gly Gln Glu Ile Ile Lys Pro Leu Ser His Pro Ile Lys 385 390 395 400 Ala Asn Gly His Leu Gln Ile Leu Tyr Gly Ser Leu Ala Pro Gly Gly                 405 410 415 Ala Val Gly Lys Ile Thr Gly Lys Gly Gly Thr Tyr Phe Lys Gly Arg             420 425 430 Ala Arg Val Phe Glu Glu Glu Gly Ala Phe Ile Glu Ala Leu Glu Arg         435 440 445 Gly Glu Ile Lys Lys Gly Glu Lys Thr Val Val Ile Arg Tyr Glu     450 455 460 Gly Pro Arg Gly Ala Pro Gly Met Pro Glu Met Leu Lys Pro Ser Ser 465 470 475 480 Ala Leu Met Gly Tyr Gly Leu Gly Lys Asp Val Ala Leu Leu Thr Asp                 485 490 495 Gly Arg Phe Ser Gly Gly Ser His Gly Phe Leu Ile Gly His Ile Val             500 505 510 Pro Glu Ala Ala Glu Gly Gly Pro Ile Gly Leu Val Arg Asp Gly Asp         515 520 525 Glu Ile Ile Asp Asp Ala Asp Asn Asn Lys Ile Asp Leu Leu Val Ser     530 535 540 Asp Lys Glu Met Ala Gln Arg Lys Gln Ser Trp Val Ala Pro Pro Pro 545 550 555 560 Arg Tyr Thr Arg Gly Thr Leu Ser Lys Tyr Ala Lys Leu Val Ser Asn                 565 570 575 Ala Ser Asn Gly Cys Val Leu Asp Ala             580 585 <210> 20 <211> 1131 <212> DNA <213> Saccharomyces cerevisiae <400> 20 atgaccttgg cacccctaga cgcctccaaa gttaagataa ctaccacaca acatgcatct 60 aagccaaaac cgaacagtga gttagtgttt ggcaagagct tcacggacca catgttaact 120 gcggaatgga cagctgaaaa agggtggggt accccagaga ttaaacctta tcaaaatctg 180 tctttagacc cttccgcggt ggttttccat tatgcttttg agctattcga agggatgaag 240 gcttacagaa cggtggacaa caaaattaca atgtttcgtc cagatatgaa tatgaagcgc 300 atgaataagt ctgctcagag aatctgtttg ccaacgttcg acccagaaga gttgattacc 360 ctaattggga aactgatcca gcaagataag tgcttagttc ctgaaggaaa aggttactct 420 ttatatatca ggcctacatt aatcggcact acggccggtt taggggtttc cacgcctgat 480 agagccttgc tatatgtcat ttgctgccct gtgggtcctt attacaaaac tggatttaag 540 gcggtcagac tggaagccac tgattatgcc acaagagctt ggccaggagg ctgtggtgac 600 aagaaactag gtgcaaacta cgccccctgc gtcctgccac aattgcaagc tgcttcaagg 660 ggttaccaac aaaatttatg gctatttggt ccaaataaca acattactga agtcggcacc 720 atgaatgctt ttttcgtgtt taaagatagt aaaacgggca agaaggaact agttactgct 780 ccactagacg gtaccatttt ggaaggtgtt actagggatt ccattttaaa tcttgctaaa 840 gaaagactcg aaccaagtga atggaccatt agtgaacgct acttcactat aggcgaagtt 900 actgagagat ccaagaacgg tgaactactt gaagcctttg gttctggtac tgctgcgatt 960 gtttccccca ttaaggaaat cggctggaaa ggcgaacaaa ttaatattcc gttgttgccc 1020 ggcgaacaaa ccggtccatt ggccaaagaa gttgcacaat ggattaatgg aatccaatat 1080 ggcgagactg agcatggcaa ttggtcaagg gttgttactg atttgaactg a 1131 <210> 21 <211> 550 <212> PRT <213> Methanococcus maripaludis <400> 21 Met Ile Ser Asp Asn Val Lys Lys Gly Val Ile Arg Thr Pro Asn Arg 1 5 10 15 Ala Leu Leu Lys Ala Cys Gly Tyr Thr Asp Glu Asp Met Glu Lys Pro             20 25 30 Phe Ile Gly Ile Val Asn Ser Phe Thr Glu Val Val Pro Gly His Ile         35 40 45 His Leu Arg Thr Leu Ser Glu Ala Ala Lys His Gly Val Tyr Ala Asn     50 55 60 Gly Gly Thr Pro Phe Glu Phe Asn Thr Ile Gly Ile Cys Asp Gly Ile 65 70 75 80 Ala Met Gly His Glu Gly Met Lys Tyr Ser Leu Pro Ser Arg Glu Ile                 85 90 95 Ile Ala Asp Ala Val Glu Ser Met Ala Arg Ala His Gly Phe Asp Gly             100 105 110 Leu Val Leu Ile Pro Thr Cys Asp Lys Ile Val Pro Gly Met Ile Met         115 120 125 Gly Ala Leu Arg Leu Asn Ile Pro Phe Ile Val Val Thr Gly Gly Pro     130 135 140 Met Leu Pro Gly Glu Phe Gln Gly Lys Lys Tyr Glu Leu Ile Ser Leu 145 150 155 160 Phe Glu Gly Val Gly Glu Tyr Gln Val Gly Lys Ile Thr Glu Glu Glu                 165 170 175 Leu Lys Cys Ile Glu Asp Cys Ala Cys Ser Gly Ala Gly Ser Cys Ala             180 185 190 Gly Leu Tyr Thr Ala Asn Ser Ala Cys Leu Thr Glu Ala Leu Gly         195 200 205 Leu Ser Leu Pro Met Cys Ala Thr Thr His Ala Val Asp Ala Gln Lys     210 215 220 Val Arg Leu Ala Lys Lys Ser Gly Ser Lys Ile Val Asp Met Val Lys 225 230 235 240 Glu Asp Leu Lys Pro Thr Asp Ile Leu Thr Lys Glu Ala Phe Glu Asn                 245 250 255 Ala Ile Leu Val Asp Leu Ala Leu Gly Gly Ser Thr Asn Thr Thr Leu             260 265 270 His Ile Pro Ala Ile Ala Asn Glu Ile Glu Asn Lys Phe Ile Thr Leu         275 280 285 Asp Asp Phe Asp Arg Leu Ser Asp Glu Val Pro His Ile Ala Ser Ile     290 295 300 Lys Pro Gly Gly Glu His Tyr Met Ile Asp Leu His Asn Ala Gly Gly 305 310 315 320 Ile Pro Ala Val Leu Asn Val Leu Lys Glu Lys Ile Arg Asp Thr Lys                 325 330 335 Thr Val Asp Gly Arg Ser Ile Leu Glu Ile Ala Glu Ser Val Lys Tyr             340 345 350 Ile Asn Tyr Asp Val Ile Arg Lys Val Glu Ala Pro Val His Glu Thr         355 360 365 Ala Gly Leu Arg Val Leu Lys Gly Asn Leu Ala Pro Asn Gly Cys Val     370 375 380 Val Lys Ile Gly Ala Val His Pro Lys Met Tyr Lys His Asp Gly Pro 385 390 395 400 Ala Lys Val Tyr Asn Ser Glu Asp Glu Ala Ile Ser Ala Ile Leu Gly                 405 410 415 Gly Lys Ile Val Glu Gly Asp Val Ile Val Ile Arg Tyr Glu Gly Pro             420 425 430 Ser Gly Gly Pro Gly Met Gly Met Leu Ser Pro Thr Ser Ala Ile         435 440 445 Cys Gly Met Gly Leu Asp Asp Ser Val Ala Leu Ile Thr Asp Gly Arg     450 455 460 Phe Ser Gly Gly Ser Arg Gly Pro Cys Ile Gly His Val Ser Pro Glu 465 470 475 480 Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Gla Asp Ile Ile                 485 490 495 Lys Ile Asp Met Ile Glu Lys Glu Ile Asn Val Asp Leu Asp Glu Ser             500 505 510 Val Ile Lys Glu Arg Leu Ser Lys Leu Gly Glu Phe Glu Pro Lys Ile         515 520 525 Lys Lys Gly Tyr Leu Ser Ser Tyr Ser Lys Leu Val Ser Ser Ala Asp     530 535 540 Glu Gly Ala Val Leu Lys 545 550 <210> 22 <211> 1653 <212> DNA <213> Methanococcus maripaludis <400> 22 atgataagtg ataacgtcaa aaagggagtt ataagaactc caaaccgagc tcttttaaag 60 gcttgcggat atacagacga agacatggaa aaaccattta ttggaattgt aaacagcttt 120 acagaagttg ttcccggcca cattcactta agaacattat cagaagcggc taaacatggt 180 gtttatgcaa acggtggaac accatttgaa tttaatacca ttggaatttg cgacggtatt 240 gcaatgggcc acgaaggtat gaaatactct ttaccttcaa gagaaattat tgcagacgct 300 gttgaatcaa tggcaagagc acatggattt gatggtcttg ttttaattcc tacgtgtgat 360 aaaatcgttc ctggaatgat aatgggtgct ttaagactaa acattccatt tattgtagtt 420 actggaggac caatgcttcc cggagaattc caaggtaaaa aatacgaact tatcagcctt 480 tttgaaggtg tcggagaata ccaagttgga aaaattactg aagaagagtt aaagtgcatt 540 gaagactgtg catgttcagg tgctggaagt tgtgcagggc tttacactgc aaacagtatg 600 gcctgcctta cagaagcttt gggactctct cttccaatgt gtgcaacaac gcatgcagtt 660 gatgcccaaa aagttaggct tgctaaaaaa agtggctcaa aaattgttga tatggtaaaa 720 gaagacctaa aaccaacaga catattaaca aaagaagctt ttgaaaatgc tattttagtt 780 gaccttgcac ttggtggatc aacaaacaca acattacaca ttcctgcaat tgcaaatgaa 840 attgaaaata aattcataac tctcgatgac tttgacaggt taagcgatga agttccacac 900 attgcatcaa tcaaaccagg tggagaacac tacatgattg atttacacaa tgctggaggt 960 attcctgcgg tattgaacgt tttaaaagaa aaaattagag atacaaaaac agttgatgga 1020 agaagcattt tggaaatcgc agaatctgtt aaatacataa attacgacgt tataagaaaa 1080 gtggaagctc cggttcacga aactgctggt ttaagggttt taaagggaaa tcttgctcca 1140 aacggttgcg ttgtaaaaat cggtgcagta catccgaaaa tgtacaaaca cgatggacct 1200 gcaaaagttt acaattccga agatgaagca atttctgcga tacttggcgg aaaaattgta 1260 gaaggggacg ttatagtaat cagatacgaa ggaccatcag gaggccctgg aatgagagaa 1320 atgctctccc caacttcagc aatctgtgga atgggtcttg atgacagcgt tgcattgatt 1380 actgatggaa gattcagtgg tggaagtagg ggcccatgta tcggacacgt ttctccagaa 1440 gctgcagctg gcggagtaat tgctgcaatt gaaaacgggg atatcatcaa aatcgacatg 1500 attgaaaaag aaataaatgt tgatttagat gaatcagtca ttaaagaaag actctcaaaa 1560 ctgggagaat ttgagcctaa aatcaaaaaa ggctatttat caagatactc aaaacttgtc 1620 tcatctgctg acgaaggggc agttttaaaa taa 1653 <210> 23 <211> 558 <212> PRT <213> Bacillus subtilis <400> 23 Met Ala Glu Leu Arg Ser Asn Met Ile Thr Gln Gly Ile Asp Arg Ala 1 5 10 15 Pro His Arg Ser Leu Leu Arg Ala Ala Gly Val Lys Glu Glu Asp Phe             20 25 30 Gly Lys Pro Phe Ile Ala Val Cys Asn Ser Tyr Ile Asp Ile Val Pro         35 40 45 Gly His Val His Leu Gln Glu Phe Gly Lys Ile Val Lys Glu Ala Ile     50 55 60 Arg Glu Ala Gly Gly Val Pro Phe Glu Phe Asn Thr Ile Gly Val Asp 65 70 75 80 Asp Gly Ile Ala Met Gly His Ile Gly Met Arg Tyr Ser Leu Pro Ser                 85 90 95 Arg Glu Ile Ile Ala Asp Ser Val Glu Thr Val Val Ser Ala His Trp             100 105 110 Phe Asp Gly Met Val Cys Ile Pro Asn Cys Asp Lys Ile Thr Pro Gly         115 120 125 Met Leu Met Ala Ala Met Arg Ile Asn Ile Pro Thr Ile Phe Val Ser     130 135 140 Gly Gly Pro Met Ala Ala Gly Arg Thr Ser Asp Gly Arg Lys Ile Ser 145 150 155 160 Leu Ser Ser Val Phe Glu Gly Val Gly Ala Tyr Gln Ala Gly Lys Ile                 165 170 175 Asn Glu Asn Glu Leu Gln Glu Leu Glu Gln Phe Gly Cys Pro Thr Cys             180 185 190 Gly Ser Cys Ser Gly Met Phe Thr Ala Asn Ser Met Asn Cys Leu Ser         195 200 205 Glu Ala Leu Gly Leu Ala Leu Pro Gly Asn Gly     210 215 220 Ser Pro Glu Arg Lys Glu Phe Val Arg Lys Ser Ala Gln Leu Met 225 230 235 240 Glu Thr Ile Arg Lys Asp Ile Lys Pro Arg Asp Ile Val Thr Val Lys                 245 250 255 Ala Ile Asp Asn Ala Phe Ala Leu Asp Met Ala Leu Gly Gly Ser Thr             260 265 270 Asn Thr Val Leu His Thr Leu Ala Leu Ala Asn Glu Ala Gly Val Glu         275 280 285 Tyr Ser Leu Glu Arg Ile Asn Glu Val Ala Glu Arg Val Val His Leu     290 295 300 Ala Lys Leu Ala Pro Ala Ser Asp Val Phe Ile Glu Asp Leu His Glu 305 310 315 320 Ala Gly Gly Val Ser Ala Ala Leu Asn Glu Leu Ser Lys Lys Glu Gly                 325 330 335 Ala Leu His Leu Asp Ala Leu Thr Val Thr Gly Lys Thr Leu Gly Glu             340 345 350 Thr Ile Ala Gly His Glu Val Lys Asp Tyr Asp Val Ile His Pro Leu         355 360 365 Asp Gln Pro Phe Thr Glu Lys Gly Gly Leu Ala Val Leu Phe Gly Asn     370 375 380 Leu Ala Pro Asp Gly Ala Ile Lys Thr Gly Gly Val Gln Asn Gly 385 390 395 400 Ile Thr Arg His Glu Gly Pro Ala Val Val Phe Asp Ser Gln Asp Glu                 405 410 415 Ala Leu Asp Gly Ile Ile Asn Arg Lys Val Lys Glu Gly Asp Val Val             420 425 430 Ile Ile Arg Tyr Glu Gly Pro Lys Gly Gly Pro Gly Met Pro Glu Met         435 440 445 Leu Ala Pro Thr Ser Gln Ile Val Gly Met Gly Leu Gly Pro Lys Val     450 455 460 Ala Leu Ile Thr Asp Gly Arg Phe Ser Gly Ala Ser Arg Gly Leu Ser 465 470 475 480 Ile Gly His Val Ser Pro Glu Ala Ala Glu Gly Gly Pro Leu Ala Phe                 485 490 495 Val Glu Asn Gly Asp His Ile Ile Val Asp Ile Glu Lys Arg Ile Leu             500 505 510 Asp Val Gln Val Pro Glu Glu Glu Trp Glu Lys Arg Lys Ala Asn Trp         515 520 525 Lys Gly Phe Glu Pro Lys Val Lys Thr Gly Tyr Leu Ala Arg Tyr Ser     530 535 540 Lys Leu Val Thr Ser Ala Asn Thr Gly Gly Ile Met Lys Ile 545 550 555 <210> 24 <211> 1677 <212> DNA <213> Bacillus subtilis <400> 24 atggcagaat tacgcagtaa tatgatcaca caaggaatcg atagagctcc gcaccgcagt 60 ttgcttcgtg cagcaggggt aaaagaagag gatttcggca agccgtttat tgcggtgtgt 120 aattcataca ttgatatcgt tcccggtcat gttcacttgc aggagtttgg gaaaatcgta 180 aaagaagcaa tcagagaagc agggggcgtt ccgtttgaat ttaataccat tggggtagat 240 gatggcatcg caatggggca tatcggtatg agatattcgc tgccaagccg tgaaattatc 300 gcagactctg tggaaacggt tgtatccgca cactggtttg acggaatggt ctgtattccg 360 aactgcgaca aaatcacacc gggaatgctt atggcggcaa tgcgcatcaa cattccgacg 420 atttttgtca gcggcggacc gatggcggca ggaagaacaa gttacgggcg aaaaatctcc 480 ctttcctcag tattcgaagg ggtaggcgcc taccaagcag ggaaaatcaa cgaaaacgag 540 cttcaagaac tagagcagtt cggatgccca acgtgcgggt cttgctcagg catgtttacg 600 gcgaactcaa tgaactgtct gtcagaagca cttggtcttg ctttgccggg taatggaacc 660 attctggcaa catctccgga acgcaaagag tttgtgagaa aatcggctgc gcaattaatg 720 gaaacgattc gcaaagatat caaaccgcgt gatattgtta cagtaaaagc gattgataac 780 gcgtttgcac tcgatatggc gctcggaggt tctacaaata ccgttcttca tacccttgcc 840 cttgcaaacg aagccggcgt tgaatactct ttagaacgca ttaacgaagt cgctgagcgc 900 gtgccgcact tggctaagct ggcgcctgca tcggatgtgt ttattgaaga tcttcacgaa 960 gcgggcggcg tttcagcggc tctgaatgag ctttcgaaga aagaaggagc gcttcattta 1020 gatgcgctga ctgttacagg aaaaactctt ggagaaacca ttgccggaca tgaagtaaag 1080 gattatgacg tcattcaccc gctggatcaa ccattcactg aaaagggagg ccttgctgtt 1140 ttattcggta atctagctcc ggacggcgct atcattaaaa caggcggcgt acagaatggg 1200 attacaagac acgaagggcc ggctgtcgta ttcgattctc aggacgaggc gcttgacggc 1260 attatcaacc gaaaagtaaa agaaggcgac gttgtcatca tcagatacga agggccaaaa 1320 ggcggacctg gcatgccgga aatgctggcg ccaacatccc aaatcgttgg aatgggactc 1380 gggccaaaag tggcattgat tacggacgga cgtttttccg gagcctcccg tggcctctca 1440 atcggccacg tatcacctga ggccgctgag ggcgggccgc ttgcctttgt tgaaaacgga 1500 gaccatatta tcgttgatat tgaaaaacgc atcttggatg tacaagtgcc agaagaagag 1560 tgggaaaaac gaaaagcgaa ctggaaaggt tttgaaccga aagtgaaaac cggctacctg 1620 gcacgttatt ctaaacttgt gacaagtgcc aacaccggcg gtattatgaa aatctag 1677 <210> 25 <211> 547 <212> PRT <213> Lactococcus lactis <400> 25 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 Gly Asp Tyr Asn Leu Gln Phe Leu             20 25 30 Asp Gln Ile Ile Ser Arg Glu Asp Met Lys Trp Ile 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 Ile 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 Asp 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 Tyr     130 135 140 Glu Ile Asp Arg Val Leu Ser Gln 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 Ala Leu Ser Leu Glu Lys Glu Ser Ser Thr Thr Asn Thr Thr Glu Gln             180 185 190 Val Ile Leu Ser Lys Ile Glu Glu Ser Leu Lys Asn Ala Gln Lys Pro         195 200 205 Val Val Ile Ala Gly His Glu Val Ile Ser Phe Gly Leu Glu Lys Thr     210 215 220 Val Thr Gln Phe Val Ser Glu Thr Lys Leu Pro Ile Thr Thr Leu Asn 225 230 235 240 Phe Gly Lys Ser Ala Val Asp Glu Ser Leu Pro Ser Phe Leu Gly Ile                 245 250 255 Tyr Asn Gly Lys Leu Ser Glu Ile Ser Leu Lys Asn 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 Asp Glu Asn Lys Met Ile Ser Leu Asn     290 295 300 Ile Asp Glu Gly Ile Ile Phe Asn Lys Val Val Glu Asp Phe Asp Phe 305 310 315 320 Arg Ala Val Val Ser Ser Leu Ser Glu Leu Lys Gly Ile Glu Tyr Glu                 325 330 335 Gly Gln Tyr Ile Asp Lys Gln Tyr Glu Glu Phe Ile Pro Ser Ser Ala             340 345 350 Pro Leu Ser Gln Asp Arg Leu Trp Gln Ala Val Glu Ser 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 Thr Ile Phe Leu Lys Ser Asn Ser Arg 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 Ser Ile Arg Glu Lys Leu 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 Thr Gln Ser Tyr Asn Asp Ile Pro Met Trp Asn Tyr 465 470 475 480 Ser Lys Leu Pro Glu Thr 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 Val Asn Arg Met Tyr Trp Ile Glu Leu Val Leu Glu Lys         515 520 525 Glu Asp Ala Pro Lys Leu Leu Lys Lys Met Gly Lys Leu Phe Ala Glu     530 535 540 Gln Asn Lys 545 <210> 26 <211> 1828 <212> DNA <213> Lactococcus lactis <400> 26 tttaaataag tcaatatcgt tgacttattt agaagaaaga gttattcttt aaatgtcaag 60 ttagttgact aaattaaata taaaatatgg aggaatgtga tgtatacagt aggagattac 120 ctgttagacc gattacacga gttgggaatt gaagaaattt ttggagttcc tggtgactat 180 aacttacaat ttttagatca aattatttca cgcgaagata tgaaatggat tggaaatgct 240 aatgaattaa atgcttctta tatggctgat ggttatgctc gtactaaaaa agctgccgca 300 tttctcacca catttggagt cggcgaattg agtgcgatca atggactggc aggaagttat 360 gccgaaaatt taccagtagt agaaattgtt ggttcaccaa cttcaaaagt acaaaatgac 420 ggaaaatttg tccatcatac actagcagat ggtgatttta aacactttat gaagatgcat 480 gaacctgtta cagcagcgcg gactttactg acagcagaaa atgccacata tgaaattgac 540 cgagtacttt ctcaattact aaaagaaaga aaaccagtct atattaactt accagtcgat 600 gttgctgcag caaaagcaga gaagcctgca ttatctttag aaaaagaaag ctctacaaca 660 aatacaactg aacaagtgat tttgagtaag attgaagaaa gtttgaaaaa tgcccaaaaa 720 ccagtagtga ttgcaggaca cgaagtaatt agttttggtt tagaaaaaac ggtaactcag 780 tttgtttcag aaacaaaact accgattacg acactaaatt ttggtaaaag tgctgttgat 840 gaatctttgc cctcattttt aggaatatat aacgggaaac tttcagaaat cagtcttaaa 900 aattttgtgg agtccgcaga ctttatccta atgcttggag tgaagcttac ggactcctca 960 acaggtgcat tcacacatca tttagatgaa aataaaatga tttcactaaa catagatgaa 1020 ggaataattt tcaataaagt ggtagaagat tttgatttta gagcagtggt ttcttcttta 1080 tcagaattaa aaggaataga atatgaagga caatatattg ataagcaata tgaagaattt 1140 attccatcaa gtgctccctt atcacaagac cgtctatggc aggcagttga aagtttgact 1200 caaagcaatg aaacaatcgt tgctgaacaa ggaacctcat tttttggagc ttcaacaatt 1260 ttcttaaaat caaatagtcg ttttattgga caacctttat ggggttctat tggatatact 1320 tttccagcgg ctttaggaag ccaaattgcg gataaagaga gcagacacct tttatttatt 1380 ggtgatggtt cacttcaact taccgtacaa gaattaggac tatcaatcag agaaaaactc 1440 aatccaattt gttttatcat aaataatgat ggttatacag ttgaaagaga aatccacgga 1500 cctactcaaa gttataacga cattccaatg tggaattact cgaaattacc agaaacattt 1560 ggagcaacag aagatcgtgt agtatcaaaa attgttagaa cagagaatga atttgtgtct 1620 gtcatgaaag aagcccaagc agatgtcaat agaatgtatt ggatagaact agttttggaa 1680 aaagaagatg cgccaaaatt actgaaaaaa atgggtaaat tatttgctga gcaaaataaa 1740 tagatatcaa cggatgatga aaagtaaaat agacaaagtc caataatttt ataaaaagta 1800 aaaacattag gattttccta atgttttt 1828 <210> 27 <211> 548 <212> PRT <213> Lactococcus lactis <400> 27 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 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> 28 <211> 1954 <212> DNA <213> Lactococcus lactis <400> 28 ctagagtttt ctttagtcat aattcactcc ttttattagt ctattatact tgataattca 60 aataagtcaa tatcgttgac ttatttaaag aaaagcgtta ttctataaat gtcaagttga 120 ttgaccaata tataataaaa tatggaggaa tgcgatgtat acagtaggag attacctatt 180 agaccgatta cacgagttag gaattgaaga aatttttgga gtccctggag actataactt 240 acaattttta gatcaaatta tttcccacaa ggatatgaaa tgggtcggaa atgctaatga 300 attaaatgct tcatatatgg ctgatggcta tgctcgtact aaaaaagctg ccgcatttct 360 tacaaccttt ggagtaggtg aattgagtgc agttaatgga ttagcaggaa gttacgccga 420 aaatttacca gtagtagaaa tagtgggatc acctacatca aaagttcaaa atgaaggaaa 480 atttgttcat catacgctgg ctgacggtga ttttaaacac tttatgaaaa tgcacgaacc 540 tgttacagca gctcgaactt tactgacagc agaaaatgca accgttgaaa ttgaccgagt 600 actttctgca ctattaaaag aaagaaaacc tgtctatatc aacttaccag ttgatgttgc 660 tgctgcaaaa gcagagaaac cctcactccc tttgaaaaag gaaaactcaa cttcaaatac 720 aagtgaccaa gaaattttga acaaaattca agaaagcttg aaaaatgcca aaaaaccaat 780 cgtgattaca ggacatgaaa taattagttt tggcttagaa aaaacagtca ctcaatttat 840 ttcaaagaca aaactaccta ttacgacatt aaactttggt aaaagttcag ttgatgaagc 900 cctcccttca tttttaggaa tctataatgg tacactctca gagcctaatc ttaaagaatt 960 cgtggaatca gccgacttca tcttgatgct tggagttaaa ctcacagact cttcaacagg 1020 agccttcact catcatttaa atgaaaataa aatgatttca ctgaatatag atgaaggaaa 1080 aatatttaac gaaagaatcc aaaattttga ttttgaatcc ctcatctcct ctctcttaga 1140 cctaagcgaa atagaataca aaggaaaata tatcgataaa aagcaagaag actttgttcc 1200 atcaaatgcg cttttatcac aagaccgcct atggcaagca gttgaaaacc taactcaaag 1260 caatgaaaca atcgttgctg aacaagggac atcattcttt ggcgcttcat caattttctt 1320 aaaatcaaag agtcatttta ttggtcaacc cttatgggga tcaattggat atacattccc 1380 agcagcatta ggaagccaaa ttgcagataa agaaagcaga caccttttat ttattggtga 1440 tggttcactt caacttacag tgcaagaatt aggattagca atcagagaaa aaattaatcc 1500 aatttgcttt attatcaata atgatggtta tacagtcgaa agagaaattc atggaccaaa 1560 tcaaagctac aatgatattc caatgtggaa ttactcaaaa ttaccagaat cgtttggagc 1620 aacagaagat cgagtagtct caaaaatcgt tagaactgaa aatgaatttg tgtctgtcat 1680 gaaagaagct caagcagatc caaatagaat gtactggatt gagttaattt tggcaaaaga 1740 aggtgcacca aaagtactga aaaaaatggg caaactattt gctgaacaaa ataaatcata 1800 atttataaat agtaaaaaac attaggaaat acctaatgtt tttttgttga ctaaatcaat 1860 ccctctttat atagaaaacc ttagtttctc aaagacaact taattaagcc tgccaaattg 1920 gaactcgcaa aatgtaatct atcctctgct ccta 1954 <210> 29 <211> 550 <212> PRT <213> Salmonella typhimurium <400> 29 Met Gln Asn Pro Tyr Thr Val Ala Asp Tyr Leu Leu Asp Arg Leu Ala 1 5 10 15 Gly Cys Gly Ile Gly His Leu Phe Gly Val Pro Gly Asp Tyr Asn Leu             20 25 30 Gln Phe Leu Asp His Val Ile Asp His Pro Thr Leu Arg Trp Val Gly         35 40 45 Cys Ala Asn Glu Leu Asn Ala Ala Tyr Ala Ala Asp Gly Tyr Ala Arg     50 55 60 Met Ser Gly Ala Gly Ala Leu Leu Thr Thr Phe Gly Val Gly Glu Leu 65 70 75 80 Ser Ala Ile Asn Gly Ile Ala Gly Ser Tyr Ala Glu Tyr Val                 85 90 95 Leu His Ile Val Gly Ala Pro Cys Ser Ala Ala Gln Gln Arg Gly Glu             100 105 110 Leu Met His His Thr Leu Gly Asp Gly Asp Phe Arg His Phe Tyr Arg         115 120 125 Met Ser Gln Ala Ile Ser Ala Ala Ser Ala Ile Leu Asp Glu Gln Asn     130 135 140 Ala Cys Phe Glu Ile Asp Arg Val Leu Gly Glu Met Leu Ala Ala Arg 145 150 155 160 Arg Pro Gly Tyr Ile Met Leu Pro Ala Asp Val Ala Lys Lys Thr Ala                 165 170 175 Ile Pro Pro Thr Gln Ala Leu Ala Leu Pro Val His Glu Ala Gln Ser             180 185 190 Gly Val Glu Thr Ala Phe Arg Tyr His Ala Arg Gln Cys Leu Met Asn         195 200 205 Ser Arg Arg Ile Ala Leu Leu Ala Asp Phe Leu Ala Gly Arg Phe Gly     210 215 220 Leu Arg Pro Leu Leu Gln Arg Trp Met Ala Glu Thr Pro Ile Ala His 225 230 235 240 Ala Thr Leu Leu Met Gly Lys Gly Leu Phe Asp Glu Gln His Pro Asn                 245 250 255 Phe Val Gly Thr Tyr Ser Ala Gly Ala Ser Ser Lys Glu Val Arg Gln             260 265 270 Ala Ile Glu Asp Ala Asp Arg Val Ile Cys Val Gly Thr Arg Phe Val         275 280 285 Asp Thr Leu Thr Ala Gly Phe Thr Gln Gln Leu Pro Ala Glu Arg Thr     290 295 300 Leu Glu Ile Gln Pro Tyr Ala Ser Arg Ile Gly Glu Thr Trp Phe Asn 305 310 315 320 Leu Pro Met Ala Gln Ala Val Ser Thr Leu Arg Glu Leu Cys Leu Glu                 325 330 335 Cys Ala Phe Ala Pro Pro Thr Arg Ser Ala Gly Gln Pro Val Arg             340 345 350 Ile Asp Lys Gly Glu Leu Thr Gln Glu Ser Phe Trp Gln Thr Leu Gln         355 360 365 Gln Tyr Leu Lys Pro Gly Asp Ile Ile Leu Val Asp Gln Gly Thr Ala     370 375 380 Ala Phe Gly Ala Ala Ala Leu Ser Leu Pro Asp Gly Ala Glu Val Val 385 390 395 400 Leu Gln Pro Leu Trp Gly Ser Ile Gly Tyr Ser Leu Pro Ala Ala Phe                 405 410 415 Gly Ala Gln Thr Ala Cys Pro Asp Arg Arg Val Ile Leu Ile Ile Gly             420 425 430 Asp Gly Ala Ala Gln Leu Thr Ile Gln Glu Met Gly Ser Met Leu Arg         435 440 445 Asp Gly Gln Ala Pro Val Ile Leu Leu Leu Asn Asn Asp Gly Tyr Thr     450 455 460 Val Glu Arg Ala Ile His Gly Ala Ala Gln Arg Tyr Asn Asp Ile Ala 465 470 475 480 Ser Trp Asn Trp Thr Gln Ile Pro Pro Ala Leu Asn Ala Ala Gln Gln                 485 490 495 Ala Glu Cys Trp Arg Val Thr Gln Ala Ile Gln Leu Ala Glu Val Leu             500 505 510 Glu Arg Leu Ala Arg Pro Gln Arg Leu Ser Phe Ile Glu Val Met Leu         515 520 525 Pro Lys Ala Asp Leu Pro Glu Leu Leu Arg Thr Val Thr Arg Ala Leu     530 535 540 Glu Ala Arg Asn Gly Gly 545 550 <210> 30 <211> 1653 <212> DNA <213> Salmonella typhimurium <400> 30 ttatcccccg ttgcgggctt ccagcgcccg ggtcacggta cgcagtaatt ccggcagatc 60 ggcttttggc aacatcactt caataaatga cagacgttgt gggcgcgcca accgttcgag 120 gacctctgcc agttggatag cctgcgtcac ccgccagcac tccgcctgtt gcgccgcgtt 180 tagcgccggt ggtatctgcg tccagttcca gctcgcgatg tcgttatacc gctgggccgc 240 gccgtgaatg gcgcgctcta cggtatagcc gtcattgttg agcagcagga tgaccggcgc 300 ctgcccgtcg cgtaacatcg agcccatctc ctgaatcgtg agctgcgccg cgccatcgcc 360 gataatcaga atcacccgcc gatcgggaca ggcggtttgc gcgccaaacg cggcgggcaa 420 ggaatagccg atagaccccc acagcggctg taacacaact tccgcgccgt caggaagcga 480 cagcgcggca gcgccaaaag ctgctgtccc ctggtcgaca aggataatat ctccgggttt 540 gagatactgc tgtaaggttt gccagaagct ttcctgggtc agttctcctt tatcaatccg 600 cactggctgt ccggcggaac gcgtcggcgg cggcgcaaaa gcgcattcca ggcacagttc 660 gcgcagcgta gacaccgcct gcgccatcgg gaggttgaac caggtttcgc cgatgcgcga 720 cgcgtaaggc tgaatctcca gcgtgcgttc cgccggtaat tgttgggtaa atccggccgt 780 aagggtatcg acaaaacggg tgccgacgca gataacccta tcggcgtcct ctatggcctg 840 acgcacttct ttgctgctgg cgccagcgct ataggtgcca acgaagttcg ggtgctgttc 900 atcaaaaagc cccttcccca tcagtagtgt cgcatgagcg atgggcgttt ccgccatcca 960 gcgctgcaac agtggtcgta aaccaaaacg cccggcaaga aagtcggcca atagcgcaat 1020 gcgccgactg ttcatcaggc actgacgggc gtgataacga aaggccgtct ccacgccgct 1080 ttgcgcttca tgcacgggca acgccagcgc ctgcgtaggt gggatggccg tttttttcgc 1140 cacatcggcg ggcaacatga tgtatcctgg cctgcgtgcg gcaagcattt cacccaacac 1200 gcggtcaatc tcgaaacagg cgttctgttc atctaatatt gcgctggcag cggatatcgc 1260 ctgactcatg cgataaaaat gacgaaaatc gccgtcaccg agggtatggt gcatcaattc 1320 gccacgctgc tgcgcagcgc tacagggcgc gccgacgata tgcaagaccg ggacatattc 1380 cgcgtaactg cccgcgatac cgttaatagc gctaagttct cccacgccaa aggtggtgag 1440 tagcgctcca gcgcccgaca tgcgcgcata gccgtccgcg gcataagcgg cgttcagctc 1500 attggcgcat cccacccaac gcagggtcgg gtggtcaatc acatggtcaa gaaactgcaa 1560 gttataatcg cccggtacgc caaaaagatg gccaatgccg catcctgcca gtctgtccag 1620 caaatagtcg gccacggtat aggggttttg cat 1653 <210> 31 <211> 554 <212> PRT <213> Clostridium acetobutylicum <400> 31 Met Lys Ser Glu Tyr Thr Ile Gly Arg Tyr Leu Leu Asp Arg Leu Ser 1 5 10 15 Glu Leu Gly Ile Arg His Ile Phe Gly Val Pro Gly Asp Tyr Asn Leu             20 25 30 Ser Phe Leu Asp Tyr Ile Met Glu Tyr Lys Gly Ile Asp Trp Val Gly         35 40 45 Asn Cys Asn Glu Leu Asn Ala Gly Tyr Ala Ala Asp Gly Tyr Ala Arg     50 55 60 Ile Asn Gly Ile Gly Ala Ile Leu Thr Thr Phe Gly Val Gly Glu Leu 65 70 75 80 Ser Ala Ile Asl Ale Ile Ala Gly Ala Tyr Ala Glu                 85 90 95 Val Lys Ile Thr Gly Ile Pro Thr Ala Lys Val Arg Asp Asn Gly Leu             100 105 110 Tyr Val His His Thr Leu Gly Asp Gly Arg Phe Asp His Phe Phe Glu         115 120 125 Met Phe Arg Glu Val Thr Val Ala Glu Ala Leu Leu Ser Glu Glu Asn     130 135 140 Ala Ala Gln Glu Ile Asp Arg Val Leu Ile Ser Cys Trp Arg Gln Lys 145 150 155 160 Arg Pro Val Leu Ile Asn Leu Pro Ile Asp Val Tyr Asp Lys Pro Ile                 165 170 175 Asn Lys Pro Leu Lys Pro Leu Leu Asp Tyr Thr Ile Ser Ser Asn Lys             180 185 190 Glu Ala Ala Cys Glu Phe Val Thr Glu Ile Val Pro Ile Ile Asn Arg         195 200 205 Ala Lys Lys Pro Val Ile Leu Ala Asp Tyr Gly Val Tyr Arg Tyr Gln     210 215 220 Val Gln His Val Leu Lys Asn Leu Ala Glu Lys Thr Gly Phe Pro Val 225 230 235 240 Ala Thr Leu Ser Met Gly Lys Gly Val Phe Asn Glu Ala His Pro Gln                 245 250 255 Phe Ile Gly Val Tyr Asn Gly Asp Val Ser Ser Pro Tyr Leu Arg Gln             260 265 270 Arg Val Asp Glu Ala Asp Cys Ile Ile Ser Val Gly Val Lys Leu Thr         275 280 285 Asp Ser Thr Thr Gly Gly Phe Ser His Gly Phe Ser Lys Arg Asn Val     290 295 300 Ile His Ile Asp Pro Phe Ser Ile Lys Ala Lys Gly Lys Lys Tyr Ala 305 310 315 320 Pro Ile Thr Met Lys Asp Ala Leu Thr Glu Leu Thr Ser Lys Ile Glu                 325 330 335 His Arg Asn Phe Glu Asp Leu Asp Ile Lys Pro Tyr Lys Ser Asp Asn             340 345 350 Gln Lys Tyr Phe Ala Lys Glu Lys Pro Ile Thr Gln Lys Arg Phe Phe         355 360 365 Glu Arg Ile Ala His Phe Ile Lys Glu Lys Asp Val Leu Leu Ala Glu     370 375 380 Gln Gly Thr Cys Phe Phe Gly Ala Ser Thr Ile Gln Leu Pro Lys Asp 385 390 395 400 Ala Thr Phe Ile Gly Gln Pro Leu Trp Gly Ser Ile Gly Tyr Thr Leu                 405 410 415 Pro Ala Leu Leu Gly Ser Gln Leu Ala Asp Gln Lys Arg Arg Asn Ile             420 425 430 Leu Leu Ile Gly Asp Gly Ala Phe Gln Met Thr Ala Gln Glu Ile Ser         435 440 445 Thr Met Leu Arg Leu Gln Ile Lys Pro Ile Ile Phe Leu Ile Asn Asn     450 455 460 Asp Gly Tyr Thr Ile Glu Arg Ala Ile His Gly Arg Glu Gln Val Tyr 465 470 475 480 Asn Asn Ile Gln Met Trp Arg Tyr His Asn Val Pro Lys Val Leu Gly                 485 490 495 Pro Lys Glu Cys Ser Leu Thr Phe Lys Val Gln Ser Glu Thr Glu Leu             500 505 510 Glu Lys Ala Leu Leu Val Ala Asp Lys Asp Cys Glu His Leu Ile Phe         515 520 525 Ile Glu Val Val Met Asp Arg Tyr Asp Lys Pro Glu Pro Leu Glu Arg     530 535 540 Leu Ser Lys Arg Phe Ala Asn Gln Asn Asn 545 550 <210> 32 <211> 1665 <212> DNA <213> Clostridium acetobutylicum <400> 32 ttgaagagtg aatacacaat tggaagatat ttgttagacc gtttatcaga gttgggtatt 60 cggcatatct ttggtgtacc tggagattac aatctatcct ttttagacta tataatggag 120 tacaaaggga tagattgggt tggaaattgc aatgaattga atgctgggta tgctgctgat 180 ggatatgcaa gaataaatgg aattggagcc atacttacaa catttggtgt tggagaatta 240 agtgccatta acgcaattgc tggggcatac gctgagcaag ttccagttgt taaaattaca 300 ggtatcccca cagcaaaagt tagggacaat ggattatatg tacaccacac attaggtgac 360 ggaaggtttg atcacttttt tgaaatgttt agagaagtaa cagttgctga ggcattacta 420 agcgaagaaa atgcagcaca agaaattgat cgtgttctta tttcatgctg gagacaaaaa 480 cgtcctgttc ttataaattt accgattgat gtatatgata aaccaattaa caaaccatta 540 aagccattac tcgattatac tatttcaagt aacaaagagg ctgcatgtga atttgttaca 600 gaaatagtac ctataataaa tagggcaaaa aagcctgtta ttcttgcaga ttatggagta 660 tatcgttacc aagttcaaca tgtgcttaaa aacttggccg aaaaaaccgg atttcctgtg 720 gctacactaa gtatgggaaa aggtgttttc aatgaagcac accctcaatt tattggtgtt 780 tataatggtg atgtaagttc tccttattta aggcagcgag ttgatgaagc agactgcatt 840 attagcgttg gtgtaaaatt gacggattca accacagggg gattttctca tggattttct 900 aaaaggaatg taattcacat tgatcctttt tcaataaagg caaaaggtaa aaaatatgca 960 cctattacga tgaaagatgc tttaacagaa ttaacaagta aaattgagca tagaaacttt 1020 gaggatttag atataaagcc ttacaaatca gataatcaaa agtattttgc aaaagagaag 1080 ccaattacac aaaaacgttt ttttgagcgt attgctcact ttataaaaga aaaagatgta 1140 ttattagcag aacagggtac atgctttttt ggtgcgtcaa ccatacaact acccaaagat 1200 gcaactttta ttggtcaacc tttatgggga tctattggat acacacttcc tgctttatta 1260 ggttcacaat tagctgatca aaaaaggcgt aatattcttt taattgggga tggtgcattt 1320 caaatgacag cacaagaaat ttcaacaatg cttcgtttac aaatcaaacc tattattttt 1380 ttaattaata acgatggtta tacaattgaa cgtgctattc atggtagaga acaagtatat 1440 aacaatattc aaatgtggcg atatcataat gttccaaagg ttttaggtcc taaagaatgc 1500 agcttaacct ttaaagtaca aagtgaaact gaacttgaaa aggctctttt agtggcagat 1560 aaggattgtg aacatttgat ttttatagaa gttgttatgg atcgttatga taaacccgag 1620 cctttagaac gtctttcgaa acgttttgca aatcaaaata attag 1665 <210> 33 <211> 1641 <212> DNA <213> Clostridium acetobutylicum <400> 33 atgaaacaac gtatcgggca atacttgatc gatgccctac acgttaatgg tgtcgataag 60 atctttggag tcccaggtga tttcacttta gcctttttgg acgatatcat aagacatgac 120 aacgtggaat gggtgggaaa tactaatgag ttgaacgccg cttacgccgc tgatggttac 180 gctagagtta atggattagc cgctgtatct accacttttg gggttggcga gttatctgct 240 gtgaatggta ttgctggaag ttacgcagag cgtgttcctg taatcaaaat ctcaggcggt 300 ccttcatcag ttgctcaaca agagggtaga tatgtccacc attcattggg tgaaggaatc 360 tttgattcat attcaaagat gtacgctcac ataaccgcaa caactacaat cttatccgtt 420 gacaacgcag tcgacgaaat tgatagagtt attcattgtg ctttgaagga aaagaggcca 480 gtgcatattc atttgcctat tgacgtagcc ttaactgaga ttgaaatccc tcatgcacca 540 aaagtttaca cacacgaatc ccagaacgtc gatgcttaca ttcaagctgt tgagaaaaag 600 ttaatgtctg caaaacaacc agtaatcata gcaggtcatg aaatcaattc attcaagttg 660 cacgaacaac tggaacagtt tgtcaatcag acaaacatcc ctgttgcaca actttccttg 720 ggtaagtctg ctttcaatga agagaatgaa cattaccttg gtatctacga tggcaaaatc 780 gcaaaggaaa atgtgagaga gtacgtcgac aatgctgatg tcatattgaa cataggtgcc 840 aaactgactg attctgctac agctggattt tcctacaagt tcgatacaaa caacataatc 900 tacattaacc ataatgactt caaagctgaa gatgtgattt ctgataatgt ttcactgatt 960 gatcttgtga atggcctgaa ttctattgac tatagaaatg aaacacacta cccatcttat 1020 caaagatctg atatgaaata cgaattgaat gacgcaccac ttacacaatc taactatttc 1080 aaaatgatga acgcttttct agaaaaagat gacatcctac tagctgaaca aggtacatcc 1140 tttttcggcg catatgactt atccctatac aagggaaatc agtttatcgg tcagccttta 1200 tgggggtcaa tagggtatac ttttccatct ttactaggaa gtcaactagc agacatgcat 1260 aggagaaaca ttttgcttat aggcgatggt agtttacaac ttactgttca agccctaagt 1320 acaatgatta gaaaggatat caaaccaatc attttcgtta tcaataacga cggttacacc 1380 gtcgaaagac ttatccacgg catggaagag ccatacaatg atatccaaat gtggaactac 1440 aagcaattgc cagaagtatt tggtggaaaa gatactgtaa aagttcatga tgctaaaacc 1500 tccaacgaac tgaaaactgt aatggattct gttaaagcag acaaagatca catgcatttc 1560 attgaagtgc atatggcagt agaggacgcc ccaaagaagt tgattgatat agctaaagcc 1620 tttagtgatg ctaacaagta a 1641 <210> 34 <211> 1647 <212> DNA <213> Listeria grayi <400> 34 atgtacaccg tcggccaata cttagtagac cgcttagaag agatcggcat cgataaggtt 60 tttggtgtcc cgggtgacta caacctgacc tttttggact acatccagaa ccacgaaggt 120 ctgagctggc aaggtaatac gaatgaactg aatgccgcgt acgcagctga tggctatgct 180 cgtgaacgcg gtgttagcgc tttggtcacg accttcggcg ttggtgagct gtccgcaatc 240 aatggcaccg caggtagctt cgcggagcaa gttccggtga ttcatatcgt gggcagcccg 300 accatgaatg ttcagagcaa caagaaactg gttcatcaca gcctgggtat gggcaacttt 360 cacaacttca gcgagatggc gaaagaagtc accgccgcaa ccacgatgct gacggaagag 420 aatgcggcgt cggagattga tcgtgttctg gaaaccgccc tgctggagaa acgcccagtg 480 tacatcaatc tgccgatcga cattgctcac aaggcgatcg tcaagccggc gaaagccctg 540 caaaccgaga agagctctgg cgagcgtgag gcacaactgg cggagatcat tctgagccat 600 ctggagaagg ctgcacagcc gattgtgatt gcgggtcacg agatcgcgcg cttccagatc 660 cgtgagcgtt tcgagaattg gattaatcaa acgaaactgc cggtgaccaa tctggcctac 720 ggcaagggta gcttcaacga agaaaacgag catttcattg gtacctatta tcctgcattt 780 agcgataaga acgtgctgga ctacgtggat aactccgact ttgtcctgca ctttggtggt 840 aaaatcattg ataacagcac ctccagcttc tcccaaggct tcaaaaccga gaacaccctg 900 actgcggcga acgatatcat tatgctgccg gacggtagca cgtattctgg tattagcctg 960 aatggcctgc tggccgagct ggaaaaactg aatttcacgt ttgccgacac cgcagcaaag 1020 caggcggagt tggcggtgtt tgagccgcag gctgaaaccc cgttgaaaca ggaccgtttt 1080 caccaggcgg tgatgaattt tctgcaagct gacgatgtcc tggttacgga acagggcacc 1140 tcttcttttg gcttgatgct ggcgcctctg aaaaagggta tgaacttgat ctcgcaaacg 1200 ctgtggggta gcattggtta cacgttgccg gcgatgattg gtagccaaat tgcggcaccg 1260 gagcgtcgtc atatcctgag cattggtgat ggtagctttc agctgactgc gcaggaaatg 1320 agcaccattt tccgtgagaa actgacccca gtcatcttca tcattaacaa tgatggctat 1380 accgttgagc gtgcgatcca tggcgaagat gaaagctata acgacattcc gacgtggaac 1440 ttgcaactgg tggcggaaac cttcggtggt gacgccgaaa ccgtcgacac tcacaatgtg 1500 ttcacggaga ctgatttcgc caacaccctg gcggcaattg acgcgacgcc gcagaaagca 1560 cacgttgtgg aagttcacat ggaacaaatg gatatgccgg agagcctgcg ccagatcggt 1620 ctggcactgt ccaagcagaa tagctaa 1647 <210> 35 <211> 312 <212> PRT <213> Saccharomyces cerevisiae <400> 35 Met Pro Ala Thr Leu Lys Asn Ser Ser Ala Thr Leu Lys Leu Asn Thr 1 5 10 15 Gly Ala Ser Ile Pro Val Leu Gly Phe Gly Thr Trp Arg Ser Val Asp             20 25 30 Asn Asn Gly Tyr His Ser Val Ile Ala Ala Leu Lys Ala Gly Tyr Arg         35 40 45 His Ile Asp Ala Ala Ile Tyr Leu Asn Glu Glu Glu Val Gly Arg     50 55 60 Ala Ile Lys Asp Ser Gly Val Pro Arg Glu Glu Ile Phe Ile Thr Thr 65 70 75 80 Lys Leu Trp Gly Thr Glu Gln Arg Asp Pro Glu Ala Ala Leu Asn Lys                 85 90 95 Ser Leu Lys Arg Leu Gly Leu Asp Tyr Val Asp Leu Tyr Leu Met His             100 105 110 Trp Pro Val Pro Leu Lys Thr Asp Arg Val Thr Asp Gly Asn Val Leu         115 120 125 Cys Ile Pro Thr Leu Glu Asp Gly Thr Val Asp Ile Asp Thr Lys Glu     130 135 140 Trp Asn Phe Ile Lys Thr Trp Glu Leu Met Gln Glu Leu Pro Lys Thr 145 150 155 160 Gly Lys Thr Lys Ala Val Gly Val Ser Asn Phe Ser Ile Asn Asn Ile                 165 170 175 Lys Glu Leu Leu Glu Ser Pro Asn Asn Lys Val Val Pro Ala Thr Asn             180 185 190 Gln Ile Glu Ile His Pro Leu Leu Pro Gln Asp Glu Leu Ile Ala Phe         195 200 205 Cys Lys Glu Lys Gly Ile Val Val Glu Ala Tyr Ser Pro Phe Gly Ser     210 215 220 Ala Asn Ala Pro Leu Leu Lys Glu Gln Ala Ile Asp Asp Met Ala Lys 225 230 235 240 Lys His Gly Val Glu Pro Ala Gln Leu Ile Ile Ser Trp Ser Ile Gln                 245 250 255 Arg Gly Tyr Val Val Leu Ala Lys Ser Val Asn Pro Glu Arg Ile Val             260 265 270 Ser Asn Phe Lys Ile Phe Thr Leu Pro Glu Asp Asp Phe Lys Thr Ile         275 280 285 Ser Asn Leu Ser Lys Val His Gly Thr Lys Arg Val Val Asp Met Lys     290 295 300 Trp Gly Ser Phe Pro Ile Phe Gln 305 310 <210> 36 <211> 939 <212> DNA <213> Saccharomyces cerevisiae <400> 36 atgcctgcta cgttaaagaa ttcttctgct acattaaaac taaatactgg tgcctccatt 60 ccagtgttgg gtttcggcac ttggcgttcc gttgacaata acggttacca ttctgtaatt 120 gcagctttga aagctggata cagacacatt gatgctgcgg ctatctattt gaatgaagaa 180 gaagttggca gggctattaa agattccgga gtccctcgtg aggaaatttt tattactact 240 aagctttggg gtacggaaca acgtgatccg gaagctgctc taaacaagtc tttgaaaaga 300 ctaggcttgg attatgttga cctatatctg atgcattggc cagtgccttt gaaaaccgac 360 agagttactg atggtaacgt tctgtgcatt ccaacattag aagatggcac tgttgacatc 420 gatactaagg aatggaattt tatcaagacg tgggagttga tgcaagagtt gccaaagacg 480 ggcaaaacta aagccgttgg tgtctctaat ttttctatta acaacattaa agaattatta 540 gaatctccaa ataacaaggt ggtaccagct actaatcaaa ttgaaattca tccattgcta 600 ccacaagacg aattgattgc cttttgtaag gaaaagggta ttgttgttga agcctactca 660 ccatttggga gtgctaatgc tcctttacta aaagagcaag caattattga tatggctaaa 720 aagcacggcg ttgagccagc acagcttatt atcagttgga gtattcaaag aggctacgtt 780 gttctggcca aatcggttaa tcctgaaaga attgtatcca attttaagat tttcactctg 840 cctgaggatg atttcaagac tattagtaac ctatccaaag tgcatggtac aaagagagtc 900 gttgatatga agtggggatc cttcccaatt ttccaatga 939 <210> 37 <211> 360 <212> PRT <213> Saccharomyces cerevisiae <400> 37 Met Ser Tyr Pro Glu Lys Phe Glu Gly Ile Ala Ile Gln Ser His Glu 1 5 10 15 Asp Trp Lys Asn Pro Lys Lys Thr Lys Tyr Asp Pro Lys Pro Phe Tyr             20 25 30 Asp His Asp Ile Asp Ile Lys Ile Glu Ala Cys Gly Val Cys Gly Ser         35 40 45 Asp Ile His Cys Ala Ala Gly His Trp Gly Asn Met Lys Met Pro Leu     50 55 60 Val Val Gly His Glu Ile Val Gly Lys Val Val Lys Leu Gly Pro Lys 65 70 75 80 Ser Asn Ser Gly Leu Lys Val Gly Gln Arg Val Gly Val Gly Ala Gln                 85 90 95 Val Phe Ser Cys Leu Glu Cys Asp Arg Cys Lys Asn Asp Asn Glu Pro             100 105 110 Tyr Cys Thr Lys Phe Val Thr Thr Tyr Ser Gln Pro Tyr Glu Asp Gly         115 120 125 Tyr Val Ser Gln Gly Gly Tyr Ala Asn Tyr Val Val Val His Glu His     130 135 140 Phe Val Val Pro Ile Pro Glu Asn Ile Pro Ser His Leu Ala Ala Pro 145 150 155 160 Leu Leu Cys Gly Gly Leu Thr Val Tyr Ser Pro Leu Val Arg Asn Gly                 165 170 175 Cys Gly Pro Gly Lys Lys Val Gly Ile Val Gly Leu Gly Gly Ile Gly             180 185 190 Ser Met Gly Thr Leu Ile Ser Lys Ala Met Gly Ala Glu Thr Tyr Val         195 200 205 Ile Ser Arg Ser Ser Arg Lys Arg Glu Asp Ala Met Lys Met Gly Ala     210 215 220 Asp His Tyr Ile Ala Thr Leu Glu Glu Gly Asp Trp Gly Glu Lys Tyr 225 230 235 240 Phe Asp Thr Phe Asp Leu Ile Val Val Cys Ala Ser Ser Leu Thr Asp                 245 250 255 Ile Asp Phe Asn Ile Met Pro Lys Ala Met Lys Val Gly Gly Arg Ile             260 265 270 Val Ser Ile Ser Ile Pro Glu Gln His Glu Met Leu Ser Leu Lys Pro         275 280 285 Tyr Gly Leu Lys Ala Val Ser Ile Ser Tyr Ser Ala Leu Gly Ser Ile     290 295 300 Lys Glu Leu Asn Gln Leu Leu Lys Leu Val Ser Glu Lys Asp Ile Lys 305 310 315 320 Ile Trp Val Glu Thr Leu Pro Val Gly Glu Ala Gly Val His Glu Ala                 325 330 335 Phe Glu Arg Met Glu Lys Gly Asp Val Arg Tyr Arg Phe Thr Leu Val             340 345 350 Gly Tyr Asp Lys Glu Phe Ser Asp         355 360 <210> 38 <211> 1083 <212> DNA <213> Saccharomyces cerevisiae <400> 38 ctagtctgaa aattctttgt cgtagccgac taaggtaaat ctatatctaa cgtcaccctt 60 ttccatcctt tcgaaggctt catggacgcc ggcttcacca acaggtaatg tttccaccca 120 aattttgata tctttttcag agactaattt caagagttgg ttcaattctt tgatggaacc 180 taaagcactg taagaaatgg agacagcctt taagccatat ggctttagcg ataacatttc 240 gtgttgttct ggtatagaga ttgagacaat tctaccacca accttcatag cctttggcat 300 aatgttgaag tcaatgtcgg taagggagga agcacagact acaatcaggt cgaaggtgtc 360 aaagtacttt tcaccccaat caccttcttc taatgtagca atgtagtgat cggcgcccat 420 cttcattgca tcttctcttt ttctcgaaga acgagaaata acatacgtct ctgcccccat 480 ggctttggaa atcaatgtac ccatactgcc gataccacca agaccaacta taccaacttt 540 tttacctgga ccgcaaccgt tacgaaccaa tggagagtac acagtcaaac caccacataa 600 tagtggagca gccaaatgtg atggaatatt ctctgggata ggcaccacaa aatgttcatg 660 aactctgacg tagtttgcat agccaccctg cgacacatag ccgtcttcat aaggctgact 720 gtatgtggta acaaacttgg tgcagtatgg ttcattatca ttcttacaac ggtcacattc 780 caagcatgaa aagacttgag cacctacacc aacacgttga ccgactttca acccactgtt 840 tgacttgggc cctagcttga caactttacc aacgatttca tgaccaacga ctagcggcat 900 cttcatattg ccccaatgac cagctgcaca atgaatatca ctaccgcaga caccacatgc 960 ttcgatctta atgtcaatgt catgatcgta aaatggtttt gggtcatact ttgtcttctt 1020 tgggtttttc caatcttcgt gtgattgaat agcgatacct tcaaatttct caggataaga 1080 cat 1083 <210> 39 <211> 387 <212> PRT <213> Escherichia coli <400> 39 Met Asn Asn Phe Asn Leu His Thr Pro Thr Arg Ile Leu Phe Gly Lys 1 5 10 15 Gly Ala Ile Ala Gly Leu Arg Glu Gln Ile Pro His Asp Ala Arg Val             20 25 30 Leu Ile Thr Tyr Gly Gly Gly Ser Val Lys Lys Thr Gly Val Leu Asp         35 40 45 Gln Val Leu Asp Ala Leu Lys Gly Met Asp Val Leu Glu Phe Gly Gly     50 55 60 Ile Glu Pro Asn Pro Ala Tyr Glu Thr Leu Met Asn Ala Val Lys Leu 65 70 75 80 Val Arg Glu Gln Lys Val Thr Phe Leu Leu Ala Val Gly Gly Gly Ser                 85 90 95 Val Leu Asp Gly Thr Lys Phe Ile Ala Ala Ala Ala Asn Tyr Pro Glu             100 105 110 Asn Ile Asp Pro Trp His Ile Leu Gln Thr Gly Gly Lys Glu Ile Lys         115 120 125 Ser Ala Ile Pro Met Gly Cys Val Leu Thr Leu Pro Ala Thr Gly Ser     130 135 140 Glu Ser Asn Ala Gly Ala Val Ile Ser Arg Lys Thr Thr Gly Asp Lys 145 150 155 160 Gln Ala Phe His Ser Ala His Val Gln Pro Val Phe Ala Val Leu Asp                 165 170 175 Pro Val Tyr Thr Tyr Thr Leu Pro Pro Arg Gln Val Ala Asn Gly Val             180 185 190 Val Asp Ala Phe Val His Thr Val Glu Gln Tyr Val Thr Lys Pro Val         195 200 205 Asp Ala Lys Ile Gln Asp Arg Phe Ala Glu Gly Ile Leu Leu Thr Leu     210 215 220 Ile Glu Asp Gly Pro Lys Ala Leu Lys Glu Pro Glu Asn Tyr Asp Val 225 230 235 240 Arg Ala Asn Val Met Trp Ala Ala Thr Gln Ala Leu Asn Gly Leu Ile                 245 250 255 Gly Ala Gly Val Pro Gln Asp Trp Ala Thr His Met Leu Gly His Glu             260 265 270 Leu Thr Ala Met His Gly Leu Asp His Ala Gln Thr Leu Ala Ile Val         275 280 285 Leu Pro Ala Leu Trp Asn Glu Lys Arg Asp Thr Lys Arg Ala Lys Leu     290 295 300 Leu Gln Tyr Ala Glu Arg Val Trp Asn Ile Thr Glu Gly Ser Asp Asp 305 310 315 320 Glu Arg Ile Asp Ala Ile Ala Ala Thr Arg Asn Phe Phe Glu Gln                 325 330 335 Leu Gly Val Pro Thr His Leu Ser Asp Tyr Gly Leu Asp Gly Ser Ser             340 345 350 Ile Pro Ala Leu Leu Lys Lys Leu Glu Glu His Gly Met Thr Gln Leu         355 360 365 Gly Glu Asn His Asp Ile Thr Leu Asp Val Ser Arg Arg Ile Tyr Glu     370 375 380 Ala Ala Arg 385 <210> 40 <211> 387 <212> PRT <213> Escherichia coli <400> 40 Met Asn Asn Phe Asn Leu His Thr Pro Thr Arg Ile Leu Phe Gly Lys 1 5 10 15 Gly Ala Ile Ala Gly Leu Arg Glu Gln Ile Pro His Asp Ala Arg Val             20 25 30 Leu Ile Thr Tyr Gly Gly Gly Ser Val Lys Lys Thr Gly Val Leu Asp         35 40 45 Gln Val Leu Asp Ala Leu Lys Gly Met Asp Val Leu Glu Phe Gly Gly     50 55 60 Ile Glu Pro Asn Pro Ala Tyr Glu Thr Leu Met Asn Ala Val Lys Leu 65 70 75 80 Val Arg Glu Gln Lys Val Thr Phe Leu Leu Ala Val Gly Gly Gly Ser                 85 90 95 Val Leu Asp Gly Thr Lys Phe Ile Ala Ala Ala Ala Asn Tyr Pro Glu             100 105 110 Asn Ile Asp Pro Trp His Ile Leu Gln Thr Gly Gly Lys Glu Ile Lys         115 120 125 Ser Ala Ile Pro Met Gly Cys Val Leu Thr Leu Pro Ala Thr Gly Ser     130 135 140 Glu Ser Asn Ala Gly Ala Val Ile Ser Arg Lys Thr Thr Gly Asp Lys 145 150 155 160 Gln Ala Phe His Ser Ala His Val Gln Pro Val Phe Ala Val Leu Asp                 165 170 175 Pro Val Tyr Thr Tyr Thr Leu Pro Pro Arg Gln Val Ala Asn Gly Val             180 185 190 Val Asp Ala Phe Val His Thr Val Glu Gln Tyr Val Thr Lys Pro Val         195 200 205 Asp Ala Lys Ile Gln Asp Arg Phe Ala Glu Gly Ile Leu Leu Thr Leu     210 215 220 Ile Glu Asp Gly Pro Lys Ala Leu Lys Glu Pro Glu Asn Tyr Asp Val 225 230 235 240 Arg Ala Asn Val Met Trp Ala Ala Thr Gln Ala Leu Asn Gly Leu Ile                 245 250 255 Gly Ala Gly Val Pro Gln Asp Trp Ala Thr His Met Leu Gly His Glu             260 265 270 Leu Thr Ala Met His Gly Leu Asp His Ala Gln Thr Leu Ala Ile Val         275 280 285 Leu Pro Ala Leu Trp Asn Glu Lys Arg Asp Thr Lys Arg Ala Lys Leu     290 295 300 Leu Gln Tyr Ala Glu Arg Val Trp Asn Ile Thr Glu Gly Ser Asp Asp 305 310 315 320 Glu Arg Ile Asp Ala Ile Ala Ala Thr Arg Asn Phe Phe Glu Gln                 325 330 335 Leu Gly Val Pro Thr His Leu Ser Asp Tyr Gly Leu Asp Gly Ser Ser             340 345 350 Ile Pro Ala Leu Leu Lys Lys Leu Glu Glu His Gly Met Thr Gln Leu         355 360 365 Gly Glu Asn His Asp Ile Thr Leu Asp Val Ser Arg Arg Ile Tyr Glu     370 375 380 Ala Ala Arg 385 <210> 41 <211> 389 <212> PRT <213> Clostridium acetobutylicum <400> 41 Met Leu Ser Phe Asp Tyr Ser Ile Pro Thr Lys Val Phe Phe Gly Lys 1 5 10 15 Gly Lys Ile Asp Val Ile Gly Glu Glu Ile Lys Lys Tyr Gly Ser Arg             20 25 30 Val Leu Ile Val Tyr Gly Gly Gly Ser Ile Lys Arg Asn Gly Ile Tyr         35 40 45 Asp Arg Ala Thr Ala Ile Leu Lys Glu Asn Asn Ile Ala Phe Tyr Glu     50 55 60 Leu Ser Gly Val Glu Pro Asn Pro Arg Ile Thr Thr Val Lys Lys Gly 65 70 75 80 Ile Glu Ile Cys Arg Glu Asn Asn Val Asp Leu Val Leu Ala Ile Gly                 85 90 95 Gly Gly Ser Ala Ile Asp Cys Ser Lys Val Ile Ala Ala Gly Val Tyr             100 105 110 Tyr Asp Gly Asp Thr Trp Asp Met Val Lys Asp Pro Ser Lys Ile Thr         115 120 125 Lys Val Leu Pro Ile Ala Ser Ile Leu Thr Leu Ser Ala Thr Gly Ser     130 135 140 Glu Met Asp Gln Ile Ala Val Ile Ser Asn Met Glu Thr Asn Glu Lys 145 150 155 160 Leu Gly Val Gly His Asp Asp Met Arg Pro Lys Phe Ser Val Leu Asp                 165 170 175 Pro Thr Tyr Thr Phe Thr Val Pro Lys Asn Gln Thr Ala Ala Gly Thr             180 185 190 Ala Asp Ile Met Ser His Thr Phe Glu Ser Tyr Phe Ser Gly Val Glu         195 200 205 Gly Ala Tyr Val Gln Asp Gly Ile Ala Glu Ala Ile Leu Arg Thr Cys     210 215 220 Ile Lys Tyr Gly Lys Ile Ala Met Glu Lys Thr Asp Asp Tyr Glu Ala 225 230 235 240 Arg Ala Asn Leu Met Trp Ala Ser Ser Leu Ala Ile Asn Gly Leu Leu                 245 250 255 Ser Leu Gly Lys Asp Arg Lys Trp Ser Cys His Pro Met Glu His Glu             260 265 270 Leu Ser Ala Tyr Tyr Asp Ile Thr His Gly Val Gly Leu Ala Ile Leu         275 280 285 Thr Pro Asn Trp Met Glu Tyr Ile Leu Asn Asp Asp Thr Leu His Lys     290 295 300 Phe Val Ser Tyr Gly Ile Asn Val Trp Gly Ile Asp Lys Asn Lys Asp 305 310 315 320 Asn Tyr Glu Ile Ala Arg Glu Ala Ile Lys Asn Thr Arg Glu Tyr Phe                 325 330 335 Asn Ser Leu Gly Ile Pro Ser Lys Leu Arg Glu Val Gly Ile Gly Lys             340 345 350 Asp Lys Leu Glu Leu Met Ala Lys Gln Ala Val Arg Asn Ser Gly Gly         355 360 365 Thr Ile Gly Ser Leu Arg Pro Ile Asn Ala Glu Asp Val Leu Glu Ile     370 375 380 Phe Lys Lys Ser Tyr 385 <210> 42 <211> 1170 <212> DNA <213> Clostridium acetobutylicum <400> 42 ttaataagat tttttaaata tctcaagaac atcctctgca tttattggtc ttaaacttcc 60 tattgttcct ccagaatttc taacagcttg ctttgccatt agttctagtt tatcttttcc 120 tattccaact tctctaagct ttgaaggaat acccaatgaa ttaaagtatt ctctcgtatt 180 tttaatagcc tctcgtgcta tttcatagtt atctttgttc ttgtctattc cccaaacatt 240 tattccataa gaaacaaatt tatgaagtgt atcgtcattt agaatatatt ccatccaatt 300 aggtgttaaa attgcaagtc ctacaccatg tgttatatca taatatgcac ttaactcgtg 360 ttccatagga tgacaactcc attttctatc cttaccaagt gataatagac catttatagc 420 taaacttgaa gcccacatca aattagctct agcctcgtaa tcatcagtct tctccattgc 480 tatttttcca tactttatac atgttcttaa gattgcttct gctataccgt cctgcacata 540 agcaccttca acaccactaa agtaagattc aaaggtgtga ctcataatgt cagctgttcc 600 cgctgctgtt tgatttttag gtactgtaaa agtatatgta ggatctaaca ctgaaaattt 660 aggtctcata tcatcatgtc ctactccaag cttttcatta gtctccatat ttgaaattac 720 tgcaatttga tccatttcag accctgttgc tgaaagagta agtatacttg caattggaag 780 aactttagtt attttagatg gatctttaac catgtcccat gtatcgccat cataataaac 840 tccagctgca attaccttag aacagtctat tgcacttcct ccccctattg ctaatactaa 900 atccacatta ttttctctac atatttctat gccttttttt actgttgtta tcctaggatt 960 tggctctact cctgaaagtt catagaaagc tatattgttt tcttttaata tagctgttgc 1020 tctatcatat ataccgttcc tttttatact tcctccgcca taaactataa gcactcttga 1080 gt; agttggtatt gaataatcaa aacttagcat 1170 <210> 43 <211> 390 <212> PRT <213> Clostridium acetobutylicum <400> 43 Met Val Asp Phe Glu Tyr Ser Ile Pro Thr Arg Ile Phe Phe Gly Lys 1 5 10 15 Asp Lys Ile Asn Val Leu Gly Arg Glu Leu Lys Lys Tyr Gly Ser Lys             20 25 30 Val Leu Ile Val Tyr Gly Gly Gly Ser Ile Lys Arg Asn Gly Ile Tyr         35 40 45 Asp Lys Ala Val Ser Ile Leu Glu Lys Asn Ser Ile Lys Phe Tyr Glu     50 55 60 Leu Ala Gly Val Glu Pro Asn Pro Arg Val Thr Thr Val Glu Lys Gly 65 70 75 80 Val Lys Ile Cys Arg Glu Asn Gly Val Glu Val Val Leu Ala Ile Gly                 85 90 95 Gly Gly Ser Ala Ile Asp Cys Ala Lys Val Ile Ala Ala Ala Cys Glu             100 105 110 Tyr Asp Gly Asn Pro Trp Asp Ile Val Leu Asp Gly Ser Lys Ile Lys         115 120 125 Arg Val Leu Pro Ile Ala Ser Ile Leu Thr Ile Ala Ala Thr Gly Ser     130 135 140 Glu Met Asp Thr Trp Ala Val Ile Asn Asn Met Asp Thr Asn Glu Lys 145 150 155 160 Leu Ile Ala Ala His Pro Asp Met Ala Pro Lys Phe Ser Ile Leu Asp                 165 170 175 Pro Thr Tyr Thr Thyr Thr Val Thr Asn Gln Thr Ala Ala Gly Thr             180 185 190 Ala Asp Ile Met Ser His Ile Phe Glu Val Tyr Phe Ser Asn Thr Lys         195 200 205 Thr Ala Tyr Leu Gln Asp Arg Met Ala Glu Ala Leu Leu Arg Thr Cys     210 215 220 Ile Lys Tyr Gly Gly Ile Ala Leu Glu Lys Pro Asp Asp Tyr Glu Ala 225 230 235 240 Arg Ala Asn Leu Met Trp Ala Ser Ser Leu Ala Ile Asn Gly Leu Leu                 245 250 255 Thr Tyr Gly Lys Asp Thr Asn Trp Ser Val His Leu Met Glu His Glu             260 265 270 Leu Ser Ala Tyr Tyr Asp Ile Thr His Gly Val Gly Leu Ala Ile Leu         275 280 285 Thr Pro Asn Trp Met Glu Tyr Ile Leu Asn Asn Asp Thr Val Tyr Lys     290 295 300 Phe Val Glu Tyr Gly Val Asn Val Trp Gly Ile Asp Lys Glu Lys Asn 305 310 315 320 His Tyr Asp Ile Ala His Gln Ala Ile Gln Lys Thr Arg Asp Tyr Phe                 325 330 335 Val Asn Val Leu Gly Leu Pro Ser Arg Leu Arg Asp Val Gly Ile Glu             340 345 350 Glu Glu Lys Leu Asp Ile Met Ala Lys Glu Ser Val Lys Leu Thr Gly         355 360 365 Gly Thr Ile Gly Asn Leu Arg Pro Val Asn Ala Ser Glu Val Leu Gln     370 375 380 Ile Phe Lys Lys Ser Val 385 390 <210> 44 <211> 1173 <212> DNA <213> Clostridium acetobutylicum <400> 44 gtggttgatt tcgaatattc aataccaact agaatttttt tcggtaaaga taagataaat 60 gtacttggaa gagagcttaa aaaatatggt tctaaagtgc ttatagttta tggtggagga 120 agtataaaga gaaatggaat atatgataaa gctgtaagta tacttgaaaa aaacagtatt 180 aaattttatg aacttgcagg agtagagcca aatccaagag taactacagt tgaaaaagga 240 gttaaaatat gtagagaaaa tggagttgaa gtagtactag ctataggtgg aggaagtgca 300 atagattgcg caaaggttat agcagcagca tgtgaatatg atggaaatcc atgggatatt 360 gtgttagatg gctcaaaaat aaaaagggtg cttcctatag ctagtatatt aaccattgct 420 gcaacaggat cagaaatgga tacgtgggca gtaataaata atatggatac aaacgaaaaa 480 ctaattgcgg cacatccaga tatggctcct aagttttcta tattagatcc aacgtatacg 540 tataccgtac ctaccaatca aacagcagca ggaacagctg atattatgag tcatatattt 600 gaggtgtatt ttagtaatac aaaaacagca tatttgcagg atagaatggc agaagcgtta 660 ttaagaactt gtattaaata tggaggaata gctcttgaga agccggatga ttatgaggca 720 agagccaatc taatgtgggc ttcaagtctt gcgataaatg gacttttaac atatggtaaa 780 gacactaatt ggagtgtaca cttaatggaa catgaattaa gtgcttatta cgacataaca 840 cacggcgtag ggcttgcaat tttaacacct aattggatgg agtatatttt aaataatgat 900 acagtgtaca agtttgttga atatggtgta aatgtttggg gaatagacaa agaaaaaaat 960 cactatgaca tagcacatca agcaatacaa aaaacaagag attactttgt aaatgtacta 1020 ggtttaccat ctagactgag agatgttgga attgaagaag aaaaattgga cataatggca 1080 aaggaatcag taaagcttac agagagacc ataggaaacc taagaccagt aaacgcctcc 1140 gaagtcctac aaatattcaa aaaatctgtg taa 1173 <210> 45 <211> 330 <212> PRT <213> Bacillus subtilis <400> 45 Met Ser Thr Asn Arg His Gln Ala Leu Gly Leu Thr Asp Gln Glu Ala 1 5 10 15 Val Asp Met Tyr Arg Thr Met Leu Leu Ala Arg Lys Ile Asp Glu Arg             20 25 30 Met Trp Leu Leu Asn Arg Ser Gly Lys Ile Pro Phe Val Ile Ser Cys         35 40 45 Gln Gly Gln Gly Ala Glad Val Gly Ala Ala Phe Ala Leu Asp Arg     50 55 60 Glu Met Asp Tyr Val Leu Pro Tyr Tyr Arg Asp Met Gly Val Val Leu 65 70 75 80 Ala Phe Gly Met Thr Ala Lys Asp Leu Met Met Ser Gly Phe Ala Lys                 85 90 95 Ala Ala Asp Pro Asn Ser Gly Gly Arg Gln Met Pro Gly His Phe Gly             100 105 110 Gln Lys Lys Asn Arg Ile Val Thr Gly Ser Ser Pro Val Thr Thr Gln         115 120 125 Val Pro His Ala Val Gly Ile Ala Leu Ala Gly Arg Met Glu Lys Lys     130 135 140 Asp Ile Ala Phe Val Thr Phe Gly Glu Gly Ser Ser Asn Gln Gly 145 150 155 160 Asp Phe His Glu Gly Ala Asn Phe Ala Ala Val His Lys Leu Pro Val                 165 170 175 Ile Phe Met Cys Glu Asn Asn Lys Tyr Ala Ile Ser Val Pro Tyr Asp             180 185 190 Lys Gln Val Ala Cys Glu Asn Ile Ser Asp Arg Ala Ile Gly Tyr Gly         195 200 205 Met Pro Gly Val Thr Val Asn Gly Asn Asp Pro Leu Glu Val Tyr Gln     210 215 220 Ala Val Lys Glu Ala Arg Glu Arg Ala Arg Arg Gly Glu Gly Pro Thr 225 230 235 240 Leu Ile Glu Thr Ile Ser Tyr Arg Leu Thr Pro His Ser Ser Asp Asp                 245 250 255 Asp Asp Ser Ser Tyr Arg Gly Arg Glu Glu Val Glu Glu Ala Lys Lys             260 265 270 Ser Asp Pro Leu Leu Thr Tyr Gln Ala Tyr Leu Lys Glu Thr Gly Leu         275 280 285 Leu Ser Asp Glu Ile Glu Gln Thr Met Leu Asp Glu Ile Met Ala Ile     290 295 300 Val Asn Glu Ala Thr Asp Glu Ala Glu Asn Ala Pro Tyr Ala Ala Pro 305 310 315 320 Glu Ser Ala Leu Asp Tyr Val Tyr Ala Lys                 325 330 <210> 46 <211> 993 <212> DNA <213> Bacillus subtilis <400> 46 atgagtacaa accgacatca agcactaggg ctgactgatc aggaagccgt tgatatgtat 60 agaaccatgc tgttagcaag aaaaatcgat gaaagaatgt ggctgttaaa ccgttctggc 120 aaaattccat ttgtaatctc ttgtcaagga caggaagcag cacaggtagg agcggctttc 180 gcacttgacc gtgaaatgga ttatgtattg ccgtactaca gagacatggg tgtcgtgctc 240 gcgtttggca tgacagcaaa ggacttaatg atgtccgggt ttgcaaaagc agcagatccg 300 aactcaggag gccgccagat gccgggacat ttcggacaaa agaaaaaccg cattgtgacg 360 gt; atggagaaaa aggatatcgc agcctttgtt acattcgggg aagggtcttc aaaccaaggc 480 gatttccatg aaggggcaaa ctttgccgct gtccataagc tgccggttat tttcatgtgt 540 gaaaacaaca aatacgcaat ctcagtgcct tacgataagc aagtcgcatg tgagaacatt 600 tccgaccgtg ccataggcta tgggatgcct ggcgtaactg tgaatggaaa tgatccgctg 660 gaagtttatc aagcggttaa agaagcacgc gaaagggcac gcagaggaga aggcccgaca 720 ttaattgaaa cgatttctta ccgccttaca ccacattcca gtgatgacga tgacagcagc 780 tacagaggcc gtgaagaagt agaggaagcg aaaaaaagtg atcccctgct tacttatcaa 840 gcttacttaa aggaaacagg cctgctgtcc gatgagatag aacaaaccat gctggatgaa 900 attatggcaa tcgtaaatga agcgacggat gaagcggaga acgccccata tgcagctcct 960 gagtcagcgc ttgattatgt ttatgcgaag tag 993 <210> 47 <211> 327 <212> PRT <213> Bacillus subtilis <400> 47 Met Ser Val Met Ser Tyr Ile Asp Ala Ile Asn Leu Ala Met Lys Glu 1 5 10 15 Glu Met Glu Arg Asp Ser Arg Val Phe Val Leu Gly Glu Asp Val Gly             20 25 30 Arg Lys Gly Gly Val Phe Lys Ala Thr Ala Gly Leu Tyr Glu Gln Phe         35 40 45 Gly Glu Glu Arg Val Met Asp Thr Pro Leu Ala Glu Ser Ala Ile Ala     50 55 60 Gly Val Gly Ile Gly Ala Ala Met Tyr Gly Met Arg Ile Ala Glu 65 70 75 80 Met Gln Phe Ala Asp Phe Ile Met Pro Ala Val Asn Gln Ile Ile Ser                 85 90 95 Glu Ala Ala Lys Ile Arg Tyr Arg Ser Asn Asn Asp Trp Ser Cys Pro             100 105 110 Ile Val Val Arg Ala Pro Tyr Gly Gly Gly Val His Gly Ala Leu Tyr         115 120 125 His Ser Gln Ser Val Glu Ala Ile Phe Ala Asn Gln Pro Gly Leu Lys     130 135 140 Ile Val Met Pro Ser Thr Pro Tyr Asp Ala Lys Gly Leu Leu Lys Ala 145 150 155 160 Ala Val Arg Asp Glu Asp Pro Val Leu Phe Phe Glu His Lys Arg Ala                 165 170 175 Tyr Arg Leu Ile Lys Gly Glu Val Pro Ala Asp Asp Tyr Val Leu Pro             180 185 190 Ile Gly Lys Ala Asp Val Lys Arg Glu Gly Asp Asp Ile Thr Val Ile         195 200 205 Thr Tyr Gly Leu Cys Val His Phe Ala Leu Gln Ala Ala Glu Arg Leu     210 215 220 Glu Lys Asp Gly Ile Ser Ala His Val Val Asp Leu Arg Thr Val Tyr 225 230 235 240 Pro Leu Asp Lys Glu Ala Ile Glu Ala Ala Ser Lys Thr Gly Lys                 245 250 255 Val Leu Leu Val Thr Glu Asp Thr Lys Glu Gly Ser Ile Met Ser Glu             260 265 270 Val Ala Ila Ile Ser Glu His Cys Leu Phe Asp Leu Asp Ala Pro         275 280 285 Ile Lys Arg Leu Ala Gly Pro Asp Ile Pro Ala Met Pro Tyr Ala Pro     290 295 300 Thr Met Glu Lys Tyr Phe Met Val Asn Pro Asp Lys Val Glu Ala Ala 305 310 315 320 Met Arg Glu Leu Ala Glu Phe                 325 <210> 48 <211> 984 <212> DNA <213> Bacillus subtilis <400> 48 atgtcagtaa tgtcatatat tgatgcaatc aatttggcga tgaaagaaga aatggaacga 60 gattctcgcg ttttcgtcct tggggaagat gtaggaagaa aaggcggtgt gtttaaagcg 120 acagcgggac tctatgaaca atttggggaa gagcgcgtta tggatacgcc gcttgctgaa 180 tctgcaatcg caggagtcgg tatcggagcg gcaatgtacg gaatgagacc gattgctgaa 240 atgcagtttg ctgatttcat tatgccggca gtcaaccaaa ttatttctga agcggctaaa 300 atccgctacc gcagcaacaa tgactggagc tgtccgattg tcgtcagagc gccatacggc 360 ggaggcgtgc acggagccct gtatcattct caatcagtcg aagcaatttt cgccaaccag 420 cccggactga aaattgtcat gccatcaaca ccatatgacg cgaaagggct cttaaaagcc 480 gcagttcgtg acgaagaccc cgtgctgttt tttgagcaca agcgggcata ccgtctgata 540 aagggcgagg ttccggctga tgattatgtc ctgccaatcg gcaaggcgga cgtaaaaagg 600 gaaggcgacg acatcacagt gatcacatac ggcctgtgtg tccacttcgc cttacaagct 660 gcagaacgtc tcgaaaaaga tggcatttca gcgcatgtgg tggatttaag aacagtttac 720 ccgcttgata aagaagccat catcgaagct gcgtccaaaa ctggaaaggt tcttttggtc 780 acagaagata caaaagaagg cagcatcatg agcgaagtag ccgcaattat atccgagcat 840 tgtctgttcg acttagacgc gccgatcaaa cggcttgcag gtcctgatat tccggctatg 900 ccttatgcgc cgacaatgga aaaatacttt atggtcaacc ctgataaagt ggaagcggcg 960 atgagagaat tagcggagtt ttaa 984 <210> 49 <211> 424 <212> PRT <213> Bacillus subtilis <400> 49 Met Ale Ile Glu Gln Met Thr Met Pro Gln Leu Gly Glu Ser Val Thr 1 5 10 15 Glu Gly Thr Ile Ser Lys Trp Leu Val Ala Pro Gly Asp Lys Val Asn             20 25 30 Lys Tyr Asp Pro Ile Ala Glu Val Met Thr Asp Lys Val Asn Ala Glu         35 40 45 Val Ser Ser Phe Thr Gly Thr Ile Thr Glu Leu Val Gly Glu Glu     50 55 60 Gly Gln Thr Leu Gln Val Gly Glu Met Ile Cys Lys Ile Glu Thr Glu 65 70 75 80 Gly Ala Asn Pro Ala Glu Gln Lys Gln Glu Gln Pro Ala Ala Ser Glu                 85 90 95 Ala Ala Glu Asn Pro Ala Lys Ser Ala Gly Ala Ala Asp Gln Pro             100 105 110 Asn Lys Lys Arg Tyr Ser Pro Ala Val Leu Arg Leu Ala Gly Glu His         115 120 125 Gly Ile Asp Leu Asp Gln Val Thr Gly Thr Gly Ala Gly Gly Arg Ile     130 135 140 Thr Arg Lys Asp Ile Gln Arg Leu Ile Glu Thr Gly Gly Val Gln Glu 145 150 155 160 Gln Asn Pro Glu Glu Leu Lys Thr Ala Ala Pro Ala Pro Lys Ser Ala                 165 170 175 Ser Lys Pro Glu Pro Lys Glu Glu Thr Ser Tyr Pro Ala Ser Ala Ala             180 185 190 Gly Asp Lys Glu Ile Pro Val Thr Gly Val Arg Lys Ala Ile Ala Ser         195 200 205 Asn Met Lys Arg Ser Lys Thr Glu Ile Pro His Ala Trp Thr Met Met     210 215 220 Glu Val Asp Val Thr Asn Met Val Ala Tyr Arg Asn Ser Ile Lys Asp 225 230 235 240 Ser Phe Lys Lys Thr Glu Gly Phe Asn Leu Thr Phe Phe Ala Phe Phe                 245 250 255 Val Lys Ala Val Ala Gln Ala Leu Lys Glu Phe Pro Gln Met Asn Ser             260 265 270 Met Trp Ala Gly Asp Lys Ile Ile Gln Lys Lys Asp Ile Asn Ile Ser         275 280 285 Ile Ala Val Ala Thr Glu Asp Ser Leu Phe Val Pro Ile Lys Asn     290 295 300 Ala Asp Glu Lys Thr Ile Lys Gly Ile Ala Lys Asp Ile Thr Gly Leu 305 310 315 320 Ala Lys Lys Val Arg Asp Gly Lys Leu Thr Ala Asp Asp Met Gln Gly                 325 330 335 Gly Thr Phe Thr Val Asn Asn Thr Gly Ser Phe Gly Ser Val Gln Ser             340 345 350 Met Gly Ile Ile Asn Tyr Pro Gln Ala Ile Leu Gln Val Glu Ser         355 360 365 Ile Val Lys Arg Pro Val Val Met Asp Asn Gly Met Ile Ala Val Arg     370 375 380 Asp Met Val Asn Leu Cys Leu Ser Leu Asp His Arg Val Leu Asp Gly 385 390 395 400 Leu Val Cys Gly Arg Phe Leu Gly Arg Val Lys Gln Ile Leu Glu Ser                 405 410 415 Ile Asp Glu Lys Thr Ser Val Tyr             420 <210> 50 <211> 1275 <212> DNA <213> Bacillus subtilis <400> 50 atggcaattg aacaaatgac gatgccgcag cttggagaaa gcgtaacaga ggggacgatc 60 agcaaatggc ttgtcgcccc cggtgataaa gtgaacaaat acgatccgat cgcggaagtc 120 atgacagata aggtaaatgc agaggttccg tcttctttta ctggtacgat aacagagctt 180 gtgggagaag aaggccaaac cctgcaagtc ggagaaatga tttgcaaaat tgaaacagaa 240 ggcgcgaatc cggctgaaca aaaacaagaa cagccagcag catcagaagc cgctgagaac 300 cctgttgcaa aaagtgctgg agcagccgat cagcccaata aaaagcgcta ctcgccagct 360 gttctccgtt tggccggaga gcacggcatt gacctcgatc aagtgacagg aactggtgcc 420 ggcgggcgca tcacacgaaa agatattcag cgcttaattg aaacaggcgg cgtgcaagaa 480 cagaatcctg aggagctgaa aacagcagct cctgcaccga agtctgcatc aaaacctgag 540 ccaaaagaag agacgtcata tcctgcgtct gcagccggtg ataaagaaat ccctgtcaca 600 ggtgtaagaa aagcaattgc ttccaatatg aagcgaagca aaacagaaat tccgcatgct 660 tggacgatga tggaagtcga cgtcacaaat atggttgcat atcgcaacag tataaaagat 720 tcttttaaga agacagaagg ctttaattta acgttcttcg ccttttttgt aaaagcggtc 780 gctcaggcgt taaaagaatt cccgcaaatg aatagcatgt gggcggggga caaaattatt 840 cagaaaaagg atatcaatat ttcaattgca gttgccacag aggattcttt atttgttccg 900 gtgattaaaa acgctgatga aaaaacaatt aaaggcattg cgaaagacat taccggccta 960 gctaaaaaag taagagacgg aaaactcact gcagatgaca tgcagggagg cacgtttacc 1020 gtcaacaaca caggttcgtt cgggtctgtt cagtcgatgg gcattatcaa ctaccctcag 1080 gctgcgattc ttcaagtaga atccatcgtc aaacgcccgg ttgtcatgga caatggcatg 1140 attgctgtca gagacatggt taatctgtgc ctgtcattag atcacagagt gcttgacggt 1200 ctcgtgtgcg gacgattcct cggacgagtg aaacaaattt tagaatcgat tgacgagaag 1260 acatctgttt actaa 1275 <210> 51 <211> 474 <212> PRT <213> Bacillus subtilis <400> 51 Met Ala Thr Glu Tyr Asp Val Valle Leu Gly Gly Gly Thr Gly Gly 1 5 10 15 Tyr Val Ala Ala Ile Arg Ala Ala Gln Leu Gly Leu Lys Thr Ala Val             20 25 30 Val Glu Lys Glu Lys Leu Gly Gly Thr Cys Leu His Lys Gly Cys Ile         35 40 45 Pro Ser Lys Ala Leu Leu Arg Ser Ala Glu Val Tyr Arg Thr Ala Arg     50 55 60 Glu Ala Asp Gln Phe Gly Val Glu Thr Ala Gly Val Ser Leu Asn Phe 65 70 75 80 Glu Lys Val Gln Gln Arg Lys Gln Ala Val Val Asp Lys Leu Ala Ala                 85 90 95 Gly Val Asn His Leu Met Lys Lys Gly Lys Ile Asp Val Tyr Thr Gly             100 105 110 Tyr Gly Arg Ile Leu Gly Pro Ser Ile Phe Ser Pro Leu Pro Gly Thr         115 120 125 Ile Ser Val Glu Arg Gly Asn Gly Glu Glu Asn Asp Met Leu Ile Pro     130 135 140 Lys Gln Val Ile Ile Ala Thr Gly Ser Arg Pro Arg Met Leu Pro Gly 145 150 155 160 Leu Glu Val Asp Gly Lys Ser Val Leu Thr Ser Asp Glu Ala Leu Gln                 165 170 175 Met Glu Glu Leu Pro Gln Ser Ile Ile Ile Val Gly Gly Gly Val Ile             180 185 190 Gly Ile Glu Trp Ala Ser Met Leu His Asp Phe Gly Val Lys Val Thr         195 200 205 Val Ile Glu Tyr Ala Asp Arg Ile Leu Pro Thr Glu Asp Leu Glu Ile     210 215 220 Ser Lys Glu Met Glu Ser Leu Leu Lys Lys Lys Gly Ile Gln Phe Ile 225 230 235 240 Thr Gly Ala Lys Val Leu Pro Asp Thr Met Thr Lys Thr Ser Asp Asp                 245 250 255 Ile Ser Ile Gln Ala Glu Lys Asp Gly Glu Thr Val Thr Tyr Ser Ala             260 265 270 Glu Lys Met Leu Val Ser Ile Gly Arg Gln Ala Asn Ile Glu Gly Ile         275 280 285 Gly Leu Glu Asn Thr Asp Ile Val Thr Glu Asn Gly Met Ile Ser Val     290 295 300 Asn Glu Ser Cys Gln Thr Lys Glu Ser His Ile Tyr Ala Ile Gly Asp 305 310 315 320 Val Ile Gly Gly Leu Gln Leu Ala His Val Ale Ser His Glu Gly Ile                 325 330 335 Ile Ala Val Glu His Phe Ala Gly Leu Asn Pro His Pro Leu Asp Pro             340 345 350 Thr Leu Val Pro Lys Cys Ile Tyr Ser Ser Pro Glu Ala Ala Ser Val         355 360 365 Gly Leu Thr Glu Asp Glu Ala Lys Ala Asn Gly His Asn Val Lys Ile     370 375 380 Gly Lys Phe Pro Phe Met Ala Ile Gly Lys Ala Leu Val Tyr Gly Glu 385 390 395 400 Ser Asp Gly Phe Val Lys Ile Val Ala Asp Arg Asp Thr Asp Asp Ile                 405 410 415 Leu Gly Val His Met Ile Gly Pro His Val Thr Asp Met Ile Ser Glu             420 425 430 Ala Gly Leu Ala Lys Val Leu Asp Ala Thr Pro Trp Glu Val Gly Gln         435 440 445 Thr Ile His Pro Th Pro Thr Leu Ser Glu Ala Ile Gly Glu Ala Ala     450 455 460 Leu Ala Ala Asp Gly Lys Ala Ile His Phe 465 470 <210> 52 <211> 1425 <212> DNA <213> Bacillus subtilis <400> 52 atggcaactg agtatgacgt agtcattctg ggcggcggta ccggcggtta tgttgcggcc 60 atcagagccg ctcagctcgg cttaaaaaca gccgttgtgg aaaaggaaaa actcggggga 120 acatgtctgc ataaaggctg tatcccgagt aaagcgctgc ttagaagcgc agaggtatac 180 cggacagctc gtgaagccga tcaattcgga gtggaaacgg ctggcgtgtc cctcaacttt 240 gaaaaagtgc agcagcgtaa gcaagccgtt gttgataagc ttgcagcggg tgtaaatcat 300 ttaatgaaaa aaggaaaaat tgacgtgtac accggatatg gacgtatcct tggaccgtca 360 atcttctctc cgctgccggg aacaatttct gttgagcggg gaaatggcga agaaaatgac 420 atgctgatcc cgaaacaagt gatcattgca acaggatcaa gaccgagaat gcttccgggt 480 cttgaagtgg acggtaagtc tgtactgact tcagatgagg cgctccaaat ggaggagctg 540 ccacagtcaa tcatcattgt cggcggaggg gttatcggta tcgaatgggc gtctatgctt 600 catgattttg gcgttaaggt aacggttatt gaatacgcgg atcgcatatt gccgactgaa 660 gatctagaga tttcaaaaga aatggaaagt cttcttaaga aaaaaggcat ccagttcata 720 acaggggcaa aagtgctgcc tgacacaatg acaaaaacat cagacgatat cagcatacaa 780 gcggaaaaag acggagaaac cgttacctat tctgctgaga aaatgcttgt ttccatcggc 840 agacaggcaa atatcgaagg catcggccta gagaacaccg atattgttac tgaaaatggc 900 atgatttcag tcaatgaaag ctgccaaacg aaggaatctc atatttatgc aatcggagac 960 gtaatcggtg gcctgcagtt agctcacgtt gcttcacatg agggaattat tgctgttgag 1020 cattttgcag gtctcaatcc gcatccgctt gatccgacgc ttgtgccgaa gtgcatttac 1080 tcaagccctg aagctgccag tgtcggctta accgaagacg aagcaaaggc gaacgggcat 1140 aatgtcaaaa tcggcaagtt cccatttatg gcgattggaa aagcgcttgt atacggtgaa 1200 agcgacggtt ttgtcaaaat cgtggctgac cgagatacag atgatattct cggcgttcat 1260 atgattggcc cgcatgtcac cgacatgatt tctgaagcgg gtcttgccaa agtgctggac 1320 gcaacaccgt gggaggtcgg gcaaacgatt cacccgcatc caacgctttc tgaagcaatt 1380 ggagaagctg cgcttgccgc agatggcaaa gccattcatt tttaa 1425 <210> 53 <211> 410 <212> PRT <213> Pseudomonas putida <400> 53 Met Asn Glu Tyr Ala Pro Leu Arg Leu His Val Pro Glu Pro Thr Gly 1 5 10 15 Arg Pro Gly Cys Gln Thr Asp Phe Ser Tyr Leu Arg Leu Asn Asp Ala             20 25 30 Gly Gln Ala Arg Lys Pro Pro Val Asp Val Asp Ala Ala Asp Thr Ala         35 40 45 Asp Leu Ser Tyr Ser Leu Val Arg Val Leu Asp Glu Gln Gly Asp Ala     50 55 60 Gln Gly Pro Trp Ala Glu Asp Ile Asp Pro Gln Ile Leu Arg Gln Gly 65 70 75 80 Met Arg Ala Met Leu Lys Thr Arg Ile Phe Asp Ser Arg Met Val Val                 85 90 95 Ala Gln Arg Gln Lys Lys Met Ser Phe Tyr Met Gln Ser Leu Gly Glu             100 105 110 Glu Ala Ile Gly Ser Gly Ale Leu Asn Arg Thr Asp Met         115 120 125 Cys Phe Pro Thr Tyr Arg Gln Gin Ser Ile Leu Met Ala Arg Asp Val     130 135 140 Ser Leu Val Glu Met Ile Cys Gln Leu Leu Ser Asn Glu Arg Asp Pro 145 150 155 160 Leu Lys Gly Arg Gln Leu Pro Ile Met Tyr Ser Val Arg Glu Ala Gly                 165 170 175 Phe Phe Thr Ile Ser Gly Asn Leu Ala Thr Gln Phe Val Gln Ala Val             180 185 190 Gly Trp Ala Met Ala Ser Ala Ile Lys Gly Asp Thr Lys Ile Ala Ser         195 200 205 Ala Trp Ile Gly Asp Gly Ala Thr Ala Glu Ser Asp Phe His Thr Ala     210 215 220 Leu Thr Phe Ala His Val Tyr Arg Ala Pro Val Ile Leu Asn Val Val 225 230 235 240 Asn Asn Gln Trp Ala Ile Ser Thr Phe Gln Ala Ile Ala Gly Gly Glu                 245 250 255 Ser Thr Thr Phe Ala Gly Arg Gly Val Gly Cys Gly Ile Ala Ser Leu             260 265 270 Arg Val Asp Gly Asn Asp Phe Val Ala Val Tyr Ala Ala Ser Arg Trp         275 280 285 Ala Ala Glu Arg Ala Arg Arg Gly Leu Gly Pro Ser Leu Ile Glu Trp     290 295 300 Val Thr Tyr Arg Ala Gly Pro His Ser Thr Ser Asp Ser Ser Ser 305 310 315 320 Tyr Arg Pro Ala Asp Asp Trp Ser His Phe Pro Leu Gly Asp Pro Ile                 325 330 335 Ala Arg Leu Lys Gln His Leu Ile Lys Ile Gly His Trp Ser Glu Glu             340 345 350 Glu His Gln Ala Thr Thr Ala Glu Phe Glu Ala Ala Val Ile Ala Ala         355 360 365 Gln Lys Glu Ala Glu Gln Tyr Gly Thr Leu Ala Asn Gly His Ile Pro     370 375 380 Ser Ala Ala Ser Met Phe Glu Asp Val Tyr Lys Glu Met Pro Asp His 385 390 395 400 Leu Arg Arg Gln Arg Gln Glu Leu Gly Val                 405 410 <210> 54 <211> 6643 <212> DNA <213> Pseudomonas putida <400> 54 gcatgcctgc aggccgccga tgaaatggtg gaaggtatcg gtaggctggc cctgctcatc 60 gctgaacacg ttacgcccgc tgccggtatc gaccaggctc tggtgaatat gcatggaact 120 gccaggcgtg cgcgccagcg gtttggccat gcacaccacg gtcagcccgt gcttgagtgc 180 cacttccttg agcaggtgtt tgaacaggaa ggtctggtcg gccagcagca gcgggtcgcc 240 atgtagcaag ttgatctcga actggctgac gcccatttcg tgcatgaagg tgtcgcgcgg 300 caggccgagc gcggccatgc actggtacac ctcattgaag aacgggcgca ggccgttgtt 360 ggaactgaca ctgaacgccg aatggcccag ctcgcggcgg ccgtcggtgc ccagcggtgg 420 ctggaacggc tgctgcgggt cactgttggg ggcaaacacg aagaactcaa gctcggtcgc 480 cactaccggt gccagaccca acgctgcgta gcgggcgatc acggccttca gctggccccg 540 ggtggacagt gccgagggcc ggccatccag ttcattggca tcgcagatgg ccagggcgcg 600 accgtcatcg ctccagggca agcgatgaac ctggctgggt tccgctacca acgccaggtc 660 gccgtcgtcg cagccgtaga atttcgccgg cgggtagccg cccatgatgc attgcagcag 720 caccccacgg gccatctgca ggcggcggcc ttcgagaaag ccttcggcgg tcatcacctt 780 gccgcgtggg acgccgttga ggtcgggggt gacgcattcg atttcatcga tgccctggag 840 ctgagcgatg ctcatgacgc ttgtccttgt tgttgtaggc tgacaacaac ataggctggg 900 ggtgtttaaa atatcaagca gcctctcgaa cgcctggggc ctcttctatt cgcgcaaggt 960 catgccattg gccggcaacg gcaaggctgt cttgtagcgc acctgtttca aggcaaaact 1020 cgagcggata ttcgccacac ccggcaaccg ggtcaggtaa tcgagaaacc gctccagcgc 1080 ctggatactc ggcagcagta cccgcaacag gtagtccggg tcgcccgtca tcaggtagca 1140 ctccatcacc tcgggccgtt cggcaatttc ttcctcgaag cggtgcagcg actgctctac 1200 ctgtttttcc aggctgacat ggatgaacac attcacatcc agccccaacg cctcgggcga 1260 caacaaggtc acctgctggc ggatcacccc cagttcttcc atggcccgca cccggttgaa 1320 acagggcgtg ggcgacaggt tgaccgagcg tgccagctcg gcgttggtga tgcgggcgtt 1380 ttcctgcagg ctgttgagaa tgccgatatc ggtacgatcg agtttgcgca tgagacaaaa 1440 tcaccggttt tttgtgttta tgcggaatgt ttatctgccc cgctcggcaa aggcaatcaa 1500 cttgagagaa aaattctcct gccggaccac taagatgtag gggacgctga cttaccagtc 1560 acaagccggt actcagcggc ggccgcttca gagctcacaa aaacaaatac ccgagcgagc 1620 gtaaaaagca tgaacgagta cgcccccctg cgtttgcatg tgcccgagcc caccggccgg 1680 ccaggctgcc agaccgattt ttcctacctg cgcctgaacg atgcaggtca agcccgtaaa 1740 ccccctgtcg atgtcgacgc tgccgacacc gccgacctgt cctacagcct ggtccgcgtg 1800 ctcgacgagc aaggcgacgc ccaaggcccg tgggctgaag acatcgaccc gcagatcctg 1860 cgccaaggca tgcgcgccat gctcaagacg cggatcttcg acagccgcat ggtggttgcc 1920 cagcgccaga agaagatgtc cttctacatg cagagcctgg gcgaagaagc catcggcagc 1980 ggccaggcgc tggcgcttaa ccgcaccgac atgtgcttcc ccacctaccg tcagcaaagc 2040 atcctgatgg cccgcgacgt gtcgctggtg gagatgatct gccagttgct gtccaacgaa 2100 cgcgaccccc tcaagggccg ccagctgccg atcatgtact cggtacgcga ggccggcttc 2160 ttcaccatca gcggcaacct ggcgacccag ttcgtgcagg cggtcggctg ggccatggcc 2220 tcggcgatca agggcgatac caagattgcc tcggcctgga tcggcgacgg cgccactgcc 2280 gaatcggact tccacaccgc cctcaccttt gcccacgttt accgcgcccc ggtgatcctc 2340 aacgtggtca acaaccagtg ggccatctca accttccagg ccatcgccgg tggcgagtcg 2400 accaccttcg ccggccgtgg cgtgggctgc ggcatcgctt cgctgcgggt ggacggcaac 2460 gacttcgtcg ccgtttacgc cgcttcgcgc tgggctgccg aacgtgcccg ccgtggtttg 2520 ggcccgagcc tgatcgagtg ggtcacctac cgtgccggcc cgcactcgac ctcggacgac 2580 ccgtccaagt accgccctgc cgatgactgg agccacttcc cgctgggtga cccgatcgcc 2640 cgcctgaagc agcacctgat caagatcggc cactggtccg aagaagaaca ccaggccacc 2700 acggccgagt tcgaagcggc cgtgattgct gcgcaaaaag aagccgagca gtacggcacc 2760 ctggccaacg gtcacatccc gagcgccgcc tcgatgttcg aggacgtgta caaggagatg 2820 cccgaccacc tgcgccgcca acgccaggaa ctgggggttt gagatgaacg accacaacaa 2880 cagcatcaac ccggaaaccg ccatggccac cactaccatg accatgatcc aggccctgcg 2940 ctcggccatg gatgtcatgc ttgagcgcga cgacaatgtg gtggtgtacg gccaggacgt 3000 cggctacttc ggcggcgtgt tccgctgcac cgaaggcctg cagaccaagt acggcaagtc 3060 ccgcgtgttc gacgcgccca tctctgaaag cggcatcgtc ggcaccgccg tgggcatggg 3120 tgcctacggc ctgcgcccgg tggtggaaat ccagttcgct gactacttct acccggcctc 3180 cgaccagatc gtttctgaaa tggcccgcct gcgctaccgt tcggccggcg agttcatcgc 3240 cccgctgacc ctgcgtatgc cctgcggtgg cggtatctat ggcggccaga cacacagcca 3300 gagcccggaa gcgatgttca ctcaggtgtg cggcctgcgc accgtaatgc catccaaccc 3360 gtacgacgcc aaaggcctgc tgattgcctc gatcgaatgc gacgacccgg tgatcttcct 3420 ggagcccaag cgcctgtaca acggcccgtt cgacggccac catgaccgcc cggttacgcc 3480 gtggtcgaaa cacccgcaca gcgccgtgcc cgatggctac tacaccgtgc cactggacaa 3540 ggccgccatc acccgccccg gcaatgacgt gagcgtgctc acctatggca ccaccgtgta 3600 cgtggcccag gtggccgccg aagaaagtgg cgtggatgcc gaagtgatcg acctgcgcag 3660 cctgtggccg ctagacctgg acaccatcgt cgagtcggtg aaaaagaccg gccgttgcgt 3720 ggtagtacac gaggccaccc gtacttgtgg ctttggcgca gaactggtgt cgctggtgca 3780 ggagcactgc ttccaccacc tggaggcgcc gatcgagcgc gtcaccggtt gggacacccc 3840 ctaccctcac gcgcaggaat gggcttactt cccagggcct tcgcgggtag gtgcggcatt 3900 gaaaaaggtc atggaggtct gaatgggcac gcacgtcatc aagatgccgg acattggcga 3960 aggcatcgcg caggtcgaat tggtggaatg gttcgtcaag gtgggcgaca tcatcgccga 4020 ggaccaagtg gtagccgacg tcatgaccga caaggccacc gtggaaatcc cgtcgccggt 4080 cagcggcaag gtgctggccc tgggtggcca gccaggtgaa gtgatggcgg tcggcagtga 4140 gctgatccgc atcgaagtgg aaggcagcgg caaccatgtg gatgtgccgc aagccaagcc 4200 ggccgaagtg cctgcggcac cggtagccgc taaacctgaa ccacagaaag acgttaaacc 4260 ggcggcgtac caggcgtcag ccagccacga ggcagcgccc atcgtgccgc gccagccggg 4320 cgacaagccg ctggcctcgc cggcggtgcg caaacgcgcc ctcgatgccg gcatcgaatt 4380 gcgttatgtg cacggcagcg gcccggccgg gcgcatcctg cacgaagacc tcgacgcgtt 4440 catgagcaaa ccgcaaagcg ctgccgggca aacccccaat ggctatgcca ggcgcaccga 4500 cagcgagcag gtgccggtga tcggcctgcg ccgcaagatc gcccagcgca tgcaggacgc 4560 caagcgccgg gtcgcgcact tcagctatgt ggaagaaatc gacgtcaccg ccctggaagc 4620 cctgcgccag cagctcaaca gcaagcacgg cgacagccgc ggcaagctga cactgctgcc 4680 gttcctggtg cgcgccctgg tcgtggcact gcgtgacttc ccgcagataa acgccaccta 4740 cgatgacgaa gcgcagatca tcacccgcca tggcgcggtg catgtgggca tcgccaccca 4800 aggtgacaac ggcctgatgg tacccgtgct gcgccacgcc gaagcgggca gcctgtgggc 4860 caatgccggt gagatttcac gcctggccaa cgctgcgcgc aacaacaagg ccagccgcga 4920 agagctgtcc ggttcgacca ttaccctgac cagcctcggc gccctgggcg gcatcgtcag 4980 cacgccggtg gtcaacaccc cggaagtggc gatcgtcggt gtcaaccgca tggttgagcg 5040 gcccgtggtg atcgacggcc agatcgtcgt gcgcaagatg atgaacctgt ccagctcgtt 5100 cgaccaccgc gtggtcgatg gcatggacgc cgccctgttc atccaggccg tgcgtggcct 5160 gctcgaacaa cccgcctgcc tgttcgtgga gtgagcatgc aacagactat ccagacaacc 5220 ctgttgatca tcggcggcgg ccctggcggc tatgtggcgg ccatccgcgc cgggcaactg 5280 ggcatcccta ccgtgctggt ggaaggccag gcgctgggcg gtacctgcct gaacatcggc 5340 tgcattccgt ccaaggcgct gatccatgtg gccgagcagt tccaccaggc ctcgcgcttt 5400 accgaaccct cgccgctggg catcagcgtg gcttcgccac gcctggacat cggccagagc 5460 gtggcctgga aagacggcat cgtcgatcgc ctgaccactg gtgtcgccgc cctgctgaaa 5520 aagcacgggg tgaaggtggt gcacggctgg gccaaggtgc ttgatggcaa gcaggtcgag 5580 gtggatggcc agcgcatcca gtgcgagcac ctgttgctgg ccacgggctc cagcagtgtc 5640 gaactgccga tgctgccgtt gggtgggccg gtgatttcct cgaccgaggc cctggcaccg 5700 aaagccctgc cgcaacacct ggtggtggtg ggcggtggct acatcggcct ggagctgggt 5760 atcgcctacc gcaagctcgg cgcgcaggtc agcgtggtgg aagcgcgcga gcgcatcctg 5820 ccgacttacg acagcgaact gaccgccccg gtggccgagt cgctgaaaaa gctgggtatc 5880 gccctgcacc ttggccacag cgtcgaaggt tacgaaaatg gctgcctgct ggccaacgat 5940 ggcaagggcg gacaactgcg cctggaagcc gaccgggtgc tggtggccgt gggccgccgc 6000 ccacgcacca agggcttcaa cctggaatgc ctggacctga agatgaatgg tgccgcgatt 6060 gccatcgacg agcgctgcca gaccagcatg cacaacgtct gggccatcgg cgacgtggcc 6120 ggcgaaccga tgctggcgca ccgggccatg gcccagggcg agatggtggc cgagatcatc 6180 gccggcaagg cacgccgctt cgaacccgct gcgatagccg ccgtgtgctt caccgacccg 6240 gaagtggtcg tggtcggcaa gacgccggaa caggccagtc agcaaggcct ggactgcatc 6300 gtcgcgcagt tcccgttcgc cgccaacggc cgggccatga gcctggagtc gaaaagcggt 6360 ttcgtgcgcg tggtcgcgcg gcgtgacaac cacctgatcc tgggctggca agcggttggc 6420 gtggcggttt ccgagctgtc cacggcgttt gcccagtcgc tggagatggg tgcctgcctg 6480 gaggatgtgg ccggtaccat ccatgcccac ccgaccctgg gtgaagcggt acaggaagcg 6540 gcactgcgtg ccctgggcca cgccctgcat atctgacact gaagcggccg aggccgattt 6600 ggcccgccgc gccgagaggc gctgcgggtc ttttttatac ctg 6643 <210> 55 <211> 352 <212> PRT <213> Pseudomonas putida <400> 55 Met Asn Asp His Asn Asn Ser Ile Asn Pro Glu Thr Ala Met Ala Thr 1 5 10 15 Thr Thr Met Thr Met Ile Gln Ala Leu Arg Ser Ala Met Asp Val Met             20 25 30 Leu Glu Arg Asp Asp Val Val Val Tyr Gly Gln Asp Val Gly Tyr         35 40 45 Phe Gly Gly Val Phe Arg Cys Thr Glu Gly Leu Gln Thr Lys Tyr Gly     50 55 60 Lys Ser Arg Val Phe Asp Ala Pro Ile Ser Glu Ser Gly Ile Val Gly 65 70 75 80 Thr Ala Val Gly Met Gly Ala Tyr Gly Leu Arg Pro Val Val Glu Ile                 85 90 95 Gln Phe Ala Asp Tyr Phe Tyr Pro Ala Ser Asp Gln Ile Val Ser Glu             100 105 110 Met Ala Arg Leu Arg Tyr Arg Ser Ala Gly Glu Phe Ile Ala Pro Leu         115 120 125 Thr Leu Arg Met Pro Cys Gly Gly Gly Ile Tyr Gly Gly Gln Thr His     130 135 140 Ser Gln Ser Pro Glu Ala Met Phe Thr Gln Val Cys Gly Leu Arg Thr 145 150 155 160 Val Met Pro Ser Asn Pro Tyr Asp Ala Lys Gly Leu Leu Ile Ala Ser                 165 170 175 Ile Glu Cys Asp Asp Pro Val Ile Phe Leu Glu Pro Lys Arg Leu Tyr             180 185 190 Asn Gly Pro Phe Asp Gly His His Asp Arg Pro Val Thr Pro Trp Ser         195 200 205 Lys His Pro His Ser Ala Val Pro Asp Gly Tyr Tyr Thr Val Pro Leu     210 215 220 Asp Lys Ala Ala Ile Thr Arg Pro Gly Asn Asp Val Ser Val Leu Thr 225 230 235 240 Tyr Gly Thr Thr Val Tyr Val Ala Gln Val Ala Ala Glu Glu Ser Gly                 245 250 255 Val Asp Ala Glu Val Ile Asp Leu Arg Ser Leu Trp Pro Leu Asp Leu             260 265 270 Asp Thr Ile Val Glu Ser Val Lys Lys Thr Gly Arg Cys Val Val Val         275 280 285 His Glu Ala Thr Arg Thr Cys Gly Phe Gly Ala Glu Leu Val Ser Leu     290 295 300 Val Glu Glu His Cys Phe His His Leu Glu Ala Pro Ile Glu Arg Val 305 310 315 320 Thr Gly Trp Asp Thr Pro Tyr Pro His Ala Gln Glu Trp Ala Tyr Phe                 325 330 335 Pro Gly Pro Ser Arg Val Gly Ala Ala Leu Lys Lys Val Met Glu Val             340 345 350 <210> 56 <211> 6643 <212> DNA <213> Pseudomonas putida <400> 56 gcatgcctgc aggccgccga tgaaatggtg gaaggtatcg gtaggctggc cctgctcatc 60 gctgaacacg ttacgcccgc tgccggtatc gaccaggctc tggtgaatat gcatggaact 120 gccaggcgtg cgcgccagcg gtttggccat gcacaccacg gtcagcccgt gcttgagtgc 180 cacttccttg agcaggtgtt tgaacaggaa ggtctggtcg gccagcagca gcgggtcgcc 240 atgtagcaag ttgatctcga actggctgac gcccatttcg tgcatgaagg tgtcgcgcgg 300 caggccgagc gcggccatgc actggtacac ctcattgaag aacgggcgca ggccgttgtt 360 ggaactgaca ctgaacgccg aatggcccag ctcgcggcgg ccgtcggtgc ccagcggtgg 420 ctggaacggc tgctgcgggt cactgttggg ggcaaacacg aagaactcaa gctcggtcgc 480 cactaccggt gccagaccca acgctgcgta gcgggcgatc acggccttca gctggccccg 540 ggtggacagt gccgagggcc ggccatccag ttcattggca tcgcagatgg ccagggcgcg 600 accgtcatcg ctccagggca agcgatgaac ctggctgggt tccgctacca acgccaggtc 660 gccgtcgtcg cagccgtaga atttcgccgg cgggtagccg cccatgatgc attgcagcag 720 caccccacgg gccatctgca ggcggcggcc ttcgagaaag ccttcggcgg tcatcacctt 780 gccgcgtggg acgccgttga ggtcgggggt gacgcattcg atttcatcga tgccctggag 840 ctgagcgatg ctcatgacgc ttgtccttgt tgttgtaggc tgacaacaac ataggctggg 900 ggtgtttaaa atatcaagca gcctctcgaa cgcctggggc ctcttctatt cgcgcaaggt 960 catgccattg gccggcaacg gcaaggctgt cttgtagcgc acctgtttca aggcaaaact 1020 cgagcggata ttcgccacac ccggcaaccg ggtcaggtaa tcgagaaacc gctccagcgc 1080 ctggatactc ggcagcagta cccgcaacag gtagtccggg tcgcccgtca tcaggtagca 1140 ctccatcacc tcgggccgtt cggcaatttc ttcctcgaag cggtgcagcg actgctctac 1200 ctgtttttcc aggctgacat ggatgaacac attcacatcc agccccaacg cctcgggcga 1260 caacaaggtc acctgctggc ggatcacccc cagttcttcc atggcccgca cccggttgaa 1320 acagggcgtg ggcgacaggt tgaccgagcg tgccagctcg gcgttggtga tgcgggcgtt 1380 ttcctgcagg ctgttgagaa tgccgatatc ggtacgatcg agtttgcgca tgagacaaaa 1440 tcaccggttt tttgtgttta tgcggaatgt ttatctgccc cgctcggcaa aggcaatcaa 1500 cttgagagaa aaattctcct gccggaccac taagatgtag gggacgctga cttaccagtc 1560 acaagccggt actcagcggc ggccgcttca gagctcacaa aaacaaatac ccgagcgagc 1620 gtaaaaagca tgaacgagta cgcccccctg cgtttgcatg tgcccgagcc caccggccgg 1680 ccaggctgcc agaccgattt ttcctacctg cgcctgaacg atgcaggtca agcccgtaaa 1740 ccccctgtcg atgtcgacgc tgccgacacc gccgacctgt cctacagcct ggtccgcgtg 1800 ctcgacgagc aaggcgacgc ccaaggcccg tgggctgaag acatcgaccc gcagatcctg 1860 cgccaaggca tgcgcgccat gctcaagacg cggatcttcg acagccgcat ggtggttgcc 1920 cagcgccaga agaagatgtc cttctacatg cagagcctgg gcgaagaagc catcggcagc 1980 ggccaggcgc tggcgcttaa ccgcaccgac atgtgcttcc ccacctaccg tcagcaaagc 2040 atcctgatgg cccgcgacgt gtcgctggtg gagatgatct gccagttgct gtccaacgaa 2100 cgcgaccccc tcaagggccg ccagctgccg atcatgtact cggtacgcga ggccggcttc 2160 ttcaccatca gcggcaacct ggcgacccag ttcgtgcagg cggtcggctg ggccatggcc 2220 tcggcgatca agggcgatac caagattgcc tcggcctgga tcggcgacgg cgccactgcc 2280 gaatcggact tccacaccgc cctcaccttt gcccacgttt accgcgcccc ggtgatcctc 2340 aacgtggtca acaaccagtg ggccatctca accttccagg ccatcgccgg tggcgagtcg 2400 accaccttcg ccggccgtgg cgtgggctgc ggcatcgctt cgctgcgggt ggacggcaac 2460 gacttcgtcg ccgtttacgc cgcttcgcgc tgggctgccg aacgtgcccg ccgtggtttg 2520 ggcccgagcc tgatcgagtg ggtcacctac cgtgccggcc cgcactcgac ctcggacgac 2580 ccgtccaagt accgccctgc cgatgactgg agccacttcc cgctgggtga cccgatcgcc 2640 cgcctgaagc agcacctgat caagatcggc cactggtccg aagaagaaca ccaggccacc 2700 acggccgagt tcgaagcggc cgtgattgct gcgcaaaaag aagccgagca gtacggcacc 2760 ctggccaacg gtcacatccc gagcgccgcc tcgatgttcg aggacgtgta caaggagatg 2820 cccgaccacc tgcgccgcca acgccaggaa ctgggggttt gagatgaacg accacaacaa 2880 cagcatcaac ccggaaaccg ccatggccac cactaccatg accatgatcc aggccctgcg 2940 ctcggccatg gatgtcatgc ttgagcgcga cgacaatgtg gtggtgtacg gccaggacgt 3000 cggctacttc ggcggcgtgt tccgctgcac cgaaggcctg cagaccaagt acggcaagtc 3060 ccgcgtgttc gacgcgccca tctctgaaag cggcatcgtc ggcaccgccg tgggcatggg 3120 tgcctacggc ctgcgcccgg tggtggaaat ccagttcgct gactacttct acccggcctc 3180 cgaccagatc gtttctgaaa tggcccgcct gcgctaccgt tcggccggcg agttcatcgc 3240 cccgctgacc ctgcgtatgc cctgcggtgg cggtatctat ggcggccaga cacacagcca 3300 gagcccggaa gcgatgttca ctcaggtgtg cggcctgcgc accgtaatgc catccaaccc 3360 gtacgacgcc aaaggcctgc tgattgcctc gatcgaatgc gacgacccgg tgatcttcct 3420 ggagcccaag cgcctgtaca acggcccgtt cgacggccac catgaccgcc cggttacgcc 3480 gtggtcgaaa cacccgcaca gcgccgtgcc cgatggctac tacaccgtgc cactggacaa 3540 ggccgccatc acccgccccg gcaatgacgt gagcgtgctc acctatggca ccaccgtgta 3600 cgtggcccag gtggccgccg aagaaagtgg cgtggatgcc gaagtgatcg acctgcgcag 3660 cctgtggccg ctagacctgg acaccatcgt cgagtcggtg aaaaagaccg gccgttgcgt 3720 ggtagtacac gaggccaccc gtacttgtgg ctttggcgca gaactggtgt cgctggtgca 3780 ggagcactgc ttccaccacc tggaggcgcc gatcgagcgc gtcaccggtt gggacacccc 3840 ctaccctcac gcgcaggaat gggcttactt cccagggcct tcgcgggtag gtgcggcatt 3900 gaaaaaggtc atggaggtct gaatgggcac gcacgtcatc aagatgccgg acattggcga 3960 aggcatcgcg caggtcgaat tggtggaatg gttcgtcaag gtgggcgaca tcatcgccga 4020 ggaccaagtg gtagccgacg tcatgaccga caaggccacc gtggaaatcc cgtcgccggt 4080 cagcggcaag gtgctggccc tgggtggcca gccaggtgaa gtgatggcgg tcggcagtga 4140 gctgatccgc atcgaagtgg aaggcagcgg caaccatgtg gatgtgccgc aagccaagcc 4200 ggccgaagtg cctgcggcac cggtagccgc taaacctgaa ccacagaaag acgttaaacc 4260 ggcggcgtac caggcgtcag ccagccacga ggcagcgccc atcgtgccgc gccagccggg 4320 cgacaagccg ctggcctcgc cggcggtgcg caaacgcgcc ctcgatgccg gcatcgaatt 4380 gcgttatgtg cacggcagcg gcccggccgg gcgcatcctg cacgaagacc tcgacgcgtt 4440 catgagcaaa ccgcaaagcg ctgccgggca aacccccaat ggctatgcca ggcgcaccga 4500 cagcgagcag gtgccggtga tcggcctgcg ccgcaagatc gcccagcgca tgcaggacgc 4560 caagcgccgg gtcgcgcact tcagctatgt ggaagaaatc gacgtcaccg ccctggaagc 4620 cctgcgccag cagctcaaca gcaagcacgg cgacagccgc ggcaagctga cactgctgcc 4680 gttcctggtg cgcgccctgg tcgtggcact gcgtgacttc ccgcagataa acgccaccta 4740 cgatgacgaa gcgcagatca tcacccgcca tggcgcggtg catgtgggca tcgccaccca 4800 aggtgacaac ggcctgatgg tacccgtgct gcgccacgcc gaagcgggca gcctgtgggc 4860 caatgccggt gagatttcac gcctggccaa cgctgcgcgc aacaacaagg ccagccgcga 4920 agagctgtcc ggttcgacca ttaccctgac cagcctcggc gccctgggcg gcatcgtcag 4980 cacgccggtg gtcaacaccc cggaagtggc gatcgtcggt gtcaaccgca tggttgagcg 5040 gcccgtggtg atcgacggcc agatcgtcgt gcgcaagatg atgaacctgt ccagctcgtt 5100 cgaccaccgc gtggtcgatg gcatggacgc cgccctgttc atccaggccg tgcgtggcct 5160 gctcgaacaa cccgcctgcc tgttcgtgga gtgagcatgc aacagactat ccagacaacc 5220 ctgttgatca tcggcggcgg ccctggcggc tatgtggcgg ccatccgcgc cgggcaactg 5280 ggcatcccta ccgtgctggt ggaaggccag gcgctgggcg gtacctgcct gaacatcggc 5340 tgcattccgt ccaaggcgct gatccatgtg gccgagcagt tccaccaggc ctcgcgcttt 5400 accgaaccct cgccgctggg catcagcgtg gcttcgccac gcctggacat cggccagagc 5460 gtggcctgga aagacggcat cgtcgatcgc ctgaccactg gtgtcgccgc cctgctgaaa 5520 aagcacgggg tgaaggtggt gcacggctgg gccaaggtgc ttgatggcaa gcaggtcgag 5580 gtggatggcc agcgcatcca gtgcgagcac ctgttgctgg ccacgggctc cagcagtgtc 5640 gaactgccga tgctgccgtt gggtgggccg gtgatttcct cgaccgaggc cctggcaccg 5700 aaagccctgc cgcaacacct ggtggtggtg ggcggtggct acatcggcct ggagctgggt 5760 atcgcctacc gcaagctcgg cgcgcaggtc agcgtggtgg aagcgcgcga gcgcatcctg 5820 ccgacttacg acagcgaact gaccgccccg gtggccgagt cgctgaaaaa gctgggtatc 5880 gccctgcacc ttggccacag cgtcgaaggt tacgaaaatg gctgcctgct ggccaacgat 5940 ggcaagggcg gacaactgcg cctggaagcc gaccgggtgc tggtggccgt gggccgccgc 6000 ccacgcacca agggcttcaa cctggaatgc ctggacctga agatgaatgg tgccgcgatt 6060 gccatcgacg agcgctgcca gaccagcatg cacaacgtct gggccatcgg cgacgtggcc 6120 ggcgaaccga tgctggcgca ccgggccatg gcccagggcg agatggtggc cgagatcatc 6180 gccggcaagg cacgccgctt cgaacccgct gcgatagccg ccgtgtgctt caccgacccg 6240 gaagtggtcg tggtcggcaa gacgccggaa caggccagtc agcaaggcct ggactgcatc 6300 gtcgcgcagt tcccgttcgc cgccaacggc cgggccatga gcctggagtc gaaaagcggt 6360 ttcgtgcgcg tggtcgcgcg gcgtgacaac cacctgatcc tgggctggca agcggttggc 6420 gtggcggttt ccgagctgtc cacggcgttt gcccagtcgc tggagatggg tgcctgcctg 6480 gaggatgtgg ccggtaccat ccatgcccac ccgaccctgg gtgaagcggt acaggaagcg 6540 gcactgcgtg ccctgggcca cgccctgcat atctgacact gaagcggccg aggccgattt 6600 ggcccgccgc gccgagaggc gctgcgggtc ttttttatac ctg 6643 <210> 57 <211> 423 <212> PRT <213> Pseudomonas putida <400> 57 Met Gly Thr His Val Ile Lys Met Pro Asp Ile Gly Glu Gly Ile Ala 1 5 10 15 Gln Val Glu Leu Val Glu Trp Phe Val Lys Val Gly Asp Ile Ile Ala             20 25 30 Glu Asp Gln Val Val Ala Asp Val Met Thr Asp Lys Ala Thr Val Glu         35 40 45 Ile Pro Ser Ser Val Gly Lys Val Leu Ala Leu Gly Gly Gln Pro     50 55 60 Gly Glu Val Met Ala Val Gly Ser Glu Leu Ile Arg Ile Glu Val Glu 65 70 75 80 Gly Ser Gly Asn His Val Asp Val Pro Gln Ala Lys Pro Ala Glu Val                 85 90 95 Pro Ala Ala Pro Val Ala Lys Pro Glu Pro Gln Lys Asp Val Lys             100 105 110 Pro Ala Ala Tyr Gln Ala Ser Ala Ser His Glu Ala Ala Pro Ile Val         115 120 125 Pro Arg Gln Pro Gly Asp Lys Pro Leu Ala Ser Pro Ala Val Arg Lys     130 135 140 Arg Ala Leu Asp Ala Gly Ile Glu Leu Arg Tyr Val His Gly Ser Gly 145 150 155 160 Pro Ala Gly Arg Ile Leu His Glu Asp Leu Asp Ala Phe Met Ser Lys                 165 170 175 Pro Gln Ser Ala Ala Gly Gln Thr Pro Asn Gly Tyr Ala Arg Arg Thr             180 185 190 Asp Ser Glu Gln Val Pro Val Ile Gly Leu Arg Arg Lys Ile Ala Gln         195 200 205 Arg Met Gln Asp Ala Lys Arg Arg Val Ala His Phe Ser Tyr Val Glu     210 215 220 Glu Ile Asp Val Thr Ala Leu Glu Ala Leu Arg Gln Gln Leu Asn Ser 225 230 235 240 Lys His Gly Asp Ser Arg Gly Lys Leu Thr Leu Leu Pro Phe Leu Val                 245 250 255 Arg Ala Leu Val Val Ala Leu Arg Asp Phe Pro Gln Ile Asn Ala Thr             260 265 270 Tyr Asp Asp Glu Ala Gln Ile Ile Thr Arg His Gly Ala Val His Val         275 280 285 Gly Ile Ala Thr Gln Gly Asp Asn Gly Leu Met Val Val Val Leu Arg     290 295 300 His Ala Glu Ala Gly Ser Leu Trp Ala Asn Ala Gly Glu Ile Ser Arg 305 310 315 320 Leu Ala Asn Ala Ala Arg Asn Asn Lys Ala Ser Arg Glu Glu Leu Ser                 325 330 335 Gly Ser Thr Ile Thr Leu Thr Ser Leu Gly Ala Leu Gly Gly Ile Val             340 345 350 Ser Thr Pro Val Val Asn Thr Pro Glu Val Ala Val Val Gly Val Asn         355 360 365 Arg Met Val Glu Arg Pro Val Val Ile Asp Gly Gln Ile Val Val Arg     370 375 380 Lys Met Met Asn Leu Ser Ser Ser Phe Asp His Arg Val Val Asp Gly 385 390 395 400 Met Asp Ala Ala Leu Phe Ile Gln Ala Val Arg Gly Leu Leu Glu Gln                 405 410 415 Pro Ala Cys Leu Phe Val Glu             420 <210> 58 <211> 6643 <212> DNA <213> Pseudomonas putida <400> 58 gcatgcctgc aggccgccga tgaaatggtg gaaggtatcg gtaggctggc cctgctcatc 60 gctgaacacg ttacgcccgc tgccggtatc gaccaggctc tggtgaatat gcatggaact 120 gccaggcgtg cgcgccagcg gtttggccat gcacaccacg gtcagcccgt gcttgagtgc 180 cacttccttg agcaggtgtt tgaacaggaa ggtctggtcg gccagcagca gcgggtcgcc 240 atgtagcaag ttgatctcga actggctgac gcccatttcg tgcatgaagg tgtcgcgcgg 300 caggccgagc gcggccatgc actggtacac ctcattgaag aacgggcgca ggccgttgtt 360 ggaactgaca ctgaacgccg aatggcccag ctcgcggcgg ccgtcggtgc ccagcggtgg 420 ctggaacggc tgctgcgggt cactgttggg ggcaaacacg aagaactcaa gctcggtcgc 480 cactaccggt gccagaccca acgctgcgta gcgggcgatc acggccttca gctggccccg 540 ggtggacagt gccgagggcc ggccatccag ttcattggca tcgcagatgg ccagggcgcg 600 accgtcatcg ctccagggca agcgatgaac ctggctgggt tccgctacca acgccaggtc 660 gccgtcgtcg cagccgtaga atttcgccgg cgggtagccg cccatgatgc attgcagcag 720 caccccacgg gccatctgca ggcggcggcc ttcgagaaag ccttcggcgg tcatcacctt 780 gccgcgtggg acgccgttga ggtcgggggt gacgcattcg atttcatcga tgccctggag 840 ctgagcgatg ctcatgacgc ttgtccttgt tgttgtaggc tgacaacaac ataggctggg 900 ggtgtttaaa atatcaagca gcctctcgaa cgcctggggc ctcttctatt cgcgcaaggt 960 catgccattg gccggcaacg gcaaggctgt cttgtagcgc acctgtttca aggcaaaact 1020 cgagcggata ttcgccacac ccggcaaccg ggtcaggtaa tcgagaaacc gctccagcgc 1080 ctggatactc ggcagcagta cccgcaacag gtagtccggg tcgcccgtca tcaggtagca 1140 ctccatcacc tcgggccgtt cggcaatttc ttcctcgaag cggtgcagcg actgctctac 1200 ctgtttttcc aggctgacat ggatgaacac attcacatcc agccccaacg cctcgggcga 1260 caacaaggtc acctgctggc ggatcacccc cagttcttcc atggcccgca cccggttgaa 1320 acagggcgtg ggcgacaggt tgaccgagcg tgccagctcg gcgttggtga tgcgggcgtt 1380 ttcctgcagg ctgttgagaa tgccgatatc ggtacgatcg agtttgcgca tgagacaaaa 1440 tcaccggttt tttgtgttta tgcggaatgt ttatctgccc cgctcggcaa aggcaatcaa 1500 cttgagagaa aaattctcct gccggaccac taagatgtag gggacgctga cttaccagtc 1560 acaagccggt actcagcggc ggccgcttca gagctcacaa aaacaaatac ccgagcgagc 1620 gtaaaaagca tgaacgagta cgcccccctg cgtttgcatg tgcccgagcc caccggccgg 1680 ccaggctgcc agaccgattt ttcctacctg cgcctgaacg atgcaggtca agcccgtaaa 1740 ccccctgtcg atgtcgacgc tgccgacacc gccgacctgt cctacagcct ggtccgcgtg 1800 ctcgacgagc aaggcgacgc ccaaggcccg tgggctgaag acatcgaccc gcagatcctg 1860 cgccaaggca tgcgcgccat gctcaagacg cggatcttcg acagccgcat ggtggttgcc 1920 cagcgccaga agaagatgtc cttctacatg cagagcctgg gcgaagaagc catcggcagc 1980 ggccaggcgc tggcgcttaa ccgcaccgac atgtgcttcc ccacctaccg tcagcaaagc 2040 atcctgatgg cccgcgacgt gtcgctggtg gagatgatct gccagttgct gtccaacgaa 2100 cgcgaccccc tcaagggccg ccagctgccg atcatgtact cggtacgcga ggccggcttc 2160 ttcaccatca gcggcaacct ggcgacccag ttcgtgcagg cggtcggctg ggccatggcc 2220 tcggcgatca agggcgatac caagattgcc tcggcctgga tcggcgacgg cgccactgcc 2280 gaatcggact tccacaccgc cctcaccttt gcccacgttt accgcgcccc ggtgatcctc 2340 aacgtggtca acaaccagtg ggccatctca accttccagg ccatcgccgg tggcgagtcg 2400 accaccttcg ccggccgtgg cgtgggctgc ggcatcgctt cgctgcgggt ggacggcaac 2460 gacttcgtcg ccgtttacgc cgcttcgcgc tgggctgccg aacgtgcccg ccgtggtttg 2520 ggcccgagcc tgatcgagtg ggtcacctac cgtgccggcc cgcactcgac ctcggacgac 2580 ccgtccaagt accgccctgc cgatgactgg agccacttcc cgctgggtga cccgatcgcc 2640 cgcctgaagc agcacctgat caagatcggc cactggtccg aagaagaaca ccaggccacc 2700 acggccgagt tcgaagcggc cgtgattgct gcgcaaaaag aagccgagca gtacggcacc 2760 ctggccaacg gtcacatccc gagcgccgcc tcgatgttcg aggacgtgta caaggagatg 2820 cccgaccacc tgcgccgcca acgccaggaa ctgggggttt gagatgaacg accacaacaa 2880 cagcatcaac ccggaaaccg ccatggccac cactaccatg accatgatcc aggccctgcg 2940 ctcggccatg gatgtcatgc ttgagcgcga cgacaatgtg gtggtgtacg gccaggacgt 3000 cggctacttc ggcggcgtgt tccgctgcac cgaaggcctg cagaccaagt acggcaagtc 3060 ccgcgtgttc gacgcgccca tctctgaaag cggcatcgtc ggcaccgccg tgggcatggg 3120 tgcctacggc ctgcgcccgg tggtggaaat ccagttcgct gactacttct acccggcctc 3180 cgaccagatc gtttctgaaa tggcccgcct gcgctaccgt tcggccggcg agttcatcgc 3240 cccgctgacc ctgcgtatgc cctgcggtgg cggtatctat ggcggccaga cacacagcca 3300 gagcccggaa gcgatgttca ctcaggtgtg cggcctgcgc accgtaatgc catccaaccc 3360 gtacgacgcc aaaggcctgc tgattgcctc gatcgaatgc gacgacccgg tgatcttcct 3420 ggagcccaag cgcctgtaca acggcccgtt cgacggccac catgaccgcc cggttacgcc 3480 gtggtcgaaa cacccgcaca gcgccgtgcc cgatggctac tacaccgtgc cactggacaa 3540 ggccgccatc acccgccccg gcaatgacgt gagcgtgctc acctatggca ccaccgtgta 3600 cgtggcccag gtggccgccg aagaaagtgg cgtggatgcc gaagtgatcg acctgcgcag 3660 cctgtggccg ctagacctgg acaccatcgt cgagtcggtg aaaaagaccg gccgttgcgt 3720 ggtagtacac gaggccaccc gtacttgtgg ctttggcgca gaactggtgt cgctggtgca 3780 ggagcactgc ttccaccacc tggaggcgcc gatcgagcgc gtcaccggtt gggacacccc 3840 ctaccctcac gcgcaggaat gggcttactt cccagggcct tcgcgggtag gtgcggcatt 3900 gaaaaaggtc atggaggtct gaatgggcac gcacgtcatc aagatgccgg acattggcga 3960 aggcatcgcg caggtcgaat tggtggaatg gttcgtcaag gtgggcgaca tcatcgccga 4020 ggaccaagtg gtagccgacg tcatgaccga caaggccacc gtggaaatcc cgtcgccggt 4080 cagcggcaag gtgctggccc tgggtggcca gccaggtgaa gtgatggcgg tcggcagtga 4140 gctgatccgc atcgaagtgg aaggcagcgg caaccatgtg gatgtgccgc aagccaagcc 4200 ggccgaagtg cctgcggcac cggtagccgc taaacctgaa ccacagaaag acgttaaacc 4260 ggcggcgtac caggcgtcag ccagccacga ggcagcgccc atcgtgccgc gccagccggg 4320 cgacaagccg ctggcctcgc cggcggtgcg caaacgcgcc ctcgatgccg gcatcgaatt 4380 gcgttatgtg cacggcagcg gcccggccgg gcgcatcctg cacgaagacc tcgacgcgtt 4440 catgagcaaa ccgcaaagcg ctgccgggca aacccccaat ggctatgcca ggcgcaccga 4500 cagcgagcag gtgccggtga tcggcctgcg ccgcaagatc gcccagcgca tgcaggacgc 4560 caagcgccgg gtcgcgcact tcagctatgt ggaagaaatc gacgtcaccg ccctggaagc 4620 cctgcgccag cagctcaaca gcaagcacgg cgacagccgc ggcaagctga cactgctgcc 4680 gttcctggtg cgcgccctgg tcgtggcact gcgtgacttc ccgcagataa acgccaccta 4740 cgatgacgaa gcgcagatca tcacccgcca tggcgcggtg catgtgggca tcgccaccca 4800 aggtgacaac ggcctgatgg tacccgtgct gcgccacgcc gaagcgggca gcctgtgggc 4860 caatgccggt gagatttcac gcctggccaa cgctgcgcgc aacaacaagg ccagccgcga 4920 agagctgtcc ggttcgacca ttaccctgac cagcctcggc gccctgggcg gcatcgtcag 4980 cacgccggtg gtcaacaccc cggaagtggc gatcgtcggt gtcaaccgca tggttgagcg 5040 gcccgtggtg atcgacggcc agatcgtcgt gcgcaagatg atgaacctgt ccagctcgtt 5100 cgaccaccgc gtggtcgatg gcatggacgc cgccctgttc atccaggccg tgcgtggcct 5160 gctcgaacaa cccgcctgcc tgttcgtgga gtgagcatgc aacagactat ccagacaacc 5220 ctgttgatca tcggcggcgg ccctggcggc tatgtggcgg ccatccgcgc cgggcaactg 5280 ggcatcccta ccgtgctggt ggaaggccag gcgctgggcg gtacctgcct gaacatcggc 5340 tgcattccgt ccaaggcgct gatccatgtg gccgagcagt tccaccaggc ctcgcgcttt 5400 accgaaccct cgccgctggg catcagcgtg gcttcgccac gcctggacat cggccagagc 5460 gtggcctgga aagacggcat cgtcgatcgc ctgaccactg gtgtcgccgc cctgctgaaa 5520 aagcacgggg tgaaggtggt gcacggctgg gccaaggtgc ttgatggcaa gcaggtcgag 5580 gtggatggcc agcgcatcca gtgcgagcac ctgttgctgg ccacgggctc cagcagtgtc 5640 gaactgccga tgctgccgtt gggtgggccg gtgatttcct cgaccgaggc cctggcaccg 5700 aaagccctgc cgcaacacct ggtggtggtg ggcggtggct acatcggcct ggagctgggt 5760 atcgcctacc gcaagctcgg cgcgcaggtc agcgtggtgg aagcgcgcga gcgcatcctg 5820 ccgacttacg acagcgaact gaccgccccg gtggccgagt cgctgaaaaa gctgggtatc 5880 gccctgcacc ttggccacag cgtcgaaggt tacgaaaatg gctgcctgct ggccaacgat 5940 ggcaagggcg gacaactgcg cctggaagcc gaccgggtgc tggtggccgt gggccgccgc 6000 ccacgcacca agggcttcaa cctggaatgc ctggacctga agatgaatgg tgccgcgatt 6060 gccatcgacg agcgctgcca gaccagcatg cacaacgtct gggccatcgg cgacgtggcc 6120 ggcgaaccga tgctggcgca ccgggccatg gcccagggcg agatggtggc cgagatcatc 6180 gccggcaagg cacgccgctt cgaacccgct gcgatagccg ccgtgtgctt caccgacccg 6240 gaagtggtcg tggtcggcaa gacgccggaa caggccagtc agcaaggcct ggactgcatc 6300 gtcgcgcagt tcccgttcgc cgccaacggc cgggccatga gcctggagtc gaaaagcggt 6360 ttcgtgcgcg tggtcgcgcg gcgtgacaac cacctgatcc tgggctggca agcggttggc 6420 gtggcggttt ccgagctgtc cacggcgttt gcccagtcgc tggagatggg tgcctgcctg 6480 gaggatgtgg ccggtaccat ccatgcccac ccgaccctgg gtgaagcggt acaggaagcg 6540 gcactgcgtg ccctgggcca cgccctgcat atctgacact gaagcggccg aggccgattt 6600 ggcccgccgc gccgagaggc gctgcgggtc ttttttatac ctg 6643 <210> 59 <211> 459 <212> PRT <213> Pseudomonas putida <400> 59 Met Gln Gln Thr Ile Gln Thr Thr Leu Leu Ile Ile Gly Gly Gly Pro 1 5 10 15 Gly Gly Tyr Val Ala Ala Ile Arg Ala Gly Gln Leu Gly Ile Pro Thr             20 25 30 Val Leu Val Glu Gly Gln Ala Leu Gly Gly Thr Cys Leu Asn Ile Gly         35 40 45 Cys Ile Pro Ser Lys Ala Leu Ile His Val Ala Glu Gln Phe His Gln     50 55 60 Ala Ser Arg Phe Thr Glu Pro Ser Pro Leu Gly Ile Ser Val Ala Ser 65 70 75 80 Pro Arg Leu Asp Ile Gly Gln Ser Val Ala Trp Lys Asp Gly Ile Val                 85 90 95 Asp Arg Leu Thr Thr Gly Val Ala Leu Leu Lys Lys His Gly Val             100 105 110 Lys Val Val His Gly Trp Ala Lys Val Leu Asp Gly Lys Gln Val Glu         115 120 125 Val Asp Gly Gln Arg Ile Gln Cys Glu His Leu Leu Leu Ala Thr Gly     130 135 140 Ser Ser Ser Val Glu Leu Pro Met Leu Pro Leu Gly Gly Pro Val Ile 145 150 155 160 Ser Ser Thr Glu Ala Leu Ala Pro Lys Ala Leu Pro Gln His Leu Val                 165 170 175 Val Val Gly Gly Gly Tyr Ile Gly Leu Glu Leu Gly Ile Ala Tyr Arg             180 185 190 Lys Leu Gly Ala Gln Val Ser Val Val Glu Ala Arg Glu Arg Ile Leu         195 200 205 Pro Thr Tyr Asp Ser Glu Leu Thr Ala Pro Val Ala Glu Ser Leu Lys     210 215 220 Lys Leu Gly Ile Ala Leu His Leu Gly His Ser Val Glu Gly Tyr Glu 225 230 235 240 Asn Gly Cys Leu Leu Ala Asn Asp Gly Lys Gly Gly Gln Leu Arg Leu                 245 250 255 Glu Ala Asp Arg Val Leu Val Ala Val Gly Arg Arg Pro Arg Thr Lys             260 265 270 Gly Phe Asn Leu Glu Cys Leu Asp Leu Lys Met Asn Gly Ala Ala Ile         275 280 285 Ala Ile Asp Glu Arg Cys Gln Thr Ser Met His Asn Val Trp Ala Ile     290 295 300 Gly Asp Val Ala Gly Glu Pro Met Leu Ala His Arg Ala Met Ala Gln 305 310 315 320 Gly Glu Met Val Ala Glu Ile Ile Ala Gly Lys Ala Arg Arg Phe Glu                 325 330 335 Pro Ala Ala Ile Ala Ala Val Cys Phe Thr Asp Pro Glu Val Val Val             340 345 350 Val Gly Lys Thr Pro Glu Gln Ala Ser Gln Gln Gly Leu Asp Cys Ile         355 360 365 Val Ala Gln Phe Pro Phe Ala Ala Asn Gly Arg Ala Met Ser Leu Glu     370 375 380 Ser Lys Ser Gly Phe Val Arg Val Val Ala Arg Arg Asp Asn His Leu 385 390 395 400 Ile Leu Gly Trp Gln Ala Val Gly Val Ala Val Ser Glu Leu Ser Thr                 405 410 415 Ala Phe Ala Gln Ser Leu Glu Met Gly Ala Cys Leu Glu Asp Val Ala             420 425 430 Gly Thr Ile His Ala His Pro Thr Leu Gly Glu Ala Val Gln Glu Ala         435 440 445 Ala Leu Arg Ala Leu Gly His Ala Leu His Ile     450 455 <210> 60 <211> 6643 <212> DNA <213> Pseudomonas putida <400> 60 gcatgcctgc aggccgccga tgaaatggtg gaaggtatcg gtaggctggc cctgctcatc 60 gctgaacacg ttacgcccgc tgccggtatc gaccaggctc tggtgaatat gcatggaact 120 gccaggcgtg cgcgccagcg gtttggccat gcacaccacg gtcagcccgt gcttgagtgc 180 cacttccttg agcaggtgtt tgaacaggaa ggtctggtcg gccagcagca gcgggtcgcc 240 atgtagcaag ttgatctcga actggctgac gcccatttcg tgcatgaagg tgtcgcgcgg 300 caggccgagc gcggccatgc actggtacac ctcattgaag aacgggcgca ggccgttgtt 360 ggaactgaca ctgaacgccg aatggcccag ctcgcggcgg ccgtcggtgc ccagcggtgg 420 ctggaacggc tgctgcgggt cactgttggg ggcaaacacg aagaactcaa gctcggtcgc 480 cactaccggt gccagaccca acgctgcgta gcgggcgatc acggccttca gctggccccg 540 ggtggacagt gccgagggcc ggccatccag ttcattggca tcgcagatgg ccagggcgcg 600 accgtcatcg ctccagggca agcgatgaac ctggctgggt tccgctacca acgccaggtc 660 gccgtcgtcg cagccgtaga atttcgccgg cgggtagccg cccatgatgc attgcagcag 720 caccccacgg gccatctgca ggcggcggcc ttcgagaaag ccttcggcgg tcatcacctt 780 gccgcgtggg acgccgttga ggtcgggggt gacgcattcg atttcatcga tgccctggag 840 ctgagcgatg ctcatgacgc ttgtccttgt tgttgtaggc tgacaacaac ataggctggg 900 ggtgtttaaa atatcaagca gcctctcgaa cgcctggggc ctcttctatt cgcgcaaggt 960 catgccattg gccggcaacg gcaaggctgt cttgtagcgc acctgtttca aggcaaaact 1020 cgagcggata ttcgccacac ccggcaaccg ggtcaggtaa tcgagaaacc gctccagcgc 1080 ctggatactc ggcagcagta cccgcaacag gtagtccggg tcgcccgtca tcaggtagca 1140 ctccatcacc tcgggccgtt cggcaatttc ttcctcgaag cggtgcagcg actgctctac 1200 ctgtttttcc aggctgacat ggatgaacac attcacatcc agccccaacg cctcgggcga 1260 caacaaggtc acctgctggc ggatcacccc cagttcttcc atggcccgca cccggttgaa 1320 acagggcgtg ggcgacaggt tgaccgagcg tgccagctcg gcgttggtga tgcgggcgtt 1380 ttcctgcagg ctgttgagaa tgccgatatc ggtacgatcg agtttgcgca tgagacaaaa 1440 tcaccggttt tttgtgttta tgcggaatgt ttatctgccc cgctcggcaa aggcaatcaa 1500 cttgagagaa aaattctcct gccggaccac taagatgtag gggacgctga cttaccagtc 1560 acaagccggt actcagcggc ggccgcttca gagctcacaa aaacaaatac ccgagcgagc 1620 gtaaaaagca tgaacgagta cgcccccctg cgtttgcatg tgcccgagcc caccggccgg 1680 ccaggctgcc agaccgattt ttcctacctg cgcctgaacg atgcaggtca agcccgtaaa 1740 ccccctgtcg atgtcgacgc tgccgacacc gccgacctgt cctacagcct ggtccgcgtg 1800 ctcgacgagc aaggcgacgc ccaaggcccg tgggctgaag acatcgaccc gcagatcctg 1860 cgccaaggca tgcgcgccat gctcaagacg cggatcttcg acagccgcat ggtggttgcc 1920 cagcgccaga agaagatgtc cttctacatg cagagcctgg gcgaagaagc catcggcagc 1980 ggccaggcgc tggcgcttaa ccgcaccgac atgtgcttcc ccacctaccg tcagcaaagc 2040 atcctgatgg cccgcgacgt gtcgctggtg gagatgatct gccagttgct gtccaacgaa 2100 cgcgaccccc tcaagggccg ccagctgccg atcatgtact cggtacgcga ggccggcttc 2160 ttcaccatca gcggcaacct ggcgacccag ttcgtgcagg cggtcggctg ggccatggcc 2220 tcggcgatca agggcgatac caagattgcc tcggcctgga tcggcgacgg cgccactgcc 2280 gaatcggact tccacaccgc cctcaccttt gcccacgttt accgcgcccc ggtgatcctc 2340 aacgtggtca acaaccagtg ggccatctca accttccagg ccatcgccgg tggcgagtcg 2400 accaccttcg ccggccgtgg cgtgggctgc ggcatcgctt cgctgcgggt ggacggcaac 2460 gacttcgtcg ccgtttacgc cgcttcgcgc tgggctgccg aacgtgcccg ccgtggtttg 2520 ggcccgagcc tgatcgagtg ggtcacctac cgtgccggcc cgcactcgac ctcggacgac 2580 ccgtccaagt accgccctgc cgatgactgg agccacttcc cgctgggtga cccgatcgcc 2640 cgcctgaagc agcacctgat caagatcggc cactggtccg aagaagaaca ccaggccacc 2700 acggccgagt tcgaagcggc cgtgattgct gcgcaaaaag aagccgagca gtacggcacc 2760 ctggccaacg gtcacatccc gagcgccgcc tcgatgttcg aggacgtgta caaggagatg 2820 cccgaccacc tgcgccgcca acgccaggaa ctgggggttt gagatgaacg accacaacaa 2880 cagcatcaac ccggaaaccg ccatggccac cactaccatg accatgatcc aggccctgcg 2940 ctcggccatg gatgtcatgc ttgagcgcga cgacaatgtg gtggtgtacg gccaggacgt 3000 cggctacttc ggcggcgtgt tccgctgcac cgaaggcctg cagaccaagt acggcaagtc 3060 ccgcgtgttc gacgcgccca tctctgaaag cggcatcgtc ggcaccgccg tgggcatggg 3120 tgcctacggc ctgcgcccgg tggtggaaat ccagttcgct gactacttct acccggcctc 3180 cgaccagatc gtttctgaaa tggcccgcct gcgctaccgt tcggccggcg agttcatcgc 3240 cccgctgacc ctgcgtatgc cctgcggtgg cggtatctat ggcggccaga cacacagcca 3300 gagcccggaa gcgatgttca ctcaggtgtg cggcctgcgc accgtaatgc catccaaccc 3360 gtacgacgcc aaaggcctgc tgattgcctc gatcgaatgc gacgacccgg tgatcttcct 3420 ggagcccaag cgcctgtaca acggcccgtt cgacggccac catgaccgcc cggttacgcc 3480 gtggtcgaaa cacccgcaca gcgccgtgcc cgatggctac tacaccgtgc cactggacaa 3540 ggccgccatc acccgccccg gcaatgacgt gagcgtgctc acctatggca ccaccgtgta 3600 cgtggcccag gtggccgccg aagaaagtgg cgtggatgcc gaagtgatcg acctgcgcag 3660 cctgtggccg ctagacctgg acaccatcgt cgagtcggtg aaaaagaccg gccgttgcgt 3720 ggtagtacac gaggccaccc gtacttgtgg ctttggcgca gaactggtgt cgctggtgca 3780 ggagcactgc ttccaccacc tggaggcgcc gatcgagcgc gtcaccggtt gggacacccc 3840 ctaccctcac gcgcaggaat gggcttactt cccagggcct tcgcgggtag gtgcggcatt 3900 gaaaaaggtc atggaggtct gaatgggcac gcacgtcatc aagatgccgg acattggcga 3960 aggcatcgcg caggtcgaat tggtggaatg gttcgtcaag gtgggcgaca tcatcgccga 4020 ggaccaagtg gtagccgacg tcatgaccga caaggccacc gtggaaatcc cgtcgccggt 4080 cagcggcaag gtgctggccc tgggtggcca gccaggtgaa gtgatggcgg tcggcagtga 4140 gctgatccgc atcgaagtgg aaggcagcgg caaccatgtg gatgtgccgc aagccaagcc 4200 ggccgaagtg cctgcggcac cggtagccgc taaacctgaa ccacagaaag acgttaaacc 4260 ggcggcgtac caggcgtcag ccagccacga ggcagcgccc atcgtgccgc gccagccggg 4320 cgacaagccg ctggcctcgc cggcggtgcg caaacgcgcc ctcgatgccg gcatcgaatt 4380 gcgttatgtg cacggcagcg gcccggccgg gcgcatcctg cacgaagacc tcgacgcgtt 4440 catgagcaaa ccgcaaagcg ctgccgggca aacccccaat ggctatgcca ggcgcaccga 4500 cagcgagcag gtgccggtga tcggcctgcg ccgcaagatc gcccagcgca tgcaggacgc 4560 caagcgccgg gtcgcgcact tcagctatgt ggaagaaatc gacgtcaccg ccctggaagc 4620 cctgcgccag cagctcaaca gcaagcacgg cgacagccgc ggcaagctga cactgctgcc 4680 gttcctggtg cgcgccctgg tcgtggcact gcgtgacttc ccgcagataa acgccaccta 4740 cgatgacgaa gcgcagatca tcacccgcca tggcgcggtg catgtgggca tcgccaccca 4800 aggtgacaac ggcctgatgg tacccgtgct gcgccacgcc gaagcgggca gcctgtgggc 4860 caatgccggt gagatttcac gcctggccaa cgctgcgcgc aacaacaagg ccagccgcga 4920 agagctgtcc ggttcgacca ttaccctgac cagcctcggc gccctgggcg gcatcgtcag 4980 cacgccggtg gtcaacaccc cggaagtggc gatcgtcggt gtcaaccgca tggttgagcg 5040 gcccgtggtg atcgacggcc agatcgtcgt gcgcaagatg atgaacctgt ccagctcgtt 5100 cgaccaccgc gtggtcgatg gcatggacgc cgccctgttc atccaggccg tgcgtggcct 5160 gctcgaacaa cccgcctgcc tgttcgtgga gtgagcatgc aacagactat ccagacaacc 5220 ctgttgatca tcggcggcgg ccctggcggc tatgtggcgg ccatccgcgc cgggcaactg 5280 ggcatcccta ccgtgctggt ggaaggccag gcgctgggcg gtacctgcct gaacatcggc 5340 tgcattccgt ccaaggcgct gatccatgtg gccgagcagt tccaccaggc ctcgcgcttt 5400 accgaaccct cgccgctggg catcagcgtg gcttcgccac gcctggacat cggccagagc 5460 gtggcctgga aagacggcat cgtcgatcgc ctgaccactg gtgtcgccgc cctgctgaaa 5520 aagcacgggg tgaaggtggt gcacggctgg gccaaggtgc ttgatggcaa gcaggtcgag 5580 gtggatggcc agcgcatcca gtgcgagcac ctgttgctgg ccacgggctc cagcagtgtc 5640 gaactgccga tgctgccgtt gggtgggccg gtgatttcct cgaccgaggc cctggcaccg 5700 aaagccctgc cgcaacacct ggtggtggtg ggcggtggct acatcggcct ggagctgggt 5760 atcgcctacc gcaagctcgg cgcgcaggtc agcgtggtgg aagcgcgcga gcgcatcctg 5820 ccgacttacg acagcgaact gaccgccccg gtggccgagt cgctgaaaaa gctgggtatc 5880 gccctgcacc ttggccacag cgtcgaaggt tacgaaaatg gctgcctgct ggccaacgat 5940 ggcaagggcg gacaactgcg cctggaagcc gaccgggtgc tggtggccgt gggccgccgc 6000 ccacgcacca agggcttcaa cctggaatgc ctggacctga agatgaatgg tgccgcgatt 6060 gccatcgacg agcgctgcca gaccagcatg cacaacgtct gggccatcgg cgacgtggcc 6120 ggcgaaccga tgctggcgca ccgggccatg gcccagggcg agatggtggc cgagatcatc 6180 gccggcaagg cacgccgctt cgaacccgct gcgatagccg ccgtgtgctt caccgacccg 6240 gaagtggtcg tggtcggcaa gacgccggaa caggccagtc agcaaggcct ggactgcatc 6300 gtcgcgcagt tcccgttcgc cgccaacggc cgggccatga gcctggagtc gaaaagcggt 6360 ttcgtgcgcg tggtcgcgcg gcgtgacaac cacctgatcc tgggctggca agcggttggc 6420 gtggcggttt ccgagctgtc cacggcgttt gcccagtcgc tggagatggg tgcctgcctg 6480 gaggatgtgg ccggtaccat ccatgcccac ccgaccctgg gtgaagcggt acaggaagcg 6540 gcactgcgtg ccctgggcca cgccctgcat atctgacact gaagcggccg aggccgattt 6600 ggcccgccgc gccgagaggc gctgcgggtc ttttttatac ctg 6643 <210> 61 <211> 468 <212> PRT <213> Clostridium beijerinckii <400> 61 Met Asn Lys Asp Thr Leu Ile Pro Thr Thr Lys Asp Leu Lys Leu Lys 1 5 10 15 Thr Asn Val Glu Asn Ile Asn Leu Lys Asn Tyr Lys Asp Asn Ser Ser             20 25 30 Cys Phe Gly Val Phe Glu Asn Val Glu Asn Ala Ile Asn Ser Ala Val         35 40 45 His Ala Gln Lys Ile Leu Ser Leu His Tyr Thr Lys Glu Gln Arg Glu     50 55 60 Lys Ile Ile Thr Glu Ile Arg Lys Ala Ala Leu Glu Asn Lys Glu Val 65 70 75 80 Leu Ala Thr Met Ile Leu Glu Glu Thr His Met Gly Arg Tyr Glu Asp                 85 90 95 Lys Ile Leu Lys His Glu Leu Val Ala Lys Tyr Thr Pro Gly Thr Glu             100 105 110 Asp Leu Thr Thr Thr Ala Trp Ser Gly Asp Asn Gly Leu Thr Val Val         115 120 125 Glu Met Ser Pro Tyr Gly Val Ile Gly Ala Ile Thr Pro Ser Thr Asn     130 135 140 Pro Thr Glu Thr Val Ile Cys Asn Ser Ile Gly Met Ile Ala Ala Gly 145 150 155 160 Asn Ala Val Val Phe Asn Gly His Pro Gly Ala Lys Lys Cys Val Ala                 165 170 175 Phe Ala Ile Glu Met Ile Asn Lys Ala Ile Ser Ser Cys Gly Gly Pro             180 185 190 Glu Asn Leu Val Thr Thr Ile Lys Asn Pro Thr Met Glu Ser Leu Asp         195 200 205 Ala Ile Ile Lys His Pro Leu Ile Lys Leu Leu Cys Gly Thr Gly Gly     210 215 220 Pro Gly Met Val Lys Thr Leu Leu Asn Ser Gly Lys Lys Ala Ile Gly 225 230 235 240 Ala Gly Ala Gly Asn Pro Pro Val Ile Val Asp Asp Thr Ala Asp Ile                 245 250 255 Glu Lys Ala Gly Lys Ser Ile Glu Gly Cys Ser Phe Asp Asn Asn             260 265 270 Leu Pro Cys Ile Ala Glu Lys Glu Val Phe Val Phe Glu Asn Val Ala         275 280 285 Asp Asp Leu Ile Ser Asn Met Leu Lys Asn Asn Ala Val Ile Ile Asn     290 295 300 Glu Asp Gln Val Ser Lys Leu Ile Asp Leu Val Leu Gln Lys Asn Asn 305 310 315 320 Glu Thr Gln Glu Tyr Phe Ile Asn Lys Lys Trp Val Gly Lys Asp Ala                 325 330 335 Lys Leu Phe Ser Asp Glu Ile Asp Val Glu Ser Pro Ser Asn Ile Lys             340 345 350 Cys Ile Val Cys Glu Val Asn Ala Asn His Pro Phe Val Met Thr Glu         355 360 365 Leu Met Met Pro Ile Leu Pro Ile Val Arg Val Lys Asp Ile Asp Glu     370 375 380 Ala Val Lys Tyr Thr Lys Ile Ala Glu Gln Asn Arg Lys His Ser Ala 385 390 395 400 Tyr Ile Tyr Ser Lys Asn Ile Asp Asn Leu Asn Arg Phe Glu Arg Glu                 405 410 415 Ile Asp Thr Thr Ile Phe Val Lys Asn Ala Lys Ser Phe Ala Gly Val             420 425 430 Gly Tyr Glu Ala Glu Gly Phe Thr Thr Phe Thr Ile Ala Gly Ser Thr         435 440 445 Gly Glu Ile Thr Ser Ala Arg Asn Phe Thr Arg Gln Arg Arg Cys     450 455 460 Val Leu Ala Gly 465 <210> 62 <211> 6558 <212> DNA <213> Clostridium beijerinckii <400> 62 aagcttaaaa tatccatagg ctattgttaa taagactata gcgcttaata ctctaagcgc 60 accatctaaa aaattataca taggaattgg ataaactcca atcttctcca taatactttt 120 cataggtgaa attgatatta taattaaggc tgcataaagc aaactcatat ctccaataaa 180 tgtcactatc ataggtaatt tccttttcat gttcacatac tcccccgtat tctataataa 240 tttacaataa acttccatca caaataaatt ataacatata ttgtaaatat agttttatta 300 ttcgcatatt tatagataaa caatatataa cttagactta tgcaataacc taccgaaagt 360 aaaaacattg ttatttcaca ggactatgaa aatttcgctg aaagtactaa attgcaggtt 420 gcaccactaa tgcttgctcc caatttcatt gtgacaagca ttagttgaac aacctacaat 480 taagagcctt taacagctca tttccaatgc ctgctacata aaaatgtttc tactttctaa 540 tatggtattt acttcaaagt gtaaatcaaa ttcttaagtt gtttatctat atagggttcc 600 atattgataa caatacatat ctactgtttc aatttcatga ataccagcga tattgaaatt 660 ttgtaagttt gaatatcatt gtgaaaccct atatatcaaa tacaatctca aaattataca 720 aaaagatccc aacttcacaa tatgaatttg agatcttcta tcttaacttc ttcaatattt 780 ttaagattat ttatactgcg tgcaaatctt ctataagtat caattatcag ctatgtacat 840 tgatagtgga cttgtaatct gaacagctta tatatttaca cttttaagtt ctcttccact 900 aacccctgct ttgaaacttc tcataaacaa atcgaaagta cctttaatta gctgtgcatt 960 ctcaaactca gaatttaact taagtacttc aattgccgct tcgatagtat agagctctcc 1020 ttctgaagca ccttttctta aagtatattc tgatttatta attggattta atgaaattct 1080 tggaagcttc tttaagtagt cgctctttct tagtatcttc tctgcttctt tccatgtgcc 1140 atctaagata ataaatgctg gaattttttc tgaatcttta catttagact ttctttctag 1200 aggttcatca tcatccatag gaaataatac acgtatttca taatcatcgc tattaatata 1260 ttcaattaat ttttcaggag tctttactct ctcccaaaga attaactcag ttgattctgg 1320 attcaccaat ttcaataatc tagcggtatt tgaaggccta ctaaattctc tttctgttga 1380 taatatcaat atctttgctt ttgtctctat tttaggcaca atatcgcaga tacaatttat 1440 tattggcaac ccacatttat tgcagctctc atataactta gtaatttgct taactttaaa 1500 ttcagactcc attttacctc cattattagt tggttagtgt gtcatatctt cttgctatta 1560 ctaactgatt ataacatatg tattcaatat atcactccta gttttcaaag cactggcaat 1620 acgaattaca aattaatttc tggatttatg tcagtatttc attaataaaa ggtcggactt 1680 ttaagatact tgttttagct attgatcata tttattaaag actatgcatt taatgtataa 1740 ttataatgaa tattatcaat aatatttatt ttatattaca atcttacagt ctttattcta 1800 aatttcactc aaataccaaa cgagctttat tcataaacaa tatataacaa taattccaaa 1860 ataatacgat attttatctg taacagccat ataaaaaaaa tatcatatag tcttgtcatt 1920 tgataacgtt ttgtcttcct tatatttact ttttcggttt aataggttga ttctgtaaat 1980 tttagtgata acatatattt gatgacatta aaaatttaat atttcatata aatttttaat 2040 gtctattaat ttttaaatca caaggaggaa tagttcatga ataaagacac actaatacct 2100 acaactaaag atttaaaatt aaaaacaaat gttgaaaaca ttaatttaaa gaactacaag 2160 gataattctt catgtttcgg agtattcgaa aatgttgaaa atgctataaa cagcgctgta 2220 cacgcgcaaa agatattatc ccttcattat acaaaagaac aaagagaaaa aatcataact 2280 gagataagaa aggccgcatt agaaaataaa gaggttttag ctaccatgat tctggaagaa 2340 acacatatgg gaaggtatga agataaaata ttaaagcatg aattagtagc taaatatact 2400 cctggtacag aagatttaac tactactgct tggtcaggtg ataatggtct tacagttgta 2460 gaaatgtctc catatggcgt tataggtgca ataactcctt ctacgaatcc aactgaaact 2520 gtaatatgta atagcatcgg catgatagct gctggaaatg ctgtagtatt taacggacac 2580 ccaggcgcta aaaaatgtgt tgcttttgct attgaaatga taaataaagc aattatttca 2640 tgtggcggtc ctgagaattt agtaacaact ataaaaaatc caactatgga atccctagat 2700 gcaattatta agcatccttt aataaaactt ctttgcggaa ctggaggtcc aggaatggta 2760 aaaaccctct taaattctgg caagaaagct ataggtgctg gtgctggaaa tccaccagtt 2820 attgtagatg ataccgctga tatagaaaag gctggtaaga gtatcattga aggctgttct 2880 tttgataata atttaccttg tattgcagaa aaagaagtat ttgtttttga gaatgttgca 2940 gatgatttaa tatctaacat gctaaaaaat aatgctgtaa ttataaatga agatcaagta 3000 tcaaaattaa tagatttagt attacaaaaa aataatgaaa ctcaagaata ctttataaac 3060 aaaaaatggg taggaaaaga tgcaaaatta ttctcagatg aaatagatgt tgagtctcct 3120 tcaaatatta aatgcatagt ctgcgaagta aatgcaaatc atccatttgt catgacagaa 3180 ctcatgatgc caatattacc aattgtaaga gttaaagata tagatgaagc tgttaaatat 3240 acaaagatag cagaacaaaa tagaaaacat agtgcctata tttattctaa aaatatagac 3300 aacctaaata gatttgaaag agaaattgat actactattt ttgtaaagaa tgctaaatct 3360 tttgctggtg ttggttatga agctgaagga tttacaactt tcactattgc tggatctact 3420 ggtgaaggca taacctctgc aagaaatttt acaagacaaa gaagatgtgt acttgccggc 3480 taacttcttg ctaaatttat acatttattc acataacttt aatatgcaat gttcccacaa 3540 aatattaaaa actatttaga agggagatat taaatgaata aattagtaaa attaacagat 3600 ttaaagcgca ttttcaaaga tggtatgaca attatggttg ggggtttttt agattgtgga 3660 actcctgaaa atattataga tatgctagtt gatttaaata taaaaaatct gactattata 3720 agcaatgata cagcttttcc taataaagga ataggaaaac ttattgtaaa tggtcaagtt 3780 tctaaagtaa ttgcttcaca tattggaact aatcctgaaa ctgggaaaaa aatgagctct 3840 ggtgaactta aagttgagct ttctccacaa ggaacactga tcgaaagaat tcgtgcagct 3900 ggatctggac tcggaggtgt attaactcca accggacttg ggactatcgt tgaagaaggt 3960 aagaaaaaag ttactatcgg tggcaaagaa tatctattag aacttccttt atccgctgat 4020 gtttcattaa taaaaggtag cattgtagat gaatttggaa ataccttcta tagagctgct 4080 actaaaaatt tcaatccata tatggcaatg gctgcaaaaa cagttatagt tgaagcagaa 4140 aatttagtta aatgtgaaga tttaaaaaga gatgccataa tgactcctgg cgtattagta 4200 gattatatcg ttaaggaggc ggcttaattg attgtagata aagttttagc aaaagagata 4260 attgccaaaa gagttgcaaa agaactaaaa aaaggccaac tcgtaaacct tggaatagga 4320 cttccaactt tagtagctaa ttatgtgcca aaagaaatga acattacttt cgaatcagaa 4380 aatggcatgg ttggcatggc acaaatggcc tcatcaggtg aaaatgaccc agatataata 4440 aatgctggtg gggaatatgt aacattatta cctcaaggtg cattttttga tagttcaacg 4500 tcttttgcac taataagagg aggacatgtt gatgttgctg ttcttggtgc tctagaagtt 4560 gatgaagaag gtaatttagc taactggatt gttccaaata aaattgtccc aggtatggga 4620 ggcgccatgg atttggcaat aggcgcaaaa aaaataatag tggcaatgca acatacakga 4680 aaaggtaaac ctaaaatcgt aaaaaaatgt actctcccac ttactgctaa ggctcaggta 4740 gatttaattg ttacagaact ttgtgtaatt gatgtaacaa atgatggttt acttttcaga 4800 gaaattcata aagatacaac tattgatgaa ataaaatttt taacagatgc agatttaatt 4860 tattoo aaaaatctta tgtattaaaa actaagaaaa gaggttgatt attttatgtt agaaagtgaa 4980 gtatctaaac aaattacaac tccacttgct gctccagcgt ttcctagagg accatataga 5040 tttcacaata gagaatatct aaacattatt tatcgaactg atttagatgc tcttcgaaaa 5100 atagtaccag agccacttga attagatgga gcatatgtta ggtttgagat gatggctatg 5160 cctgatacaa ccggactagg ctcatatact gagtgtggtc aagccattcc agtaaaatat 5220 aatgaggtta aaggtgacta cttgcatatg atgtacctag ataatgaacc tgctattgct 5280 gttggaagag aaagcagtgc ttatcccaaa aagttcggct atccaaagct atttgttgat 5340 tcagacgccc tagttggcgc ccttaagtat ggtgcattac cggtagttac tgcgacgatg 5400 ggatataagc atgagcccct agatcttaaa gaagcctata ctcaaattgc aagacccaat 5460 ttcatgctaa aaatcattca aggttatgat ggtaagccaa gaatttgtga actcatctgt 5520 gcagaaaata ctgatataac tatccacggt gcttggactg gaagtgcacg cctacaatta 5580 tttagccatg cactagctcc tcttgctgat ttacctgtat tagagatcgt atcagcatct 5640 catatcctaa cagatttaac tcttggaaca cctaaggttg tacatgatta tctttcagta 5700 aaataaaagc aatatagaat aaccactaca aaagtagtgg ttattctata ttttaaatca 5760 aactgtaaaa cttaagtttt atagtaccta ataatatttt actaccagca ttagattagt 5820 taaaatacaa agtttgtggt aaaagtattt tagattgcat aatagccttc tatactttta 5880 acaatataac caattgctca ccatctgctt agaatatgct tctttaagct ctaaaataca 5940 tataaaaaag taggaatttc ttattaaaat tcctacttat attatatata aatttaatcg 6000 ttaggtttta ttcgcattgt tcctctttaa tttatctctt ataacatttt attataattg 6060 ttcatataat taattcaata tactattata tattttcaag cattaataat tattcagcat 6120 ctgtcattac atatgcttcc atactttgac ttcttattaa atcatagcta atccatccat 6180 agccattgat tccccagtct ttaccccatg aatttattat ttttacagct tttttactat 6240 catcataacc aactacgcaa actgcatgac cacctctatt ttctccatca atctggtcat 6300 aaattggatt atcagaattt aaattatcaa aatctggata tactgatatt ccaataacta 6360 ctggatttcc agctgctatt tgtgccttta ttgcattata gtcaccatct ggaagttgac 6420 tccaactttt tgctttatat ttggctgcat tagccttttg ttcatctgta ggtgtaacct 6480 cccaactata ttcactacca tcataaggca tatcagataa tgtagtacaa ccttgttctt 6540 ctaataattt aaatgcat 6558 <210> 63 <211> 862 <212> PRT <213> Clostridium acetobutylicum <400> 63 Met Lys Val Thr Thr Val Lys Glu Leu Asp Glu Lys Leu Lys Val Ile 1 5 10 15 Lys Glu Ala Gln Lys Lys Phe Ser Cys Tyr Ser Gln Glu Met Val Asp             20 25 30 Glu Ile Phe Arg Asn Ala Ala Met Ala Ala Ile Asp Ala Arg Ile Glu         35 40 45 Leu Ala Lys Ala Ala Val Leu Glu Thr Gly Met Gly Leu Val Glu Asp     50 55 60 Lys Val Ile Lys Asn His Phe Ala Gly Glu Tyr Ile Tyr Asn Lys Tyr 65 70 75 80 Lys Asp Glu Lys Thr Cys Gly Ile Ile Glu Arg Asn Glu Pro Tyr Gly                 85 90 95 Ile Thr Lys Ile Ala Glu Pro Ile Gly Val Val Ala Ala Ile Ile Pro             100 105 110 Val Thr Asn Pro Thr Ser Thr Thr Ile Phe Lys Ser Leu Ile Ser Leu         115 120 125 Lys Thr Arg Asn Gly Ile Phe Phe Ser Pro His Pro Arg Ala Lys Lys     130 135 140 Ser Thr Ile Leu Ala Ala Lys Thr Ile Leu Asp Ala Ala Val Lys Ser 145 150 155 160 Gly Ala Pro Glu Asn Ile Ile Gly Trp Ile Asp Glu Pro Ser Ile Glu                 165 170 175 Leu Thr Gln Tyr Leu Met Gln Lys Ala Asp Ile Thr Leu Ala Thr Gly             180 185 190 Gly Pro Ser Leu Val Lys Ser Ala Tyr Ser Ser Gly Lys Pro Ala Ile         195 200 205 Gly Val Gly Pro Gly Asn Thr Pro Val Ile Ile Asp Glu Ser Ala His     210 215 220 Ile Lys Met Ala Val Ser Ser Ile Ile Leu Ser Lys Thr Tyr Asp Asn 225 230 235 240 Gly Val Ile Cys Ala Ser Glu Gln Ser Val Ile Val Leu Lys Ser Ile                 245 250 255 Tyr Asn Lys Val Lys Asp Glu Phe Gln Glu Arg Gly Ala Tyr Ile Ile             260 265 270 Lys Lys Asn Glu Leu Asp Lys Val Arg Glu Val Ile Phe Lys Asp Gly         275 280 285 Ser Val Asn Pro Lys Ile Val Gly Gln Ser Ala Tyr Thr Ile Ala Ala     290 295 300 Met Ala Gly Ile Lys Val Lys Thr Thr Arg Ile Leu Ile Gly Glu 305 310 315 320 Val Thr Ser Leu Gly Glu Glu Glu Pro Phe Ala His Glu Lys Leu Ser                 325 330 335 Pro Val Leu Ala Met Tyr Glu Ala Asp Asn Phe Asp Asp Ala Leu Lys             340 345 350 Lys Ala Val Thr Leu Ile Asn Leu Gly Gly Leu Gly His Thr Ser Gly         355 360 365 Ile Tyr Ala Asp Glu Ile Lys Ala Arg Asp Lys Ile Asp Arg Phe Ser     370 375 380 Ser Ala Met Lys Thr Val Arg Thr Phe Val Asn Ile Pro Thr Ser Gln 385 390 395 400 Gly Ala Ser Gly Asp Leu Tyr Asn Phe Arg Ile Pro Pro Ser Phe Thr                 405 410 415 Leu Gly Cys Gly Phe Trp Gly Gly Asn Ser Val Ser Glu Asn Val Gly             420 425 430 Pro Lys His Leu Leu Asn Ile Lys Thr Val Ala Glu Arg Arg Glu Asn         435 440 445 Met Leu Trp Phe Arg Val Pro His Lys Val Tyr Phe Lys Phe Gly Cys     450 455 460 Leu Gln Phe Ala Leu Lys Asp Leu Lys Asp Leu Lys Lys Lys Arg Ala 465 470 475 480 Phe Ile Val Thr Asp Ser Asp Pro Tyr Asn Leu Asn Tyr Val Asp Ser                 485 490 495 Ile Ile Lys Ile Leu Glu His Leu Asp Ile Asp Phe Lys Val Phe Asn             500 505 510 Lys Val Gly Arg Glu Ala Asp Leu Lys Thr Ile Lys Lys Ala Thr Glu         515 520 525 Glu Met Ser Ser Phe Met Pro Asp Thr Ile Ile Ala Leu Gly Gly Thr     530 535 540 Pro Glu Met Ser Ser Ala Lys Leu Met Trp Val Leu Tyr Glu His Pro 545 550 555 560 Glu Val Lys Phe Glu Asp Leu Ala Ile Lys Phe Met Asp Ile Arg Lys                 565 570 575 Arg Ile Tyr Thr Phe Pro Lys Leu Gly Lys Lys Ala Met Leu Val Ala             580 585 590 Ile Thr Thr Ser Ala Gly Ser Gly Ser Glu Val Thr Pro Phe Ala Leu         595 600 605 Val Thr Asp Asn Asn Thr Gly Asn Lys Tyr Met Leu Ala Asp Tyr Glu     610 615 620 Met Thr Pro Asn Met Ala Ile Val Asp Ala Glu Leu Met Met Lys Met 625 630 635 640 Pro Lys Gly Leu Thr Ala Tyr Ser Gly Ile Asp Ala Leu Val Asn Ser                 645 650 655 Ile Glu Ala Tyr Thr Ser Val Tyr Ala Ser Glu Tyr Thr Asn Gly Leu             660 665 670 Ala Leu Glu Ala Ile Arg Leu Ile Phe Lys Tyr Leu Pro Glu Ala Tyr         675 680 685 Lys Asn Gly Arg Thr Asn Glu Lys Ala Arg Glu Lys Met Ala His Ala     690 695 700 Ser Thr Met Ala Gly Met Ala Ser Ala Asn Ala Phe Leu Gly Leu Cys 705 710 715 720 His Ser Met Ala Ile Lys Leu Ser Ser Glu His Asn Ile Pro Ser Gly                 725 730 735 Ile Ala Asn Ala Leu Leu Ile Glu Glu Val Ile Lys Phe Asn Ala Val             740 745 750 Asp Asn Pro Val Lys Gln Ala Pro Cys Pro Gln Tyr Lys Tyr Pro Asn         755 760 765 Thr Ile Phe Arg Tyr Ala Arg Ile Asp Tyr Ile Lys Leu Gly Gly     770 775 780 Asn Thr Asp Glu Glu Lys Val Asp Leu Leu Ile Asn Lys Ile His Glu 785 790 795 800 Leu Lys Lys Ala Leu Asn Ile Pro Thr Ser Ile Lys Asp Ala Gly Val                 805 810 815 Leu Glu Glu Asn Phe Tyr Ser Ser Leu Asp Arg Ile Ser Glu Leu Ala             820 825 830 Leu Asp Asp Gln Cys Thr Gly Ala Asn Pro Arg Phe Pro Leu Thr Ser         835 840 845 Glu Ile Lys Glu Met Tyr Ile Asn Cys Phe Lys Lys Gln Pro     850 855 860 <210> 64 <211> 1665 <212> DNA <213> Clostridium acetobutylicum <400> 64 ttgaagagtg aatacacaat tggaagatat ttgttagacc gtttatcaga gttgggtatt 60 cggcatatct ttggtgtacc tggagattac aatctatcct ttttagacta tataatggag 120 tacaaaggga tagattgggt tggaaattgc aatgaattga atgctgggta tgctgctgat 180 ggatatgcaa gaataaatgg aattggagcc atacttacaa catttggtgt tggagaatta 240 agtgccatta acgcaattgc tggggcatac gctgagcaag ttccagttgt taaaattaca 300 ggtatcccca cagcaaaagt tagggacaat ggattatatg tacaccacac attaggtgac 360 ggaaggtttg atcacttttt tgaaatgttt agagaagtaa cagttgctga ggcattacta 420 agcgaagaaa atgcagcaca agaaattgat cgtgttctta tttcatgctg gagacaaaaa 480 cgtcctgttc ttataaattt accgattgat gtatatgata aaccaattaa caaaccatta 540 aagccattac tcgattatac tatttcaagt aacaaagagg ctgcatgtga atttgttaca 600 gaaatagtac ctataataaa tagggcaaaa aagcctgtta ttcttgcaga ttatggagta 660 tatcgttacc aagttcaaca tgtgcttaaa aacttggccg aaaaaaccgg atttcctgtg 720 gctacactaa gtatgggaaa aggtgttttc aatgaagcac accctcaatt tattggtgtt 780 tataatggtg atgtaagttc tccttattta aggcagcgag ttgatgaagc agactgcatt 840 attagcgttg gtgtaaaatt gacggattca accacagggg gattttctca tggattttct 900 aaaaggaatg taattcacat tgatcctttt tcaataaagg caaaaggtaa aaaatatgca 960 cctattacga tgaaagatgc tttaacagaa ttaacaagta aaattgagca tagaaacttt 1020 gaggatttag atataaagcc ttacaaatca gataatcaaa agtattttgc aaaagagaag 1080 ccaattacac aaaaacgttt ttttgagcgt attgctcact ttataaaaga aaaagatgta 1140 ttattagcag aacagggtac atgctttttt ggtgcgtcaa ccatacaact acccaaagat 1200 gcaactttta ttggtcaacc tttatgggga tctattggat acacacttcc tgctttatta 1260 ggttcacaat tagctgatca aaaaaggcgt aatattcttt taattgggga tggtgcattt 1320 caaatgacag cacaagaaat ttcaacaatg cttcgtttac aaatcaaacc tattattttt 1380 ttaattaata acgatggtta tacaattgaa cgtgctattc atggtagaga acaagtatat 1440 aacaatattc aaatgtggcg atatcataat gttccaaagg ttttaggtcc taaagaatgc 1500 agcttaacct ttaaagtaca aagtgaaact gaacttgaaa aggctctttt agtggcagat 1560 aaggattgtg aacatttgat ttttatagaa gttgttatgg atcgttatga taaacccgag 1620 cctttagaac gtctttcgaa acgttttgca aatcaaaata attag 1665 <210> 65 <211> 858 <212> PRT <213> Clostridium acetobutylicum <400> 65 Met Lys Val Thr Asn Gln Lys Glu Leu Lys Gln Lys Leu Asn Glu Leu 1 5 10 15 Arg Glu Ala Gln Lys Lys Phe Ala Thr Tyr Thr Gln Glu Gln Val Asp             20 25 30 Lys Ile Phe Lys Gln Cys Ala Ile Ala Ala Ala Lys Glu Arg Ile Asn         35 40 45 Leu Ala Lys Leu Ala Val Glu Glu Thr Gly Ile Gly Leu Val Glu Asp     50 55 60 Lys Ile Ile Lys Asn His Phe Ala Ala Glu Tyr Ile Tyr Asn Lys Tyr 65 70 75 80 Lys Asn Glu Lys Thr Cys Gly Ile Ile Asp His Asp Asp Ser Leu Gly                 85 90 95 Ile Thr Lys Val Ala Glu Pro Ile Gly Ile Val Ala Ala Ile Val Pro             100 105 110 Thr Asn Pro Thr Ser Thr Ala Ile Phe Lys Ser Leu Ile Ser Leu         115 120 125 Lys Thr Arg Asn Ala Ile Phe Phe Ser Pro His Pro Arg Ala Lys Lys     130 135 140 Ser Thr Ile Ala Ala Lys Leu Ile Leu Asp Ala Ala Val Lys Ala 145 150 155 160 Gly Ala Pro Lys Asn Ile Ile Gly Trp Ile Asp Glu Pro Ser Ile Glu                 165 170 175 Leu Ser Gln Asp Leu Met Ser Glu Ala Asp Ile Ile Leu Ala Thr Gly             180 185 190 Gly Pro Ser Met Val Lys Ala Ala Tyr Ser Ser Gly Lys Pro Ala Ile         195 200 205 Gly Val Gly Ala Gly Asn Thr Pro Ala Ile Ile Asp Glu Ser Ala Asp     210 215 220 Ile Asp Met Ala Val Ser Ser Ile Ile Leu Ser Lys Thr Tyr Asp Asn 225 230 235 240 Gly Val Ile Cys Ala Ser Glu Gln Ser Ile Leu Val Met Asn Ser Ile                 245 250 255 Tyr Glu Lys Val Lys Glu Glu Phe Val Lys Arg Gly Ser Tyr Ile Leu             260 265 270 Asn Gln Asn Glu Ile Ala Lys Ile Lys Glu Thr Met Phe Lys Asn Gly         275 280 285 Ala Ile Asl Ala Asp Ile Val Gly Lys Ser Ala Tyr Ile Ile Ala Lys     290 295 300 Met Ala Gly Ile Glu Val Pro Gln Thr Thr Lys Ile Leu Ile Gly Glu 305 310 315 320 Val Gln Ser Val Glu Lys Ser Glu Leu Phe Ser His Glu Lys Leu Ser                 325 330 335 Pro Val Leu Ala Met Tyr Lys Val Lys Asp Phe Asp Glu Ala Leu Lys             340 345 350 Lys Ala Gln Arg Leu Ile Glu Leu Gly Gly Ser Gly His Thr Ser Ser         355 360 365 Leu Tyr Ile Asp Ser Gln Asn Asn Lys Asp Lys Val Lys Glu Phe Gly     370 375 380 Leu Ala Met Lys Thr Ser Arg Thr Phe Ile Asn Met Pro Ser Ser Gln 385 390 395 400 Gly Ala Ser Gly Asp Leu Tyr Asn Phe Ala Ile Ala Pro Ser Phe Thr                 405 410 415 Leu Gly Cys Gly Thr Trp Gly Gly Asn Ser Val Ser Gln Asn Val Glu             420 425 430 Pro Lys His Leu Leu Asn Ile Lys Ser Val Ala Glu Arg Arg Glu Asn         435 440 445 Met Leu Trp Phe Lys Val Pro Gln Lys Ile Tyr Phe Lys Tyr Gly Cys     450 455 460 Leu Arg Phe Ala Leu Lys Glu Leu Lys Asp Met Asn Lys Lys Arg Ala 465 470 475 480 Phe Ile Val Thr Asp Lys Asp Leu Phe Lys Leu Gly Tyr Val Asn Lys                 485 490 495 Ile Thr Lys Val Leu Asp Glu Ile Asp Ile Lys Tyr Ser Ile Phe Thr             500 505 510 Asp Ile Lys Ser Asp Pro Thr Ile Asp Ser Val Lys Lys Gly Ala Lys         515 520 525 Glu Met Leu Asn Phe Glu Pro Asp Thr Ile Ile Ser Ile Gly Gly Gly     530 535 540 Ser Pro Met Asp Ala Ala Lys Val Met His Leu Leu Tyr Glu Tyr Pro 545 550 555 560 Glu Ala Glu Ile Glu Asn Leu Ala Ile Asn Phe Met Asp Ile Arg Lys                 565 570 575 Arg Ile Cys Asn Phe Pro Lys Leu Gly Thr Lys Ala Ile Ser Val Ala             580 585 590 Ile Pro Thr Thr Ala Gly Thr Gly Ser Glu Ala Thr Pro Phe Ala Val         595 600 605 Ile Thr Asn Asp Glu Thr Gly Met Lys Tyr Pro Leu Thr Ser Tyr Glu     610 615 620 Leu Thr Pro As Met Ale Ile Asle Asp Thr Glu Leu Met Leu Asn Met 625 630 635 640 Pro Arg Lys Leu Thr Ala Ala Thr Gly Ile Asp Ala Leu Val His Ala                 645 650 655 Ile Glu Ala Tyr Val Ser Val Ala Thr Asp Tyr Thr Asp Glu Leu             660 665 670 Ala Leu Arg Ala Ile Lys Met Ile Phe Lys Tyr Leu Pro Arg Ala Tyr         675 680 685 Lys Asn Gly Thr Asn Asp Ile Glu Ala Arg Glu Lys Met Ala His Ala     690 695 700 Ser Asn Ile Ala Gly Met Ala Phe Ala Asn Ala Phe Leu Gly Val Cys 705 710 715 720 His Ser Met Ala His Lys Leu Gly Ala Met His His Val Pro His Gly                 725 730 735 Ile Ala Cys Ala Val Leu Ile Glu Glu Val Ile Lys Tyr Asn Ala Thr             740 745 750 Asp Cys Pro Thr Lys Gln Thr Ala Phe Pro Gln Tyr Lys Ser Pro Asn         755 760 765 Ala Lys Arg Lys Tyr Ala Glu Ile Ala Glu Tyr Leu Asn Leu Lys Gly     770 775 780 Thr Ser Asp Thr Glu Lys Val Thr Ala Leu Ile Glu Ala Ile Ser Lys 785 790 795 800 Leu Lys Ile Asp Leu Ser Ile Pro Gln Asn Ile Ser Ala Ala Gly Ile                 805 810 815 Asn Lys Lys Asp Phe Tyr Asn Thr Leu Asp Lys Met Ser Glu Leu Ala             820 825 830 Phe Asp Gln Cys Thr Thr Ala Asn Pro Arg Tyr Pro Leu Ile Ser         835 840 845 Glu Leu Lys Asp Ile Tyr Ile Lys Ser Phe     850 855 <210> 66 <211> 2589 <212> DNA <213> Clostridium acetobutylicum <400> 66 atgaaagtca caacagtaaa ggaattagat gaaaaactca aggtaattaa agaagctcaa 60 aaaaaattct cttgttactc gcaagaaatg gttgatgaaa tctttagaaa tgcagcaatg 120 gcagcaatcg acgcaaggat agagctagca aaagcagctg ttttggaaac cggtatgggc 180 ttagttgaag acaaggttat aaaaaatcat tttgcaggcg aatacatcta taacaaatat 240 aaggatgaaa aaacctgcgg tataattgaa cgaaatgaac cctacggaat tacaaaaata 300 gcagaaccta taggagttgt agctgctata atccctgtaa caaaccccac atcaacaaca 360 atatttaaat ccttaatatc ccttaaaact agaaatggaa ttttcttttc gcctcaccca 420 agggcaaaaa aatccacaat actagcagct aaaacaatac ttgatgcagc cgttaagagt 480 ggtgccccgg aaaatataat aggttggata gatgaacctt caattgaact aactcaatat 540 ttaatgcaaa aagcagatat aacccttgca actggtggtc cctcactagt taaatctgct 600 tattcttccg gaaaaccagc aataggtgtt ggtccgggta acaccccagt aataattgat 660 gaatctgctc atataaaaat ggcagtaagt tcaattatat tatccaaaac ctatgataat 720 gt; aaagatgagt tccaagaaag aggagcttat ataataaaga aaaacgaatt ggataaagtc 840 cgtgaagtga tttttaaaga tggatccgta aaccctaaaa tagtcggaca gtcagcttat 900 actatagcag ctatggctgg cataaaagta cctaaaacca caagaatatt aataggagaa 960 gttacctcct taggtgaaga agaacctttt gcccacgaaa aactatctcc tgttttggct 1020 atgtatgagg ctgacaattt tgatgatgct ttaaaaaaag cagtaactct aataaactta 1080 ggaggcctcg gccatacctc aggaatatat gcagatgaaa taaaagcacg agataaaata 1140 gatagattta gtagtgccat gaaaaccgta agaacctttg taaatatccc aacctcacaa 1200 ggtgcaagtg gagatctata taattttaga ataccacctt ctttcacgct tggctgcgga 1260 ttttggggag gaaattctgt ttccgagaat gttggtccaa aacatctttt gaatattaaa 1320 accgtagctg aaaggagaga aaacatgctt tggtttagag ttccacataa agtatatttt 1380 aagttcggtt gtcttcaatt tgctttaaaa gatttaaaag atctaaagaa aaaaagagcc 1440 tttatagtta ctgatagtga cccctataat ttaaactatg ttgattcaat aataaaaata 1500 cttgagcacc tagatattga ttttaaagta tttaataagg ttggaagaga agctgatctt 1560 aaaaccataa aaaaagcaac tgaagaaatg tcctccttta tgccagacac tataatagct 1620 ttaggtggta cccctgaaat gagctctgca aagctaatgt gggtactata tgaacatcca 1680 gaagtaaaat ttgaagatct tgcaataaaa tttatggaca taagaaagag aatatatact 1740 ttcccaaaac tcggtaaaaa ggctatgtta gttgcaatta caacttctgc tggttccggt 1800 tctgaggtta ctccttttgc tttagtaact gacaataaca ctggaaataa gtacatgtta 1860 gcagattatg aaatgacacc aaatatggca attgtagatg cagaacttat gatgaaaatg 1920 ccaaagggat taaccgctta ttcaggtata gatgcactag taaatagtat agaagcatac 1980 acatccgtat atgcttcaga atacacaaac ggactagcac tagaggcaat acgattaata 2040 tttaaatatt tgcctgaggc ttacaaaaac ggaagaacca atgaaaaagc aagagagaaa 2100 atggctcacg cttcaactat ggcaggtatg gcatccgcta atgcatttct aggtctatgt 2160 cattccatgg caataaaatt aagttcagaa cacaatattc ctagtggcat tgccaatgca 2220 ttactaatag aagaagtaat aaaatttaac gcagttgata atcctgtaaa acaagcccct 2280 tgcccacaat ataagtatcc aaacaccata tttagatatg ctcgaattgc agattatata 2340 aagcttggag gaaatactga tgaggaaaag gtagatctct taattaacaa aatacatgaa 2400 ctaaaaaaag ctttaaatat accaacttca ataaaggatg caggtgtttt ggaggaaaac 2460 ttctattcct cccttgatag aatatctgaa cttgcactag atgatcaatg cacaggcgct 2520 aatcctagat ttcctcttac aagtgagata aaagaaatgt atataaattg ttttaaaaaa 2580 caaccttaa 2589 <210> 67 <211> 307 <212> PRT <213> Pseudomonas putida <400> 67 Met Ser Lys Lys Leu Lys Ala Ala Ile Ile Gly Pro Gly Asn Ile Gly 1 5 10 15 Thr Asp Leu Val Met Lys Met Leu Arg Ser Glu Trp Ile Glu Pro Val             20 25 30 Trp Met Val Gly Ile Asp Pro Asn Ser Asp Gly Leu Lys Arg Ala Arg         35 40 45 Asp Phe Gly Met Lys Thr Thr Ala Glu Gly Val Asp Gly Leu Leu Pro     50 55 60 His Val Leu Asp Asp Asp Ile Arg Ile Ala Phe Asp Ala Thr Ser Ala 65 70 75 80 Tyr Val His Ala Glu Asn Ser Arg Lys Leu Asn Ala Leu Gly Val Leu                 85 90 95 Met Val Asp Leu Thr Pro Ala Ala Ile Gly Pro Tyr Cys Val Pro Pro             100 105 110 Val Asn Leu Lys Gln His Val Gly Arg Leu Glu Met Asn Val Asn Met         115 120 125 Val Thr Cys Gly Gly Gln Ala Thr Ile Pro Met Val Ala Ala Val Ser     130 135 140 Arg Val Gln Pro Val Ala Tyr Ala Glu Ile Val Ala Thr Val Ser Ser 145 150 155 160 Arg Ser Val Gly Pro Gly Thr Arg Lys Asn Ile Asp Glu Phe Thr Arg                 165 170 175 Thr Thr Ala Gly Ala Ile Glu Gln Val Gly Gly Ala Arg Glu Gly Lys             180 185 190 Ala Ile Ile Val Ile Asn Pro Ala Glu Pro Pro Leu Met Met Arg Asp         195 200 205 Thr Ile His Cys Leu Thr Asp Ser Glu Pro Asp Gln Ala Ala Ile Thr     210 215 220 Ala Ser Val His Ala Met Ile Ala Glu Val Gln Lys Tyr Val Pro Gly 225 230 235 240 Tyr Arg Leu Lys Asn Gly Pro Val Phe Asp Gly Asn Arg Val Ser Ile                 245 250 255 Phe Met Glu Val Glu Gly Leu Gly Asp Tyr Leu Pro Lys Tyr Ala Gly             260 265 270 Asn Leu Asp Ile Met Thr Ala Ala Ala Leu Arg Thr Gly Glu Met Phe         275 280 285 Ala Glu Glu Ile Ala Ala Gly Thr Ile Gln Leu Pro Arg Arg Asp Ile     290 295 300 Ala Leu Ala 305 <210> 68 <211> 2180 <212> DNA <213> Pseudomonas putida <400> 68 ggtacccctg gagccggtca aggccggcga cttcatgcgc gtcgagatcg gcggcatcgg 60 cagcgcctcc gtgcgcttca cctgatcgaa cagaggacaa acccatgagc aagaaactca 120 aggcggccat cataggcccc ggcaatatcg gtaccgatct ggtgatgaag atgctccgtt 180 ccgagtggat tgagccggtg tggatggtcg gcatcgaccc caactccgac ggcctcaaac 240 gcgcccgcga tttcggcatg aagaccacag ccgaaggcgt cgacggcctg ctcccgcacg 300 tgctggacga cgacatccgc atcgccttcg acgccacctc ggcctatgtg catgccgaga 360 atagccgcaa gctcaacgcg cttggcgtgc tgatggtcga cctgaccccg gcggccatcg 420 gcccctactg cgtgccgccg gtcaacctca agcagcatgt cggccgcctg gaaatgaacg 480 tcaacatggt cacctgcggc ggccaggcca ccatccccat ggtcgccgcg gtgtcccgcg 540 tgcagccggt ggcctacgcc gagatcgtcg ccaccgtctc ctcgcgctcg gtcggcccgg 600 gcacgcgcaa gaacatcgac gagttcaccc gcaccaccgc cggcgccatc gagcaggtcg 660 gcggcgccag ggaaggcaag gcgatcatcg tcatcaaccc ggccgagccg ccgctgatga 720 tgcgcgacac catccactgc ctgaccgaca gcgagccgga ccaggctgcg atcaccgctt 780 cggttcacgc gatgatcgcc gaggtgcaga aatacgtgcc cggctaccgc ctgaagaacg 840 gcccggtgtt cgacggcaac cgcgtgtcga tcttcatgga agtcgaaggc ctgggcgact 900 acctgcccaa gtacgccggc aacctcgaca tcatgaccgc cgccgcgctg cgtaccggcg 960 agatgttcgc cgaggaaatc gccgccggca ccattcaact gccgcgtcgc gacatcgcgc 1020 tggcttgagg agtagcacca tgaatttgca cggcaagagc gtcatcctgc acgacatgag 1080 cctgcgcgac ggcatgcacg ccaagcgcca ccagatcagc ctggagcaga tggtcgcggt 1140 cgccaccggc ctcgatcaag ccggtatgcc gctgatcgag atcacccacg gcgacggcct 1200 cggcggtcgt tcgatcaact acggcttccc ggcccacagt gacgaggagt acctgcgcgc 1260 ggtgatcccg cagctcaagc aggccaaagt ctcggcgctg ctgctgcccg gcatcggcac 1320 cgtcgaccac ctgaagatgg ccctggactg cggcgtctcg actattcgcg tggccaccca 1380 ctgtaccgag gcggatgtct ccgagcagca catcggcatg gcgcgcaagc tgggggtcga 1440 caccgtcggc ttcctgatga tggcgcacat gatcagcgcc gagaaagtcc tggagcaggc 1500 caagctgatg gaaagctatg gtgccaactg catctactgc accgactcgg ccggctacat 1560 gctgcctgat gaagtcagcg agaaaatcgg cctcctgcgc gccgagctga acccggccac 1620 cgaagtcggc ttccacggcc accacaacat gggcatggct atcgccaact cgctggccgc 1680 catcgaagcc ggtgccgcgc gcatcgacgg ctcggtcgcc ggcctcggcg ccggtgccgg 1740 caacaccccg ctggaagtgt tcgtcgcagt gtgcaaacgc atgggcgtgg agaccggcat 1800 cgacctgtac aagatcatgg acgtggccga ggacctggtg gtgccgatga tggatcagcc 1860 gatccgcgtc gaccgcgacg ccctgaccct gggctacgcc ggggtgtaca gctcgttcct 1920 gctgttcgcc cagcgcgccg agaagaaata tggcgtgtcg gcccgcgaca tcctggtcga 1980 actgggccgg cgcggcaccg tcggtggcca ggaagacatg atcgaagacc tcgccctgga 2040 catggcccgg gcccgtcagc agcagaaggt gagcgcatga accgtaccct gacccgcgaa 2100 caggtgctgg ccctggccga gcacatcgaa aacgccgagc tgaatgtcca cgacatcggc 2160 aaggtgacca acgattttcc 2180 <210> 69 <211> 307 <212> PRT <213> Thermus thermophilus <400> 69 Met Ser Glu Arg Val Lys Val Ala Leu Gly Ser Gly Asn Ile Gly 1 5 10 15 Thr Asp Leu Met Tyr Lys Leu Leu Lys Asn Pro Gly His Met Glu Leu             20 25 30 Val Ala Val Val Gly Ile Asp Pro Lys Ser Glu Gly Leu Ala Arg Ala         35 40 45 Arg Ala Leu Gly Leu Glu Ala Ser His Glu Gly Ile Ala Tyr Ile Leu     50 55 60 Glu Arg Pro Glu Ile Lys Ile Val Phe Asp Ala Thr Ser Ala Lys Ala 65 70 75 80 His Val Arg His Ala Lys Leu Leu Arg Glu Ala Gly Lys Ile Ala Ile                 85 90 95 Asp Leu Thr Pro Ala Ala Arg Gly Pro Tyr Val Val Pro Pro Val Asn             100 105 110 Leu Lys Glu His Leu Asp Lys Asp Asn Val Asn Leu Ile Thr Cys Gly         115 120 125 Gly Gln Ala Thr Ile Pro Leu Val Tyr Ala Val His Arg Val Ala Pro     130 135 140 Val Leu Tyr Ala Glu Met Val Ser Thr Val Ala Ser Arg Ser Ala Gly 145 150 155 160 Pro Gly Thr Arg Gln Asn Ile Asp Glu Phe Thr Phe Thr Thr Ala Arg                 165 170 175 Gly Leu Gly Ala Ile Gly Aly Lys Aly Ily Ily Ily             180 185 190 Leu Asn Pro Ala Glu Pro Pro Ile Leu Met Thr Asn Thr Val Arg Cys         195 200 205 Ile Pro Glu Asp Glu Gly Phe Asp Arg Glu Ala Val Val Ala Ser Val     210 215 220 Arg Ala Met Glu Arg Glu Val Gln Ala Tyr Val Pro Gly Tyr Arg Leu 225 230 235 240 Lys Ala Asp Pro Val Phe Glu Arg Leu Pro Thr Pro Trp Gly Glu Arg                 245 250 255 Thr Val Val Ser Met Leu Leu Glu Val Glu Gly Ala Gly Asp Tyr Leu             260 265 270 Pro Lys Tyr Ala Gly Asn Leu Asp Ile Met Thr Ala Ser Ala Arg Arg         275 280 285 Val Gly Glu Val Phe Ala Gln His Leu Leu Gly Lys Pro Val Glu Glu     290 295 300 Val Val Ala 305 <210> 70 <211> 924 <212> DNA <213> Thermus thermophilus <400> 70 atgtccgaaa gggttaaggt agccatcctg ggctccggca acatcgggac ggacctgatg 60 tacaagctcc tgaagaaccc gggccacatg gagcttgtgg cggtggtggg gatagacccc 120 aagtccgagg gcctggcccg ggcgcgggcc ttagggttag aggcgagcca cgaagggatc 180 gcctacatcc tggagaggcc ggagatcaag atcgtctttg acgccaccag cgccaaggcc 240 cacgtgcgcc acgccaagct cctgagggag gcggggaaga tcgccataga cctcacgccg 300 gcggcccggg gcccttacgt ggtgcccccg gtgaacctga aggaacacct ggacaaggac 360 aacgtgaacc tcatcacctg cggggggcag gccaccatcc ccctggtcta cgcggtgcac 420 cgggtggccc ccgtgctcta cgcggagatg gtctccacgg tggcctcccg ctccgcgggc 480 cccggcaccc ggcagaacat cgacgagttc accttcacca ccgcccgggg cctggaggcc 540 atcggggggg ccaagaaggg gaaggccatc atcatcctga acccggcgga accccccatc 600 ctcatgacca acaccgtgcg ctgcatcccc gaggacgagg gctttgaccg ggaggccgtg 660 gtggcgagcg tccgggccat ggagcgggag gtccaggcct acgtgcccgg ctaccgcctg 720 aaggcggacc cggtgtttga gaggcttccc accccctggg gggagcgcac cgtggtctcc 780 atgctcctgg aggtggaggg ggcgggggac tatttgccca aatacgccgg caacctggac 840 atcatgacgg cttctgcccg gagggtgggg gaggtcttcg cccagcacct cctggggaag 900 cccgtggagg aggtggtggc gtga 924 <210> 71 <211> 417 <212> PRT <213> Escherichia coli <400> 71 Met Thr Phe Ser Leu Phe Gly Asp Lys Phe Thr Arg His Ser Gly Ile 1 5 10 15 Thr Leu Leu Met Glu Asp Leu Asn Asp Gly Leu Arg Thr Pro Gly Ala             20 25 30 Ile Met Leu Gly Gly Gly Asn Pro Ala Gln Ile Pro Glu Met Gln Asp         35 40 45 Tyr Phe Gln Thr Leu Leu Thr Asp Met Leu Glu Ser Gly Lys Ala Thr     50 55 60 Asp Ala Leu Cys Asn Tyr Asp Gly Pro Gln Gly Lys Thr Glu Leu Leu 65 70 75 80 Thr Leu Leu Ala Gly Met Leu Arg Glu Lys Leu Gly Trp Asp Ile Glu                 85 90 95 Pro Gln Asn Ile Ala Leu Thr Asn Gly Ser Gln Ser Ala Phe Phe Tyr             100 105 110 Leu Phe Asn Leu Phe Ala Gly Arg Arg Ala Asp Gly Arg Val Lys Lys         115 120 125 Val Leu Phe Pro Leu Ala Pro Glu Tyr Ile Gly Tyr Ala Asp Ala Gly     130 135 140 Leu Glu Glu Asp Leu Phe Val Ser Ala Arg Pro Asn Ile Glu Leu Leu 145 150 155 160 Pro Glu Gly Gln Phe Lys Tyr His Val Asp Phe Glu His Leu His Ile                 165 170 175 Gly Glu Glu Thr Gly Met Ile Cys Val Ser Arg Pro Thr Asn Pro Thr             180 185 190 Gly Asn Val Ile Thr Asp Glu Glu Leu Leu Lys Leu Asp Ala Leu Ala         195 200 205 Asn Gln His Gly Ile Pro Leu Val Ile Asp Asn Ala Tyr Gly Val Pro     210 215 220 Phe Pro Gly Ile Ile Phe Ser Glu Ala Arg Pro Leu Trp Asn Pro Asn 225 230 235 240 Ile Val Leu Cys Met Ser Leu Ser Lys Leu Gly Leu Pro Gly Ser Arg                 245 250 255 Cys Gly Ile Ile Ile Ala Asn Glu Lys Ile Ile Thr Ala Ile Thr Asn             260 265 270 Met Asn Gly Ile Ser Ser Leu Ala Pro Gly Gly Ile Gly Pro Ala Met         275 280 285 Met Cys Glu Met Ile Lys Arg Asn Asp Leu Leu Arg Leu Ser Glu Thr     290 295 300 Val Ile Lys Pro Phe Tyr Tyr Gln Arg Val Gln Glu Thr Ile Ala Ile 305 310 315 320 Ile Arg Arg Tyr Leu Pro Glu Asn Arg Cys Leu Ile His Lys Pro Glu                 325 330 335 Gly Ala Ile Phe Leu Trp Leu Trp Phe Lys Asp Leu Pro Ile Thr Thr             340 345 350 Lys Gln Leu Tyr Gln Arg Leu Lys Ala Arg Gly Val Leu Met Val Pro         355 360 365 Gly His Asn Phe Phe Pro Gly Leu Asp Lys Pro Trp Pro His Thr His     370 375 380 Gln Cys Met Arg Met Met Asn Tyr Val Pro Glu Pro Glu Lys Ile Glu Ala 385 390 395 400 Gly Val Lys Ile Leu Ala Glu Glu Ile Glu Arg Ala Trp Ala Glu Ser                 405 410 415 His      <210> 72 <211> 417 <212> PRT <213> Escherichia coli <400> 72 Met Thr Phe Ser Leu Phe Gly Asp Lys Phe Thr Arg His Ser Gly Ile 1 5 10 15 Thr Leu Leu Met Glu Asp Leu Asn Asp Gly Leu Arg Thr Pro Gly Ala             20 25 30 Ile Met Leu Gly Gly Gly Asn Pro Ala Gln Ile Pro Glu Met Gln Asp         35 40 45 Tyr Phe Gln Thr Leu Leu Thr Asp Met Leu Glu Ser Gly Lys Ala Thr     50 55 60 Asp Ala Leu Cys Asn Tyr Asp Gly Pro Gln Gly Lys Thr Glu Leu Leu 65 70 75 80 Thr Leu Leu Ala Gly Met Leu Arg Glu Lys Leu Gly Trp Asp Ile Glu                 85 90 95 Pro Gln Asn Ile Ala Leu Thr Asn Gly Ser Gln Ser Ala Phe Phe Tyr             100 105 110 Leu Phe Asn Leu Phe Ala Gly Arg Arg Ala Asp Gly Arg Val Lys Lys         115 120 125 Val Leu Phe Pro Leu Ala Pro Glu Tyr Ile Gly Tyr Ala Asp Ala Gly     130 135 140 Leu Glu Glu Asp Leu Phe Val Ser Ala Arg Pro Asn Ile Glu Leu Leu 145 150 155 160 Pro Glu Gly Gln Phe Lys Tyr His Val Asp Phe Glu His Leu His Ile                 165 170 175 Gly Glu Glu Thr Gly Met Ile Cys Val Ser Arg Pro Thr Asn Pro Thr             180 185 190 Gly Asn Val Ile Thr Asp Glu Glu Leu Leu Lys Leu Asp Ala Leu Ala         195 200 205 Asn Gln His Gly Ile Pro Leu Val Ile Asp Asn Ala Tyr Gly Val Pro     210 215 220 Phe Pro Gly Ile Ile Phe Ser Glu Ala Arg Pro Leu Trp Asn Pro Asn 225 230 235 240 Ile Val Leu Cys Met Ser Leu Ser Lys Leu Gly Leu Pro Gly Ser Arg                 245 250 255 Cys Gly Ile Ile Ile Ala Asn Glu Lys Ile Ile Thr Ala Ile Thr Asn             260 265 270 Met Asn Gly Ile Ser Ser Leu Ala Pro Gly Gly Ile Gly Pro Ala Met         275 280 285 Met Cys Glu Met Ile Lys Arg Asn Asp Leu Leu Arg Leu Ser Glu Thr     290 295 300 Val Ile Lys Pro Phe Tyr Tyr Gln Arg Val Gln Glu Thr Ile Ala Ile 305 310 315 320 Ile Arg Arg Tyr Leu Pro Glu Asn Arg Cys Leu Ile His Lys Pro Glu                 325 330 335 Gly Ala Ile Phe Leu Trp Leu Trp Phe Lys Asp Leu Pro Ile Thr Thr             340 345 350 Lys Gln Leu Tyr Gln Arg Leu Lys Ala Arg Gly Val Leu Met Val Pro         355 360 365 Gly His Asn Phe Phe Pro Gly Leu Asp Lys Pro Trp Pro His Thr His     370 375 380 Gln Cys Met Arg Met Met Asn Tyr Val Pro Glu Pro Glu Lys Ile Glu Ala 385 390 395 400 Gly Val Lys Ile Leu Ala Glu Glu Ile Glu Arg Ala Trp Ala Glu Ser                 405 410 415 His      <210> 73 <211> 425 <212> PRT <213> Bacillus licheniformis <400> 73 Met Lys Pro Pro Leu Ser Lys Ile Gly Glu Lys Met Ile Glu Lys Thr 1 5 10 15 Gly Val Arg Ala Val Met Ser Asp Ile Gln Glu Val Leu Ala Gly Gly             20 25 30 Glu Arg Ser Tyr Ile Asn Leu Ser Ala Gly Asn Pro Met Ile Leu Pro         35 40 45 Gly Val Ser Ala Met Trp Lys Ser Ala Leu Ala Asp Leu Leu Asp Asp     50 55 60 Asp Arg Phe Ser Ser Val Ile Gly Gln Tyr Gly Ser Ser Tyr Gly Thr 65 70 75 80 Asp Glu Leu Ile Ala Ser Val Val Arg Phe Phe Ser Glu Arg Tyr Ser                 85 90 95 Ala Gly Ile Arg Lys Glu Asn Val Leu Ile Thr Ala Gly Ser Gln Gln             100 105 110 Leu Phe Phe Leu Ala Ile Asn Ser Phe Cys Gly Met Gly Ser Gly Ser         115 120 125 Val Met Lys Lys Ala Leu Ile Pro Met Leu Pro Asp Tyr Ser Gly Tyr     130 135 140 Ser Gly Ala Ala Leu Glu Glu Ile Pro Pro Leu 145 150 155 160 Ile Ser Lys Leu Asp Asp His Thr Phe Arg Tyr Glu Leu Asp Arg Lys                 165 170 175 Gly Phe Leu Glu Arg Met Ile Gly Ala Val Leu Leu Ser Arg Pro             180 185 190 Asn Asn Pro Cys Gly Asn Ile Leu Pro Lys Glu Asp Val Ala Phe Ile         195 200 205 Ser Asp Ala Cys Arg Glu Ala Asn Val Pro Leu Phe Ile Asp Ser Ala     210 215 220 Tyr Ala Pro Pro Phe Pro Ala Ile His Phe Ile Asp Met Glu Pro Ile 225 230 235 240 Phe Asn Glu Gln Ile Ile His Cys Met Ser Leu Ser Lys Ala Gly Leu                 245 250 255 Pro Gly Glu Arg Ile Gly Ile Ala Ile Gly Pro Ser Arg Tyr Ile Gln             260 265 270 Ala Met Glu Ala Phe Gln Ser Asn Ala Ala Ile His Ser Ser Arg         275 280 285 Gly Gln Tyr Met Ala Ala Ser Val Leu Asn Asp Gly Arg Leu Ala Asp     290 295 300 Val Ser Leu Asn Glu Val Arg Pro Tyr Tyr Arg Asn Lys Phe Met Leu 305 310 315 320 Leu Lys Glu Thr Leu Leu Cys Lys Met Pro Glu Asp Ile Lys Trp Tyr                 325 330 335 Leu His Gln Gly Glu Gly Ser Leu Phe Gly Trp Leu Trp Phe Glu Asp             340 345 350 Leu Pro Val Thr Asp Ala Leu Tyr Glu Tyr Met Lys Ala Asp Gly         355 360 365 Val Ile Ile Val Pro Gly Ser Ser Phe Phe His Arg Gln Ser Arg Arg     370 375 380 Leu Ala His Ser His Gln Cys Ile Arg Ile Ser Leu Thr Ala Ala Asp 385 390 395 400 Glu Asp Ile Ile Arg Gly Ile Asp Val Leu Ala Lys Ile Ala Lys Gly                 405 410 415 Val Tyr Glu Lys Gln Val Glu Tyr Leu             420 425 <210> 74 <211> 1278 <212> DNA <213> Bacillus licheniformis <400> 74 ttataagtat tcaacctgtt tctcatatac acccttcgca attttagcta aaacatcgat 60 tccccttata atatcttcat ccgccgcggt taggctgatt cgtatacact ggtgtgaatg 120 cgccaggcgc cgggattgac ggtgaaagaa agatgatccg ggaacgataa tgactccatc 180 cgctttcata tactcataca gcgctgcatc ggtcaccggc aggtcttcaa accacagcca 240 tccgaaaagc gatccttccc cttgatgcag ataccatttg atgtcttcag gcatcttgca 300 taaaagcgtt tccttgagca gcatgaattt attgcggtaa tatggcctga cttcattcag 360 cgacacgtcg gcgaggcgcc cgtcattcaa tactgatgca gccatatact gccccagcct 420 tgaagaatgg atcgccgcat tcgactgaaa agcttccatt gcctgaatat accgggacgg 480 cccgatggcg attccgatcc tttcgccagg caggccggct tttgaaaggc tcatacagtg 540 aatgatctgc tcgttgaaaa tcggttccat gtcgataaag tgaatcgccg gaaaaggcgg 600 agcatatgcg gaatcaatga acagcggaac attcgcttct cggcatgcgt ctgaaatgaa 660 tgctacatct tctttaggca agatgtttcc gcaaggattg ttcgggcgcg atagcaagac 720 agcaccgatg cgcatcctct ctaaaaaccc cttacggtcg agctcatatc gaaacgtatg 780 atcatccaat ttcgatatga gcggagggat cccctcaatc atctcccgct ccagtgccgc 840 cccgctgtat cccgaatagt caggcagcat cgggatcaag gcttttttca tcacagatcc 900 gcttcccatt ccgcaaaacg aattgatcgc cagaaaaaac agctgctggc ttccggctgt 960 aatcaacacg ttctcttttc gaatgccggc gctataccgc tctgaaaaga agcggacaac 1020 acttgcaatc agttcatcgg ttccatagct cgatccgtat tggccgatca ccgaagaaaa 1080 cctgtcatcg tcaaggagat cggcaagagc cgacttccac atggctgaca cgccgggcaa 1140 aatcatcgga ttgcccgcac ttaaattaat gtatgaccgt tcaccgccgg ccaggacttc 1200 ctgaatatcg ctcatcacag ccctgacccc tgttttctca atcattttct ctccgatttt 1260 gcttaatggc ggcttcac 1278 <210> 75 <211> 309 <212> PRT <213> Escherichia coli <400> 75 Met Thr Thr Lys Lys Ala Asp Tyr Ile Trp Phe Asn Gly Glu Met Val 1 5 10 15 Arg Trp Glu Asp Ala Lys Val His Val Met Ser His Ala Leu His Tyr             20 25 30 Gly Thr Ser Val Phe Glu Gly Ile Arg Cys Tyr Asp Ser His Lys Gly         35 40 45 Pro Val Val Phe Arg His Arg Glu His Met Gln Arg Leu His Asp Ser     50 55 60 Ala Lys Ile Tyr Arg Phe Pro Val Ser Gln Ser Ile Asp Glu Leu Met 65 70 75 80 Glu Ala Cys Arg Asp Val Ile Arg Lys Asn Asn Leu Thr Ser Ala Tyr                 85 90 95 Ile Arg Pro Leu Ile Phe Val Gly Asp Val Gly Met Gly Val Asn Pro             100 105 110 Pro Ala Gly Tyr Ser Thr Asp Val Ile Ile Ala Ala Phe Pro Trp Gly         115 120 125 Ala Tyr Leu Gly Ala Glu Ala Leu Glu Gln Gly Ile Asp Ala Met Val     130 135 140 Ser Ser Trp Asn Arg Ala Ala Pro Asn Thr Ile Pro Thr Ala Ala Lys 145 150 155 160 Ala Gly Gly Asn Tyr Leu Ser Ser Leu Leu Val Gly Ser Glu Ala Arg                 165 170 175 Arg His Gly Tyr Gln Glu Gly Ile Ala Leu Asp Val Asn Gly Tyr Ile             180 185 190 Ser Glu Gly Ala Gly Glu Asn Leu Phe Glu Val Lys Asp Gly Val Leu         195 200 205 Phe Thr Pro Pro Phe Thr Ser Ser Ala Leu Pro Gly Ile Thr Arg Asp     210 215 220 Ala Ile Ile Lys Leu Ala Lys Glu Leu Gly Ile Glu Val Arg Glu Gln 225 230 235 240 Val Leu Ser Arg Glu Ser Leu Tyr Leu Ala Asp Glu Val Phe Met Ser                 245 250 255 Gly Thr Ala Ala Glu Ile Thr Pro Val Arg Ser Val Asp Gly Ile Gln             260 265 270 Val Gly Glu Gly Arg Cys Gly Pro Val Thr Lys Arg Ile Gln Gln Ala         275 280 285 Phe Phe Gly Leu Phe Thr Gly Glu Thr Glu Asp Lys Trp Gly Trp Leu     290 295 300 Asp Gln Val Asn Gln 305 <210> 76 <211> 1476 <212> DNA <213> Escherichia coli <400> 76 atggctaact acttcaatac actgaatctg cgccagcagc tggcacagct gggcaaatgt 60 cgctttatgg gccgcgatga attcgccgat ggcgcgagct accttcaggg taaaaaagta 120 gtcatcgtcg gctgtggcgc acagggtctg aaccagggcc tgaacatgcg tgattctggt 180 ctcgatatct cctacgctct gcgtaaagaa gcgattgccg agaagcgcgc gtcctggcgt 240 aaagcgaccg aaaatggttt taaagtgggt acttacgaag aactgatccc acaggcggat 300 ctggtgatta acctgacgcc ggacaagcag cactctgatg tagtgcgcac cgtacagcca 360 ctgatgaaag acggcgcggc gctgggctac tcgcacggtt tcaacatcgt cgaagtgggc 420 gagcagatcc gtaaagatat caccgtagtg atggttgcgc cgaaatgccc aggcaccgaa 480 gtgcgtgaag agtacaaacg tgggttcggc gtaccgacgc tgattgccgt tcacccggaa 540 aacgatccga aaggcgaagg catggcgatt gccaaagcct gggcggctgc aaccggtggt 600 caccgtgcgg gtgtgctgga atcgtccttc gttgcggaag tgaaatctga cctgatgggc 660 gagcaaacca tcctgtgcgg tatgttgcag gctggctctc tgctgtgctt cgacaagctg 720 gtggaagaag gtaccgatcc agcatacgca gaaaaactga ttcagttcgg ttgggaaacc 780 atcaccgaag cactgaaaca gggcggcatc accctgatga tggaccgtct ctctaacccg 840 gcgaaactgc gtgcttatgc gctttctgaa cagctgaaag agatcatggc acccctgttc 900 cagaaacata tggacgacat catctccggc gaattctctt ccggtatgat ggcggactgg 960 gccaacgatg ataagaaact gctgacctgg cgtgaagaga ccggcaaaac cgcgtttgaa 1020 accgcgccgc agtatgaagg caaaatcggc gagcaggagt acttcgataa aggcgtactg 1080 atgattgcga tggtgaaagc gggcgttgaa ctggcgttcg aaaccatggt cgattccggc 1140 atcattgaag agtctgcata ttatgaatca ctgcacgagc tgccgctgat tgccaacacc 1200 atcgcccgta agcgtctgta cgaaatgaac gtggttatct ctgataccgc tgagtacggt 1260 aactatctgt tctcttacgc ttgtgtgccg ttgctgaaac cgtttatggc agagctgcaa 1320 ccgggcgacc tgggtaaagc tattccggaa ggcgcggtag ataacgggca actgcgtgat 1380 gtgaacgaag cgattcgcag ccatgcgatt gagcaggtag gtaagaaact gcgcggctat 1440 atgacagata tgaaacgtat tgctgttgcg ggttaa 1476 <210> 77 <211> 376 <212> PRT <213> Saccharomyces cerevisiae <400> 77 Met Thr Leu Ala Pro Leu Asp Ala Ser Lys Val Lys Ile Thr Thr Thr 1 5 10 15 Gln His Ala Ser Lys Pro Lys Pro Asn Ser Glu Leu Val Phe Gly Lys             20 25 30 Ser Phe Thr Asp His Met Leu Thr Ala Glu Trp Thr Ala Glu Lys Gly         35 40 45 Trp Gly Thr Pro Glu Ile Lys Pro Tyr Gln Asn Leu Ser Leu Asp Pro     50 55 60 Ser Ala Val Val Phe His Tyr Ala Phe Glu Leu Phe Glu Gly Met Lys 65 70 75 80 Ala Tyr Arg Thr Val Asp Asn Lys Ile Thr Met Phe Arg Pro Asp Met                 85 90 95 Asn Met Lys Arg Met Asn Lys Ser Ala Gln Arg Ile Cys Leu Pro Thr             100 105 110 Phe Asp Pro Glu Glu Leu Ile Thr Leu Ile Gly Lys Leu Ile Gln Gln         115 120 125 Asp Lys Cys Leu Val Pro Glu Gly Lys Gly Tyr Ser Leu Tyr Ile Arg     130 135 140 Pro Thr Leu Ile Gly Thr Thr Ala Gly Leu Gly Val Ser Thr Pro Asp 145 150 155 160 Arg Ala Leu Leu Tyr Val Ile Cys Cys Pro Val Gly Pro Tyr Tyr Lys                 165 170 175 Thr Gly Phe Lys Ala Val Arg Leu Glu Ala Thr Asp Tyr Ala Thr Arg             180 185 190 Ala Trp Pro Gly Gly Cys Gly Asp Lys Lys Leu Gly Ala Asn Tyr Ala         195 200 205 Pro Cys Val Leu Pro Gln Leu Gln Ala Ala Ser Arg Gly Tyr Gln Gln     210 215 220 Asn Leu Trp Leu Phe Gly Pro Asn Asn Ile Thr Glu Val Gly Thr 225 230 235 240 Met Asn Ala Phe Phe Val Phe Lys Asp Ser Lys Thr Gly Lys Lys Glu                 245 250 255 Leu Val Thr Ala Pro Leu Asp Gly Thr Ile Leu Glu Gly Val Thr Arg             260 265 270 Asp Ser Ile Leu Asn Leu Ala Lys Glu Arg Leu Glu Pro Ser Glu Trp         275 280 285 Thr Ile Ser Glu Arg Tyr Phe Thr Ile Gly Glu Val Thr Glu Arg Ser     290 295 300 Lys Asn Gly Glu Leu Leu Glu Ala Phe Gly Ser Gly Thr Ala Ala Ile 305 310 315 320 Val Ser Pro Ile Lys Glu Ile Gly Trp Lys Gly Glu Gln Ile Asn Ile                 325 330 335 Pro Leu Leu Pro Gly Glu Gln Thr Gly Pro Leu Ala Lys Glu Val Ala             340 345 350 Gln Trp Ile Asn Gly Ile Gln Tyr Gly Glu Thr Glu His Gly Asn Trp         355 360 365 Ser Arg Val Val Thr Asp Leu Asn     370 375 <210> 78 <211> 376 <212> PRT <213> Saccharomyces cerevisiae <400> 78 Met Thr Leu Ala Pro Leu Asp Ala Ser Lys Val Lys Ile Thr Thr Thr 1 5 10 15 Gln His Ala Ser Lys Pro Lys Pro Asn Ser Glu Leu Val Phe Gly Lys             20 25 30 Ser Phe Thr Asp His Met Leu Thr Ala Glu Trp Thr Ala Glu Lys Gly         35 40 45 Trp Gly Thr Pro Glu Ile Lys Pro Tyr Gln Asn Leu Ser Leu Asp Pro     50 55 60 Ser Ala Val Val Phe His Tyr Ala Phe Glu Leu Phe Glu Gly Met Lys 65 70 75 80 Ala Tyr Arg Thr Val Asp Asn Lys Ile Thr Met Phe Arg Pro Asp Met                 85 90 95 Asn Met Lys Arg Met Asn Lys Ser Ala Gln Arg Ile Cys Leu Pro Thr             100 105 110 Phe Asp Pro Glu Glu Leu Ile Thr Leu Ile Gly Lys Leu Ile Gln Gln         115 120 125 Asp Lys Cys Leu Val Pro Glu Gly Lys Gly Tyr Ser Leu Tyr Ile Arg     130 135 140 Pro Thr Leu Ile Gly Thr Thr Ala Gly Leu Gly Val Ser Thr Pro Asp 145 150 155 160 Arg Ala Leu Leu Tyr Val Ile Cys Cys Pro Val Gly Pro Tyr Tyr Lys                 165 170 175 Thr Gly Phe Lys Ala Val Arg Leu Glu Ala Thr Asp Tyr Ala Thr Arg             180 185 190 Ala Trp Pro Gly Gly Cys Gly Asp Lys Lys Leu Gly Ala Asn Tyr Ala         195 200 205 Pro Cys Val Leu Pro Gln Leu Gln Ala Ala Ser Arg Gly Tyr Gln Gln     210 215 220 Asn Leu Trp Leu Phe Gly Pro Asn Asn Ile Thr Glu Val Gly Thr 225 230 235 240 Met Asn Ala Phe Phe Val Phe Lys Asp Ser Lys Thr Gly Lys Lys Glu                 245 250 255 Leu Val Thr Ala Pro Leu Asp Gly Thr Ile Leu Glu Gly Val Thr Arg             260 265 270 Asp Ser Ile Leu Asn Leu Ala Lys Glu Arg Leu Glu Pro Ser Glu Trp         275 280 285 Thr Ile Ser Glu Arg Tyr Phe Thr Ile Gly Glu Val Thr Glu Arg Ser     290 295 300 Lys Asn Gly Glu Leu Leu Glu Ala Phe Gly Ser Gly Thr Ala Ala Ile 305 310 315 320 Val Ser Pro Ile Lys Glu Ile Gly Trp Lys Gly Glu Gln Ile Asn Ile                 325 330 335 Pro Leu Leu Pro Gly Glu Gln Thr Gly Pro Leu Ala Lys Glu Val Ala             340 345 350 Gln Trp Ile Asn Gly Ile Gln Tyr Gly Glu Thr Glu His Gly Asn Trp         355 360 365 Ser Arg Val Val Thr Asp Leu Asn     370 375 <210> 79 <211> 330 <212> PRT <213> Methanobacterium thermoautotrophicum <400> 79 Met Arg Leu Trp Arg Ala Leu Tyr Arg Pro Pro Thr Ile Thr Tyr Pro 1 5 10 15 Ser Lys Ser Pro Glu Val Ile Ile Met Ser Cys Glu Ala Ser Gly Lys             20 25 30 Ile Trp Leu Asn Gly Glu Met Val Glu Trp Glu Glu Ala Thr Val His         35 40 45 Val Leu Ser His Val Val His Tyr Gly Ser Ser Val Phe Glu Gly Ile     50 55 60 Arg Cys Tyr Arg Asn Ser Lys Gly Ser Ala Ile Phe Arg Leu Arg Glu 65 70 75 80 His Val Lys Arg Leu Phe Asp Ser Ala Lys Ile Tyr Arg Met Asp Ile                 85 90 95 Pro Tyr Thr Gln Glu Gln Ile Cys Asp Ala Ile Val Glu Thr Val Arg             100 105 110 Glu Asn Gly Leu Glu Glu Cys Tyr Ile Arg Pro Val Val Phe Arg Gly         115 120 125 Tyr Gly Glu Met Gly Val His Pro Val Asn Cys Pro Val Asp Val Ala     130 135 140 Val Ala Ala Trp Glu Trp Gly Ala Tyr Leu Gly Ala Glu Ala Leu Glu 145 150 155 160 Val Gly Val Asp Ala Gly Val Ser Thr Trp Arg Arg Met Ala Pro Asn                 165 170 175 Thr Met Pro Asn Met Ala Lys Ala Gly Gly Asn Tyr Leu Asn Ser Gln             180 185 190 Leu Ala Lys Met Glu Ala Val Arg His Gly Tyr Asp Glu Ala Ile Met         195 200 205 Leu Asp Tyr His Gly Tyr Ile Ser Glu Gly Ser Gly Glu Asn Ile Phe     210 215 220 Leu Val Ser Glu Gly Glu Ile Tyr Thr Pro Ser Val Ser Ser Leu 225 230 235 240 Leu Arg Gly Ile Thr Arg Asp Ser Val Ile Lys Ile Ala Arg Thr Glu                 245 250 255 Gly Val Thr Val His Glu Glu Pro Ile Thr Arg Glu Met Leu Tyr Ile             260 265 270 Ala Asp Glu Ala Phe Phe Thr Gly Thr Ala Ala Glu Ile Thr Pro Ile         275 280 285 Arg Ser Val Asp Gly Ile Glu Ile Gly Ala Gly Arg Arg Gly Pro Val     290 295 300 Thr Lys Leu Leu Gln Asp Glu Phe Phe Arg Ile Ile Arg Ala Glu Thr 305 310 315 320 Glu Asp Ser Phe Gly Trp Leu Thr Tyr Ile                 325 330 <210> 80 <211> 993 <212> DNA <213> Methanobacterium thermoautotrophicum <400> 80 tcagatgtag gtgagccatc cgaagctgtc ctctgtctct gccctgatta tcctgaagaa 60 ctcatcctgc agcagctttg taacgggacc ccttcgcccg gcacctatct ctataccatc 120 aactgatctg atgggtgtta tctctgcggc tgtacctgtg aagaaggcct catctgcgat 180 gtagagcatc tccctggtta tgggttcctc atgcacggta acaccctcgg tcctggctat 240 ctttattacg gagtcccttg ttatccccct cagaagggat gatgaaacag ggggggtgta 300 aatttcaccc tcactgacga ggaatatgtt ctccccgcta ccctcactta tgtagccatg 360 gtagtccagc attatggcct catcatagcc gtgtctcaca gcctccatct tggcaagctg 420 tgagttgagg tagttaccgc cggcctttgc catgttgggc attgtgtttg gtgccatcct 480 ccgccaggtt gaaacaccag catcgacacc aacctcaagg gcctctgcac ccagataggc 540 cccccattcc caggcagcca cagcgacgtc cactgggcag ttcaccgggt gaacacccat 600 ctcaccgtat cccctgaata ccacgggtct tatatagcac tcctcaagtc cgttctccct 660 gacggtctca actatggcat cacatatctg ctcctgggtg tagggtatgt ccatccggta 720 tatctttgca gaatcaaaaa ggcgtttaac atgctcccgc aaacggaaga tggctgaccc 780 cttactgttc ctgtagcacc ttattccctc aaagacagat gatccataat gcacaacatg 840 tgagagtacg tggacggtgg cttcttccca ttcaaccatt tcaccgttta accatatctt 900 tccactggct tcgcatgaca tgataataac ctcaggtgat ttactaggat aggttatggt 960 tggaggccta tataatgctc tccataaccg caa 993 <210> 81 <211> 364 <212> PRT <213> Streptomyces coelicolor <400> 81 Met Thr Asp Val Asn Gly Ala Pro Ala Asp Val Leu His Thr Leu Phe 1 5 10 15 His Ser Asp Gln Gly Gly His Glu Gln Val Val Leu Cys Gln Asp Arg             20 25 30 Ala Ser Gly Leu Lys Ala Le         35 40 45 Pro Ala Leu Gly Gly Thr Arg Phe Tyr Pro Tyr Ala Ser Glu Ala Glu     50 55 60 Ala Val Ala Asp Ala Leu Asn Leu Ala Arg Gly Met Ser Tyr Lys Asn 65 70 75 80 Ala Met Ala Gly Leu Asp His Gly Gly Gly Lys Ala Val Ile Ile Gly                 85 90 95 Asp Pro Glu Gln Ile Lys Ser Glu Glu Leu Leu Leu Ala Tyr Gly Arg             100 105 110 Phe Val Ala Ser Leu Gly Gly Arg Tyr Val Thr Ala Cys Asp Val Gly         115 120 125 Thr Tyr Val Ala Asp Met Asp Val Val Ala Arg Glu Cys Arg Trp Thr     130 135 140 Thr Gly Arg Ser Pro Glu Asn Gly Gly Ala Gly Asp Ser Ser Val Leu 145 150 155 160 Thr Ser Phe Gly Val Tyr Gln Gly Met Arg Ala Ala Gln His Leu                 165 170 175 Trp Gly Asp Pro Thr Leu Arg Asp Arg Thr Val Gly Ile Ala Gly Val             180 185 190 Gly Lys Val Gly His His Leu Val Glu His Leu Leu Ala Glu Gly Ala         195 200 205 His Val Val Thr Asp Val Arg Lys Asp Val Val Arg Gly Ile Thr     210 215 220 Glu Arg His Pro Ser Val Val Ala Val Ala Asp Thr Asp Ala Leu Ile 225 230 235 240 Arg Val Glu Asn Leu Asp Ile Tyr Ala Pro Cys Ala Leu Gly Gly Ala                 245 250 255 Leu Asn Asp Asp Thr Val Pro Val Leu Thr Ala Lys Val Val Cys Gly             260 265 270 Ala Ala Asn Asn Gln Leu Ala His Pro Gly Val Glu Lys Asp Leu Ala         275 280 285 Asp Arg Gly Ile Leu Tyr Ala Pro Asp Tyr Val Val Asn Ala Gly Gly     290 295 300 Val Ile Gln Val Ala Asp Glu Leu His Gly Phe Asp Phe Asp Arg Cys 305 310 315 320 Lys Ala Lys Ala Ser Lys Ile Tyr Asp Thr Thr Leu Ala Ile Phe Ala                 325 330 335 Arg Ala Lys Glu Asp Gly Ile Pro Pro Ala Ala Ala Ala Asp Arg Ile             340 345 350 Ala Glu Gln Arg Met Ala Glu Ala Arg Pro Arg Pro         355 360 <210> 82 <211> 1095 <212> DNA <213> Streptomyces coelicolor <400> 82 tcacggccgg ggacgggcct ccgccatccg ctgctcggcg atccggtcgg ccgccgcggc 60 cggcggaata ccgtcctcct tcgcacgtgc gaatatggcc agcgtggtgt cgtagatctt 120 cgaggccttc gccttgcacc ggtcgaagtc gaacccgtgc agctcgtcgg cgacctggat 180 gacaccgccg gcgttcacca catagtccgg cgcgtagagg atcccgcggt cggcgaggtc 240 cttctcgacg cccgggtggg cgagctggtt gttggccgcg ccgcacacca ccttggcggt 300 cagcaccggc acggtgtcgt cgttcagcgc gccgccgagc gcgcagggcg cgtagatgtc 360 caggttctcc acccggatca gcgcgtcggt gtcggcgacg gcgaccaccg acgggtgccg 420 ctccgtgatc ccgcgcacca cgtccttgcg cacgtccgtg acgacgacgt gggcgccctc 480 ggcgagcagg tgctcgacca ggtggtggcc gaccttgccg acgcccgcga tgccgacggt 540 gcggtcgcgc agcgtcgggt cgccccacag gtgctgggcg gcggcccgca tgccctggta 600 gacgccgaag gaggtgagca cggaggagtc gcccgcgccg ccgttctccg gggaacgccc 660 ggtcgtccag cggcactcgc gggccacgac gtccatgtcg gcgacgtagg tgccgacgtc 720 gcacgcggtg acgtagcggc cgcccagcga ggcgacgaac cggccgtagg cgaggagcag 780 ctcctcgctc ttgatctgct ccggatcgcc gatgatcacg gccttgccgc caccgtggtc 840 cagaccggcc atggcgttct tgtacgacat cccgcgggcg aggttcagcg cgtcggcgac 900 ggcctccgcc tcgctcgcgt acgggtagaa gcgggtaccg ccgagcgccg ggcccagggc 960 ggtggagtgg agggcgatca cggccttgag gccgctggca cggtcctggc agagcacgac 1020 ttgctcatgt cccccctgat ccgagtggaa cagggtgtgc agtacatcag caggtgcgcc 1080 gtttacgtcg gtcac 1095 <210> 83 <211> 364 <212> PRT <213> Bacillus subtilis <400> 83 Met Glu Leu Phe Lys Tyr Met Glu Lys Tyr Asp Tyr Glu Gln Leu Val 1 5 10 15 Phe Cys Gln Asp Glu Gln Ser Gly Leu Lys Ala Ile Ala Ile His             20 25 30 Asp Thr Thr Leu Gly Pro Ala Leu Gly Gly Thr Arg Met Trp Thr Tyr         35 40 45 Glu Asn Glu Glu Ala Glu Asp Ala Leu Arg Leu Ala Arg Gly     50 55 60 Met Thr Tyr Lys Asn Ala Ala Gly Leu Asn Leu Gly Gly Gly Lys 65 70 75 80 Thr Val Ile Ile Gly Asp Pro Arg Lys Asp Lys Asn Glu Glu Met Phe                 85 90 95 Arg Ala Phe Gly Arg Tyr Ile Gln Gly Leu Asn Gly Arg Tyr Ile Thr             100 105 110 Ala Glu Asp Val Gly Thr Thr Val Asp Met Asp Ile Ile His Asp         115 120 125 Glu Thr Asp Tyr Val Thr Gly Ile Ser Pro Ala Phe Gly Ser Ser Gly     130 135 140 Asn Pro Ser Pro Val Thr Ala Tyr Gly Val Tyr Arg Gly Met Lys Ala 145 150 155 160 Ala Ala Lys Ala Ala Phe Gly Thr Asp Ser Leu Glu Gly Lys Thr Ile                 165 170 175 Ala Val Gln Gly Val Gly Asn Val Ala Tyr Asn Leu Cys Arg His Leu             180 185 190 His Glu Glu Gly Ala Asn Leu Ile Val Thr Asp Ile Asn Lys Gln Ser         195 200 205 Val Gln Arg Ala Val Glu Asp Phe Gly Ala Arg Ala Val Asp Pro Asp     210 215 220 Asp Ile Tyr Ser Gln Asp Cys Asp Ile Tyr Ala Pro Cys Ala Leu Gly 225 230 235 240 Ala Thr Ile Asn Asp Asp Thr Ile Lys Gln Leu Lys Ala Lys Val Ile                 245 250 255 Ala Gly Ala Ala Asn Asn Gln Leu Lys Glu Thr Arg His Gly Asp Gln             260 265 270 Ile His Glu Met Gly Ile Val Tyr Ala Pro Asp Tyr Val Ile Asn Ala         275 280 285 Gly Gly Val Ile Asn Val Ala Asp Glu Leu Tyr Gly Tyr Asn Ala Glu     290 295 300 Arg Ala Leu Lys Lys Val Glu Gly Ile Tyr Gly Asn Ile Glu Arg Val 305 310 315 320 Leu Glu Ile Ser Gln Arg Asp Gly Ile Pro Ala Tyr Leu Ala Ala Asp                 325 330 335 Arg Leu Ala Glu Glu Arg Ile Glu Arg Met Arg Arg Ser Ser Arg Ser Gln             340 345 350 Phe Leu Gln Asn Gly His Ser Val Leu Ser Arg Arg         355 360 <210> 84 <211> 364 <212> PRT <213> Bacillus subtilis <400> 84 Met Glu Leu Phe Lys Tyr Met Glu Lys Tyr Asp Tyr Glu Gln Leu Val 1 5 10 15 Phe Cys Gln Asp Glu Gln Ser Gly Leu Lys Ala Ile Ala Ile His             20 25 30 Asp Thr Thr Leu Gly Pro Ala Leu Gly Gly Thr Arg Met Trp Thr Tyr         35 40 45 Glu Asn Glu Glu Ala Glu Asp Ala Leu Arg Leu Ala Arg Gly     50 55 60 Met Thr Tyr Lys Asn Ala Ala Gly Leu Asn Leu Gly Gly Gly Lys 65 70 75 80 Thr Val Ile Ile Gly Asp Pro Arg Lys Asp Lys Asn Glu Glu Met Phe                 85 90 95 Arg Ala Phe Gly Arg Tyr Ile Gln Gly Leu Asn Gly Arg Tyr Ile Thr             100 105 110 Ala Glu Asp Val Gly Thr Thr Val Asp Met Asp Ile Ile His Asp         115 120 125 Glu Thr Asp Tyr Val Thr Gly Ile Ser Pro Ala Phe Gly Ser Ser Gly     130 135 140 Asn Pro Ser Pro Val Thr Ala Tyr Gly Val Tyr Arg Gly Met Lys Ala 145 150 155 160 Ala Ala Lys Ala Ala Phe Gly Thr Asp Ser Leu Glu Gly Lys Thr Ile                 165 170 175 Ala Val Gln Gly Val Gly Asn Val Ala Tyr Asn Leu Cys Arg His Leu             180 185 190 His Glu Glu Gly Ala Asn Leu Ile Val Thr Asp Ile Asn Lys Gln Ser         195 200 205 Val Gln Arg Ala Val Glu Asp Phe Gly Ala Arg Ala Val Asp Pro Asp     210 215 220 Asp Ile Tyr Ser Gln Asp Cys Asp Ile Tyr Ala Pro Cys Ala Leu Gly 225 230 235 240 Ala Thr Ile Asn Asp Asp Thr Ile Lys Gln Leu Lys Ala Lys Val Ile                 245 250 255 Ala Gly Ala Ala Asn Asn Gln Leu Lys Glu Thr Arg His Gly Asp Gln             260 265 270 Ile His Glu Met Gly Ile Val Tyr Ala Pro Asp Tyr Val Ile Asn Ala         275 280 285 Gly Gly Val Ile Asn Val Ala Asp Glu Leu Tyr Gly Tyr Asn Ala Glu     290 295 300 Arg Ala Leu Lys Lys Val Glu Gly Ile Tyr Gly Asn Ile Glu Arg Val 305 310 315 320 Leu Glu Ile Ser Gln Arg Asp Gly Ile Pro Ala Tyr Leu Ala Ala Asp                 325 330 335 Arg Leu Ala Glu Glu Arg Ile Glu Arg Met Arg Arg Ser Ser Arg Ser Gln             340 345 350 Phe Leu Gln Asn Gly His Ser Val Leu Ser Arg Arg         355 360 <210> 85 <211> 594 <212> PRT <213> Streptomyces viridifaciens <400> 85 Met Ser Thr Ser Ser Ala Ser Ser Gly Pro Asp Leu Pro Phe Gly Pro 1 5 10 15 Glu Asp Thr Pro Trp Gln Lys Ala Phe Ser Arg Leu Arg Ala Val Asp             20 25 30 Gly Val Pro Arg Val Thr Ala Pro Ser Ser Asp Pro Arg Glu Val Tyr         35 40 45 Met Asp Ile Pro Glu Ile Pro Phe Ser Lys Val Gln Ile Pro Pro Asp     50 55 60 Gly Met Asp Glu Gln Gln Tyr Ala Glu Ala Glu Ser Leu Phe Arg Arg 65 70 75 80 Tyr Val Asp Ala Gln Thr Arg Asn Phe Ala Gly Tyr Gln Val Thr Ser                 85 90 95 Asp Leu Asp Tyr Gln His Leu Ser His Tyr Leu Asn Arg His Leu Asn             100 105 110 Asn Val Gly Asp Pro Tyr Glu Ser Ser Ser Tyr Thr Leu Asn Ser Lys         115 120 125 Val Leu Glu Arg Ala Val Leu Asp Tyr Phe Ala Ser Leu Trp Asn Ala     130 135 140 Lys Trp Pro His Asp Ala Ser Asp Pro Glu Thr Tyr Trp Gly Tyr Val 145 150 155 160 Leu Thr Met Gly Ser Ser Glu Gly Asn Leu Tyr Gly Leu Trp Asn Ala                 165 170 175 Arg Asp Tyr Leu Ser Gly Lys Leu Leu Arg Arg Gln His Arg Glu Ala             180 185 190 Gly Gly Asp Lys Ala Ser Val Val Tyr Thr Gln Ala Leu Arg His Glu         195 200 205 Gly Gln Ser Pro His Ala Tyr Glu Pro Val Ala Phe Ser Ser Gln Asp     210 215 220 Thr His Tyr Ser Leu Thr Lys Ala Val Arg Val Leu Gly Ile Asp Thr 225 230 235 240 Phe His Ser Ile Gly Ser Ser Arg Tyr Pro Asp Glu Asn Pro Leu Gly                 245 250 255 Pro Gly Thr Pro Trp Pro Thr Gly Val Pro Ser Val Asp Gly Ala Ile             260 265 270 Asp Val Asp Lys Leu Ala Ser Leu Val Arg Phe Phe Ala Ser Lys Gly         275 280 285 Tyr Pro Ile Leu Val Ser Leu Asn Tyr Gly Ser Thr Phe Lys Gly Ala     290 295 300 Tyr Asp Asp Val Pro Ala Val Ala Gln Ala Val Arg Asp Ile Cys Thr 305 310 315 320 Glu Tyr Gly Leu Asp Arg Arg Arg Val Tyr His Asp Arg Ser Lys Asp                 325 330 335 Ser Asp Phe Asp Glu Arg Ser Gly Phe Trp Ile His Ile Asp Ala Ala             340 345 350 Leu Gly Ala Gly Tyr Ala Pro Tyr Leu Gln Met Ala Arg Asp Ala Gly         355 360 365 Met Val Glu Glu Ala Pro Pro Val Phe Asp Phe Arg Leu Pro Glu Val     370 375 380 His Ser Leu Thr Met Ser Gly His Lys Trp Met Gly Thr Pro Trp Ala 385 390 395 400 Cys Gly Val Tyr Met Thr Arg Thr Gly Leu Gln Met Thr Pro Pro Lys                 405 410 415 Ser Ser Glu Tyr Ile Gly Ala Ala Asp Thr Thr Phe Ala Gly Ser Arg             420 425 430 Asn Gly Phe Ser Leu Leu Leu Trp Asp Tyr Leu Ser Arg His Ser         435 440 445 Tyr Asp Asp Leu Val Arg Leu Ala Ala Asp Cys Asp Arg Leu Ala Gly     450 455 460 Tyr Ala His Asp Arg Leu Leu Thr Leu Gln Asp Lys Leu Gly Met Asp 465 470 475 480 Leu Trp Val Ala Arg Ser Pro Gln Ser Leu Thr Val Arg Phe Arg Gln                 485 490 495 Pro Cys Ala Asp Ile Val Arg Lys Tyr Ser Leu Ser Cys Glu Thr Val             500 505 510 Tyr Glu Asp Asn Glu Gln Arg Thr Tyr Val His Leu Tyr Ala Val Pro         515 520 525 His Leu Thr Arg Glu Leu Val Asp Glu Leu Val Arg Asp Leu Arg Gln     530 535 540 Pro Gly Ala Phe Thr Asn Ala Glu Ala Leu Glu Gly Glu Ala Trp Ala 545 550 555 560 Gly Val Ile Asp Ala Leu Gly Arg Pro Asp Pro Asp Gly Thr Tyr Ala                 565 570 575 Gly Ala Leu Ser Ala Pro Ala Ser Gly Pro Arg Ser Glu Asp Gly Gly             580 585 590 Gly Ser          <210> 86 <211> 1785 <212> DNA <213> Streptomyces viridifaciens <400> 86 gtgtcaactt cctccgcttc ttccgggccg gacctcccct tcgggcccga ggacacgcca 60 tggcagaagg ccttcagcag gctgcgggcg gtggatggcg tgccgcgcgt caccgcgccg 120 tccagtgatc cgcgtgaggt ctacatggac atcccggaga tccccttctc caaggtccag 180 atccccccgg acggaatgga cgagcagcag tacgcagagg ccgagagcct cttccgccgc 240 tacgtagacg cccagacccg caacttcgcg ggataccagg tcaccagcga cctcgactac 300 cagcacctca gtcactatct caaccggcat ctgaacaacg tcggcgatcc ctatgagtcc 360 agctcctaca cgctgaactc caaggtcctt gagcgagccg ttctcgacta cttcgcctcc 420 ctgtggaacg ccaagtggcc ccatgacgca agcgatccgg aaacgtactg gggttacgtg 480 ctgaccatgg gctccagcga aggcaacctg tacgggttgt ggaacgcacg ggactatctg 540 tcgggcaagc tgctgcggcg ccagcaccgg gaggccggcg gcgacaaggc ctcggtcgtc 600 tacacgcaag cgctgcgaca cgaagggcag agtccgcatg cctacgagcc ggtggcgttc 660 ttctcgcagg acacgcacta ctcgctcacg aaggccgtgc gggttctggg catcgacacc 720 ttccacagca tcggcagcag tcggtatccg gacgagaacc cgctgggccc cggcactccg 780 tggccgaccg aagtgccctc ggttgacggt gccatcgatg tcgacaaact cgcctcgttg 840 gtccgcttct tcgccagcaa gggctacccg atactggtca gcctcaacta cgggtcaacg 900 ttcaagggcg cctacgacga cgtcccggcc gtggcacagg ccgtgcggga catctgcacg 960 gaatacggtc tggatcggcg gcgggtatac cacgaccgca gtaaggacag tgacttcgac 1020 gagcgcagcg gcttctggat ccacatcgat gccgccctgg gggcgggcta cgctccctac 1080 ctgcagatgg cccgggatgc cggcatggtc gaggaggcgc cgcccgtttt cgacttccgg 1140 ctcccggagg tgcactcgct gaccatgagc ggccacaagt ggatgggaac accgtgggca 1200 tgcggtgtct acatgacacg gaccgggctg cagatgaccc cgccgaagtc gtccgagtac 1260 atcggggcgg ccgacaccac cttcgcgggc tcccgcaacg gcttctcgtc actgctgctg 1320 tgggactacc tgtcccggca ttcgtatgac gatctggtgc gcctggccgc cgactgcgac 1380 cggctggccg gctacgccca cgaccggttg ctgaccttgc aggacaaact cggcatggat 1440 ctgtgggtcg cccgcagccc gcagtccctc acggtgcgct tccgtcagcc atgtgcagac 1500 atcgtccgca agtactcgct gtcgtgtgag acggtctacg aagacaacga gcaacggacc 1560 tacgtacatc tctacgccgt tccccacctc actcgggaac tcgtggatga gctcgtgcgc 1620 gatctgcgcc agcccggagc cttcaccaac gctggtgcac tggaggggga ggcctgggcc 1680 ggggtgatcg atgccctcgg ccgcccggac cccgacggaa cctatgccgg cgccttgagc 1740 gctccggctt ccggcccccg ctccgaggac ggcggcggga gctga 1785 <210> 87 <211> 440 <212> PRT <213> Alcaligenes denitrificans <400> 87 Met Ser Ala Ala Lys Leu Pro Asp Leu Ser His Leu Trp Met Pro Phe 1 5 10 15 Thr Ala Asn Arg Gln Phe Lys Ala Asn Pro Arg Leu Leu Ala Ser Ala             20 25 30 Lys Gly Met Tyr Tyr Thr Ser Phe Asp Gly Arg Gln Ile Leu Asp Gly         35 40 45 Thr Ala Gly Leu Trp Cys Val Asn Ala Gly His Cys Arg Glu Glu Ile     50 55 60 Val Ser Ala Ile Ala Ser Gln Ala Gly Val Met Asp Tyr Ala Pro Gly 65 70 75 80 Phe Gln Leu Gly His Pro Leu Ala Phe Glu Ala Ala Thr Ala Val Ala                 85 90 95 Gly Leu Met Pro Gln Gly Leu Asp Arg Val Phe Phe Thr Asn Ser Gly             100 105 110 Ser Glu Ser Val Asp Thr Ala Leu Lys Ile Ala Leu Ala Tyr His Arg         115 120 125 Ala Arg Gly Glu Ala Gln Arg Thr Arg Leu Ile Gly Arg Glu Arg Gly     130 135 140 Tyr His Gly Val Gly Phe Gly Gly Ile Ser Val Gly Gly Ile Ser Pro 145 150 155 160 Asn Arg Lys Thr Phe Ser Gly Ala Leu Leu Pro Ala Val Asp His Leu                 165 170 175 Pro His Thr His Ser Leu Glu His Asn Ala Phe Thr Arg Gly Gln Pro             180 185 190 Glu Trp Gly Ala His Leu Ala Asp Glu Leu Glu Arg Ile Ile Ala Leu         195 200 205 His Asp Ala Ser Thr Ile Ala Ala Val Ile Val Glu Pro Met Ala Gly     210 215 220 Ser Thr Gly Val Leu Val Pro Lys Gly Tyr Leu Glu Lys Leu Arg 225 230 235 240 Glu Ile Thr Ala Arg His Gly Ile Leu Leu Ile Phe Asp Glu Val Ile                 245 250 255 Thr Ala Tyr Gly Arg Glu Aly Ala Ala Tyr Phe Gly             260 265 270 Val Thr Pro Asp Leu Ile Thr Met Ala Lys Gly Val Ser Asn Ala Ala         275 280 285 Val Pro Ala Gly Ala Val Ala Val Arg Arg Glu Val His Asp Ala Ile     290 295 300 Val Asn Gly Pro Gln Gly Gly Ile Glu Phe Phe His Gly Tyr Thr Tyr 305 310 315 320 Ser Ala His Pro Leu Ala Ala Ala Val Leu Ala Thr Leu Asp Ile                 325 330 335 Tyr Arg Arg Glu Asp Leu Phe Ala Arg Ala Arg Lys Leu Ser Ala Ala             340 345 350 Phe Glu Glu Ala Ala His Ser Leu Lys Gly Ala Pro His Val Ile Asp         355 360 365 Val Arg Asn Ile Gly Leu Val Ala Gly Ile Glu Leu Ser Pro Arg Glu     370 375 380 Gly Ala Pro Gly Ala Arg Ala Ala Glu Ala Phe Gln Lys Cys Phe Asp 385 390 395 400 Thr Gly Leu Met Val Arg Tyr Thr Gly Asp Ile Leu Ala Val Ser Pro                 405 410 415 Pro Leu Ile Val Asp Glu Asn Gln Ile Gly Gln Ile Phe Glu Gly Ile             420 425 430 Gly Lys Val Leu Lys Glu Val Ala         435 440 <210> 88 <211> 1947 <212> DNA <213> Alcaligenes denitrificans <400> 88 ttcgatggcg cgctgcacgg cggccaccag ctgctccacc aggggtgggc gcctgcccgc 60 gcgcgcggtc gggctggaaa tcgatcatgg atgaatctat acagttgtca tgattgcaac 120 tatacagtta gcccgttttg cggcaattgt atattttcat tcgctcgtgg acgtccgaga 180 atcggtttga tcgcgccgcc cgcccctttc cgcgcagcgg cgtttctttt cctccggagt 240 ctccccatga gcgctgccaa actgcccgac ctgtcccacc tctggatgcc ctttaccgcc 300 aaccggcagt tcaaggcgaa cccccgcctg ctggcctcgg ccaagggcat gtactacacg 360 tctttcgacg gccgccagat cctggacggc acggccggcc tgtggtgcgt gaacgccggc 420 cactgccgcg aagaaatcgt ctccgccatc gccagccagg ccggcgtcat ggactacgcg 480 ccggggttcc agctcggcca cccgctggcc ttcgaggccg ccaccgccgt ggccggcctg 540 atgccgcagg gcctggaccg cgtgttcttc accaattcgg gctccgaatc ggtggacacc 600 gcgctgaaga tcgccctggc ctaccaccgc gcgcgcggcg aggcgcagcg cacccgcctc 660 atcgggcgcg agcgcggcta ccacggcgtg ggcttcggcg gcatttccgt gggcggcatc 720 tcgcccaacc gcaagacctt ctccggcgcg ctgctgccgg ccgtggacca cctgccgcac 780 acccacagcc tggaacacaa cgccttcacg cgcggccagc ccgagtgggg cgcgcacctg 840 gccgacgagt tggaacgcat catcgccctg cacgacgcct ccaccatcgc ggccgtgatc 900 gtcgagccca tggccggctc caccggcgtg ctcgtcccgc ccaagggcta tctcgaaaaa 960 ctgcgcgaaa tcaccgcccg ccacggcatt ctgctgatct tcgacgaagt catcaccgcg 1020 tacggccgcc tgggcgaggc caccgccgcg gcctatttcg gcgtaacgcc cgacctcatc 1080 accatggcca agggcgtgag caacgccgcc gttccggccg gcgccgtcgc ggtgcgccgc 1140 gaagtgcatg acgccatcgt caacggaccg caaggcggca tcgagttctt ccacggctac 1200 acctactcgg cccacccgct ggccgccgcc gccgtgctcg ccacgctgga catctaccgc 1260 cgcgaagacc tgttcgcccg cgcccgcaag ctgtcggccg cgttcgagga agccgcccac 1320 agcctcaagg gcgcgccgca cgtcatcgac gtgcgcaaca tcggcctggt ggccggcatc 1380 gagctgtcgc cgcgcgaagg cgccccgggc gcgcgcgccg ccgaagcctt ccagaaatgc 1440 ttcgacaccg gcctcatggt gcgctacacg ggcgacatcc tcgcggtgtc gcctccgctc 1500 atcgtcgacg aaaaccagat cggccagatc ttcgagggca tcggcaaggt gctcaaggaa 1560 gtggcttagg gtgaacacgc cctgagccgg ccccggcagg aaacgcgccg ccgcgcggcg 1620 gcgcgtccat cgaactcccg catcgagctt ttgcattcat gaagaaaatc acgcatttca 1680 tcaacggcca gccccacgaa ggccgcagca accgctacac cgagggcttc aacccggcca 1740 cgggcgagtc gtctcctcga tctgcctggg cggggccgaa gaagtggacc tggccgtggc 1800 ggccgcccgc gcggcctttc ccgcctggtc cgaaacgccg gcgctcaagc gcgcgcgcgt 1860 gctgttcaac ttcaaggcgc tgctggacaa gcaccaggac gagctggccg cgctcatcac 1920 gcgcgagcac ggcaaggtgt tttccga 1947 <210> 89 <211> 443 <212> PRT <213> Ralstonia eutropha <400> 89 Met Asp Ala Ala Lys Thr Val Ile Pro Asp Leu Asp Ala Leu Trp Met 1 5 10 15 Pro Phe Thr Ala Asn Arg Gln Tyr Lys Ala Ala Pro Arg Leu Leu Ala             20 25 30 Ser Ala Ser Gly Met Tyr Tyr Thr Thr His Asp Gly Arg Gln Ile Leu         35 40 45 Asp Gly Cys Ala Gly Leu Trp Cys Val Ala Gly His Cys Arg Lys     50 55 60 Glu Ile Ala Glu Ala Val Ala Arg Gln Ala Ala Thr Leu Asp Tyr Ala 65 70 75 80 Pro Pro Phe Gln Met Gly His Pro Leu Ser Phe Glu Ala Ala Thr Lys                 85 90 95 Val Ala Ile Met Pro Gln Gly Leu Asp Arg Ile Phe Phe Thr Asn             100 105 110 Ser Gly Ser Glu Ser Val Asp Thr Ala Leu Lys Ile Ala Leu Ala Tyr         115 120 125 His Arg Ala Arg Gly Glu Gly Gln Arg Thr Arg Phe Ile Gly Arg Glu     130 135 140 Arg Gly Tyr His Gly Val Gly Phe Gly Gly Met Ala Val Gly Gly Ile 145 150 155 160 Gly Pro Asn Arg Lys Ala Phe Ser Ala Asn Leu Met Pro Gly Thr Asp                 165 170 175 His Leu Pro Ala Thr Leu Asn Ile Ala Glu Ala Ala Phe Ser Lys Gly             180 185 190 Gln Pro Thr Trp Gly Ala His Leu Ala Asp Glu Leu Glu Arg Ile Val         195 200 205 Ala Leu His Asp Pro Ser Thr Ile Ala Ala Val Ile Val Glu Pro Leu     210 215 220 Ala Gly Ser Ala Gly Val Leu Val Pro Pro Val Gly Tyr Leu Asp Lys 225 230 235 240 Leu Arg Glu Ile Thr Thr Lys His Gly Ile Leu Leu Ile Phe Asp Glu                 245 250 255 Val Ile Thr Ala Phe Gly Arg Leu Gly Thr Ala Thr Ala Ala Glu Arg             260 265 270 Phe Lys Val Thr Pro Asp Leu Ile Thr Met Ala Lys Ala Ile Asn Asn         275 280 285 Ala Ala Val Met Gly Ala Val Ala Val Arg Arg Glu Val His Asp     290 295 300 Thr Val Val Asn Ser Ala Ala Pro Gly Ala Ile Glu Leu Ala His Gly 305 310 315 320 Tyr Thr Tyr Ser Gly His Pro Leu Ala Ala Ala Ala Ile Ala Thr                 325 330 335 Leu Asp Leu Tyr Gln Arg Glu Asn Leu Phe Gly Arg Ala Ala Glu Leu             340 345 350 Ser Pro Val Phe Glu Ala Ala Val His Ser Val Arg Ser Ala Pro His         355 360 365 Val Lys Asp Ile Arg Asn Leu Gly Met Val Ala Gly Ile Glu Leu Glu     370 375 380 Pro Arg Pro Gly Gln Pro Gly Ala Arg Ala Tyr Glu Ala Phe Leu Lys 385 390 395 400 Cys Leu Glu Arg Gly Val Leu Val Arg Tyr Thr Gly Asp Ile Leu Ala                 405 410 415 Phe Ser Pro Pro Leu Ile Ile Ser Glu Ala Gln Ile Ala Glu Leu Phe             420 425 430 Asp Thr Val Lys Gln Ala Leu Gln Glu Val Gln         435 440 <210> 90 <211> 1341 <212> DNA <213> Ralstonia eutropha <400> 90 atggccgact cacccaacaa cctcgctcac gaacatcctt cacttgaaca ctattggatg 60 ccttttaccg ccaatcgcca attcaaagcg agccctcgtt tactcgccca agctgaaggt 120 atgtattaca cagatatcaa tggcaacaag gtattagact ctacagcggg cttatggtgt 180 tgtaatgctg gccatggtcg ccgtgagatc agtgaagccg tcagcaaaca aattcggcag 240 atggattacg ctccctcctt ccaaatgggc catcccatcg cttttgaact ggccgaacgt 300 ttaaccgaac tcagcccaga aggactcaac aaagtattct ttaccaactc aggctctgag 360 tcggttgata ccgcgctaaa aatggctctt tgctaccata gagccaatgg ccaagcgtca 420 cgcacccgct ttattggccg tgaaatgggt taccatggcg taggatttgg tgggatctcg 480 gtgggtggtt taagcaataa ccgtaaagcc ttcagcggcc agctattgca aggcgtggat 540 cacctgcccc acaccttaga cattcaacat gccgccttta gtcgtggctt accgagcctc 600 ggtgctgaaa aagctgaggt attagaacaa ttagtcacac tccatggcgc cgaaaatatt 660 gccgccgtta ttgttgaacc catgtcaggt tctgcagggg taattttacc acctcaaggc 720 tacttaaaac gcttacgtga aatcactaaa aaacacggca tcttattgat tttcgatgaa 780 gtcattaccg catttggccg tgtaggtgca gcattcgcca gccaacgttg gggcgttatt 840 ccagacataa tcaccacggc taaagccatt aataatggcg ccatccccat gggcgcagtg 900 tttgtacagg attatatcca cgatacttgc atgcaagggc caaccgaact gattgaattt 960 ttccacggtt atacctattc gggccaccca gtcgccgcag cagcagcact cgccacgctc 1020 tccatctacc aaaacgagca actgtttgag cgcagttttg agcttgagcg gtatttcgaa 1080 gaagccgttc atagcctcaa agggttaccg aatgtgattg atattcgcaa caccggatta 1140 gtcgcgggtt tccagctagc accgaatagc caaggtgttg gtaaacgcgg atacagcgtg 1200 ttcgagcatt gtttccatca aggcacactc gtgcgggcaa cgggcgatat tatcgccatg 1260 tccccaccac tcattgttga gaaacatcag attgaccaaa tggtaaatag ccttagcgat 1320 gcaattcacg ccgttggatg a 1341 <210> 91 <211> 446 <212> PRT <213> Shewanella oneidensis <400> 91 Met Ala Asp Ser Pro Asn Asn Leu Ala His Glu His Pro Ser Leu Glu 1 5 10 15 His Tyr Trp Met Pro Phe Thr Ala Asn Arg Gln Phe Lys Ala Ser Pro             20 25 30 Arg Leu Leu Ala Gln Ala Glu Gly Met Tyr Tyr Thr Asp Ile Asn Gly         35 40 45 Asn Lys Val Leu Asp Ser Thr Ala Gly Leu Trp Cys Cys Asn Ala Gly     50 55 60 His Gly Arg Arg Glu Ile Ser Glu Ala Val Ser Lys Gln Ile Arg Gln 65 70 75 80 Met Asp Tyr Ala Pro Ser Phe Gln Met Gly His Pro Ile Ala Phe Glu                 85 90 95 Leu Ala Glu Arg Leu Thr Glu Leu Ser Pro Glu Gly Leu Asn Lys Val             100 105 110 Phe Phe Thr Asn Ser Gly Ser Glu Ser Val Asp Thr Ala Leu Lys Met         115 120 125 Ala Leu Cys Tyr His Arg Ala Asn Gly Gln Ala Ser Arg Thr Arg Phe     130 135 140 Ile Gly Arg Glu Met Gly Tyr His Gly Val Gly Phe Gly Gly Ile Ser 145 150 155 160 Val Gly Gly Leu Ser Asn Asn Arg Lys Ala Phe Ser Gly Gln Leu Leu                 165 170 175 Gln Gly Val Asp His Leu Pro His Thr Leu Asp Ile Gln His Ala Ala             180 185 190 Phe Ser Arg Gly Leu Pro Ser Leu Gly Ala Glu Lys Ala Glu Val Leu         195 200 205 Glu Gln Leu Val Thr Leu His Gly Ala Glu Asn Ile Ala Ala Val Ile     210 215 220 Val Glu Pro Met Ser Gly Ser Ala Gly Val Ile Leu Pro Pro Gln Gly 225 230 235 240 Tyr Leu Lys Arg Leu Arg Glu Ile Thr Lys Lys His Gly Ile Leu Leu                 245 250 255 Ile Phe Asp Glu Val Ile Thr Ala Phe Gly             260 265 270 Ala Ser Gln Arg Trp Gly Val Ile Pro Asp Ile Ile Thr Thr Ala Lys         275 280 285 Ala Ile Asn Asn Gly Ala Ile Pro Met Gly Ala Val Phe Val Gln Asp     290 295 300 Tyr Ile His Asp Thr Cys Met Gln Gly Pro Thr Glu Leu Ile Glu Phe 305 310 315 320 Phe His Gly Tyr Thr Tyr Ser Gly His Pro Val Ala Ala Ala Ala Ala                 325 330 335 Leu Ala Thr Leu Ser Ile Tyr Gln Asn Glu Gln Leu Phe Glu Arg Ser             340 345 350 Phe Glu Leu Glu Arg Tyr Phe Glu Glu Ala Val His Ser Leu Lys Gly         355 360 365 Leu Pro Asn Val Ile Asp Ile Arg Asn Thr Gly Leu Val Ala Gly Phe     370 375 380 Gln Leu Ala Pro Asn Ser Gln Gly Val Gly Lys Arg Gly Tyr Ser Val 385 390 395 400 Phe Glu His Cys Phe His Gln Gly Thr Leu Val Arg Ala Thr Gly Asp                 405 410 415 Ile Ile Ala Met Ser Pro Pro Leu Ile Val Glu Lys His Gln Ile Asp             420 425 430 Gln Met Val Asn Ser Leu Ser Asp Ala Ile His Ala Val Gly         435 440 445 <210> 92 <211> 1341 <212> DNA <213> Shewanella oneidensis <400> 92 atggccgact cacccaacaa cctcgctcac gaacatcctt cacttgaaca ctattggatg 60 ccttttaccg ccaatcgcca attcaaagcg agccctcgtt tactcgccca agctgaaggt 120 atgtattaca cagatatcaa tggcaacaag gtattagact ctacagcggg cttatggtgt 180 tgtaatgctg gccatggtcg ccgtgagatc agtgaagccg tcagcaaaca aattcggcag 240 atggattacg ctccctcctt ccaaatgggc catcccatcg cttttgaact ggccgaacgt 300 ttaaccgaac tcagcccaga aggactcaac aaagtattct ttaccaactc aggctctgag 360 tcggttgata ccgcgctaaa aatggctctt tgctaccata gagccaatgg ccaagcgtca 420 cgcacccgct ttattggccg tgaaatgggt taccatggcg taggatttgg tgggatctcg 480 gtgggtggtt taagcaataa ccgtaaagcc ttcagcggcc agctattgca aggcgtggat 540 cacctgcccc acaccttaga cattcaacat gccgccttta gtcgtggctt accgagcctc 600 ggtgctgaaa aagctgaggt attagaacaa ttagtcacac tccatggcgc cgaaaatatt 660 gccgccgtta ttgttgaacc catgtcaggt tctgcagggg taattttacc acctcaaggc 720 tacttaaaac gcttacgtga aatcactaaa aaacacggca tcttattgat tttcgatgaa 780 gtcattaccg catttggccg tgtaggtgca gcattcgcca gccaacgttg gggcgttatt 840 ccagacataa tcaccacggc taaagccatt aataatggcg ccatccccat gggcgcagtg 900 tttgtacagg attatatcca cgatacttgc atgcaagggc caaccgaact gattgaattt 960 ttccacggtt atacctattc gggccaccca gtcgccgcag cagcagcact cgccacgctc 1020 tccatctacc aaaacgagca actgtttgag cgcagttttg agcttgagcg gtatttcgaa 1080 gaagccgttc atagcctcaa agggttaccg aatgtgattg atattcgcaa caccggatta 1140 gtcgcgggtt tccagctagc accgaatagc caaggtgttg gtaaacgcgg atacagcgtg 1200 ttcgagcatt gtttccatca aggcacactc gtgcgggcaa cgggcgatat tatcgccatg 1260 tccccaccac tcattgttga gaaacatcag attgaccaaa tggtaaatag ccttagcgat 1320 gcaattcacg ccgttggatg a 1341 <210> 93 <211> 448 <212> PRT <213> Pseudomonas putida <400> 93 Met Asn Met Pro Glu Thr Gly Pro Ala Gly Ile Ala Ser Gln Leu Lys 1 5 10 15 Leu Asp Ala His Trp Met Pro Tyr Thr Ala Asn Arg Asn Phe Gln Arg             20 25 30 Asp Pro Arg Leu Ile Val Ala Ala Glu Gly Asn Tyr Leu Val Asp Asp         35 40 45 His Gly Arg Lys Ile Phe Asp Ala Leu Ser Gly Leu Trp Thr Cys Gly     50 55 60 Ala Gly His Thr Arg Lys Glu Ile Ala Asp Ala Val Thr Arg Gln Leu 65 70 75 80 Ser Thr Leu Asp Tyr Ser Pro Ala Phe Gln Phe Gly His Pro Leu Ser                 85 90 95 Phe Gln Leu Ala Glu Lys Ile Ala Glu Leu Val Pro Gly Asn Leu Asn             100 105 110 His Val Phe Tyr Thr Asn Ser Gly Ser Glu Cys Ala Asp Thr Ala Leu         115 120 125 Lys Met Val Arg Ala Tyr Trp Arg Leu Lys Gly Gln Ala Thr Lys Thr     130 135 140 Lys Ile Ile Gly Arg Ala Arg Gly Tyr His Gly Val Asn Ile Ala Gly 145 150 155 160 Thr Ser Leu Gly Gly Val Asn Gly Asn Arg Lys Met Phe Gly Gln Leu                 165 170 175 Leu Asp Val Asp His Leu Pro His Thr Val Leu Pro Val Asn Ala Phe             180 185 190 Ser Lys Gly Leu Pro Glu Glu Gly Gly Ile Ala Leu Ala Asp Glu Met         195 200 205 Leu Lys Leu Ile Glu Leu His Asp Ala Ser Asn Ile Ala Ala Val Ile     210 215 220 Val Glu Pro Leu Ala Gly Ser Ala Gly Val Leu Pro Pro Pro Lys Gly 225 230 235 240 Tyr Leu Lys Arg Leu Arg Glu Ile Cys Thr Gln His Asn Ile Leu Leu                 245 250 255 Ile Phe Asp Glu Val Ile Thr Gly Phe Gly Arg Met Gly Ala Met Thr             260 265 270 Gly Ser Glu Ala Phe Gly Val Thr Pro Asp Leu Met Cys Ile Ala Lys         275 280 285 Gln Val Thr Asn Gly Ala Ile Pro Gly Ala Val Ile Ala Ser Ser     290 295 300 Glu Ile Tyr Gln Thr Phe Met Asn Gln Pro Thr Pro Glu Tyr Ala Val 305 310 315 320 Glu Phe Pro His Gly Tyr Thr Tyr Ser Ala His Pro Val Ala Cys Ala                 325 330 335 Ala Gly Leu Ala Ala Leu Asp Leu Leu Gln Lys Glu Asn Leu Val Gln             340 345 350 Ser Ala Ala Glu Leu Ala Pro His Phe Glu Lys Leu Leu His Gly Val         355 360 365 Lys Gly Thr Lys Asn Ile Val Asp Ile Arg Asn Tyr Gly Leu Ala Gly     370 375 380 Ala Ile Gln Ile Ala Ala Arg Asp Gly Asp Ala Ile Val Arg Pro Tyr 385 390 395 400 Glu Ala Ala Met Lys Leu Trp Lys Ala Gly Phe Tyr Val Arg Phe Gly                 405 410 415 Gly Asp Thr Leu Gln Phe Gly Pro Thr Phe Asn Thr Lys Pro Gln Glu             420 425 430 Leu Asp Arg Leu Phe Asp Ala Val Gly Glu Thr Leu Asn Leu Ile Asp         435 440 445 <210> 94 <211> 930 <212> DNA <213> Pseudomonas putida <400> 94 atgaccacga agaaagctga ttacatttgg ttcaatgggg agatggttcg ctgggaagac 60 gcgaaggtgc atgtgatgtc gcacgcgctg cactatggca cttcggtttt tgaaggcatc 120 cgttgctacg actcgcacaa aggaccggtt gtattccgcc atcgtgagca tatgcagcgt 180 ctgcatgact ccgccaaaat ctatcgcttc ccggtttcgc agagcattga tgagctgatg 240 gt; atcttcgtcg gtgatgttgg catgggagta aacccgccag cgggatactc aaccgacgtg 360 attatcgctg ctttcccgtg gggagcgtat ctgggcgcag aagcgctgga gcaggggatc 420 gatgcgatgg tttcctcctg gaaccgcgca gcaccaaaca ccatcccgac ggcggcaaaa 480 gccggtggta actacctctc ttccctgctg gtgggtagcg aagcgcgccg ccacggttat 540 caggaaggta tcgcgctgga tgtgaacggt tatatctctg aaggcgcagg cgaaaacctg 600 tttgaagtga aagatggtgt gctgttcacc ccaccgttca cctcctccgc gctgccgggt 660 attacccgtg atgccatcat caaactggcg aaagagctgg gaattgaagt acgtgagcag 720 gtgctgtcgc gcgaatccct gtacctggcg gatgaagtgt ttatgtccgg tacggcggca 780 gaaatcacgc cagtgcgcag cgtagacggt attcaggttg gcgaaggccg ttgtggcccg 840 gttaccaaac gcattcagca agccttcttc ggcctcttca ctggcgaaac cgaagataaa 900 tggggctggt tagatcaagt taatcaataa 930 <210> 95 <211> 566 <212> PRT <213> Streptomyces cinnamonensis <400> 95 Met Asp Ala Asp Ala Ile Glu Glu Gly Arg Arg Arg Trp Gln Ala Arg 1 5 10 15 Tyr Asp Lys Ala Arg Lys Arg Asp Ala Asp Phe Thr Thr Leu Ser Gly             20 25 30 Asp Pro Val Asp Pro Val Tyr Gly Pro Arg Gly Asp Thr Tyr Asp         35 40 45 Gly Phe Glu Arg Ile Gly Trp Pro Gly Glu Tyr Pro Phe Thr Arg Gly     50 55 60 Leu Tyr Ala Thr Gly Tyr Arg Gly Arg Thr Trp Thr Ile Arg Gln Phe 65 70 75 80 Ala Gly Phe Gly Asn Ala Glu Gln Thr Asn Glu Arg Tyr Lys Met Ile                 85 90 95 Leu Ala Asn Gly Gly Gly Gly Leu Ser Val Ala Phe Asp Met Pro Thr             100 105 110 Leu Met Gly Arg Asp Ser Asp Asp Pro Arg Ser Leu Gly Glu Val Gly         115 120 125 His Cys Gly Val Ala Ile Asp Ser Ala Ala Asp Met Glu Val Leu Phe     130 135 140 Lys Asp Ile Pro Leu Gly Asp Val Thr Thr Ser Met Thr Ile Ser Gly 145 150 155 160 Pro Ala Val Pro Val Phe Cys Met Tyr Leu Val Ala Ala Glu Arg Gln                 165 170 175 Gly Val Asp Pro Ala Val Leu Asn Gly Thr Leu Gln Thr Asp Ile Phe             180 185 190 Lys Glu Tyr Ile Ala Gln Lys Glu Trp Leu Phe Gln Pro Glu Pro His         195 200 205 Leu Arg Leu Ile Gly Asp Leu Met Glu His Cys Ala Arg Asp Ile Pro     210 215 220 Ala Tyr Lys Pro Leu Ser Val Ser Gly Tyr His Ile Arg Glu Ala Gly 225 230 235 240 Ala Thr Ala Ala Gln Glu Leu Ala Tyr Thr Leu Ala Asp Gly Phe Gly                 245 250 255 Tyr Val Glu Leu Gly Leu Ser Arg Gly Leu Asp Val Asp Val Phe Ala             260 265 270 Pro Gly Leu Ser Phe Phe Phe Asp Ala His Val Asp Phe Phe Glu Glu         275 280 285 Ile Ala Lys Phe Arg Ala Arg Arg Ile Trp Ala Arg Trp Leu Arg     290 295 300 Asp Glu Tyr Gly Ala Lys Thr Glu Lys Ala Gln Trp Leu Arg Phe His 305 310 315 320 Thr Gln Thr Ala Gly Val Ser Leu Thr Ala Gln Gln Pro Tyr Asn Asn                 325 330 335 Val Val Arg Thr Ala Val Glu Ala Leu Ala Ala Val Leu Gly Gly Thr             340 345 350 Asn Ser Leu His Thr Asn Ala Leu Asp Glu Thr Leu Ala Leu Pro Ser         355 360 365 Glu Gln Ala Ala Glu Ile Ala Leu Arg Thr Gln Gln Val Leu Met Glu     370 375 380 Glu Thr Gly Val Ala Asn Val Ala Asp Pro Leu Gly Gly Ser Trp Tyr 385 390 395 400 Ile Glu Gln Leu Thr Asp Arg Ile Glu Ala Asp Ala Glu Lys Ile Phe                 405 410 415 Glu Gln Ile Arg Glu Arg Gly Arg Arg Ala Cys Pro Asp Gly Gln His             420 425 430 Pro Ile Gly Pro Ile Thr Ser Gly Ile Leu Arg Gly Ile Glu Asp Gly         435 440 445 Trp Phe Thr Gly Glu Ile Ala Glu Ser Ala Phe Gln Tyr Gln Arg Ser     450 455 460 Leu Glu Lys Gly Asp Lys Arg Val Val Gly Val Asn Cys Leu Glu Gly 465 470 475 480 Ser Val Thr Gly Asp Leu Glu Ile Leu Arg Val Ser His Glu Val Glu                 485 490 495 Arg Glu Gln Val Arg Glu Leu Ala Gly Arg Lys Gly Arg Arg Asp Asp             500 505 510 Ala Arg Val Ala Ala Ala Arg Asp Ala Met Leu Ala Ala Arg Asp         515 520 525 Gly Ser Asn Met Ile Ala Pro Met Leu Glu Ala Val Arg Ala Glu Ala     530 535 540 Thr Leu Gly Glu Ile Cys Gly Val Leu Arg Asp Glu Trp Gly Val Tyr 545 550 555 560 Val Glu Pro Pro Gly Phe                 565 <210> 96 <211> 4362 <212> DNA <213> Streptomyces cinnamonensis <400> 96 tgaggcgctg gatcgcctcg gagagcagct ggtaacggtc cgcgtggtac tcggccgggg 60 tgcagccgtc cacgatgtgc gggatcgcgt cgggctcgag gatcaccagg gcgggggcgt 120 cgccgatcgc gtcggcgaac gtgtccaccc agctccggta ggcctccgca ctggccgcgc 180 cgcccgcgga gtgctgaccg cagtcgcggt gcgggatgtt gtacgcgacg agtacggcgg 240 tgcggtcctc cttgaccgcg ccccgcgtcg ccttcgcgac gtcgggcgcc ggatcgtccc 300 cggccggcca cacggccatg gcccgttcgg agatgcgcct gagcgtctcg gcgtcctcgg 360 cgcggccctg ttcctcccac tgcctgacct ggcgcgcggc ggggctgtcg gggtcgaccc 420 agaaggtgcc ggcggggggc ccggcgctcg cggtggcggg cttgcgcacg gccgcctcct 480 ccttcgtgcc gtcggacccc gggtctgagg aggagcagcc tgccgggagc ccgagggcgg 540 cgagggccgc gagtgccgtg aacgtgcgga gcagccggtg catccagccc ccttgggcga 600 tggtgacagt gacggtcagt cagcccggca atcgttacat aaaggactat tcaagctctt 660 gtgccacacc gcctccggtg ccgagcgcga acccggcggg caccagagcc ccgccgcggc 720 cgcggagccg tacgtacgac cgaattgcga gacggggctg accaccatat gaccggcggg 780 taaggtcgat gccgtgccga agccgctcag cctccccttc gatcccatcg cccgcgccga 840 cgagctctgg aagcagcgct ggggatcggt cccggccatg ggcgcgatca cctcgatcat 900 gcgggcgcac cagatcctgc tcgccgaggt cgacgcggtc gtcaagccgt acggactgac 960 cttcgcgcgc tacgaggcgc tggtgctcct caccttctcg caggccggcg agttgccgat 1020 gtcgaagatc ggcgagcggc tcatggtgca cccgacctcg gtcacgaaca ccgtggaccg 1080 cctggtgaag tccggcctgg tcgacaagcg cccgaacccc aacgacggcc gcggcacgct 1140 cgcctccatc acggagaagg gccgcgaggt cgtcgaggcg gccacccgcg agctgatggc 1200 ggggacttc gggctcgggg tgtacgacgc ggaggagtgc ggggagatct tcgcgatgct 1260 gcggcccctg cgggtggcgg cgcgcgattt cgaggagcag tagggcccgc ccggtgagaa 1320 gtgggatcgg gtcgtcccgg tacgggcggg ggcggcgaag atcgcgtgaa aagggcggtt 1380 acgctcgtag ccatgaaacg cagcgtgctg acccgctacc gggtgatggc ctacgtcacc 1440 gccgtcatgc tcctcatcct gtgcgcctgc atggtggcca agtacggctt cgacaagggc 1500 gagggtctga ccctcgtcgt gtcgcaggtg cacggcgtgc tctacatcat ctacctgatc 1560 ttcgccttcg acctgggctc caaggcgaag tggccgttcg gcaagctgct ctgggtgctg 1620 gtctcgggca cgatcccgac cgccgccttc ttcgtcgagc gcaaggtcgc ccgtgacgtc 1680 gagccgctga tcgccgacgg ctccccggtc accgcgaagg cgtaacccgc accgccacgg 1740 acaggtccgt ggcggttggc catcgacttt tactaggacg tcctagtaaa ttcgatggta 1800 tggacgctga cgcgatcgag gaaggccgcc gacgctggca ggcccgttac gacaaggccc 1860 gcaagcgcga cgcggacttc accacgctct ccggggaccc cgtcgacccc gtctacggcc 1920 cccggcccgg ggacacgtac gacgggttcg agcggatcgg ctggccgggg gagtacccct 1980 tcacccgcgg gctctacgcc accgggtacc gcggccgcac ctggaccatc cgccagttcg 2040 ccggcttcgg caacgccgag cagacgaacg agcgctacaa gatgatcctg gccaacggcg 2100 gcggcggcct ctccgtcgcc ttcgacatgc cgaccctcat gggccgcgac tccgacgacc 2160 cgcgctcgct cggcgaggtc ggccactgcg gtgtcgccat cgactccgcc gccgacatgg 2220 aggtcctctt caaggacatc ccgctcggcg acgtcacgac gtccatgacc atcagcgggc 2280 ccgccgtgcc cgtcttctgc atgtacctcg tcgcggccga gcgccagggc gtcgacccgg 2340 ccgtcctcaa cggcacgctg cagaccgaca tcttcaagga gtacatcgcc cagaaggagt 2400 ggctcttcca gcccgagccg cacctgcgcc tcatcggcga cctgatggag cactgcgcgc 2460 gcgacatccc cgcgtacaag ccgctctcgg tctccggcta ccacatccgc gaggccgggg 2520 cgacggccgc gcaggagctc gcgtacaccc tcgcggacgg cttcgggtac gtggaactgg 2580 gcctctcgcg cggcctggac gtggacgtct tcgcgcccgg cctctccttc ttcttcgacg 2640 cgcacgtcga cttcttcgag gagatcgcga agttccgcgc cgcacgccgc atctgggcgc 2700 gctggctccg ggacgagtac ggagcgaaga ccgagaaggc acagtggctg cgcttccaca 2760 cgcagaccgc gggggtctcg ctcacggccc agcagccgta caacaacgtg gtgcggacgg 2820 cggtggaggc cctcgccgcg gtgctcggcg gcacgaactc cctgcacacc aacgctctcg 2880 acgagaccct tgccctcccc agcgagcagg ccgcggagat cgcgctgcgc acccagcagg 2940 tgctgatgga ggagaccggc gtcgccaacg tcgcggaccc gctgggcggc tcctggtaca 3000 tcgagcagct caccgaccgc atcgaggccg acgccgagaa gatcttcgag cagatcaggg 3060 agcgggggcg gcgggcctgc cccgacgggc agcacccgat cgggccgatc acctccggca 3120 tcctgcgcgg catcgaggac ggctggttca ccggcgagat cgccgagtcc gccttccagt 3180 accagcggtc cctggagaag ggcgacaagc gggtcgtcgg cgtcaactgc ctcgaaggct 3240 ccgtcaccgg cgacctggag atcctgcgcg tcagccacga ggtcgagcgc gagcaggtgc 3300 gggagcttgc ggggcgcaag gggcggcgtg acgatgcgcg ggtgcgggcc tcgctcgacg 3360 cgatgctcgc cgctgcgcgg gacgggtcga acatgattgc ccccatgctg gaggcggtgc 3420 gggccgaggc gaccctcggg gagatctgcg gggtgcttcg cgatgagtgg ggggtctacg 3480 tggagccgcc cgggttctga gggcgcgctc cctttgcctg cgggtctgct gtggctggtc 3540 gcgcagttcc ccgcacccct gaaagacccc ggcgctttcc cttcctggct cgcctcgtcg 3600 ctgtctgcgg ggccgtgggg gctggtcgcg cagttccccg cgcccctgcc cgcacctgcg 3660 ccccgccgcc tgcatgccgc ccccaccctg acgggggcgt tcggggccca ccctgacggg 3720 tgcggtcggg gcgtgccggg gtcttttagg ggcgcgggga actgcgcgag caacccccac 3780 ccacccgcag gtgcacgcgg agcggcggac gccccgcaga cgggggcaaa acgggcggag 3840 tgcccccgcc cgccgggcgg cgcgaattcg taggtttaag gggcaggggt cagggcaggc 3900 gccgagccgg tcaaccgccc ccgtcccagg agaccccgtg acctcgaccg gccacgcccg 3960 caccgccgcc atcgccatcg gagccgccac cgccaccgtc ctcggcgcgc tgctggtcgg 4020 cggctccggc gaggtgagtg cgagcccgcc gcccgagccc aaggtccagg acgacttcga 4080 ctccctcggc cccgaggtgc gcgccgcgaa gctctccgac gggcggacgg cccactactc 4140 ggacacgggc gacaaggacg gcaagccggc cctgttcatc ggcggcaccg gcacgagcgc 4200 ccgcgcctcc cacatgaccg acttcttccg ctcgacgcgc gaggacctgg gcctgcgcct 4260 catctccgtg gagcgcaacg gcttcggcga caccgcgttc gacgagaagc tgggcaccgc 4320 cgacttcgcg aaggacgccc tcgaagtcct cgaccggctc gg 4362 <210> 97 <211> 136 <212> PRT <213> Streptomyces cinnamonensis <400> 97 Met Gly Val Ala Ala Gly Pro Ile Arg Val Val Val Ala Lys Pro Gly 1 5 10 15 Leu Asp Gly His Asp Arg Gly Ala Lys Val Ile Ala Arg Ala Leu Arg             20 25 30 Asp Ala Gly Met Glu Val Ile Tyr Thr Gly Leu His Gln Thr Pro Glu         35 40 45 Gln Val Val Asp Thr Ala Ile Glu Glu Asp Ala Asp Ala Ile Gly Leu     50 55 60 Ser Ile Leu Ser Gly Ala His Asn Thr Leu Phe Ala Arg Val Leu Glu 65 70 75 80 Leu Leu Lys Glu Arg Asp Ala Glu Asp Ile Lys Val Phe Gly Gly Gly                 85 90 95 Ile Ile Pro Glu Ala Asp Ile Ala Pro Leu Lys Glu Lys Gly Val Ala             100 105 110 Glu Ile Phe Thr Pro Gly Ala Thr Thr Thr Ser Ile Val Glu Trp Val         115 120 125 Arg Gly Asn Val Arg Gln Ala Val     130 135 <210> 98 <211> 1643 <212> DNA <213> Streptomyces cinnamonensis <400> 98 gtcgacctcc cgtttggcgc acggaaggga ggctctgtcc cccgtgtgcc ctagggggag 60 tcgtggtcga ggagtcggct gtgcgatggc gatcccggcc accgccctgc ggtgactccg 120 tgcccccgtt gcatcgccga tgcgcggtgt caccacgccg tgcggctgcc ggcgcggtgg 180 cccggcgtct cgttgcggct cccctcgcgc ctggtccgga tgcggagcgt gaacccctgg 240 gttacggacg ggcgcgcagc gaacgtgtcc cacgtgtgat ttccccctcg ctctccaccg 300 cgaaactgcc gcttgcgcga tgctggggat aacgttcgtt cacttccccg gccggtgcgg 360 tgcggggtat ctgtgccggg acagactttg tcggtacgga tatcggtaca tggaggcagt 420 gatgggtgtg gcagccgggc cgatccgcgt ggtggtcgcc aagccggggc tcgacgggca 480 cgatcgcggg gccaaggtga tcgcgcgggc gttgcgtgac gcgggtatgg aggtcatcta 540 caccgggctg caccagacgc ccgagcaggt ggtggacacc gcgatccagg aggacgccga 600 cgcgatcggc ctctccatcc tctccggagc gcacaacacg ctgttcgcgc gcgtgttgga 660 gctcttgaag gagcgggacg cggaggacat caaggtgttt ggtggcggca tcatcccgga 720 ggcggacatc gcgccgctga aggagaaggg cgtcgcggag atcttcacgc ccggggccac 780 caccacgtcg atcgtggagt gggttcgggg gaacgtgcga caggccgtct gaggcattcc 840 ccgtcgcccg tctgccgtgg tcggcgtcat atcggcggac atcgtctcgg tggacgtcat 900 ggcggcgggg ggagttcgtc gcgtatcgcc gcgcggaggc gcagggtggt gaccaggcgc 960 tggaacgctt ccgaccagta gctgcccgcg ccgggtgacg cgtcctccgc ttcgtcgggg 1020 accgcggtga gcgcttccag gcggaccgcc tcggccgggt ccagacagcg ttccgccagg 1080 cccatcactc cgctgaagct ccatgggtaa ctgcccgcgt cgcgcgcgat gttcagggcg 1140 tccaccacgg cccggccgag agggccggcc cagggcaccg cgcagacgcc gagcagttgg 1200 aacgcctccg acaggccgtg tgccgctatg aaccccgcca cccagtccgc gcgctcggcg 1260 gcaggcatgg aggcgagcag tttggcccgc tcggcgaggg acacggcgcc aggccccgcc 1320 gcgtcgggtg aggcgggggc gccgagcagc gctctggacc aggcgacgtc acgctggcgt 1380 acggccgcgc ggcaccatgc ggcgtgcagt tcgccccgcc agtcgtcggc caccgggagc 1440 gccacgatc ccgccggggt gcggttgccg agccggggcg gccaggtggc gagcggggcc 1500 gattccacga gctggccgag ccaccaggag cgctcgcccc ggccggtggg gggcttcggg 1560 acgacgccgt cccgctccat gcccgcgtcg cactcgtgcg gcgcctcgac ggtgagggtc 1620 ggcgtgctcg atgtgtggtc gac 1643 <210> 99 <211> 566 <212> PRT <213> Streptomyces coelicolor <400> 99 Met Asp Ala His Ala Ile Glu Glu Gly Arg Leu Arg Trp Gln Ala Arg 1 5 10 15 Tyr Asp Ala Ala Arg Lys Arg Asp Ala Asp Phe Thr Thr Leu Ser Gly             20 25 30 Asp Pro Val Glu Pro Val Tyr Gly Pro Arg Pro Gly Asp Glu Tyr Glu         35 40 45 Gly Phe Glu Arg Ile Gly Trp Pro Gly Glu Tyr Pro Phe Thr Arg Gly     50 55 60 Leu Tyr Pro Thr Gly Tyr Arg Gly Arg Thr Trp Thr Ile Arg Gln Phe 65 70 75 80 Ala Gly Phe Gly Asn Ala Glu Gln Thr Asn Glu Arg Tyr Lys Met Ile                 85 90 95 Leu Arg Asn Gly Gly Gly Gly Leu Ser Val Ala Phe Asp Met Pro Thr             100 105 110 Leu Met Gly Arg Asp Ser Asp Asp Pro Arg Ser Leu Gly Glu Val Gly         115 120 125 His Cys Gly Val Ala Ile Asp Ser Ala Ala Asp Met Glu Val Leu Phe     130 135 140 Lys Asp Ile Pro Leu Gly Asp Val Thr Thr Ser Met Thr Ile Ser Gly 145 150 155 160 Pro Ala Val Pro Val Phe Cys Met Tyr Leu Val Ala Ala Glu Arg Gln                 165 170 175 Gly Val Asp Ala Ser Val Leu Asn Gly Thr Leu Gln Thr Asp Ile Phe             180 185 190 Lys Glu Tyr Ile Ala Gln Lys Glu Trp Leu Phe Gln Pro Glu Pro His         195 200 205 Leu Arg Leu Ile Gly Asp Leu Met Glu Tyr Cys Ala Ala Gly Ile Pro     210 215 220 Ala Tyr Lys Pro Leu Ser Val Ser Gly Tyr His Ile Arg Glu Ala Gly 225 230 235 240 Ala Thr Ala Ala Gln Glu Leu Ala Tyr Thr Leu Ala Asp Gly Phe Gly                 245 250 255 Tyr Val Glu Leu Gly Leu Ser Arg Gly Leu Asp Val Asp Val Phe Ala             260 265 270 Pro Gly Leu Ser Phe Phe Phe Asp Ala His Leu Asp Phe Phe Glu Glu         275 280 285 Ile Ala Lys Phe Arg Ala Arg Arg Ile Trp Ala Arg Trp Met Arg     290 295 300 Asp Val Tyr Gly Ala Arg Thr Asp Lys Ala Gln Trp Leu Arg Phe His 305 310 315 320 Thr Gln Thr Ala Gly Val Ser Leu Thr Ala Gln Gln Pro Tyr Asn Asn                 325 330 335 Val Val Arg Thr Ala Val Glu Ala Leu Ala Ala Val Leu Gly Gly Thr             340 345 350 Asn Ser Leu His Thr Asn Ala Leu Asp Glu Thr Leu Ala Leu Pro Ser         355 360 365 Glu Gln Ala Ala Glu Ile Ala Leu Arg Thr Gln Gln Val Leu Met Glu     370 375 380 Glu Thr Gly Val Ala Asn Val Ala Asp Pro Leu Gly Gly Ser Trp Phe 385 390 395 400 Ile Glu Gln Leu Thr Asp Arg Ile Glu Ala Asp Ala Glu Lys Ile Phe                 405 410 415 Glu Gln Ile Lys Glu Arg Gly Leu Arg Ala His Pro Asp Gly Gln His             420 425 430 Pro Val Gly Pro Ile Thr Ser Gly Leu Leu Arg Gly Ile Glu Asp Gly         435 440 445 Trp Phe Thr Gly Glu Ile Ala Glu Ser Ala Phe Arg Tyr Gln Gln Ser     450 455 460 Leu Glu Lys Asp Asp Lys Lys Val Val Gly Val Asn Val His Thr Gly 465 470 475 480 Ser Val Thr Gly Asp Leu Glu Ile Leu Arg Val Ser His Glu Val Glu                 485 490 495 Arg Glu Gln Val Arg Val Leu Gly Glu Arg Lys Asp Ala Arg Asp Asp             500 505 510 Ala Ala Val Ala Ala Leu Asp Ala         515 520 525 Gly Gly Asn Met Ile Gly Pro Met Leu Asp Ala Val Arg Ala Glu Ala     530 535 540 Thr Leu Gly Glu Ile Cys Gly Val Leu Arg Asp Glu Trp Gly Val Tyr 545 550 555 560 Thr Glu Pro Ala Gly Phe                 565 <210> 100 <211> 1701 <212> DNA <213> Streptomyces coelicolor <400> 100 atggacgctc atgccataga ggagggccgc cttcgctggc aggcccggta cgacgcggcg 60 cgcaagcgcg acgcggactt caccacgctc tccggagacc ccgtggagcc ggtgtacggg 120 ccccgccccg gggacgagta cgagggcttc gagcggatcg gctggccggg cgagtacccc 180 ttcacccgcg gcctgtatcc gaccgggtac cgggggcgta cgtggaccat ccggcagttc 240 gccgggttcg gcaacgccga gcagaccaac gagcgctaca agatgatcct ccgcaacggc 300 ggcggcgggc tctcggtcgc cttcgacatg ccgaccctga tgggccgcga ctccgacgac 360 ccgcgctcgc tgggcgaggt cgggcactgc ggggtggcca tcgactcggc cgccgacatg 420 gaagtgctgt tcaaggacat cccgctcggg gacgtgacga cctccatgac gatcagcggg 480 cccgccgtgc ccgtgttctg catgtacctc gtcgccgccg agcgccaggg cgtcgacgca 540 tccgtgctca acggcacgct gcagaccgac atcttcaagg agtacatcgc ccagaaggag 600 tggctcttcc agcccgagcc ccacctccgg ctcatcggcg acctcatgga gtactgcgcg 660 gccggcatcc ccgcctacaa gccgctctcc gtctccggct accacatccg cgaggcgggc 720 gcgacggccg cgcaggagct ggcgtacacg ctcgccgacg gcttcggata cgtggagctg 780 ggcctcagcc gcgggctcga cgtggacgtc ttcgcgcccg gcctctcctt cttcttcgac 840 gcgcacctcg acttcttcga ggagatcgcc aagttccgcg cggcccgcag gatctgggcc 900 cgctggatgc gcgacgtgta cggcgcgcgg accgacaagg cccagtggct gcggttccac 960 acccagaccg ccggagtctc gctcaccgcg cagcagccgt acaacaacgt cgtacgcacc 1020 gcggtggagg cgctggcggc cgtgctcggc ggcaccaact ccctgcacac caacgcgctc 1080 gacgagaccc tcgccctgcc cagcgagcag gccgccgaga tcgccctgcg cacccagcag 1140 gtgctgatgg aggagaccgg cgtcgccaac gtcgccgacc cgctgggcgg ttcctggttc 1200 atcgagcagc tgaccgaccg catcgaggcc gacgccgaga agatcttcga gcagatcaag 1260 gagcgggggc tgcgcgccca ccccgacggg cagcaccccg tcggaccgat cacctccggc 1320 ctgctgcgcg gcatcgagga cggctggttc accggcgaga tcgccgagtc cgccttccgc 1380 taccagcagt ccttggagaa ggacgacaag aaggtggtcg gcgtcaacgt ccacaccggc 1440 tccgtcaccg gcgacctgga gatcctgcgg gtcagccacg aggtcgagcg cgagcaggtg 1500 cgggtcctgg gcgagcgcaa ggacgcccgg gacgacgccg ccgtgcgcgg cgccctggac 1560 gccatgctgg ccgcggcccg ctccggcggc aacatgatcg ggccgatgct ggacgcggtg 1620 cgcgcggagg cgacgctggg cgagatctgc ggtgtgctgc gcgacgagtg gggggtgtac 1680 acggaaccgg cggggttctg a 1701 <210> 101 <211> 138 <212> PRT <213> Streptomyces coelicolor <400> 101 Met Gly Val Ala Ala Gly Pro Ile Arg Val Val Val Ala Lys Pro Gly 1 5 10 15 Leu Asp Gly His Asp Arg Gly Ala Lys Val Ile Ala Arg Ala Leu Arg             20 25 30 Asp Ala Gly Met Glu Val Ile Tyr Thr Gly Leu His Gln Thr Pro Glu         35 40 45 Gln Ile Val Asp Thr Ala Ile Gln Glu Asp Ala Asp Ala Ile Gly Leu     50 55 60 Ser Ile Leu Ser Gly Ala His Asn Thr Leu Phe Ala Ala Val Ile Glu 65 70 75 80 Leu Leu Arg Glu Arg Asp Ala Asp Ile Leu Val Phe Gly Gly Gly                 85 90 95 Ile Ile Pro Glu Ala Asp Ile Ala Pro Leu Lys Glu Lys Gly Val Ala             100 105 110 Glu Ile Phe Thr Pro Gly Ala Thr Thr Ala Ser Ile Val Asp Trp Val         115 120 125 Arg Ala Asn Val Arg Glu Pro Ala Gly Ala     130 135 <210> 102 <211> 417 <212> DNA <213> Streptomyces coelicolor <400> 102 atgggtgtgg cagccggtcc gatccgcgtg gtggtggcca agccggggct cgacggccac 60 gatcgcgggg ccaaggtgat cgcgagggcc ctgcgtgacg ccggtatgga ggtgatctac 120 accgggctcc accagacgcc cgagcagatc gtcgacaccg cgatccagga ggacgccgac 180 gcgatcgggc tgtccatcct ctccggtgcg cacaacacgc tcttcgccgc cgtgatcgag 240 ctgctccggg agcgggacgc cgcggacatc ctggtcttcg gcggcgggat catccccgag 300 gcggacatcg ccccgctgaa ggagaagggc gtcgcggaga tcttcacgcc cggcgccacc 360 acggcgtcca tcgtggactg ggtccgggcg aacgtgcggg agcccgcggg agcatag 417 <210> 103 <211> 566 <212> PRT <213> Streptomyces avermitilis <400> 103 Met Asp Ala Asp Ala Ile Glu Glu Gly Arg Arg Arg Trp Gln Ala Arg 1 5 10 15 Tyr Asp Ala Ser Arg Lys Arg Glu Ala Asp Phe Thr Thr Leu Ser Gly             20 25 30 Asp Pro Val Glu Pro Ala Tyr Gly Pro Arg Pro Gly Asp Ala Tyr Glu         35 40 45 Gly Phe Glu Arg Ile Gly Trp Pro Gly Glu Tyr Pro Phe Thr Arg Gly     50 55 60 Leu Tyr Pro Thr Gly Tyr Arg Gly Arg Thr Trp Thr Ile Arg Gln Phe 65 70 75 80 Ala Gly Phe Gly Asn Ala Glu Gln Thr Asn Glu Arg Tyr Lys Lys Ile                 85 90 95 Leu Ala Asn Gly Gly Gly Gly Leu Ser Val Ala Phe Asp Met Pro Thr             100 105 110 Leu Met Gly Arg Asp Ser Asp Asp Arg Arg Ala Leu Gly Glu Val Gly         115 120 125 His Cys Gly Val Ala Ile Asp Ser Ala Ala Asp Met Glu Val Leu Phe     130 135 140 Lys Asp Ile Pro Leu Gly Asp Val Thr Thr Ser Met Thr Ile Ser Gly 145 150 155 160 Pro Ala Val Pro Val Phe Cys Met Tyr Leu Val Ala Ala Glu Arg Gln                 165 170 175 Gly Val Asp Pro Ser Val Leu Asn Gly Thr Leu Gln Thr Asp Ile Phe             180 185 190 Lys Glu Tyr Ile Ala Gln Lys Glu Trp Leu Phe Gln Pro Glu Pro His         195 200 205 Leu Arg Leu Ile Gly Asp Leu Met Glu His Cys Ala Ser Lys Ile Pro     210 215 220 Ala Tyr Lys Pro Leu Ser Val Ser Gly Tyr His Ile Arg Glu Ala Gly 225 230 235 240 Ala Thr Ala Ala Gln Glu Leu Ala Tyr Thr Leu Ala Asp Gly Phe Gly                 245 250 255 Tyr Val Glu Leu Gly Leu Ser Arg Gly Leu Asp Val Asp Val Phe Ala             260 265 270 Pro Gly Leu Ser Phe Phe Phe Asp Ala His Val Asp Phe Phe Glu Glu         275 280 285 Ile Ala Lys Phe Arg Ala Arg Arg Ile Trp Ala Arg Trp Leu Arg     290 295 300 Asp Val Tyr Gly Ala Lys Ser Glu Lys Ala Gln Trp Leu Arg Phe His 305 310 315 320 Thr Gln Thr Ala Gly Val Ser Leu Thr Ala Gln Gln Pro Tyr Asn Asn                 325 330 335 Val Val Arg Thr Ala Val Glu Ala Leu Ala Ala Val Leu Gly Gly Thr             340 345 350 Asn Ser Leu His Thr Asn Ala Leu Asp Glu Thr Leu Ala Leu Pro Ser         355 360 365 Glu Gln Ala Ala Glu Ile Ala Leu Arg Thr Gln Gln Val Leu Met Glu     370 375 380 Glu Thr Gly Val Ala Asn Val Ala Asp Pro Leu Gly Gly Ser Trp Tyr 385 390 395 400 Val Glu Gln Leu Thr Asp Arg Ile Glu Ala Asp Ala Glu Lys Ile Phe                 405 410 415 Glu Gln Ile Arg Glu Arg Gly Leu Arg Ala His Pro Asp Gly Arg His             420 425 430 Pro Ile Gly Pro Ile Thr Ser Gly Ile Leu Arg Gly Ile Glu Asp Gly         435 440 445 Trp Phe Thr Gly Glu Ile Ala Glu Ser Ala Phe Gln Tyr Gln Gln Ala     450 455 460 Leu Glu Lys Gly Asp Lys Arg Val Val Gly Val Asn Val His His Gly 465 470 475 480 Ser Val Thr Gly Asp Leu Glu Ile Leu Arg Val Ser His Glu Val Glu                 485 490 495 Arg Glu Gln Val Arg Val Leu Gly Glu Arg Lys Ser Gly Arg Asp Asp             500 505 510 Thr Ala Val Thr Ala Ala Leu Asp Ala Met Leu Ala Ala Ala Arg Asp         515 520 525 Gly Ser Asn Met Ile Ala Pro Met Leu Asp Ala Val Arg Ala Glu Ala     530 535 540 Thr Leu Gly Glu Ile Cys Asp Val Leu Arg Glu Glu Trp Gly Val Tyr 545 550 555 560 Thr Glu Pro Ala Gly Phe                 565 <210> 104 <211> 1701 <212> DNA <213> Streptomyces avermitilis <400> 104 tcagaaaccg gcgggctccg tgtagacccc ccactcctcc cggaggacat cgcagatctc 60 gcccagcgtg gcctccgcgc ggaccgcgtc cagcatcggg gcgatcatgt tcgacccgtc 120 gcgcgcggcg gcgagcatcg cgtccagggc cgcggttacg gccgtgtcgt cgcgccccga 180 cttccgctcg cccagcaccc gcacctgctc gcgctccacc tcgtggctga cgcgcaggat 240 ctccaggtcg cccgtcacgg acccgtggtg gacgttgacg ccgacgaccc gcttgtcgcc 300 cttctccagc gcctgctggt actggaaggc cgactcggcg atctccccgg tgaaccagcc 360 gtcctcgatg ccgcgcagga tgccggaggt gatgggcccg atcgggtgcc gcccgtccgg 420 gtgggcccgc agcccgcgct ccctgatctg ttcgaagatc ttctcggcgt cggcctcgat 480 ccggtcggtc agctgctcca cgtaccagga accgcccagc ggatcggcca cgttggcgac 540 gcccgtctcc tccatcagca cctgctgggt gcgcagggcg atctcggccg cctgctcgga 600 cggcagggcg agggtctcgt cgagggcgtt ggtgtgcagc gagttcgtcc cgccgagcac 660 cgcggcgagg gcctccacgg ccgtccgtac gacgttgttg tacggctgct gcgcggtgag 720 cgagacgccc gcggtctggg tgtggaagcg cagccactgc gccttctccg acttcgcccc 780 gtacacgtcc cgcagccagc gcgcccagat gcgccgcgcc gcacggaact tggcgatctc 840 ctcgaagaag tcgacgtgcg cgtcgaagaa gaaggagagc ccgggcgcga acacgtccac 900 gtccaggccg cggctcagcc ccagctccac gtatccgaaa ccgtcggcga gggtgtacgc 960 cagctcctgg gcggccgtgg caccggcctc ccggatgtgg tacccggaga cggacagcgg 1020 cttgtacgcg gggatcttcg aggcgcagtg ctccatcagg tcgccgatga gccgcagatg 1080 gggctcgggc tggaagagcc actccttctg cgcgatgtac tccttgaaga tgtcggtctg 1140 gagggtgccg ttgaggacgg aggggtcgac gccctgccgc tcggccgcga ccaggtacat 1200 gcgaagacg ggcacggcgg gcccgctgat cgtcatcgac gtcgtcacgt cacccagcgg 1260 gatgtccttg aacaggacct ccatgtcggc cgccgagtcg atcgcgaccc cgcagtgccc 1320 gacctcgccg agcgcgcggc ggtcgtcgga gtcgcgcccc atgagcgtcg gcatgtcgaa 1380 ggccacggac agcccaccgc cgccgttggc gaggatcttc ttgtagcgct cgttggtctg 1440 ctcggcgttg ccgaacccgg cgaactgccg gatggtccag gtccggcccc ggtagccggt 1500 cggatacaga ccgcgcgtga aggggtactc acccggccag ccgatccgct cgaaaccctc 1560 gtacgcgtcc ccgggccggg gcccgtacgc cggctccacg ggatcgccgg agagcgtggt 1620 gaaatcggcc tcgcgcttgc gtgaggcgtc gtagcgggcc tgccagcgtc ggcggccttc 1680 ctcgatggcg tcagcgtcca t 1701 <210> 105 <211> 138 <212> PRT <213> Streptomyces avermitilis <400> 105 Met Gly Val Ala Ala Gly Pro Ile Arg Val Val Val Ala Lys Pro Gly 1 5 10 15 Leu Asp Gly His Asp Arg Gly Ala Lys Val Ile Ala Arg Ala Leu Arg             20 25 30 Asp Ala Gly Met Glu Val Ile Tyr Thr Gly Leu His Gln Thr Pro Glu         35 40 45 Gln Ile Val Gly Thr Ala Ile Gln Glu Asp Ala Asp Ala Ile Gly Leu     50 55 60 Ser Ile Leu Ser Gly Ala His Asn Thr Leu Phe Ala Ala Val Ile Asp 65 70 75 80 Leu Leu Lys Glu Arg Asp Ala Glu Asp Ile Lys Val Phe Gly Gly Gly                 85 90 95 Ile Ile Pro Glu Ala Asp Ile Ala Pro Leu Lys Glu Lys Gly Val Ala             100 105 110 Glu Ile Phe Thr Pro Gly Ala Thr Thr Ala Ser Ile Val Glu Trp Val         115 120 125 Arg Ala Asn Val Arg Gln Pro Ala Gly Ala     130 135 <210> 106 <211> 1701 <212> DNA <213> Streptomyces avermitilis <400> 106 tcagaaaccg gcgggctccg tgtagacccc ccactcctcc cggaggacat cgcagatctc 60 gcccagcgtg gcctccgcgc ggaccgcgtc cagcatcggg gcgatcatgt tcgacccgtc 120 gcgcgcggcg gcgagcatcg cgtccagggc cgcggttacg gccgtgtcgt cgcgccccga 180 cttccgctcg cccagcaccc gcacctgctc gcgctccacc tcgtggctga cgcgcaggat 240 ctccaggtcg cccgtcacgg acccgtggtg gacgttgacg ccgacgaccc gcttgtcgcc 300 cttctccagc gcctgctggt actggaaggc cgactcggcg atctccccgg tgaaccagcc 360 gtcctcgatg ccgcgcagga tgccggaggt gatgggcccg atcgggtgcc gcccgtccgg 420 gtgggcccgc agcccgcgct ccctgatctg ttcgaagatc ttctcggcgt cggcctcgat 480 ccggtcggtc agctgctcca cgtaccagga accgcccagc ggatcggcca cgttggcgac 540 gcccgtctcc tccatcagca cctgctgggt gcgcagggcg atctcggccg cctgctcgga 600 cggcagggcg agggtctcgt cgagggcgtt ggtgtgcagc gagttcgtcc cgccgagcac 660 cgcggcgagg gcctccacgg ccgtccgtac gacgttgttg tacggctgct gcgcggtgag 720 cgagacgccc gcggtctggg tgtggaagcg cagccactgc gccttctccg acttcgcccc 780 gtacacgtcc cgcagccagc gcgcccagat gcgccgcgcc gcacggaact tggcgatctc 840 ctcgaagaag tcgacgtgcg cgtcgaagaa gaaggagagc ccgggcgcga acacgtccac 900 gtccaggccg cggctcagcc ccagctccac gtatccgaaa ccgtcggcga gggtgtacgc 960 cagctcctgg gcggccgtgg caccggcctc ccggatgtgg tacccggaga cggacagcgg 1020 cttgtacgcg gggatcttcg aggcgcagtg ctccatcagg tcgccgatga gccgcagatg 1080 gggctcgggc tggaagagcc actccttctg cgcgatgtac tccttgaaga tgtcggtctg 1140 gagggtgccg ttgaggacgg aggggtcgac gccctgccgc tcggccgcga ccaggtacat 1200 gcgaagacg ggcacggcgg gcccgctgat cgtcatcgac gtcgtcacgt cacccagcgg 1260 gatgtccttg aacaggacct ccatgtcggc cgccgagtcg atcgcgaccc cgcagtgccc 1320 gacctcgccg agcgcgcggc ggtcgtcgga gtcgcgcccc atgagcgtcg gcatgtcgaa 1380 ggccacggac agcccaccgc cgccgttggc gaggatcttc ttgtagcgct cgttggtctg 1440 ctcggcgttg ccgaacccgg cgaactgccg gatggtccag gtccggcccc ggtagccggt 1500 cggatacaga ccgcgcgtga aggggtactc acccggccag ccgatccgct cgaaaccctc 1560 gtacgcgtcc ccgggccggg gcccgtacgc cggctccacg ggatcgccgg agagcgtggt 1620 gaaatcggcc tcgcgcttgc gtgaggcgtc gtagcgggcc tgccagcgtc ggcggccttc 1680 ctcgatggcg tcagcgtcca t 1701 <210> 107 <211> 139 <212> PRT <213> Saccharomyces cerevisiae <400> 107 Met Ser Glu Ile Thr Leu Gly Lys Tyr Leu Phe Glu Arg Leu Lys Gln 1 5 10 15 Val Asn Val Asn Thr Val Phe Gly Leu Pro Gly Asp Phe Asn Leu Ser             20 25 30 Leu Leu Asp Lys Ile Tyr Glu Val Glu Gly Met Arg Trp Ala Gly Asn         35 40 45 Ala Asn Glu Leu Asn Ala Ala Tyr Ala Ala Asp Gly Tyr Ala Arg Ile     50 55 60 Lys Gly Met Ser Cys Ile Ile Thr Thr Phe Gly Val Gly Glu Leu Ser 65 70 75 80 Ala Leu Asn Gly Ile Ala Gly Ser Tyr Ala Glu His Val Gly Val Leu                 85 90 95 His Val Val Gly Val Ser Ser Ser Ser Ala Gln Ala Lys Gln Leu Leu             100 105 110 Leu His His Thr Leu Gly Asn Gly Asp Phe Thr Val Phe His Arg Met         115 120 125 Ser Ala Asn Ile Ser Glu Thr Thr Ala Met Ile     130 135 <210> 108 <211> 1689 <212> DNA <213> Saccharomyces cerevisiae <400> 108 atgtctgaaa ttactttggg taaatatttg ttcgaaagat taaagcaagt caacgttaac 60 accgttttcg gtttgccagg tgacttcaac ttgtccttgt tggacaagat ctacgaagtt 120 gaaggtatga gatgggctgg taacgccaac gaattgaacg ctgcttacgc cgctgatggt 180 tacgctcgta tcaagggtat gtcttgtatc atcaccacct tcggtgtcgg tgaattgtct 240 gctttgaacg gtattgccgg ttcttacgct gaacacgtcg gtgttttgca cgttgttggt 300 gtcccatcca tctctgctca agctaagcaa ttgttgttgc accacacctt gggtaacggt 360 gacttcactg ttttccacag aatgtctgcc aacatttctg aaaccactgc tatgatcact 420 gacattgcta ccgccccagc tgaaattgac agatgtatca gaaccactta cgtcacccaa 480 agaccagtct acttaggttt gccagctaac ttggtcgact tgaacgtccc agctaagttg 540 ttgcaaactc caattgacat gtctttgaag ccaaacgatg ctgaatccga aaaggaagtc 600 attgacacca tcttggcttt ggtcaaggat gctaagaacc cagttatctt ggctgatgct 660 tgttgttcca gacacgacgt caaggctgaa actaagaagt tgattgactt gactcaattc 720 ccagctttcg tcaccccaat gggtaagggt tccattgacg aacaacaccc aagatacggt 780 ggtgtttacg tcggtacctt gtccaagcca gaagttaagg aagccgttga atctgctgac 840 ttgattttgt ctgtcggtgc tttgttgtct gatttcaaca ccggttcttt ctcttactct 900 tacaagacca agaacattgt cgaattccac tccgaccaca tgaagatcag aaacgccact 960 ttcccaggtg tccaaatgaa attcgttttg caaaagttgt tgaccactat tgctgacgcc 1020 gctaagggtt acaagccagt tgctgtccca gctagaactc cagctaacgc tgctgtccca 1080 gcttctaccc cattgaagca agaatggatg tggaaccaat tgggtaactt cttgcaagaa 1140 gt; ccaaacaaca cctacggtat ctctcaagtc ttatggggtt ccattggttt caccactggt 1260 gctaccttgg gtgctgcttt cgctgctgaa gaaattgatc caaagaagag agttatctta 1320 ttcattggtg acggttcttt gcaattgact gttcaagaaa tctccaccat gatcagatgg 1380 ggcttgaagc catacttgtt cgtcttgaac aacgatggtt acaccattga aaagttgatt 1440 cacggtccaa aggctcaata caacgaaatt caaggttggg accacctatc cttgttgcca 1500 actttcggtg ctaaggacta tgaaacccac agagtcgcta ccaccggtga atgggacaag 1560 ttgacccaag acaagtcttt caacgacaac tctaagatca gaatgattga aatcatgttg 1620 ccagtcttcg atgctccaca aaacttggtt gaacaagcta agttgactgc tgctaccaac 1680 gctaagcaa 1689 <210> 109 <211> 563 <212> PRT <213> Saccharomyces cerevisiae <400> 109 Met Ser Glu Ile Thr Leu Gly Lys Tyr Leu Phe Glu Arg Leu Ser Gln 1 5 10 15 Val Asn Cys Asn Thr Val Phe Gly Leu Pro Gly Asp Phe Asn Leu Ser             20 25 30 Leu Leu Asp Lys Leu Tyr Glu Val Lys Gly Met Arg Trp Ala Gly Asn         35 40 45 Ala Asn Glu Leu Asn Ala Ala Tyr Ala Ala Asp Gly Tyr Ala Arg Ile     50 55 60 Lys Gly Met Ser Cys Ile Ile Thr Thr Phe Gly Val Gly Glu Leu Ser 65 70 75 80 Ala Leu Asn Gly Ile Ala Gly Ser Tyr Ala Glu His Val Gly Val Leu                 85 90 95 His Val Val Gly Val Ser Ser Ser Ser Gln Ala Lys Gln Leu Leu             100 105 110 Leu His His Thr Leu Gly Asn Gly Asp Phe Thr Val Phe His Arg Met         115 120 125 Ser Ala Asn Ile Ser Glu Thr Thr Ala Met Ile Thr Asp Ile Ala Asn     130 135 140 Ala Pro Ala Glu Ile Asp Arg Cys Ile Arg Thr Thr Tyr Thr Thr Gln 145 150 155 160 Arg Pro Val Tyr Leu Gly Leu Pro Ala Asn Leu Val Asp Leu Asn Val                 165 170 175 Pro Ala Lys Leu Leu Glu Thr Pro Ile Asp Leu Ser Leu Lys Pro Asn             180 185 190 Asp Ala Glu Ala Glu Ala Glu Val Val Arg Thr Val Val Glu Leu Ile         195 200 205 Lys Asp Ala Lys Asn Pro Val Ile Leu Ala Asp Ala Cys Ala Ser Arg     210 215 220 His Asp Val Lys Ala Glu Thr Lys Lys Leu Met Asp Leu Thr Gln Phe 225 230 235 240 Pro Val Tyr Val Thr Pro Met Gly Lys Gly Ala Ile Asp Glu Gln His                 245 250 255 Pro Arg Tyr Gly Gly Val Tyr Val Gly Thr Leu Ser Arg Pro Glu Val             260 265 270 Lys Lys Ala Val Glu Ser Ala Asp Leu Ile Leu Ser Ile Gly Ala Leu         275 280 285 Leu Ser Asp Phe Asn Thr Gly Ser Phe Ser Tyr Ser Tyr Lys Thr Lys     290 295 300 Asn Ile Val Glu Phe His Ser Asp His Ile Lys Ile Arg Asn Ala Thr 305 310 315 320 Phe Pro Gly Val Gln Met Lys Phe Ala Leu Gln Lys Leu Leu Asp Ala                 325 330 335 Ile Pro Glu Val Val Lys Asp Tyr Lys Pro Val Ala Val Pro Ala Arg             340 345 350 Val Pro Ile Thr Lys Ser Thr Pro Ala Asn Thr Pro Met Lys Gln Glu         355 360 365 Trp Met Trp Asn His Leu Gly Asn Phe Leu Arg Glu Gly Asp Ile Val     370 375 380 Ile Ala Glu Thr Gly Thr Ser Ala Phe Gly Ile Asn Gln Thr Thr Phe 385 390 395 400 Pro Thr Asp Val Tyr Ala Ile Val Gln Val Leu Trp Gly Ser Ile Gly                 405 410 415 Phe Thr Val Gly Ala Leu Leu Gly Ala Thr Met Ala Ala Glu Glu Leu             420 425 430 Asp Pro Lys Lys Arg Val Ile Leu Phe Ile Gly Asp Gly Ser Leu Gln         435 440 445 Leu Thr Val Gln Glu Ile Ser Thr Met Ile Arg Trp Gly Leu Lys Pro     450 455 460 Tyr Ile Phe Val Leu Asn Asn Asn Gly Tyr Thr Ile Glu Lys Leu Ile 465 470 475 480 His Gly Pro His Ala Glu Tyr Asn Glu Ile Gln Gly Trp Asp His Leu                 485 490 495 Ala Leu Leu Pro Thr Phe Gly Ala Arg Asn Tyr Glu Thr His Arg Val             500 505 510 Ala Thr Thr Gly Glu Trp Glu Lys Leu Thr Gln Asp Lys Asp Phe Gln         515 520 525 Asp Asn Ser Lys Ile Arg Met Ile Glu Val Met Leu Pro Val Phe Asp     530 535 540 Ala Pro Gln Asn Leu Val Lys Gln Ala Gln Leu Thr Ala Ala Thr Asn 545 550 555 560 Ala Lys Gln              <210> 110 <211> 1689 <212> DNA <213> Saccharomyces cerevisiae <400> 110 atgtctgaaa taaccttagg taaatattta tttgaaagat tgagccaagt caactgtaac 60 accgtcttcg gtttgccagg tgactttaac ttgtctcttt tggataagct ttatgaagtc 120 aaaggtatga gatgggctgg taacgctaac gaattgaacg ctgcctatgc tgctgatggt 180 tacgctcgta tcaagggtat gtcctgtatt attaccacct tcggtgttgg tgaattgtct 240 gctttgaatg gtattgccgg ttcttacgct gaacatgtcg gtgttttgca cgttgttggt 300 gttccatcca tctcttctca agctaagcaa ttgttgttgc atcatacctt gggtaacggt 360 gacttcactg ttttccacag aatgtctgcc aacatttctg aaaccactgc catgatcact 420 gatattgcta acgctccagc tgaaattgac agatgtatca gaaccaccta cactacccaa 480 agaccagtct acttgggttt gccagctaac ttggttgact tgaacgtccc agccaagtta 540 ttggaaactc caattgactt gtctttgaag ccaaacgacg ctgaagctga agctgaagtt 600 gttagaactg ttgttgaatt gatcaaggat gctaagaacc cagttatctt ggctgatgct 660 tgtgcttcta gacatgatgt caaggctgaa actaagaagt tgatggactt gactcaattc 720 ccagtttacg tcaccccaat gggtaagggt gctattgacg aacaacaccc aagatacggt 780 gt; ttgatattgt ctatcggtgc tttgttgtct gatttcaata ccggttcttt ctcttactcc 900 tacaagacca aaaatatcgt tgaattccac tctgaccaca tcaagatcag aaacgccacc 960 ttcccaggtg ttcaaatgaa atttgccttg caaaaattgt tggatgctat tccagaagtc 1020 gtcaaggact acaaacctgt tgctgtccca gctagagttc caattaccaa gtctactcca 1080 gctaacactc caatgaagca agaatggatg tggaaccatt tgggtaactt cttgagagaa 1140 ggtgatattg ttattgctga aaccggtact tccgccttcg gtattaacca aactactttc 1200 ccaacagatg tatacgctat cgtccaagtc ttgtggggtt ccattggttt cacagtcggc 1260 gctctattgg gtgctactat ggccgctgaa gaacttgatc caaagaagag agttatttta 1320 ttcattggtg acggttctct acaattgact gttcaagaaa tctctaccat gattagatgg 1380 ggtttgaagc catacatttt tgtcttgaat aacaacggtt acaccattga aaaattgatt 1440 cacggtcctc atgccgaata taatgaaatt caaggttggg accacttggc cttattgcca 1500 acttttggtg ctagaaacta cgaaacccac agagttgcta ccactggtga atgggaaaag 1560 ttgactcaag acaaggactt ccaagacaac tctaagatta gaatgattga agttatgttg 1620 ccagtctttg atgctccaca aaacttggtt aaacaagctc aattgactgc cgctactaac 1680 gctaaacaa 1689 <210> 111 <211> 533 <212> PRT <213> Saccharomyces cerevisiae <400> 111 Met Ser Glu Ile Thr Leu Gly Lys Tyr Leu Phe Glu Arg Leu Lys Gln 1 5 10 15 Val Asn Val Asn Thr Ile Phe Gly Leu Pro Gly Asp Phe Asn Leu Ser             20 25 30 Leu Leu Asp Lys Ile Tyr Glu Val Asp Gly Leu Arg Trp Ala Gly Asn         35 40 45 Ala Asn Glu Leu Asn Ala Ala Tyr Ala Ala Asp Gly Tyr Ala Arg Ile     50 55 60 Lys Gly Leu Ser Val Leu Val Thr Thr Phe Gly Val Gly Glu Leu Ser 65 70 75 80 Ala Leu Asn Gly Ile Ala Gly Ser Tyr Ala Glu His Val Gly Val Leu                 85 90 95 His Val Val Gly Val Ser Ser Ser Ser Ala Gln Ala Lys Gln Leu Leu             100 105 110 Leu His His Thr Leu Gly Asn Gly Asp Phe Thr Val Phe His Arg Met         115 120 125 Ser Ala Asn Ile Ser Glu Thr Thr Ser Met Ile Thr Asp Ile Ala Thr     130 135 140 Ala Pro Ser Glu Ile Asp Arg Leu Ile Arg Thr Thr Phe Ile Thr Gln 145 150 155 160 Arg Pro Ser Tyr Leu Gly Leu Pro Ala Asn Leu Val Asp Leu Lys Val                 165 170 175 Pro Gly Ser Leu Leu Glu Lys Pro Ile Asp Leu Ser Leu Lys Pro Asn             180 185 190 Asp Pro Glu Ala Glu Lys Glu Val Ile Asp Thr Val Leu Glu Leu Ile         195 200 205 Gln Asn Ser Lys Asn Pro Val Ile Leu Ser Asp Ala Cys Ala Ser Arg     210 215 220 His Asn Val Lys Lys Glu Thr Gln Lys Leu Ile Asp Leu Thr Gln Phe 225 230 235 240 Pro Ala Phe Val Thr Pro Leu Gly Lys Gly Ser Ile Asp Glu Gln His                 245 250 255 Pro Arg Tyr Gly Gly Val Tyr Val Gly Thr Leu Ser Lys Gln Asp Val             260 265 270 Lys Gln Ala Val Glu Ser Ala Asp Leu Ile Leu Ser Val Gly Ala Leu         275 280 285 Leu Ser Asp Phe Asn Thr Gly Ser Phe Ser Tyr Ser Tyr Lys Thr Lys     290 295 300 Asn Val Val Glu Phe His Ser Asp Tyr Val Lys Val Lys Asn Ala Thr 305 310 315 320 Phe Leu Gly Val Gln Met Lys Phe Ala Leu Gln Asn Leu Leu Lys Val                 325 330 335 Ile Pro Asp Val Val Lys Gly Tyr Lys Ser Val Pro Val Thr Lys             340 345 350 Thr Pro Ala Asn Lys Gly Val Pro Ala Ser Thr Pro Leu Lys Gln Glu         355 360 365 Trp Leu Trp Asn Glu Leu Ser Lys Phe Leu Gln Glu Gly Asp Val Ile     370 375 380 Ile Ser Glu Thr Gly Thr Ser Ala Phe Gly Ile Asn Gln Thr Ile Phe 385 390 395 400 Pro Lys Asp Ala Tyr Gly Ile Ser Gln Val Leu Trp Gly Ser Ile Gly                 405 410 415 Phe Thr Thr Gly Ala Thr Leu Gly Ala Ala Phe Ala Ala Glu Glu Ile             420 425 430 Asp Pro Asn Lys Arg Val Ile Leu Phe Ile Gly Asp Gly Ser Leu Gln         435 440 445 Leu Thr Val Gln Glu Ile Ser Thr Met Ile Arg Trp Gly Leu Lys Pro     450 455 460 Tyr Leu Phe Val Leu Asn Asn Asp Gly Tyr Thr Ile Glu Lys Leu Ile 465 470 475 480 His Gly Pro His Ala Glu Tyr Asn Glu Ile Gln Thr Trp Asp His Leu                 485 490 495 Ala Leu Leu Pro Ala Phe Gly Ala Lys Lys Tyr Glu Asn His Lys Ile             500 505 510 Ala Thr Thr Gly Glu Trp Asp Ala Leu Thr Thr Asp Ser Glu Phe Gln         515 520 525 Lys Asn Ser Val Ile     530 <210> 112 <211> 1599 <212> DNA <213> Saccharomyces cerevisiae <400> 112 atgtctgaaa ttactcttgg aaaatactta tttgaaagat tgaagcaagt taatgttaac 60 accatttttg ggctaccagg cgacttcaac ttgtccctat tggacaagat ttacgaggta 120 gatggattga gatgggctgg taatgcaaat gagctgaacg ccgcctatgc cgccgatggt 180 tacgcacgca tcaagggttt atctgtgctg gtaactactt ttggcgtagg tgaattatcc 240 gccttgaatg gtattgcagg atcgtatgca gaacacgtcg gtgtactgca tgttgttggt 300 gtcccctcta tctccgctca ggctaagcaa ttgttgttgc atcatacctt gggtaacggt 360 gattttaccg tttttcacag aatgtccgcc aatatctcag aaactacatc aatgattaca 420 gacattgcta cagccccttc agaaatcgat aggttgatca ggacaacatt tataacacaa 480 aggcctagct acttggggtt gccagcgaat ttggtagatc taaaggttcc tggttctctt 540 ttggaaaaac cgattgatct atcattaaaa cctaacgatc ccgaagctga aaaggaagtt 600 attgataccg tactagaatt gatccagaat tcgaaaaacc ctgttatact atcggatgcc 660 tgtgcttcta ggcacaacgt taaaaaagaa acccagaagt taattgattt gacgcaattc 720 ccagcttttg tgacacctct aggtaaaggg tcaatagatg aacagcatcc cagatatggc 780 ggtgtttatg tgggaacgct gtccaaacaa gacgtgaaac aggccgttga gtcggctgat 840 ttgatccttt cggtcggtgc tttgctctct gattttaaca caggttcgtt ttcctactcc 900 tacaagacta aaaatgtagt ggagtttcat tccgattacg taaaggtgaa gaacgctacg 960 ttcctcggtg tacaaatgaa atttgcacta caaaacttac tgaaggttat tcccgatgtt 1020 gttaagggct acaagagcgt tcccgtacca accaaaactc ccgcaaacaa aggtgtacct 1080 gctagcacgc ccttgaaaca agagtggttg tggaacgaat tgtccaaatt cttgcaagaa 1140 ggtgatgtta tcatttccga gaccggcacg tctgccttcg gtatcaatca aactatcttt 1200 cctaaggacg cctacggtat ctcgcaggtg ttgtgggggt ccatcggttt tacaacagga 1260 gcaactttag gtgctgcctt tgccgctgag gagattgacc ccaacaagag agtcatctta 1320 ttcataggtg acgggtcttt gcagttaacc gtccaagaaa tctccaccat gatcagatgg 1380 gggttaaagc cgtatctttt tgtccttaac aacgacggct acactatcga aaagctgatt 1440 catgggcctc acgcagagta caacgaaatc cagacctggg atcacctcgc cctgttgccc 1500 gcatttggtg cgaaaaagta cgaaaatcac aagatcgcca ctacgggtga gtgggatgcc 1560 ttaaccactg attcagagtt ccagaaaaac tcggtgatc 1599 <210> 113 <211> 564 <212> PRT <213> Candida glabrata <400> 113 Met Ser Glu Ile Thr Leu Gly Arg Tyr Leu Phe Glu Arg Leu Asn Gln 1 5 10 15 Val Asp Val Lys Thr Ile Phe Gly Leu Pro Gly Asp Phe Asn Leu Ser             20 25 30 Leu Leu Asp Lys Ile Tyr Glu Val Glu Gly Met Arg Trp Ala Gly Asn         35 40 45 Ala Asn Glu Leu Asn Ala Ala Tyr Ala Ala Asp Gly Tyr Ala Arg Ile     50 55 60 Lys Gly Met Ser Cys Ile Ile Thr Thr Phe Gly Val Gly Glu Leu Ser 65 70 75 80 Ala Leu Asn Gly Ile Ala Gly Ser Tyr Ala Glu His Val Gly Val Leu                 85 90 95 His Val Val Gly Val Ser Ser Ser Ser Gln Ala Lys Gln Leu Leu             100 105 110 Leu His His Thr Leu Gly Asn Gly Asp Phe Thr Val Phe His Arg Met         115 120 125 Ser Ala Asn Ile Ser Glu Thr Thr Ala Met Val Thr Asp Ile Ala Thr     130 135 140 Ala Pro Ala Glu Ile Asp Arg Cys Ile Arg Thr Thr Tyr Ile Thr Gln 145 150 155 160 Arg Pro Val Tyr Leu Gly Leu Pro Ala Asn Leu Val Asp Leu Lys Val                 165 170 175 Pro Ala Lys Leu Leu Glu Thr Pro Ile Asp Leu Ser Leu Lys Pro Asn             180 185 190 Asp Pro Glu Ala Glu Thr Glu Val Val Asp Thr Val Leu Glu Leu Ile         195 200 205 Lys Ala Ala Lys Asn Pro Val Ile Leu Ala Asp Ala Cys Ala Ser Arg     210 215 220 His Asp Val Lys Ala Glu Thr Lys Lys Leu Ile Asp Ala Thr Gln Phe 225 230 235 240 Pro Ser Phe Val Thr Pro Met Gly Lys Gly Ser Ile Asp Glu Gln His                 245 250 255 Pro Arg Phe Gly Gly Val Tyr Val Gly Thr Leu Ser Arg Pro Glu Val             260 265 270 Lys Glu Ala Val Glu Ser Ala Asp Leu Ile Leu Ser Val Gly Ala Leu         275 280 285 Leu Ser Asp Phe Asn Thr Gly Ser Phe Ser Tyr Ser Tyr Lys Thr Lys     290 295 300 Asn Ile Val Glu Phe His Ser Asp Tyr Ile Lys Ile Arg Asn Ala Thr 305 310 315 320 Phe Pro Gly Val Gln Met Lys Phe Ala Leu Gln Lys Leu Leu Asn Ala                 325 330 335 Val Pro Glu Ala Ile Lys Gly Tyr Lys Pro Val Val Pro Ala Arg             340 345 350 Val Pro Glu Asn Lys Ser Cys Asp Pro Ala Thr Pro Leu Lys Gln Glu         355 360 365 Trp Met Trp Asn Gln Val Ser Lys Phe Leu Gln Glu Gly Asp Val Val     370 375 380 Ile Thr Glu Thr Gly Thr Ser Ala Phe Gly Ile Asn Gln Thr Pro Phe 385 390 395 400 Pro Asn Asn Ala Tyr Gly Ile Ser Gln Val Leu Trp Gly Ser Ile Gly                 405 410 415 Phe Thr Thr Gly Ala Cys Leu Gly Ala Ala Phe Ala Ala Glu Glu Ile             420 425 430 Asp Pro Lys Lys Arg Val Ile Leu Phe Ile Gly Asp Gly Ser Leu Gln         435 440 445 Leu Thr Val Gln Glu Ile Ser Thr Met Ile Arg Trp Gly Leu Lys Pro     450 455 460 Tyr Leu Phe Val Leu Asn Asn Asp Gly Tyr Thr Ile Glu Arg Leu Ile 465 470 475 480 His Gly Glu Lys Ala Gly Tyr Asn Asp Ile Gln Asn Trp Asp His Leu                 485 490 495 Ala Leu Leu Pro Thr Phe Gly Ala Lys Asp Tyr Glu Asn His Arg             500 505 510 Ala Thr Thr Gly Glu Trp Asp Lys Leu Thr Gln Asp Lys Glu Phe Asn         515 520 525 Lys Asn Ser Lys Ile Arg Met Ile Glu Val Met Leu Pro Val Met Asp     530 535 540 Ala Pro Thr Ser Leu Ile Glu Gln Ala Lys Leu Thr Ala Ser Ile Asn 545 550 555 560 Ala Lys Gln Glu                  <210> 114 <211> 1692 <212> DNA <213> Candida glabrata <400> 114 atgtctgaga ttactttggg tagatacttg ttcgagagat tgaaccaagt cgacgttaag 60 accatcttcg gtttgccagg tgacttcaac ttgtccctat tggacaagat ctacgaagtt 120 gaaggtatga gatgggctgg taacgctaac gaattgaacg ctgcttacgc tgctgacggt 180 tacgctagaa tcaagggtat gtcctgtatc atcaccacct tcggtgtcgg tgaattgtct 240 gccttgaacg gtattgccgg ttcttacgct gaacacgtcg gtgtcttgca cgtcgtcggt 300 gtcccatcca tctcctctca agctaagcaa ttgttgttgc accacacctt gggtaacggt 360 gacttcactg tcttccacag aatgtccgct aacatctctg agaccaccgc tatggtcact 420 gacatcgcta ccgctccagc tgagatcgac agatgtatca gaaccaccta catcacccaa 480 agaccagtct acttgggtct accagctaac ttggtcgacc taaaggtccc agccaagctt 540 ttggaaaccc caattgactt gtccttgaag ccaaacgacc cagaagccga aactgaagtc 600 gttgacaccg tcttggaatt gatcaaggct gctaagaacc cagttatctt ggctgatgct 660 tgtgcttcca gacacgacgt caaggctgaa accaagaagt tgattgacgc cactcaattc 720 ccatccttcg ttaccccaat gggtaagggt tccatcgacg aacaacaccc aagattcggt 780 ggtgtctacg tcggtacctt gtccagacca gaagttaagg aagctgttga atccgctgac 840 ttgatcttgt ctgtcggtgc tttgttgtcc gatttcaaca ctggttcttt ctcttactct 900 tacaagacca agaacatcgt cgaattccac tctgactaca tcaagatcag aaacgctacc 960 ttcccaggtg tccaaatgaa gttcgctttg caaaagttgt tgaacgccgt cccagaagct 1020 atcaagggtt acaagccagt ccctgtccca gctagagtcc cagaaaacaa gtcctgtgac 1080 ccagctaccc cattgaagca agaatggatg tggaaccaag tttccaagtt cttgcaagaa 1140 ggtgatgttg ttatcactga aaccggtacc tccgcttttg gtatcaacca aaccccattc 1200 ccaaacaacg cttacggtat ctcccaagtt ctatggggtt ccatcggttt caccaccggt 1260 gcttgtttgg gtgccgcttt cgctgctgaa gaaatcgacc caaagaagag agttatcttg 1320 ttcattggtg acggttcttt gcaattgact gtccaagaaa tctccaccat gatcagatgg 1380 ggcttgaagc catacttgtt cgtcttgaac aacgacggtt acaccatcga aagattgatt 1440 cacggtgaaa aggctggtta caacgacatc caaaactggg accacttggc tctattgcca 1500 accttcggtg ctaaggacta cgaaaaccac agagtcgcca ccaccggtga atgggacaag 1560 ttgacccaag acaaggaatt caacaagaac tccaagatca gaatgatcga agttatgttg 1620 ccagttatgg acgctccaac ttccttgatt gaacaagcta agttgaccgc ttccatcaac 1680 gctaagcaag aa 1692 <210> 115 <211> 596 <212> PRT <213> Pichia stipitis <400> 115 Met Ala Glu Val Ser Leu Gly Arg Tyr Leu Phe Glu Arg Leu Tyr Gln 1 5 10 15 Leu Gln Val Gln Thr Ile Phe Gly Val Pro Gly Asp Phe Asn Leu Ser             20 25 30 Leu Leu Asp Lys Ile Tyr Glu Val Glu Asp Ala His Gly Lys Asn Ser         35 40 45 Phe Arg Trp Ala Gly Asn Ala Asn Glu Leu Asn Ala Ser Tyr Ala Ala     50 55 60 Asp Gly Tyr Ser Arg Val Lys Arg Leu Gly Cys Leu Val Thr Thr Phe 65 70 75 80 Gly Val Gly Glu Leu Ser Ala Leu Asn Gly Ile Ala Gly Ser Tyr Ala                 85 90 95 Glu His Val Gly Leu Leu His Val Val Gly Val Ser Ser Ser Ser             100 105 110 Gln Ala Lys Gln Leu Leu Leu His His Thr Leu Gly Asn Gly Asp Phe         115 120 125 Thr Val Phe His Arg Met Ser Asn Asn Ile Ser Gln Thr Thr Ala Phe     130 135 140 Ile Ser Asp Ile Asn Ser Ala Pro Ala Glu Ile Asp Arg Cys Ile Arg 145 150 155 160 Glu Ala Tyr Val Lys Gln Arg Pro Val Tyr Ile Gly Leu Pro Ala Asn                 165 170 175 Leu Val Asp Leu Asn Val Pro Ala Ser Leu Leu Glu Ser Pro Ile Asn             180 185 190 Leu Ser Leu Glu Lys Asn Asp Pro Glu Ala Gln Asp Glu Val Ile Asp         195 200 205 Ser Val Leu Asp Leu Ile Lys Lys Ser Ser Asn Pro Ile Ile Leu Val     210 215 220 Asp Ala Cys Ala Ser Arg His Asp Cys Lys Ala Glu Val Thr Gln Leu 225 230 235 240 Ile Glu Gln Thr Gln Phe Pro Val Phe Val Thr Pro Met Gly Lys Gly                 245 250 255 Thr Val Asp Glu Gly Gly Val Asp Gly Glu Leu Leu Glu Asp Asp Pro             260 265 270 His Leu Ile Ala Lys Val Ala Ala Arg Leu Ser Ala Gly Lys Asn Ala         275 280 285 Ala Ser Arg Phe Gly Gly Val Tyr Val Gly Thr Leu Ser Lys Pro Glu     290 295 300 Val Lys Asp Ala Val Glu Ser Ala Asp Leu Ile Leu Ser Val Gly Ala 305 310 315 320 Leu Leu Ser Asp Phe Asn Thr Gly Ser Phe Ser Tyr Ser Tyr Arg Thr                 325 330 335 Lys Asn Ile Val Glu Phe His Ser Asp Tyr Thr Lys Ile Arg Gln Ala             340 345 350 Thr Phe Pro Gly Val Gln Met Lys Glu Ala Leu Gln Glu Leu Asn Lys         355 360 365 Lys Val Ser Ser Ala Ser Ser His Tyr Glu Val Lys Pro Val Pro Lys     370 375 380 Ile Lys Leu Ala Asn Thr Pro Ala Thr Arg Glu Val Lys Leu Thr Gln 385 390 395 400 Glu Trp Leu Trp Thr Arg Val Ser Ser Trp Phe Arg Glu Gly Asp Ile                 405 410 415 Ile Ile Thr Glu Thr Gly Thr Ser Ser Phe Gly Ile Val Gln Ser Arg             420 425 430 Phe Pro Asn Asn Thr Ile Gly Ile Ser Gln Val Leu Trp Gly Ser Ile         435 440 445 Gly Phe Ser Val Gly Ala Thr Leu Gly Ala Ala Met Ala Ala Gln Glu     450 455 460 Leu Asp Pro Asn Lys Arg Thr Ile Leu Phe Val Gly Asp Gly Ser Leu 465 470 475 480 Gln Leu Thr Val Gln Glu Ile Ser Thr Ile Ile Arg Trp Gly Thr Thr                 485 490 495 Pro Tyr Leu Phe Val Leu Asn Asn Asp Gly Tyr Thr Ile Glu Arg Leu             500 505 510 Ile His Gly Val Asn Ala Ser Tyr Asn Asp Ile Gln Pro Trp Gln Asn         515 520 525 Leu Glu Ile Leu Pro Thr Phe Ser Ala Lys Asn Tyr Asp Ala Val Arg     530 535 540 Ile Ser Asn Ile Gly Glu Ala Glu Asp Ile Leu Lys Asp Lys Glu Phe 545 550 555 560 Gly Lys Asn Ser Lys Ile Arg Leu Ile Glu Val Met Leu Pro Arg Leu                 565 570 575 Asp Ala Pro Ser Asn Leu Ala Lys Gln Ala Ala Ile Thr Ala Ala Thr             580 585 590 Asn Ala Glu Ala         595 <210> 116 <211> 1788 <212> DNA <213> Pichia stipitis <400> 116 atggctgaag tctcattagg aagatatctc ttcgagagat tgtaccaatt gcaagtgcag 60 accatcttcg gtgtccctgg tgatttcaac ttgtcgcttt tggacaagat ctacgaagtg 120 gaagatgccc atggcaagaa ttcgtttaga tgggctggta atgccaacga attgaatgca 180 tcgtacgctg ctgacggtta ctcgagagtc aagcgtttag ggtgtttggt cactaccttt 240 ggtgtcggtg aattgtctgc tttgaatggt attgccggtt cttatgccga acatgttggt 300 ttgcttcatg tcgtaggtgt tccatcgatt tcctcgcaag ctaagcaatt gttacttcac 360 cacactttgg gtaatggtga tttcactgtt ttccatagaa tgtccaacaa catttctcag 420 accacagcct ttatctccga tatcaactcg gctccagctg aaattgatag atgatatcaga 480 gaggcctacg tcaaacaaag accagtttat atcgggttac cagctaactt agttgatttg 540 aatgttccgg cctctttgct tgagtctcca atcaacttgt cgttggaaaa gaacgaccca 600 gaggctcaag atgaagtcat tgactctgtc ttagacttga tcaaaaagtc gctgaaccca 660 atcatcttgg tcgatgcctg tgcctcgaga catgactgta aggctgaagt tactcagttg 720 attgaacaaa cccaattccc agtatttgtc actccaatgg gtaaaggtac cgttgatgag 780 ggtggtgtag acggagaatt gttagaagat gatcctcatt tgattgccaa ggtcgctgct 840 aggttgtctg ctggcaagaa cgctgcctct agattcggag gtgtttatgt cggaaccttg 900 tcgaagcccg aagtcaagga cgctgtagag agtgcagatt tgattttgtc tgtcggtgcc 960 cttttgtctg atttcaacac tggttcattt tcctactcct acagaaccaa gaacatcgtc 1020 gaattccatt ctgattacac taagattaga caagccactt tcccaggtgt gcagatgaag 1080 gaagccttgc aagaattgaa caagaaagtt tcatctgctg ctagtcacta tgaagtcaag 1140 cctgtgccca agatcaagtt ggccaataca ccagccacca gagaagtcaa gttaactcag 1200 gaatggttgt ggaccagagt gtcttcgtgg ttcagagaag gtgatattat tatcaccgaa 1260 accggtacat cctccttcgg tatagttcaa tccagattcc caaacaacac catcggtatc 1320 tcccaagtat tgtggggttc tattggtttc tctgttggtg ccactttggg tgctgccatg 1380 gctgcccaag aactcgaccc taacaagaga accatcttgt ttgttggaga tggttctttg 1440 caattgaccg ttcaggaaat ctccaccata atcagatggg gtaccacacc ttaccttttc 1500 gtgttgaaca atgacggtta caccatcgag cgtttgatcc acggtgtaaa tgcctcatat 1560 aatgacatcc aaccatggca aaacttggaa atcttgccta ctttctcggc caagaactac 1620 gacgctgtga gaatctccaa catcggagaa gcagaagata tcttgaaaga caaggaattc 1680 ggaaagaact ccaagattag attgatagaa gtcatgttac caagattgga tgcaccatct 1740 aaccttgcca aacaagctgc cattacagct gccaccaacg ccgaagct 1788 <210> 117 <211> 569 <212> PRT <213> Pichia stipitis <400> 117 Met Val Ser Thr Tyr Pro Glu Ser Glu Val Thr Leu Gly Arg Tyr Leu 1 5 10 15 Phe Glu Arg Leu His Gln Leu Lys Val Asp Thr Ile Phe Gly Leu Pro             20 25 30 Gly Asp Phe Asn Leu Ser Leu Leu Asp Lys Val Tyr Glu Val Pro Asp         35 40 45 Met Arg Trp Ala Gly Asn Ala Asn Glu Leu Asn Ala Ala Tyr Ala Ala     50 55 60 Asp Gly Tyr Ser Arg Ile Lys Gly Leu Ser Cys Leu Val Thr Thr Phe 65 70 75 80 Gly Val Gly Gly Leu Ser Ala Leu Asn Gly Val Gly Gly Ala Tyr Ala                 85 90 95 Glu His Val Gly Leu Leu His Val Val Gly Val Ser Ser Ser Ser             100 105 110 Gln Ala Lys Gln Leu Leu Leu His His Thr Leu Gly Asn Gly Asp Phe         115 120 125 Thr Val Phe His Arg Met Ser Asn Ser Ile Ser Gln Thr Thr Ala Phe     130 135 140 Leu Ser Asp Ile Ser Ile Pro Gly Gln Ile Asp Arg Cys Ile Arg 145 150 155 160 Glu Ala Tyr Val His Gln Arg Pro Val Tyr Val Gly Leu Pro Ala Asn                 165 170 175 Met Val Asp Leu Lys Val Ser Ser Leu Leu Glu Thr Pro Ile Asp             180 185 190 Leu Lys Leu Lys Gln Asn Asp Pro Glu Ala Gln Glu Val Val Glu Thr         195 200 205 Val Leu Lys Leu Val Ser Gln Ala Thr Asn Pro Ile Ile Leu Val Asp     210 215 220 Ala Cys Ala Leu Arg His His Cys Lys Glu Glu Val Lys Gln Leu Val 225 230 235 240 Asp Ala Thr Asn Phe Gln Val Phe Thr Thr Pro Met Gly Lys Ser Gly                 245 250 255 Ile Ser Glu Ser His Pro Arg Leu Gly Gly Val Tyr Val Gly Thr Met             260 265 270 Ser Ser Pro Gln Val Lys Lys Ala Val Glu Asn Ala Asp Leu Ile Leu         275 280 285 Ser Val Gly Ser Leu Leu Ser Asp Phe Asn Thr Gly Ser Phe Ser Tyr     290 295 300 Ser Tyr Lys Thr Lys Asn Val Val Glu Phe His Ser Asp Tyr Met Lys 305 310 315 320 Ile Arg Gln Ala Thr Phe Pro Gly Val Gln Met Lys Glu Ala Leu Gln                 325 330 335 Gln Leu Ile Lys Arg Val Ser Ser Tyr Ile Asn Pro Ser Tyr Ile Pro             340 345 350 Thr Arg Val Pro Lys Arg Lys Gln Pro Leu Lys Ala Pro Ser Glu Ala         355 360 365 Pro Leu Thr Gln Glu Tyr Leu Trp Ser Lys Val Ser Gly Trp Phe Arg     370 375 380 Glu Gly Asp Ile Ile Val Thr Glu Thr Gly Thr Ser Ala Phe Gly Ile 385 390 395 400 Ile Gln Ser His Phe Pro Ser Asn Thr Ile Gly Ile Ser Gln Val Leu                 405 410 415 Trp Gly Ser Ile Gly Phe Thr Val Gly Ala Thr Val Gly Ala Ala Met             420 425 430 Ala Ala Gln Glu Ile Asp Pro Ser Arg Arg Val Ile Leu Phe Val Gly         435 440 445 Asp Gly Ser Leu Gln Leu Thr Val Gln Glu Ile Ser Thr Leu Cys Lys     450 455 460 Trp Asp Cys Asn Asn Thr Tyr Leu Tyr Val Leu Asn Asn Asp Gly Tyr 465 470 475 480 Thr Ile Glu Arg Leu Ile His Gly Lys Ser Ala Ser Tyr Asn Asp Ile                 485 490 495 Gln Pro Trp Asn His Leu Ser Leu Leu Arg Leu Phe Asn Ala Lys Lys             500 505 510 Tyr Gln Asn Val Arg Ser Ser Thr Ala Gly Glu Leu Asp Ser Leu Phe         515 520 525 Ser Asp Lys Lys Phe Ala Ser Pro Asp Arg Ile Arg Met Ile Glu Val     530 535 540 Met Leu Ser Arg Leu Asp Ala Pro Ala Asn Leu Val Ala Gln Ala Lys 545 550 555 560 Leu Ser Glu Arg Val Asn Leu Glu Asn                 565 <210> 118 <211> 1707 <212> DNA <213> Pichia stipitis <400> 118 atggtatcaa cctacccaga atcagaggtt actctaggaa ggtacctctt tgagcgactc 60 caccaattga aagtggacac cattttcggc ttgccgggtg acttcaacct ttccttattg 120 gacaaagtgt atgaagttcc ggatatgagg tgggctggaa atgccaacga attgaatgct 180 gcctatgctg ccgatggtta ctccagaata aagggattgt cttgcttggt cacaactttt 240 ggtgttggtg aattgtctgc tttaaacgga gttggtggtg cctatgctga acacgtagga 300 cttctacatg tcgttggagt tccatccata tcgtcacagg ctaaacagtt gttgctccac 360 cataccttgg gtaatggtga cttcactgtt tttcacagaa tgtccaatag catttctcaa 420 actacagcat ttctctcaga tatctctatt gcaccaggtc aaatagatag atgcatcaga 480 gaagcatatg ttcatcagag accagtttat gttggtttac cggcaaatat ggttgatctc 540 aaggttcctt ctagtctctt agaaactcca attgatttga aattgaaaca aaatgatcct 600 gaagctcaag aagttgttga aacagtcctg aagttggtgt cccaagctac aaaccccatt 660 atcttggtag acgcttgtgc cctcagacac aattgcaaag aggaagtcaa acaattggtt 720 gatgccacta attttcaagt ctttacaact ccaatgggta aatctggtat ctccgaatct 780 catccaagat tgggcggtgt ctatgtcggg acaatgtcga gtcctcaagt caaaaaagcc 840 gttgaaaatg ccgatcttat actatctgtt ggttcgttgt tatcggactt caatacaggt 900 tcattttcat actcctacaa gacgaagaat gttgttgaat tccactctga ctatatgaaa 960 atcagacagg ccaccttccc aggagttcaa atgaaagaag ccttgcaaca gttgataaaa 1020 agggtctctt cttacatcaa tccaagctac attcctactc gagttcctaa aaggaaacag 1080 ccattgaaag ctccatcaga agctcctttg acccaagaat atttgtggtc taaagtatcc 1140 ggctggttta gagagggtga tattatcgta accgaaactg gtacatctgc tttcggaatt 1200 attcaatccc attttcccag caacactatc ggtatatccc aagtcttgtg gggctcaatt 1260 ggtttcacag taggtgcaac agttggtgct gccatggcag cccaggaaat cgaccctagc 1320 aggagagtaa ttttgttcgt cggtgatggt tcattgcagt tgacggttca ggaaatctct 1380 acgttgtgta aatgggattg taacaatact tatctttacg tgttgaacaa tgatggttac 1440 actatagaaa ggttgatcca cggcaaaagt gccagctaca acgatataca gccttggaac 1500 catttatcct tgcttcgctt attcaatgct aagaaatacc aaaatgtcag agtatcgact 1560 gctggagaat tggactcttt gttctctgat aagaaatttg cttctccaga taggataaga 1620 atgattgagg tgatgttatc gagattggat gcaccagcaa atcttgttgc tcaagcaaag 1680 ttgtctgaac gggtaaacct tgaaaat 1707 <210> 119 <211> 563 <212> PRT <213> Kluyveromyces lactis <400> 119 Met Ser Glu Ile Thr Leu Gly Arg Tyr Leu Phe Glu Arg Leu Lys Gln 1 5 10 15 Val Glu Val Gln Thr Ile Phe Gly Leu Pro Gly Asp Phe Asn Leu Ser             20 25 30 Leu Leu Asp Asn Ile Tyr Glu Val Pro Gly Met Arg Trp Ala Gly Asn         35 40 45 Ala Asn Glu Leu Asn Ala Ala Tyr Ala Ala Asp Gly Tyr Ala Arg Leu     50 55 60 Lys Gly Met Ser Cys Ile Ile Thr Thr Phe Gly Val Gly Glu Leu Ser 65 70 75 80 Ala Leu Asn Gly Ile Ala Gly Ser Tyr Ala Glu His Val Gly Val Leu                 85 90 95 His Val Val Gly Val Ser Ser Val Ser Ser Gln Ala Lys Gln Leu Leu             100 105 110 Leu His His Thr Leu Gly Asn Gly Asp Phe Thr Val Phe His Arg Met         115 120 125 Ser Ser Asn Ile Ser Glu Thr Thr Ala Met Ile Thr Asp Ile Asn Thr     130 135 140 Ala Pro Ala Glu Ile Asp Arg Cys Ile Arg Thr Thr Tyr Val Ser Gln 145 150 155 160 Arg Pro Val Tyr Leu Gly Leu Pro Ala Asn Leu Val Asp Leu Thr Val                 165 170 175 Pro Ala Ser Leu Leu Asp Thr Pro Ile Asp Leu Ser Leu Lys Pro Asn             180 185 190 Asp Pro Glu Ala Glu Glu Glu Val Ile Glu Asn Val Leu Gln Leu Ile         195 200 205 Lys Glu Ala Lys Asn Pro Val Ile Leu Ala Asp Ala Cys Cys Ser Arg     210 215 220 His Asp Ala Lys Ala Glu Thr Lys Lys Leu Ile Asp Leu Thr Gln Phe 225 230 235 240 Pro Ala Phe Val Thr Pro Met Gly Lys Gly Ser Ile Asp Glu Lys His                 245 250 255 Pro Arg Phe Gly Gly Val Tyr Val Gly Thr Leu Ser Ser Pro Ala Val             260 265 270 Lys Glu Ala Val Glu Ser Ala Asp Leu Val Leu Ser Val Gly Ala Leu         275 280 285 Leu Ser Asp Phe Asn Thr Gly Ser Phe Ser Tyr Ser Tyr Lys Thr Lys     290 295 300 Asn Ile Val Glu Phe His Ser Asp Tyr Thr Lys Ile Arg Ser Ala Thr 305 310 315 320 Phe Pro Gly Val Gln Met Lys Phe Ala Leu Gln Lys Leu Leu Thr Lys                 325 330 335 Val Ala Asp Ala Ala Lys Gly Tyr Lys Pro Val Val Ser Ser Glu             340 345 350 Pro Glu His Asn Glu Ala Val Ala Asp Ser Thr Pro Leu Lys Gln Glu         355 360 365 Trp Val Trp Thr Gln Val Gly Glu Phe Leu Arg Glu Gly Asp Val Val     370 375 380 Ile Thr Glu Thr Gly Thr Ser Ala Phe Gly Ile Asn Gln Thr His Phe 385 390 395 400 Pro Asn Asn Thr Tyr Gly Ile Ser Gln Val Leu Trp Gly Ser Ile Gly                 405 410 415 Phe Thr Thr Gly Ala Thr Leu Gly Ala Ala Phe Ala Ala Glu Glu Ile             420 425 430 Asp Pro Lys Lys Arg Val Ile Leu Phe Ile Gly Asp Gly Ser Leu Gln         435 440 445 Leu Thr Val Gln Glu Ile Ser Thr Met Ile Arg Trp Gly Leu Lys Pro     450 455 460 Tyr Leu Phe Val Leu Asn Asn Asp Gly Tyr Thr Ile Glu Arg Leu Ile 465 470 475 480 His Gly Glu Thr Ala Gln Tyr Asn Cys Ile Gln Asn Trp Gln His Leu                 485 490 495 Glu Leu Leu Pro Thr Ply Gly Ala Lys Asp Tyr Glu Ala Val Val Arg             500 505 510 Ser Thr Thr Gly Glu Trp Asn Lys Leu Thr Thr Asp Glu Lys Phe Gln         515 520 525 Asp Asn Thr Arg Ile Arg Leu Ile Glu Val Met Leu Pro Thr Met Asp     530 535 540 Ala Pro Ser Asn Leu Val Lys Gln Ala Gln Leu Thr Ala Ala Thr Asn 545 550 555 560 Ala Lys Asn              <210> 120 <211> 1689 <212> DNA <213> Kluyveromyces lactis <400> 120 atgtctgaaa ttacattagg tcgttacttg ttcgaaagat taaagcaagt cgaagttcaa 60 accatctttg gtctaccagg tgatttcaac ttgtccctat tggacaatat ctacgaagtc 120 ccaggtatga gatgggctgg taatgccaac gaattgaacg ctgcttacgc tgctgatggt 180 tacgccagat taaagggtat gtcctgtatc atcaccacct tcggtgtcgg tgaattgtct 240 gctttgaacg gtattgccgg ttcttacgct gaacacgttg gtgtcttgca cgttgtcggt 300 gttccatccg tctcttctca agctaagcaa ttgttgttgc accacacctt gggtaacggt 360 gacttcactg ttttccacag aatgtcctcc aacatttctg aaaccactgc tatgatcacc 420 gatatcaaca ctgccccagc tgaaatcgac agatgtatca gaaccactta cgtttcccaa 480 agaccagtct acttgggttt gccagctaac ttggtcgact tgactgtccc agcttctttg 540 ttggacactc caattgattt gagcttgaag ccaaatgacc cagaagccga agaagaagtc 600 atcgaaaacg tcttgcaact gatcaaggaa gctaagaacc cagttatctt ggctgatgct 660 tgttgttcca gacacgatgc caaggctgag accaagaagt tgatcgactt gactcaattc 720 ccagccttcg ttaccccaat gggtaagggt tccattgacg aaaagcaccc aagattcggt 780 ggtgtctacg tcggtaccct atcttctcca gctgtcaagg aagccgttga atctgctgac 840 ttggttctat cggtcggtgc tctattgtcc gatttcaaca ctggttcttt ctcttactct 900 tacaagacca agaacattgt cgaattccac tctgactaca ccaagatcag aagcgctacc 960 ttcccaggtg tccaaatgaa gttcgcttta caaaaattgt tgactaaggt tgccgatgct 1020 gctaagggtt acaagccagt tccagttcca tctgaaccag aacacaacga agctgtcgct 1080 gactccactc cattgaagca agaatgggtc tggactcaag tcggtgaatt cttgagagaa 1140 ggtgatgttg ttatcactga aaccggtacc tctgccttcg gtatcaacca aactcatttc 1200 ccaaacaaca catacggtat ctctcaagtt ttatggggtt ccattggttt caccactggt 1260 gctaccttgg gtgctgcctt cgctgccgaa gaaattgatc caaagaagag agttatctta 1320 ttcattggtg acggttcttt gcaattgact gttcaagaaa tctccaccat gatcagatgg 1380 ggcttgaagc catacttgtt cgtattgaac aacgacggtt acaccattga aagattgatt 1440 cacggtgaaa ccgctcaata caactgtatc caaaactggc aacacttgga attattgcca 1500 actttcggtg ccaaggacta cgaagctgtc agagtttcca ccactggtga atggaacaag 1560 ttgaccactg acgaaaagtt ccaagacaac accagaatca gattgatcga agttatgttg 1620 ccaactatgg atgctccatc taacttggtt aagcaagctc aattgactgc tgctaccaac 1680 gctaagaac 1689 <210> 121 <211> 571 <212> PRT <213> Yarrowia lipolytica <400> 121 Met Ser Asp Ser Glu Pro Gln Met Val Asp Leu Gly Asp Tyr Leu Phe 1 5 10 15 Ala Arg Phe Lys Gln Leu Gly Val Asp Ser Val Phe Gly Val Pro Gly             20 25 30 Asp Phe Asn Leu Thr Leu Leu Asp His Val Tyr Asn Val Asp Met Arg         35 40 45 Trp Val Gly Asn Thr Asn Glu Leu Asn Ala Gly Tyr Ser Ala Asp Gly     50 55 60 Tyr Ser Arg Val Lys Arg Leu Ala Cys Leu Val Thr Thr Phe Gly Val 65 70 75 80 Gly Glu Leu Ser Ala Val Ala Ala Val Ala Gly Ser Tyr Ala Glu His                 85 90 95 Val Gly Val Val His Val Val Gly Val Ser Ser Ser Ser Ala Glu Asn             100 105 110 Lys His Leu Leu Leu His His Thr Leu Gly Asn Gly Asp Phe Arg Val         115 120 125 Phe Ala Gln Met Ser Lys Leu Ile Ser Glu Tyr Thr His His Ile Glu     130 135 140 Asp Pro Ser Glu Ala Ala Asp Val Ile Asp Thr Ala Ile Arg Ile Ala 145 150 155 160 Tyr Thr His Gln Arg Pro Val Tyr Ile Ala Val Ser Ser Asn Phe Ser                 165 170 175 Glu Val Asp Ile Ala Asp Gln Ala Arg Leu Asp Thr Pro Leu Asp Leu             180 185 190 Ser Leu Gln Pro Asn Asp Pro Glu Ser Gln Tyr Glu Val Ile Glu Glu         195 200 205 Ile Cys Ser Arg Ile Lys Ala Ala Lys Lys Pro Val Ile Leu Val Asp     210 215 220 Ala Cys Ala Ser Arg Tyr Arg Cys Val Asp Glu Thr Lys Glu Leu Ala 225 230 235 240 Lys Ile Thr Asn Phe Ala Tyr Phe Val Thr Pro Met Gly Lys Gly Ser                 245 250 255 Val Asp Glu Asp Thr Asp Arg Tyr Gly Gly Thr Tyr Val Gly Ser Leu             260 265 270 Thr Ala Pro Ala Thr Ala Glu Val Val Glu Thr Ala Asp Leu Ile Ile         275 280 285 Ser Val Gly Ala Leu Leu Ser Asp Phe Asn Thr Gly Ser Phe Ser Tyr     290 295 300 Ser Tyr Ser Thr Lys Asn Val Val Glu Leu His Ser Asp His Val Lys 305 310 315 320 Ile Lys Ser Ala Thr Tyr Asn Asn Val Gly Met Lys Met Leu Phe Pro                 325 330 335 Pro Leu Leu Glu Ala Val Lys Lys Leu Val Ala Glu Thr Pro Asp Phe             340 345 350 Ala Ser Lys Ala Leu Ala Val Pro Asp Thr Thr Pro Lys Ile Pro Glu         355 360 365 Val Pro Asp Asp His Ile Thr Thr Gln Ala Trp Leu Trp Gln Arg Leu     370 375 380 Ser Tyr Phe Leu Arg Pro Thr Asp Ile Val Val Thr Glu Thr Gly Thr 385 390 395 400 Ser Ser Phe Gly Ile Ile Gln Thr Lys Phe Pro His Asn Val Arg Gly                 405 410 415 Ile Ser Gln Val Leu Trp Gly Ser Ile Gly Tyr Ser Val Gly Ala Ala             420 425 430 Cys Gly Ala Ser Ile Ala Gln Glu Ile Asp Pro Gln Gln Arg Val         435 440 445 Ile Leu Phe Val Gly Asp Gly Ser Leu Gln Leu Thr Val Thr Glu Ile     450 455 460 Ser Cys Met Ile Arg Asn Asn Val Lys Pro Tyr Ile Phe Val Leu Asn 465 470 475 480 Asn Asp Gly Tyr Thr Ile Glu Arg Leu Ile His Gly Glu Asn Ala Ser                 485 490 495 Tyr Asn Asp Val His Met Trp Lys Tyr Ser Lys Ile Leu Asp Thr Phe             500 505 510 Asn Ala Lys Ala His Glu Ser Ile Val Val Asn Thr Lys Gly Glu Met         515 520 525 Asp Ala Leu Phe Asp Asn Glu Glu Phe Ala Lys Pro Asp Lys Ile Arg     530 535 540 Leu Ile Glu Val Met Cys Asp Lys Met Asp Ala Pro Ala Ser Leu Ile 545 550 555 560 Lys Gln Ala Glu Leu Ser Ala Lys Thr Asn Val                 565 570 <210> 122 <211> 1713 <212> DNA <213> Yarrowia lipolytica <400> 122 atgagcgact ccgaacccca aatggtcgac ctgggcgact atctctttgc ccgattcaag 60 cagctaggcg tggactccgt ctttggagtg cccggcgact tcaacctcac cctgttggac 120 cacgtgtaca atgtcgacat gcggtgggtt gggaacacaa acgagctgaa tgccggctac 180 tcggccgacg gctactcccg ggtcaagcgg ctggcatgtc ttgtcaccac ctttggcgtg 240 ggagagctgt ctgccgtggc tgctgtggca ggctcgtacg ccgagcatgt gggcgtggtg 300 catgttgtgg gcgttcccag cacctctgct gagaacaagc atctgctgct gcaccacaca 360 ctcggtaacg gcgacttccg ggtctttgcc cagatgtcca aactcatctc cgagtacacc 420 caccatattg aggaccccag cgaggctgcc gacgtaatcg acaccgccat ccgaatcgcc 480 tacacccacc agcggcccgt ttacattgct gtgccctcca acttctccga ggtcgatatt 540 gccgaccagg ctagactgga tacccccctg gacctttcgc tgcagcccaa cgaccccgag 600 agccagtacg aggtgattga ggagatttgc tcgcgtatca aggccgccaa gaagcccgtg 660 attctcgtcg acgcctgcgc ttcgcgatac agatgtgtgg acgagaccaa ggagctggcc 720 aagatcacca actttgccta ctttgtcact cccatgggta agggttctgt ggacgaggat 780 actgaccggt acggaggaac atacgtcgga tcgctgactg ctcctgctac tgccgaggtg 840 gttgagacag ctgatctcat catctccgta ggagctcttc tgtcggactt caacaccggt 900 tccttctcgt actcctactc caccaaaaac gtggtggaat tgcattcgga ccacgtcaaa 960 atcaagtccg ccacctacaa caacgtcggc atgaaatgc tgttcccgcc cctgctcgaa 1020 gccgtcaaga aactggttgc cgagacccct gactttgcat ccaaggctct ggctgttccc 1080 gacaccactc ccaagatccc cgaggtaccc gatgatcaca ttacgaccca ggcatggctg 1140 tggcagcgtc tcagttactt tctgaggccc accgacatcg tggtcaccga gaccggaacc 1200 tcgtcctttg gaatcatcca gaccaagttc ccccacaacg tccgaggtat ctcgcaggtg 1260 ctgtggggct ctattggata ctcggtggga gcagcctgtg gagcctccat tgctgcacag 1320 gagattgacc cccagcagcg agtgattctg tttgtgggcg acggctctct tcagctgacg 1380 gtgaccgaga tctcgtgcat gatccgcaac aacgtcaagc cgtacatttt tgtgctcaac 1440 aacgacggct acaccatcga gaggctcatt cacggcgaaa acgcctcgta caacgatgtg 1500 cacatgtgga agtactccaa gattctcgac acgttcaacg ccaaggccca cgagtcgatt 1560 gtggtcaaca ccaagggcga gatggacgct ctgttcgaca acgaagagtt tgccaagccc 1620 gacaagatcc ggctcattga ggtcatgtgc gacaagatgg acgcgcctgc ctcgttgatc 1680 aagcaggctg agctctctgc caagaccaac gtt 1713 <210> 123 <211> 571 <212> PRT <213> Schizosaccharomyces pombe <400> 123 Met Ser Gly Asp Ile Leu Val Gly Glu Tyr Leu Phe Lys Arg Leu Glu 1 5 10 15 Gln Leu Gly Val Lys Ser Ile Leu Gly Val Pro Gly Asp Phe Asn Leu             20 25 30 Ala Leu Leu Asp Leu Ile Glu Lys Val Gly Asp Glu Lys Phe Arg Trp         35 40 45 Val Gly Asn Thr Asn Glu Leu Asn Gly Ala Tyr Ala Ala Asp Gly Tyr     50 55 60 Ala Arg Val Asn Gly Leu Ser Ala Ile Val Thr Thr Phe Gly Val Gly 65 70 75 80 Glu Leu Ser Ala Ile Asn Gly Val Ala Gly Ser Tyr Ala Glu His Val                 85 90 95 Pro Val Val His Ile Val Gly Met Pro Ser Thr Lys Val Gln Asp Thr             100 105 110 Gly Ala Leu Leu His His Thr Leu Gly Asp Gly Asp Phe Arg Thr Phe         115 120 125 Met Asp Met Phe Lys Lys Val Ser Ala Tyr Ser Ile Met Ile Asp Asn     130 135 140 Gly Asn Asp Ala Glu Lys Ile Asp Glu Ala Leu Ser Ile Cys Tyr 145 150 155 160 Lys Lys Ala Arg Pro Val Tyr Ile Gly Ile Pro Ser Asp Ala Gly Tyr                 165 170 175 Phe Lys Ala Ser Ser Ser Asn Leu Gly Lys Arg Leu Lys Leu Glu Glu             180 185 190 Asp Thr Asn Asp Pro Ala Val Glu Gln Glu Val Ile Asn His Ile Ser         195 200 205 Glu Met Val Val Asn Ala Lys Lys Pro Val Ile Leu Ile Asp Ala Cys     210 215 220 Ala Val Arg His Arg Val Val Pro Glu Val His Glu Leu Ile Lys Leu 225 230 235 240 Thr His Phe Pro Thr Tyr Val Thr Pro Met Gly Lys Ser Ala Ile Asp                 245 250 255 Glu Thr Ser Gln Phe Phe Asp Gly Val Tyr Val Gly Ser Ser Ser Asp             260 265 270 Pro Glu Val Lys Asp Arg Ile Glu Ser Thr Asp Leu Leu Leu Ser Ile         275 280 285 Gly Ala Leu Lys Ser Asp Phe Asn Thr Gly Ser Phe Ser Tyr His Leu     290 295 300 Ser Gln Lys Asn Ala Val Glu Phe His Ser Asp His Met Arg Ile Arg 305 310 315 320 Tyr Ala Leu Tyr Pro Asn Val Ala Met Lys Tyr Ile Leu Arg Lys Leu                 325 330 335 Leu Lys Val Leu Asp Ala Ser Met Cys His Ser Lys Ala Ala Pro Thr             340 345 350 Ile Gly Tyr Asn Ile Lys Pro Lys His Ala Glu Gly Tyr Ser Ser Asn         355 360 365 Glu Ile Thr His Cys Trp Phe Trp Pro Lys Phe Ser Glu Phe Leu Lys     370 375 380 Pro Arg Asp Val Leu Ile Thr Glu Thr Gly Thr Ala Asn Phe Gly Val 385 390 395 400 Leu Asp Cys Arg Phe Pro Lys Asp Val Thr Ala Ile Ser Gln Val Leu                 405 410 415 Trp Gly Ser Ile Gly Tyr Ser Val Gly Ala Met Phe Gly Ala Val Leu             420 425 430 Ala Val His Asp Ser Lys Glu Pro Asp Arg Arg Thr Ile Leu Val Val         435 440 445 Gly Asp Gly Ser Leu Gln Leu Thr Ile Thr Glu Ile Ser Thr Cys Ile     450 455 460 Arg His Asn Leu Lys Pro Ile Ile Phe Ile Ile Asn Asn Asp Gly Tyr 465 470 475 480 Thr Ile Glu Arg Leu Ile His Gly Leu His Ala Ser Tyr Asn Glu Ile                 485 490 495 Asn Thr Lys Trp Gly Tyr Gln Gln Ile Pro Lys Phe Phe Gly Ala Ala             500 505 510 Glu Asn His Phe Arg Thr Tyr Cys Val Lys Thr Pro Thr Asp Val Glu         515 520 525 Lys Leu Phe Ser Asp Lys Glu Phe Ala Asn Ala Asp Val Ile Gln Val     530 535 540 Val Glu Leu Val Met Met Leu Asp Ala Pro Arg Val Leu Val Glu 545 550 555 560 Gln Ala Lys Leu Thr Ser Lys Ile Asn Lys Gln                 565 570 <210> 124 <211> 1713 <212> DNA <213> Schizosaccharomyces pombe <400> 124 atgagtgggg atattttagt cggtgaatat ctattcaaaa ggcttgaaca attaggggtc 60 aagtccattc ttggtgttcc aggagatttc aatttagctc tacttgactt aattgagaaa 120 gttggagatg agaaatttcg ttgggttggc aataccaatg agttgaatgg tgcttatgcc 180 gctgatggtt atgctcgtgt taatggtctt tcagccattg ttacaacgtt cggcgtggga 240 gagctttccg ctattaatgg agtggcaggt tcttatgcgg agcatgtccc agtagttcat 300 attgttggaa tgccttccac aaaggtgcaa gatactggag ctttgcttca tcatacttta 360 ggagatggag actttcgcac tttcatggat atgtttaaga aagtttctgc ctacagtata 420 atgatcgata acggaaacga tgcagctgaa aagatcgatg aagccttgtc gatttgttat 480 aaaaaggcta ggcctgttta cattggtatt ccttctgatg ctggctactt caaagcatct 540 tcatcaaatc ttgggaaaag actaaagctc gaggaggata ctaacgatcc agcagttgag 600 caagaagtca tcaatcatat ctcggaaatg gttgtcaatg caaagaaacc agtgatttta 660 attgacgctt gtgctgtaag acatcgtgtc gttccagaag tacatgagct gattaaattg 720 acccatttcc ctacatatgt aactcccatg ggtaaatctg caattgacga aacttcgcaa 780 ttttttggg gcgtttatgt tggttcaatt tcagatcctg aagttaaaga cagaattgaa 840 tccactgatc tgttgctatc catcggtgct ctcaaatcag actttaacac gggttccttc 900 tcttaccacc tcagccaaaa gaatgccgtt gagtttcatt cagaccacat gcgcattcga 960 tatgctcttt atccaaatgt agccatgaag tatattcttc gcaaactgtt gaaagtactt 1020 gatgcttcta tgtgtcattc caaggctgct cctaccattg gctacaacat caagcctaag 1080 catgcggaag gatattcttc caacgagatt actcattgct ggttttggcc taaatttagt 1140 gaatttttga agccccgaga tgttttgatc accgagactg gaactgcaaa ctttggtgtc 1200 cttgattgca ggtttccaaa ggatgtaaca gccatttccc aggtattatg gggatctatt 1260 ggatactccg ttggtgcaat gtttggtgct gttttggccg tccacgattc taaagagccc 1320 gatcgtcgta ccattcttgt agtaggtgat ggatccttac aactgacgat tacagagatt 1380 tcaacctgca ttcgccataa cctcaaacca attattttca taattaacaa cgacggttac 1440 accattgagc gtttaattca tggtttgcat gctagctata acgaaattaa cactaaatgg 1500 ggctaccaac agattcccaa gtttttcgga gctgctgaaa accacttccg cacttactgt 1560 gttaaaactc ctactgacgt tgaaaagttg tttagcgaca aggagtttgc aaatgcagat 1620 gtcattcaag tagttgagct tgtaatgcct atgttggatg cacctcgtgt cctagttgag 1680 caagccaagt tgacgtctaa gatcaataag caa 1713 <210> 125 <211> 563 <212> PRT <213> Zygosaccharomyces rouxii <400> 125 Met Ser Glu Ile Thr Leu Gly Arg Tyr Leu Phe Glu Arg Leu Lys Gln 1 5 10 15 Val Asp Thr Asn Thr Ile Phe Gly Val Pro Gly Asp Phe Asn Leu Ser             20 25 30 Leu Leu Asp Lys Val Tyr Glu Val Gln Gly Leu Arg Trp Ala Gly Asn         35 40 45 Ala Asn Glu Leu Asn Ala Ala Tyr Ala Ala Asp Gly Tyr Ala Arg Val     50 55 60 Lys Gly Leu Ala Leu Ile Thr Thr Phe Gly Val Gly Glu Leu Ser 65 70 75 80 Ala Leu Asn Gly Ile Ala Gly Ser Tyr Ala Glu His Val Gly Val Leu                 85 90 95 His Ile Val Gly Val Ser Ser Val Ser Ser Gln Ala Lys Gln Leu Leu             100 105 110 Leu His His Thr Leu Gly Asn Gly Asp Phe Thr Val Phe His Arg Met         115 120 125 Ser Ala Asn Ile Ser Glu Thr Thr Ala Met Leu Thr Asp Ile Thr Ala     130 135 140 Ala Pro Ala Glu Ile Asp Arg Cys Ile Arg Val Ala Tyr Val Asn Gln 145 150 155 160 Arg Pro Val Tyr Leu Gly Leu Pro Ala Asn Leu Val Asp Gln Lys Val                 165 170 175 Pro Ala Ser Leu Leu Asn Thr Pro Ile Asp Leu Ser Leu Lys Glu Asn             180 185 190 Asp Pro Glu Ala Glu Thr Glu Val Val Asp Thr Val Leu Glu Leu Ile         195 200 205 Lys Glu Ala Lys Asn Pro Val Ile Leu Ala Asp Ala Cys Cys Ser Arg     210 215 220 His Asp Val Lys Ala Glu Thr Lys Lys Leu Ile Asp Leu Thr Gln Phe 225 230 235 240 Pro Ser Phe Val Thr Pro Met Gly Lys Gly Ser Ile Asp Glu Gln Asn                 245 250 255 Pro Arg Phe Gly Gly Val Tyr Val Gly Thr Leu Ser Ser Pro Glu Val             260 265 270 Lys Glu Ala Val Glu Ser Ala Asp Leu Val Leu Ser Val Gly Ala Leu         275 280 285 Leu Ser Asp Phe Asn Thr Gly Ser Phe Ser Tyr Ser Tyr Lys Thr Lys     290 295 300 Asn Val Val Glu Phe His Ser Asp His Ile Lys Ile Arg Asn Ala Thr 305 310 315 320 Phe Pro Gly Val Gln Met Lys Phe Val Leu Lys Lys Leu Leu Gln Ala                 325 330 335 Val Pro Glu Ala Val Lys Asn Tyr Lys Pro Gly Pro Val Pro Ala Pro             340 345 350 Pro Ser Pro Asn Ala Glu Val Ala Asp Ser Thr Thr Leu Lys Gln Glu         355 360 365 Trp Leu Trp Arg Gln Val Gly Ser Phe Leu Arg Glu Gly Asp Val Val     370 375 380 Ile Thr Glu Thr Gly Thr Ser Ala Phe Gly Ile Asn Gln Thr His Phe 385 390 395 400 Pro Asn Gln Thr Tyr Gly Ile Ser Gln Val Leu Trp Gly Ser Ile Gly                 405 410 415 Tyr Thr Thr Gly Ser Thr Leu Gly Ala Ala Phe Ala Ala Glu Glu Ile             420 425 430 Asp Pro Lys Lys Arg Val Ile Leu Phe Ile Gly Asp Gly Ser Leu Gln         435 440 445 Leu Thr Val Gln Glu Ile Ser Thr Met Ile Arg Trp Gly Leu Lys Pro     450 455 460 Tyr Leu Phe Val Leu Asn Asn Asp Gly Tyr Thr Ile Glu Arg Leu Ile 465 470 475 480 His Gly Glu Thr Ala Glu Tyr Asn Cys Ile Gln Pro Trp Lys His Leu                 485 490 495 Glu Leu Leu Asn Thr Phe Gly Ala Lys Asp Tyr Glu Asn His Arg Val             500 505 510 Ser Thr Val Gly Glu Trp Asn Lys Leu Thr Gln Asp Pro Lys Phe Asn         515 520 525 Glu Asn Ser Arg Ile Arg Met Ile Glu Val Met Leu Glu Val Met Asp     530 535 540 Ala Pro Ser Ser Leu Val Ala Gln Ala Gln Leu Thr Ala Ala Thr Asn 545 550 555 560 Ala Lys Gln              <210> 126 <211> 1689 <212> DNA <213> Zygosaccharomyces rouxii <400> 126 atgtctgaaa ttactctagg tcgttacttg ttcgaaagat taaagcaagt tgacactaac 60 accatcttcg gtgttccagg tgacttcaac ttgtccttgt tggacaaggt ctacgaagtg 120 caaggtctaa gatgggctgg taacgctaac gaattgaacg ctgcctacgc tgctgacggt 180 tacgccagag ttaagggttt ggctgctttg atcaccacct tcggtgtcgg tgaattgtct 240 gctttgaacg gtattgcagg ttcttacgct gaacacgttg gtgttttgca cattgttggt 300 gttccatctg tctcttctca agctaagcaa ttgttgttgc accacacctt gggtaacggt 360 gacttcactg ttttccacag aatgtccgcc aacatctctg aaaccaccgc tatgttgacc 420 gacatcactg ctgctccagc tgaaattgac cgttgcatca gagttgctta cgtcaaccaa 480 agaccagtct acttgggtct accagctaac ttggttgacc aaaaggtccc agcttctttg 540 ttgaacactc caattgatct atctctaaag gagaacgacc cagaagctga aaccgaagtt 600 gttgacaccg ttttggaatt gatcaaggaa gctaagaacc cagttatctt ggctgatgct 660 tgctgctcca gacacgacgt caaggctgaa accaagaagt tgatcgactt gactcaattc 720 ccatctttcg ttactcctat gggtaagggt tccatcgacg aacaaaaccc aagattcggt 780 ggtgtctacg tcggtactct atccagccca gaagttaagg aagctgttga atctgctgac 840 ttggttctat ctgtcggtgc tctattgtcc gatttcaaca ctggttcttt ctcttactct 900 tacaagacca agaacgttgt tgaattccac tctgaccaca tcaagatcag aaacgctacc 960 ttcccaggtg ttcaaatgaa attcgttttg aagaaactat tgcaagctgt cccagaagct 1020 gtcaagaact acaagccagg tccagtccca gctccgccat ctccaaacgc tgaagttgct 1080 gactctacca ccttgaagca agaatggtta tggagacaag tcggtagctt cttgagagaa 1140 ggtgatgttg ttattaccga aactggtacc tctgctttcg gtatcaacca aactcacttc 1200 cctaaccaaa cttacggtat ctctcaagtc ttgtggggtt ctattggtta caccactggt 1260 tccactttgg gtgctgcctt cgctgctgaa gaaattgacc ctaagaagag agttatcttg 1320 ttcattggtg acggttctct acaattgacc gttcaagaaa tctccaccat gatcagatgg 1380 ggtctaaagc catacttgtt cgttttgaac aacgatggtt acaccattga aagattgatt 1440 cacggtgaaa ccgctgaata caactgtatc caaccatgga agcacttgga attgttgaac 1500 accttcggtg ccaaggacta cgaaaaccac agagtctcca ctgtcggtga atggaacaag 1560 ttgactcaag atccaaaatt caacgaaaac tctagaatta gaatgatcga agttatgctt 1620 gaagtcatgg acgctccatc ttctttggtc gctcaagctc aattgaccgc tgctactaac 1680 gctaagcaa 1689 <210> 127 <211> 267 <212> PRT <213> Saccharomyces cerevisiae <400> 127 Met Ser Gln Gly Arg Lys Ala Ala Glu Arg Leu Ala Lys Lys Thr Val 1 5 10 15 Leu Ile Thr Gly Ala Ser Ala Gly Ile Gly Lys Ala Thr Ala Leu Glu             20 25 30 Tyr Leu Glu Ala Ser Asn Gly Asp Met Lys Leu Ile Leu Ala Ala Arg         35 40 45 Arg Leu Glu Lys Leu Glu Glu Leu Lys Lys Thr Ile Asp Gln Glu Phe     50 55 60 Pro Asn Ala Lys Val Val Ala Gln Leu Asp Ile Thr Gln Ala Glu 65 70 75 80 Lys Ile Lys Pro Phe Ile Glu Asn Leu Pro Gln Glu Phe Lys Asp Ile                 85 90 95 Asp Ile Leu Val Asn Asn Ala Gly Lys Ala Leu Gly Ser Asp Arg Val             100 105 110 Gly Gln Ile Ala Thr Glu Asp Ile Gln Asp Val Phe Asp Thr Asn Val         115 120 125 Thr Ala Leu Ile Asn Ile Thr Gln     130 135 140 Lys Asn Ser Gly Asp Ile Val Asn Leu Gly Ser Ile Ala Gly Arg Asp 145 150 155 160 Ala Tyr Pro Thr Gly Ser Ile Tyr Cys Ala Ser Lys Phe Ala Val Gly                 165 170 175 Ala Phe Thr Asp Ser Leu Arg Lys Glu Leu Ile Asn Thr Lys Ile Arg             180 185 190 Val Ile Leu Ile Ala Pro Gly Leu Val Glu Thr Glu Phe Ser Leu Val         195 200 205 Arg Tyr Arg Gly Asn Glu Glu Gln Ala Lys Asn Val Tyr Lys Asp Thr     210 215 220 Thr Pro Leu Met Ala Asp Asp Val Ala Asp Leu Ile Val Tyr Ala Thr 225 230 235 240 Ser Arg Lys Gln Asn Thr Val Ile Ala Asp Thr Leu Ile Phe Pro Thr                 245 250 255 Asn Gln Ala Ser Pro His His Ile Phe Arg Gly             260 265 <210> 128 <211> 804 <212> DNA <213> Saccharomyces cerevisiae <400> 128 atgtcccaag gtagaaaagc tgcagaaaga ttggctaaga agactgtcct cattacaggt 60 gcatctgctg gtattggtaa ggcgaccgca ttagagtact tggaggcatc caatggtgat 120 atgaaactga tcttggctgc tagaagatta gaaaagctcg aggaattgaa gaagaccatt 180 gatcaagagt ttccaaacgc aaaagttcat gtggcccagc tggatatcac tcaagcagaa 240 aaaatcaagc ccttcattga aaacttgcca caagagttca aggatattga cattctggtg 300 aacaatgccg gaaaggctct tggcagtgac cgtgtgggcc agatcgcaac ggaggatatc 360 caggacgtgt ttgacaccaa cgtcacggct ttaatcaata tcacacaagc tgtactgccc 420 atattccaag ccaagaattc aggagatatt gtaaatttgg gttcaatcgc tggcagagac 480 gcatacccaa caggttctat ctattgtgcc tctaagtttg ccgtgggggc gttcactgat 540 agtttgagaa aggagctcat caacactaaa attagagtca ttctaattgc accagggcta 600 gtcgagactg aattttcact agttagatac agaggtaacg aggaacaagc caagaatgtt 660 tacaaggata ctaccccatt gatggctgat gacgtggctg atctgatcgt ctatgcaact 720 tccagaaaac aaaatactgt aattgcagac actttaatct ttccaacaaa ccaagcgtca 780 cctcatcata tcttccgtgg ataa 804

Claims (47)

Providing a feedstock slurry comprising a fermentable carbon source, an undissolved solid, and an oil;
Forming an aqueous solution comprising a fermentable carbon source, a wet cake comprising a solid, and an oil stream by separating the undissolved solids and a portion of the oil from the feedstock slurry; And
Adding an aqueous solution to a fermentation broth comprising a microorganism to produce a fermentation product. The method of claim 1, further comprising recovering an oil stream. The method of claim 1, further comprising washing the wet cake to provide an aqueous stream comprising carbohydrates. 4. The process according to claim 3, further comprising the step of adding an aqueous stream according to claim 3 to a fermentation broth. 2. The method of claim 1, wherein the aqueous solution comprises no more than about 5 wt% undissolved solids. The process according to claim 1, wherein the oil is corn oil and comprises at least one of triglycerides, fatty acids, diglycerides, monoglycerides, and phospholipids. The method of claim 1, further comprising the step of combining a portion of the wet cake with a portion of the oil to produce a wet cake comprising triglycerides, free fatty acids, diglycerides, monoglycerides, and phospholipids Gt; 2. The method of claim 1, further comprising the step of combining the aqueous solution with a portion of the wet cake to produce a mixture of aqueous solution and wet cake, and adding the mixture to the fermentation broth. The process according to claim 1, wherein separating the feedstock slurry is a single step process. The process of claim 1, wherein the undissolved solids and oil are separated from the feedstock slurry by decanter bowl centrifugation, 3-phase centrifugation, disk stack centrifugation, Filtration using screen, screen separation, grating, porous grating, flotation, centrifugal separation, centrifugal separation, centrifugal separation, filtration, vacuum filtration, beltfilter, membrane filtration, cross flow filtration, Wherein the hydrolyzate is separated by a hydrocyclone, a filter press, a screw press, a gravity settler, a vortex separator, or a combination thereof. 11. The method of claim 10, wherein the at least one adjustment parameter of the separation device is adjusted to improve the separation of the feedstock slurry. 12. The method of claim 11, wherein the at least one adjustment parameter is selected from the group consisting of differential speed, bowl speed, flow speed, impeller position, weir position, scroll pitch, volume of the fermentation product. The process according to claim 1, wherein the fermentation product is a product alcohol selected from ethanol, propanol, butanol, pentanol, hexanol, and fusel alcohol. The method of claim 1, wherein the microorganism comprises a butanol biosynthetic pathway. 15. The method of claim 14, wherein the butanol biosynthesis pathway is a 1-butanol biosynthesis pathway, a 2-butanol biosynthesis pathway, or an isobutanol biosynthesis pathway. The method of claim 1, wherein real-time measurement is used to monitor the separation of the feedstock slurry. 17. The method of claim 16, wherein the separation is performed using Fourier transform infrared spectroscopy, near-infrared spectroscopy, Raman spectroscopy, high pressure liquid chromatography, viscometer, densitometer, tensiometer, , A droplet size analyzer, a particle analyzer, or a combination thereof. a) providing a feedstock slurry comprising a fermentable carbon source and an undissolved solid;
b) producing a wet cake comprising a solid and an aqueous solution comprising a fermentable carbon source by separating at least a portion of the undissolved solids from the feedstock slurry;
c) contacting the wet cake with a liquid to form a wet cake mixture; And
d) separating at least a portion of the undissolved solids from the wet cake mixture to produce a second wet cake comprising a second aqueous solution comprising a fermentable carbon source and a solid. 19. The method of claim 18, wherein the liquid is selected from fresh water, a backset, a cook water, a process water, a lutter water, an evaporation water, or a combination thereof. 19. The method of claim 18, wherein step c) and step d) are repeated. Providing a feedstock slurry comprising a fermentable carbon source, an undissolved solid, and an oil;
Producing a wet cake comprising an aqueous solution, an oil stream, and a solid comprising a fermentable carbon source by separating at least a portion of said oil and undissolved solids from a feedstock slurry; And
c) contacting the wet cake with a liquid to form a wet cake mixture; And
d) producing a second wet cake comprising a second aqueous solution comprising a fermentable carbon source, a second oil stream, and a solid by separating at least a portion of the undissolved solids and the oil from the wet cake mixture, Methods of inclusion. 22. The method of claim 21, wherein separating the feedstock slurry is a single step process. 22. The process of claim 21 wherein the undissolved solids and oil are separated from the feedstock slurry by decanter bowl centrifugation, 3-phase centrifugation, disk stack centrifugation, filtration centrifugation, decanter centrifugation, filtration, vacuum filtration, Separation by membrane filtration, crossflow filtration, pressure filtration, screen filtration, screen separation, grating, porous grating, suspension, hydrocyclone, filter press, screw press, gravity settler, vortex separator, How to do it. 24. The method of claim 23, wherein one or more adjustment parameters of the separation device are adjusted to improve separation of the feed slurry. 25. The method of claim 24, wherein the at least one adjustment parameter is selected from differential speed, bowl speed, flow rate, impeller position, wear position, scroll pitch, residence time, and discharge volume. 22. The method of claim 21, wherein real-time measurement is used to monitor the separation of the feedstock slurry. 27. The method of claim 26, wherein the separation is monitored by Fourier transform infrared spectroscopy, near infrared spectroscopy, Raman spectroscopy, high pressure liquid chromatography, viscometer, densitometer, tensiometer, droplet size analyzer, particle analyzer, or a combination thereof. Providing a feedstock slurry comprising a fermentable carbon source, an undissolved solid, and an oil;
By separating the feedstock slurry, a stream, solid, and fermentable carbon source comprising (i) a first aqueous solution comprising a fermentable carbon source, (ii) a first wet cake comprising a solid, and (iii) To form an aqueous stream; And
Adding a first aqueous solution to a fermentation broth comprising a microorganism to produce a fermentation product. 29. The method of claim 28,
(I) a second aqueous solution comprising a fermentable carbon source, (ii) a second wet cake comprising a solid, and (iii) a second wet cake comprising a solid, ) &Lt; / RTI &gt; oil stream. 30. The method according to claim 29, wherein the first aqueous solution and the second aqueous solution are combined before being added to the fermentation broth. 30. The method of claim 29, wherein the second aqueous solution further comprises an oil. 32. The method of claim 31, wherein the oil in the second aqueous solution or a portion thereof is treated to produce an extractant. 33. The method of claim 32, wherein the oil is treated chemically or enzymatically. 30. A method according to claim 28 or 29, wherein the separation is carried out by means of decanter bowl centrifugation, 3-phase centrifugation, disk stack centrifugation, filtration centrifugation, decanter centrifugation, filtration, vacuum filtration, belt filtration, Such as filtration, screen separation, grating, porous grating, suspension, hydrocyclone, filter press, screw press, gravity settler, vortex separator, or a combination thereof. Providing a feedstock slurry comprising a fermentable carbon source, an undissolved solid, and an oil;
Separating the feedstock slurry to form a first aqueous stream comprising (i) a first aqueous solution comprising a fermentable carbon source and a solid, (ii) a first wet cake comprising a solid, and (iii) a first oil stream; And
Adding oil to the first aqueous solution to form a second aqueous solution comprising an oil layer comprising solids and a fermentable carbon source. 36. The method of claim 35, wherein the oil layer comprising solids comprises (i) a second oil stream, (ii) a second wet cake comprising a solid, and (iii) a third aqueous solution comprising a fermentable carbon source, &Lt; / RTI &gt; 37. The method of claim 36, wherein a fermentation product is produced by adding a second aqueous solution and a third aqueous solution to a fermentation broth comprising a microorganism. 36. The method of claim 35, wherein the second aqueous solution and the third aqueous solution further comprise an oil. 39. The method of claim 38, wherein the second aqueous solution and the third aqueous solution are combined and the second aqueous solution and the third aqueous solution or a portion of the oils are treated to produce an extractant. 40. The method of claim 39, wherein the oil is treated chemically or enzymatically. 37. A method according to claim 35 or 36, wherein the separation is carried out by means of decanter bowl centrifugation, 3-phase centrifugation, disk stack centrifugation, filtration centrifugation, decanter centrifugation, filtration, vacuum filtration, belt filtration, Such as filtration, screen separation, grating, porous grating, suspension, hydrocyclone, filter press, screw press, gravity settler, vortex separator, or a combination thereof. At least one liquefaction unit configured to liquefy the feedstock to produce a feedstock slurry, the liquefaction unit comprising:
An inlet for receiving said feedstock; And
An outlet for discharging the feedstock slurry, wherein the feedstock slurry comprises a liquefying unit comprising a fermentable carbon source, oil, and undissolved solids; And
At least one separation unit configured to remove the oil and undissolved solids from the feedstock slurry to produce a wet cake comprising an aqueous solution comprising a fermentable carbon source, an oil stream, and a portion of the undissolved solids, wherein the centrifuge
An inlet for receiving a feedstock slurry;
A first outlet for discharging the aqueous solution;
A second outlet for discharging the wet cake; And
And a third outlet for discharging the oil. 43. The method of claim 42,
At least one mixing unit; And
Further comprising at least one separation unit, wherein the system further comprises at least one cleaning system configured to recover a fermentable carbon source from the wet cake. 44. The method of claim 43, wherein the separation unit is selected from the group consisting of decanter bowl centrifuge, 3-phase centrifuge, disk stack centrifuge, filtration centrifuge, decanter centrifuge, filtration, vacuum filtration, belt filter, membrane filtration, A system selected from the group consisting of pressure filtration, screen filtration, screen separation, grating, porous grating, suspension, hydrocyclone, filter press, screw press, gravity settler, vortex separator, or a combination thereof. 44. The method of claim 43 further comprising at least one fermenter configured to ferment an aqueous solution to produce product alcohol,
An inlet for receiving an aqueous solution and / or a wet cake; And
And an outlet for discharging the fermentation broth comprising the product alcohol. 43. The system of claim 42, wherein the system further comprises an on-line measurement device. 47. The method of claim 46, wherein the on-line measurement device is a particle size analyzer, a Fourier transform infrared spectroscope, a near infrared ray spectroscope, a Raman spectroscope, a high pressure liquid chromatography, a viscometer, a densitometer, a tensiometer, , Or a combination thereof.

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