CN103459587A

CN103459587A - In situ expression of lipase for enzymatic production of alcohol esters during fermentation

Info

Publication number: CN103459587A
Application number: CN2012800146568A
Authority: CN
Inventors: R.迪科斯莫; A.L.克鲁科伯格; T.E.范阿肯
Original assignee: Butamax Advanced Biofuels LLC
Current assignee: Butamax Advanced Biofuels LLC
Priority date: 2011-03-23
Filing date: 2012-03-23
Publication date: 2013-12-18
Also published as: EP2689006A2; AU2012230730A1; JP2014509869A; WO2012129548A2; BR112013024329A2; CA2829941A1; ZA201306647B; WO2012129548A3; EP2689006A4

Abstract

Disclosed herein are methods of producing alcohol esters during a fermentation by providing alcohol-producing microorganisms which further comprise an engineered polynucleotide encoding a polypeptide having lipase activity.

Description

During fermentation for the expressed in situ of the lipase of enzymatic production alcohol ester

the cross reference of related application

The application relates to and requires to be filed in the U.S. Provisional Patent Application 61/466712 and the right of priority that is filed in the U.S. Provisional Patent Application 61/498,292 on June 17th, 2011 on March 23rd, 2011, and their full text contents are incorporated herein by reference.

the sequence table reference of submitting in the electronics mode

Sequence table content (the filename: 20120322_CL5145USNA_SeqList_ST25.txt of submitting to together with the application, submit in the electronics mode of ASCII text file form, file size: 656,901 bytes, and date created is: on March 22nd, 2012), it is incorporated herein by reference in full.

Technical field

The present invention relates to comprise ethanol and butanols alcohol fermentative production and adopt the situ product removing method for improving the method for alcohol fermentation.

Background technology

Alcohol serves many purposes in industry and science.For example alcohol can be used as beverage (that is, ethanol), fuel, reagent, solvent and antiseptic.For example butanols is a kind of alcohol, and it is important industrial chemical, serves many purposes, and comprises as fuel dope, is used as chemical feedstocks in plastics industry, and the extraction agent that is used as food grade in food and flavour agent industry.Therefore, height requirement alcohol is as butanols and the high-efficiency method for producing that does not rely on Nonrenewable resources.

Utilizing microorganism fermentative production alcohol is a kind of such production method, and it is used to the substrate from renewable raw materials.In production of butanol, the microorganism of some production high yield butanols also has low butanols toxicity threshold values specifically, makes when producing butanols and need to from fermentor tank, remove butanols.Therefore, exist and split hair in the effective ways of producing butanols (low butanols toxicity threshold being arranged in fermention medium although produce the microorganism of butanols) with high yield and the lasting demand of system.Situ product removes (ISPR) (be also referred to as and extract fermentation) and can be used for the fermenting container from producing it removing butanols (or other fermentable alcohol), thereby makes the microbe high-yield rate produce butanols.It is a kind of that this area had been described is that liquid-liquid extracts (U.S. Patent Application Publication 20090305370) for the ISPR method that removes fermentable alcohol.In general, comprise that about butylic fermentation is for example described the fermention medium of microorganism contacts with organic extraction agent.Described organic extraction agent and described fermention medium form two-phase mixture.Described butanols is dispensed to organic extraction agent phase, has reduced the concentration in comprising the water of microorganism, thereby has limited the exposure of microorganism in the butanols of inhibition.Liquid-liquid extracts and caused by following steps: contact extraction agent and fermentation broth are to be delivered to product alcohol in extraction agent; Separation and Extraction agent phase and water; And preferably, the recycling extraction agent, the while is in the reduction of long-time running minimization extraction agent partition ratio.

Each circulation, extraction agent can carry out and contaminated along with the time, for example by the raw material as hydrolyzable starch, was fed to the accumulation of the lipid existed in the biomass in fermenting container.For example, the liquefaction corn mash that is loaded on fermenting container in the process that is butanols in conversion of glucose can produce the fermentation broth that comprises Semen Maydis oil, and it produces (saccharification of the wine with dregs of described liquefaction occurs by the interpolation of the glucoamylase for generation of glucose during the fermentation) by saccharification and fermentation simultaneously.In the ISPR process, the Semen Maydis oil lipid is dissolved in extraction agent and can causes lipid concentration to accumulate along with the recycle of each extraction agent, this makes the partition ratio of product alcohol in extraction agent reduce, because the lipid concentration in extraction agent raises along with each recycle.

It is a kind of method that reduces the lipid amount of charging in fermenting container that the lipid existed in liquefied fermented glutinous rice is changed into to the extraction agent that can use in ISPR, it is by adding the lipase as the esterification catalyzer, the product alcohol during fermentation produced with fatty acid esterification to fermentation.These class methods announce 20110312044 and 20110312043 at for example U. S. application, and describe to some extent in PCT Shen Qing Publication WO2011/159998.

Need alternative extraction fermentation process, it also can reduce and the cost that adds lipase to be associated to fermentation always.

Summary of the invention

Method provided herein comprises: fermention medium a) is provided, it comprises the fermented carbon substrate that derives from biomass material, the alcohol that originates from the fermented carbon substrate that derives from biomass material and alcohol and produces microorganism, wherein alcohol production microorganism comprises the polynucleotide that coding has the polypeptide of lipase activity, and this microbial expression is also showed or secretes described polypeptide, making in fermention medium and have lipase activity; B) fermention medium is contacted with carboxylic acid; During wherein lipase activity is present in fermention medium with the q.s that in extracellular, at least a portion of the alcohol of microorganisms is changed into to alcohol ester.In an embodiment, alcohol production microorganism is yeast.In an embodiment, the polynucleotide through engineering approaches that coding is there is to the polypeptide of lipase activity.In an embodiment, the method also comprises and makes fermention medium contact to form the two-phase mixture that comprises water and organic phase with extraction agent.In an embodiment, extraction agent comprises described carboxylic acid.In an embodiment, product alcohol is C ₂-C ₈alkyl alcohol.In an embodiment, product alcohol is ethanol.In an embodiment, alcohol ester comprises fatty-acid ethyl ester.In an embodiment, product alcohol is butanols.In an embodiment, alcohol ester comprises fatty acid butyl ester.In an embodiment, alcohol ester also comprises fatty-acid ethyl ester.

In an embodiment, the polypeptide with lipase activity provided herein is illustrated on the microorganism surface.In an embodiment, secrete the polypeptide that to have lipase activity.In an embodiment, have sequence that the polypeptide of lipase activity comprises and SEQ ID NO:249,250,251,252,253 or their fragment in any one have at least about 70% identity, at least about 80% identity, at least about 90% identity, or at least about 95% identity.In an embodiment, coding there is sequence that the polynucleotide of polypeptide of lipase activity comprise and there is SEQ ID NO:1,3,5,7,8,9,46,48,50,52,54,255,271 or 273 polynucleotide have at least about 70% identity.In an embodiment, have sequence that the polypeptide of lipase activity comprises with have SEQ ID NO:2,4,6,256,47,49,51,53,55,241,242,243,244,245,246,247,248,272 or 274 or their polypeptide of active fragments have at least about 70% identity, at least about 80% identity, at least about 90% identity, or at least about 95% identity.In an embodiment, the polypeptide that has a lipase activity does not comprise the glycosylation motif.In an embodiment, the polypeptide that there is lipase activity be do not have glycosylated.

In an embodiment, carboxylic acid comprises free fatty acids, and described free fatty acids derives from Semen Maydis oil, Tower rape oil, plam oil, linseed oil, curcas oil or soybean oil.In an embodiment, carboxylic acid derives from and the identical biomass material of carbon substrate that can ferment.In an embodiment, carboxylic acid comprises having C ₁₂-C ₂₂the carboxylic acid of the aliphatic chain of straight chain or branching.In an embodiment, with the contacting and occur with contacting simultaneously of carboxylic acid of extraction agent.In an embodiment, be converted to alcohol ester by the alcohol of microorganisms at least about 60% effective titre.In an embodiment, at least a portion that fermention medium also comprises triglyceride level, triglyceride, monoglyceride and phosphatide or their combination and lipase activity triglyceride level, triglyceride, monoglyceride and phosphatide or their combination is hydrolyzed to form free fatty acids.

In an embodiment, effective titre of the alcohol during fermentation produced is greater than effective titre of being produced the alcohol that microorganism during fermentation produces by the alcohol of polynucleotide that does not comprise coding and have the polypeptide of lipase activity, and this microbial expression is also secreted or shows described polypeptide, make in fermention medium and have this lipase activity.In an embodiment, the effective speed of the alcohol during fermentation produced is greater than the effective speed of being produced the alcohol that microorganism during fermentation produces by the alcohol of polynucleotide that does not comprise coding and have the polypeptide of lipase activity, and this microbial expression is also secreted or shows described polypeptide, make in fermention medium and have this lipase activity.

This paper also provides recombinant host cell, the pure production ways that it comprises through engineering approaches; And the polynucleotide of through engineering approaches, its coding has the polypeptide of lipase activity.In an embodiment, have sequence that the polypeptide of lipase activity comprises and SEQ ID NO:2,4,6,256,47,49,51,53,55,241,242,243,244,245,246,247,248,272 or 274 or their active fragments have at least about 70% identity, at least about 80% identity, at least about 90% identity, or at least about 95% identity.In an embodiment, have sequence that the polypeptide of lipase activity comprises and SEQ ID NO:249,250,251,252,253 or their fragment in any one have at least about 70% identity, at least about 80% identity, at least about 90% identity, or at least about 95% identity.In an embodiment, the polypeptide that has a lipase activity does not comprise the glycosylation motif.In an embodiment, the polypeptide that there is lipase activity be do not have glycosylated.In an embodiment, coding has sequence and the SEQ ID NO:1,3,5,7,8,9,46,48,50,52,54,255,271 or 273 that the through engineering approaches polynucleotide of polypeptide of lipase activity comprise and has at least about 70% identity, at least about 80% identity, at least about 90% identity, or at least about 95% identity.

This paper also provides recombinant host cell, and it comprises pure production ways; There are the through engineering approaches polynucleotide of the polypeptide of lipase activity with coding, the sequence that wherein this polypeptide with lipase activity comprises and SEQ ID NO:2,4,6,256,47,49,51,53,55,241,242,243,244,245,246,247,248,272 or 274 or their active fragments have at least about 70% identity, at least about 80% identity, at least about 90% identity, or at least about 95% identity.In an embodiment, have sequence that the polypeptide of lipase activity also comprises and SEQ ID NO:249,250,251,252,253 or their fragment in any one have at least about 70% identity, at least about 80% identity, at least about 90% identity, or at least about 95% identity.In an embodiment, pure production ways is the production of butanol approach.In an embodiment, the production of butanol approach is the isopropylcarbinol production ways.In an embodiment, host cell also comprises pyruvic carboxylase activity reduction or that eliminate.

This paper also provides and improved alcohol and produce the method for microorganism to the tolerance of the alcohol that produces, and the method comprises: the through engineering approaches microorganism is to express and secrete or show polypeptide with lipase activity; Under the condition that makes microorganisms alcohol, make the through engineering approaches microorganism contact with triglyceride level, triglyceride, monoglyceride, phosphatide, free fatty acids or their mixture and carbon substrate.In an embodiment, the through engineering approaches microorganism is contacted with triglyceride level, triglyceride, monoglyceride and phosphatide or their combination, and wherein secretion or lipase that show change into free fatty acids by least a portion of triglyceride level, triglyceride, monoglyceride and phosphatide or their combination.In an embodiment, the formation of lipase-catalyzed alcohol ester.In an embodiment, effective titre of microorganisms alcohol be greater than by not yet through through engineering approaches to express and to secrete effective titre of the microorganisms alcohol of the polypeptide with lipid acid activity.In an embodiment, microorganism also comprises the pure biosynthetic pathway of through engineering approaches.In an embodiment, the pure biosynthetic pathway of through engineering approaches is n-butyl alcohol, 2-butanols or isopropylcarbinol biosynthetic pathway.In an embodiment, the isopropylcarbinol biosynthetic pathway comprises the conversion of following substrate to product: pyruvic acid is to acetylactis; Acetylactis to 2,3-dihydroxyl isovaleric acid; 2,3-dihydroxyl isovaleric acid is to the 2-ketoisovaleric acid; The 2-ketoisovaleric acid is to isobutyric aldehyde; And isobutyric aldehyde is to isopropylcarbinol.

This paper provides the method for during fermentation producing butyl ester, and the method comprises provides fermention medium, and described fermention medium comprises carbon substrate and triglyceride level, triglyceride, monoglyceride and phosphatide or their mixture; And making fermention medium and the alcohol that comprises the butanols biosynthetic pathway produce microorganism contacts, wherein said microorganism also comprises the through engineering approaches polynucleotide that coding has the polypeptide of lipase activity, and it is expressed and secretes or show this polypeptide, makes in fermention medium and has lipase activity.In an embodiment, fermention medium also comprises one or more carboxylic acids.In an embodiment, carbon substrate derives from biomass.In an embodiment, biomass are corn or sugarcane.In an embodiment, carbon substrate and triglyceride level, triglyceride, monoglyceride and phosphatide derive from identical biomass.

This paper provides fermention medium, it comprises alcohol and produces microorganism, butyl ester and butanols, this alcohol is produced microorganism and is comprised the butanols biosynthetic pathway and comprise the through engineering approaches polynucleotide that coding has the polypeptide of lipase activity, and described polypeptide is expressed and secretes or show.

The animal food prods that comprises microorganism as herein described also is provided.

accompanying drawing and sequence summary

The accompanying drawing and the sequence table that are incorporated to this paper and become the part of specification sheets show the present invention, and together with specification sheets, further are used for explaining principle of the present invention and making those skilled in the art can utilize the present invention.

Fig. 1 schematically shows illustrative methods of the present invention and system, wherein together with microorganism and carboxylic acid and/or natural oil, is provided to fermenting container.

Fig. 2 has described from the example biosynthetic pathway of pyruvic acid biosynthesizing isopropylcarbinol.

Fig. 3 is plasmid pRS423::TEF1 (M4)-CdLIP1 (" pNAK10 ", SEQ ID NO:45, referring to example 1) collection of illustrative plates, this plasmid carries lopsided candiyeast (Candida deformans) LIP1 lipase, it is at composing type TEF1 (M4) promotor (Nevoigt E, Kohnke J, Fischer CR, Alper H, Stahl U and Stephanopoulos G (2006), Engineering of promoter replacement cassettes for fine-tuning of gene expression in Saccharomyces cerevisiae.Appl Environ Microbiol 72:5266-5273) and the transcribing under control of CYC1 transcription terminator, be arranged in yeast-shuttle vehicle.

Fig. 4 is plasmid pRS423::TEF1 (M4)-THlip (" pTVAN2 "; SEQ ID NO:100; Referring to example 2) collection of illustrative plates, this plasmid carries dredges the thermophilic hyphomycete of cotton shape (Thermomyces lanuginosus) Tlan lipase, it is transcribed under control composing type TEF1 (M4) promotor (people such as Nevoigt E) and CYC1 transcription terminator, is arranged in yeast-shuttle vehicle.

Fig. 5 is plasmid pRS423::TEF1 (M4)-CalB (" pTVAN3 "; SEQ ID NO:; Referring to example 7) collection of illustrative plates, this plasmid carries antarctic candida (Candida antarctica) CalB lipase, it is transcribed under control composing type TEF1 (M4) promotor (people such as Nevoigt E) and CYC1 transcription terminator, is arranged in yeast-shuttle vehicle.

Fig. 6 is plasmid pYZ090 Δ alsS (SEQ ID NO:43; Referring to example) collection of illustrative plates, this plasmid carries Ketol-acid Reductoisomerase (KARI) enzyme ORF, is arranged in yeast-shuttle vehicle.

Fig. 7 plasmid pBP915 (SEQ ID NO:44; Referring to example 9 and 10) collection of illustrative plates, this plasmid carries the ORF of coding dihydroxylated acid dehydratase and alcoholdehydrogenase, is arranged in yeast-shuttle vehicle.

SEQ ID NO:1 and 2 is nucleic acid and the aminoacid sequences from the lipase B of antarctic candida (Candida antarctica) (" CalB ").

SEQ ID NO:3 and 4 is nucleic acid and the aminoacid sequences from the lipase 1 of lopsided candiyeast (Candida deformans) (" LIP1 ").

SEQ ID NO:5 and 6 is nucleic acid and the aminoacid sequences from the Tlan lipase (" Tlan ") of dredging the thermophilic hyphomycete of cotton shape (Thermomyces lanuginosus).

SEQ ID NO:255 and 256 is nucleic acid and the aminoacid sequences from the lipase 3 of Tabin aspergillus (Aspergillus tubingensis) (" lip3 ").

SEQ ID NO:7,8,9 and 257 is from antarctic candida (Candida antarctica), lopsided candiyeast (Candida deformans), dredges the encoding sequence of CalB, LIP1, Tlan and the lip3 lipase of the thermophilic hyphomycete of cotton shape (Thermomyces lanuginosus) and Tabin aspergillus (Aspergillus tubingensis), they through codon optimized with expression in Saccharomyces cerevisiae (S.cerevisiae).

SEQ ID NO:46 and 47 is nucleic acid and the aminoacid sequences with CalB variant of modifying N99A.

SEQ ID NO:48 and 49 is nucleic acid and the aminoacid sequences with LIP1 variant of modifying N146A.

SEQ ID NO:50 and 51 is nucleic acid and the aminoacid sequences with LIP1 variant of modifying N167A.

SEQ ID NO:52 and 53 is nucleic acid and the aminoacid sequences with LIP1 variant of modifying N146A and N167A.

SEQ ID NO:54 and 55 is nucleic acid and the aminoacid sequences with Tlan variant of modifying N55A.

SEQ ID NO:271 and 272 is nucleic acid and the aminoacid sequences with lip3 variant of modifying N59A.

SEQ ID NO:273 and 274 is nucleic acid and the aminoacid sequences with lip3 variant of modifying N269A.

SEQ ID NO:275 and 276 is nucleic acid and the aminoacid sequences with lip3 variant of modifying N59A and N269A.

SEQ ID NO:241 and 248 is the aminoacid sequences from the lipase of Aspergillus albicans (Aspergillus kawachii), aspergillus niger (Aspergillus niger), Yarrowia lipolytica (Yarrowia lipolytica), thermophilic ankle joint bacterium (Talaromyces thermophilus).

SEQ ID NO:249 and 254 is the aminoacid sequences in the cell surface grappling territory of Saccharomyces cerevisiae (S.cerevisiae).

SEQ ID NO:258 and 259 is the aminoacid sequences from the alcoholdehydrogenase of Achromobacter xylosoxidans (Achromobacter xylosoxidans) and India Bai Yelinke Salmonella (Beijerinkia indica).

SEQ ID NO:260 and 261 is the aminoacid sequences from the keto acid decarboxylase of Lactococcus lactis (Lactococcus lactis) and listeria grayi (Listeria grayi).

SEQ ID NO:262 and 263 is the aminoacid sequences from the dihydroxylated acid dehydratase of Streptococcus mutans (Streptococcus mutans) and Lactococcus lactis (Lactococcus lactis).

SEQ ID NO:10-45,56-144,153-238,240,264-270,277 and 278 are synthetic construct and the primer sequences described in example.

Embodiment

Unless otherwise defined, otherwise that the implication of all scientific and technical terminologies used herein and those skilled in the art understand usually is the same.As conflict, with the application's (comprising its definition), be as the criterion.In addition, unless that context separately has is required, singular references will comprise that plural number and plural term will comprise odd number.For all purposes, other bibliography that all publications, patent and this paper mention all is incorporated herein by reference in full.

In order further to limit the present invention, this paper provides following term and definition.

As used herein, term " comprises ", " comprising ", " having ", " containing " or their any other modification will be understood to mean the integer that comprises appointment or integer group but do not get rid of any other integer or integer group.For example, the composition that comprises series of elements, mixture, technique, method, goods or equipment needn't only limit to those elements, and can comprise the element that other is not clearly listed, or the intrinsic element of such composition, mixture, technique, method, goods or equipment.In addition, unless stated otherwise, otherwise "or" refer to inclusive or, rather than refer to exclusive or.For example,, any one all mean to satisfy condition A or B:A are that genuine (or existence) and B are that false (or non-existent), A are that false (or non-existent) and B are that genuine (or existence) and A and B are genuine (or existence).

In addition, number indefinite article " " or " a kind of " before element of the present invention or component who relates to element or component example (occurring) is intended to for nonrestrictive.Therefore, " one " or " a kind of " should be interpreted as and comprise one or at least one, and the word singulative of element or component also comprises that plural number refers to, unless there is numeral obviously to mean odd number.

As used herein, term " invention " or " the present invention " are non-limiting term, and are not intended to mean any independent embodiment of the present invention, but contain all possible embodiment as described as patent application.

As used herein, the term " about " that the amount of composition of the present invention or reactant of modifying is used refers to can be by the variation of the umerical amount that for example following mode occurs: in real world for generation of general measure and the liquid treatment operation of enriched material or solution; By unintentional error in these operations; For the preparation of the difference in manufacture, source or the purity of the composition of composition or manner of execution; Deng.Term " about " also comprises the different amount owing to producing from the different equilibrium conditionss of the composition of specific starting mixt.No matter whether by term " about ", modify the equal parts of the claim amount of comprising.In one embodiment, term " about " refer to the report numerical value 10% scope in, or the report numerical value 5% scope in.

As used herein, " biomass " refer to the natural product that comprises the hydrolyzable polysaccharide, described polysaccharide provides fermentable sugars, comprises and derives from natural origin as corn, sugarcane, wheat, Mierocrystalline cellulose or ligno-cellulosic materials and comprise Mierocrystalline cellulose, hemicellulose, xylogen, starch, oligose, disaccharides and/or monose and any sugar and starch of the material of their mixture.Biomass also can comprise annexing ingredient as protein and/or lipid.Biomass can derive from single source, or biomass can comprise the mixture that derives from more than one sources; For example, biomass can comprise the mixture of corn ear and maize straw, or the mixture of grass and blade.Biomass include but not limited to bioenergy crop, agricultural residue, municipal solid refuse, industrial solid rubbish, the sludge from papermaking, garden rubbish, timber and forestry rubbish.The example of biomass includes but not limited to: corn grain, corn ear, crop residue are as corn husk, maize straw, grass, wheat, rye, wheat stalk, barley, Barley straw, hay, rice straw, switchgrass, waste paper, bagasse, Chinese sorghum, soybean, component, tree, branch, root, leaf, wood chip, sawdust, shrub and the shrubbery, vegetables, fruit, flower, Animal manure and their mixture that obtain from the grinding of cereal.For example, can be by any working method known in the art, utilize the fermentation processing biological to form mash or fruit juice or molasses or hydrolysate from biomass, described method is for example ground, is processed and/or liquefies, and they comprise fermentable sugars and can comprise a certain amount of water.The hydrolysate that for example, can come by any method known to those skilled in the art processing of cellulose and/or lignocellulose biomass to comprise fermentable sugars with acquisition.Disclose low ammonia pretreatment in U.S. Patent Application Publication US20070031918A1, this patent application is incorporated herein by reference.The enzymatic saccharification of Mierocrystalline cellulose and/or lignocellulose biomass utilizes the enzyme consortium to come degraded cellulose and hemicellulose to comprise sugared hydrolysate with generation usually, and described sugar comprises glucose, wood sugar and pectinose.(saccharifying enzyme that is suitable for Mierocrystalline cellulose and/or lignocellulose biomass is summarized in Lynd, the people such as L.R. (Microbiol.Mol.Biol.Rev., 66:506-577,2002).

Mash or fruit juice or molasses or hydrolysate can comprise raw material 12 and raw slurry 16 as described herein.Water-containing material stream can be by any working method known in the art, utilize the fermentation processing biological derive from biomass or form from biomass, described method is for example ground, is processed and/or liquefies, and they comprise fermentable carbon source (for example sugar) and water.Water-containing material stream can comprise raw material 12 and raw slurry 16 as described herein.

As used herein, " product alcohol " refers to during the fermentation can be by any alcohol of microorganisms, and described microorganism utilizes the source of biomass as the carbon substrate that can ferment.Product alcohol includes but not limited to C ₁-C ₈alkyl alcohol.In an embodiment, product alcohol is C ₂-C ₈alkyl alcohol.In a further embodiment, product alcohol is C ₂-C ₅alkyl alcohol.Should be appreciated that C ₁-C ₈alkyl alcohol includes but not limited to methyl alcohol, ethanol, propyl alcohol, butanols and amylalcohol.Similarly, C ₂-C ₈alkyl alcohol includes but not limited to ethanol, propyl alcohol, butanols and amylalcohol." alcohol " this paper also is used in reference to product alcohol.

As used herein, " butanols " refers in particular to butanols isomer n-butyl alcohol (1-BuOH), 2-butanols (2-BuOH) and/or the isopropylcarbinol (iBuOH or i-BuOH or I-BUOH, also referred to as 2-methyl isophthalic acid-propyl alcohol) with independent or its form of mixtures.

As used herein, " propyl alcohol " refers to propyl alcohol isomer Virahol or 1-propyl alcohol.

As used herein, " amylalcohol " refers to amylalcohol isomer 1-amylalcohol, 3-methyl-1-butanol, 2-methyl-1-butene alcohol, 2,2-dimethyl-1-propyl alcohol, 3-amylalcohol, 2-amylalcohol, 3-methyl-2-butanols or 2-methyl-2-butanols.

As used herein, " situ product removes (ISPR) " refers to from the bioprocess such as fermentation, and when product generates, selectivity shifts out concrete tunning to control the production concentration in bioprocess.

As used herein, " fermentable carbon source " or " carbon substrate can ferment " refers to can be by microbial metabolism disclosed herein to produce the pure carbon source of fermentation.Suitable fermentable carbon source includes but not limited to monose, as glucose or fructose; Disaccharides is as lactose or sucrose; Oligose; Polysaccharide is as starch or Mierocrystalline cellulose; A carbon substrate that comprises methane; And their mixture.

As used herein, " raw material " refers to the raw material in fermenting process, described raw material packet is containing the fermentable carbon source that has or do not have undissolved solid, and if applicable, described raw material comprised fermentable carbon source before or after from starch, having discharged fermentable carbon source by further processing (for example, by liquefaction, saccharification or other method) or obtain fermentable carbon source from the complex sugar degraded.Raw material comprises or derives from biomass.Suitable raw material includes but not limited to rye, wheat, corn, sugarcane and their mixture.

As used herein, " undissolved solid ", refer to the part that can not ferment of raw material, for example, and plumule, fiber and gluten.

As used herein, " fermentation broth " refers to the mixture of all other material compositions that have in alcohol, product alcohol and the fermenting container of solid, microorganisms of water, sugar, dissolving, and wherein product alcohol by making the sugar reaction generate alcohol, water and carbonic acid gas (CO under the existence of microorganism ₂) and make.Sometimes, as used herein, term " fermention medium " and " fermenting mixture " can be used with " fermentation broth " synonym.

As used herein, " fermenting container " refers to the container that wherein carries out fermentation reaction, as butanols, by fermentation reaction, by sugar, prepared by product alcohol.

As used herein, term " effectively titre " refers to the total amount of the pure equivalent of the certain alcohols (for example butanols) that every liter of fermention medium produces by fermentation or the alcohol ester produced by pure esterification.For example, effective titre of the butanols in the unit volume fermentation comprises: (i) the butanols amount in the fermention medium; (ii) the butanols amount reclaimed by organic extraction agent; (iii) if use stripping, the butanols amount reclaimed from gas phase, and (iv) the equivalent alcohol of the butyl ester in organic phase or water.

As used herein, " saccharification " refers to that oligose is degraded into monose." synchronous saccharification and fermentation " refers to that fermentation and saccharification occur in same containers simultaneously.

As used herein, " saccharifying enzyme " refers to can Polysaccharides and/or oligose one or more enzymes of the α-Isosorbide-5-Nitrae of glycogen, starch-glycosidic link for example.Saccharifying enzyme also can comprise can hydrocellulose or the enzyme of ligno-cellulosic materials.

As used herein, " lipase activity " refers to the enzymic activity of the ester chemical bond hydrolysis in the lipid substrates of the water-insoluble or low water solubility of catalysis.Lipase is a subclass of esterase, and in itself, " lipase activity " also refers to that the catalysis ester is hydrolyzed into the enzymic activity of Carboxylic acid and alcohol, and as used herein, and " lipase activity " also refers to the alcohol and carboxylic acid esterification is become the enzymic activity of carboxylic acid alcohol ester.

As used herein, " glycosylation " is that the carbohydrate molecule enzymatic is added to biomacromolecule as on albumen, when this may occur in the albumen orientation and is secreted into extracellular.In the O-glycosylated protein, carbohydrate is connected on the hydroxyl of Serine, Threonine or tyrosine residues.In the N-glycosylated protein, carbohydrate is connected on the amide side chains of l-asparagine (N) residue in consensus sequence NXS/T, and wherein X is that any amino acid and S/T are Serine or Threonine.As used herein, " glycosylated " refers to the protein molecular with covalently bound carbohydrate.

As used herein, " liquefaction vessel " refers to the container wherein liquefied.Liquefaction is the process that wherein oligose discharges from raw material.Raw material is in the embodiment of corn therein, and oligose discharges during liquefying from the W-Gum content.

As used herein, term " separation " and " recoverys " synonym, and refer to and remove compound with compound purity or the higher compound of concentration in acquisition purity or concentration ratio original mixture from original mixture.

Term " immiscible with water " or " undissolved " refer to that chemical composition can not mix as fermentation broth with the aqueous solution with the form that forms single liquid phase as extraction agent or solvent.

As used herein, " extraction agent " or " ISPR extraction agent " refers to for extracting spawn pure as butanols, or for extracting the organic solvent from the spawn alcohol ester of product alcohol and carboxylic acid or lipid generation by catalyzer.Sometimes, as used herein, term " solvent " can be used with " extraction agent " synonym.For method as herein described, extraction agent is immiscible with water.

" natural oil " as used herein refers to be obtained from the lipid of plant (biological example matter) or animal.As used herein, " oil of plant origin " refers to the lipid obtained from plant especially." lipid " Chang Keyu " oil " and " acyl glyceride " synonym are used.Natural oil includes but not limited to tallow oil, Semen Maydis oil, Tower rape oil, capric acid/Trivent OCG, Viscotrol C, Oleum Cocois, Oleum Gossypii semen, fish oil, Jojoba oil, lard, linseed oil, neat's foot oil, oiticica oil, plam oil, peanut oil, rapeseed oil, rice oil, Thistle oil, soybean oil, sunflower oil, tung oil, curcas oil and vegetables oil blend.

As used herein, term " organic phase " refers to the nonaqueous phase in the two-phase mixture by fermentation broth contact and the immiscible organic extraction agent of water are obtained.

As used herein, term " fatty acid " " refer to there is C ₄-C ₂₈carbon atom (is C the most commonly ₁₂-C ₂₄carbon atom) carboxylic acid (for example aliphatic monocarboxylic acid), it is saturated or undersaturated.Lipid acid also can be branching or non-branching.Lipid acid can derive from or be contained in the form of esterification in fat, oil or the wax of animal or plant.Lipid acid can glyceride form natural existence in fat and fatty oil, or can be by hydrolysed fat or by synthetic acquisition.Term lipid acid can be described independent chemical substance or the mixture of lipid acid.Lipid acid can comprise mixture protonated and lipid acid non-protonization, wherein the protonated soap (for example sodium, potassium, ammonium or calcium ion salts) of the lipid acid right and wrong of non-protonization.In addition, term lipid acid can be contained free fatty acids.

As used herein, term " fatty alcohol " refers to have C ₄-C ₂₂the alcohol of the aliphatic chain of carbon atom, aliphatic chain wherein is saturated or unsaturated.

As used herein, term " alkanoic " refers to have C ₄-C ₂₂the aldehyde of the aliphatic chain of carbon atom, described aliphatic chain is saturated or unsaturated.

As used herein, term " carboxylic acid " refers to have any organic compound of general chemical formula COOH, and wherein carbon atom is combined with Sauerstoffatom and is formed carbonyl (C=O) by two keys, and by singly-bound and hydroxyl (OH) combination.Carboxylic acid can be the form of protonated carboxylic acid or carboxylate form (for example ammonium, sodium or sylvite) or protonated carboxylic acid and carboxylic acid salt mixture's form.The term carboxylic acid can be described the mixture of independent chemical substance (for example oleic acid) or carboxylic acid, and it can produce by fatty acid ester or triglyceride level, triglyceride, monoglyceride and the phosphatide in for example hydrolyzing biomass source.

As used herein, term " butanols biosynthetic pathway " or " production of butanol approach " refer to the enzymatic pathway that produces n-butyl alcohol, 2-butanols or isopropylcarbinol.

As used herein, term " n-butyl alcohol biosynthetic pathway " or " n-butyl alcohol production ways " refer to the enzymatic pathway that produces n-butyl alcohol from acetyl-coenzyme A (acetyl-CoA).

As used herein, term " 2-butanols biosynthetic pathway " or " 2-production of butanol approach " refer to the enzymatic pathway that produces the 2-butanols from pyruvic acid.

As used herein, term " isopropylcarbinol biosynthetic pathway " or " isopropylcarbinol production ways " refer to the enzymatic pathway that produces isopropylcarbinol from pyruvic acid.

As used herein, term " pure biosynthetic pathway " or " pure production ways " refer to carbon substrate are changed into to pure enzymatic pathway.The recombinant host cell that comprises " the pure production ways of through engineering approaches " refers to the host cell that comprises the modification approach, this approach with the approach from usually existing in host cell different modes produce alcohol.This type of difference comprises produces the unproductive alcohol of common host cell, or improves output or more effectively produce.

Term " gene " refers to be expressed as the nucleic acid fragment of specified protein, and it optionally comprises adjusting sequence (5 ' non-coding sequence) before encoding sequence and the adjusting sequence (3 ' non-coding sequence) after encoding sequence." natural gene " refers under state of nature and gene together with himself regulating and controlling sequence." mosaic gene " refer to non-natural any gene of comprising natively the regulation and control that exist not together and encoding sequence (that is, it be adorned from its native state or from other source).Therefore, mosaic gene can comprise regulating and controlling sequence and the encoding sequence that is derived from different sources, or is derived from identical source but arrangement mode regulating and controlling sequence and the encoding sequence different from naturally occurring arrangement mode." endogenous gene " refers to the natural gene in its natural place in the organism genome." alien gene " or " heterology gene " refer in host living beings usually as natural gene, not exist but be directed to the gene in host living beings by transgenosis.Alien gene can comprise natural gene or the mosaic gene be inserted in the non-natural organism.

As used herein, term " coding region " refers to the DNA sequence dna of coding specific amino acid sequence." suitable regulating and controlling sequence " refers to be positioned at the upstream (5 ' non-coding sequence), centre of encoding sequence or the nucleotide sequence of downstream (3 ' non-coding sequence), and it can affect the transcribing of correlative coding sequence, RNA processing or stability or translation.Regulate sequence and can comprise promotor, translation leader sequence, intron, polyadenylation recognition sequence, RNA Processing position, effector binding site and loop-stem structure.

Term " polynucleotide " is intended to comprise the nucleic acid of odd number and the nucleic acid of plural number, and refers to nucleic acid molecule or construct, for example, and messenger RNA(mRNA) (mRNA) or plasmid DNA (pDNA).As used herein, " gene " is polynucleotide.Polynucleotide can comprise the nucleotide sequence of full-length gene or cDNA sequence, or its fragment, comprise 5 of untranslated ' and 3 ' sequence and encoding sequence.Described polynucleotide can be comprised of any multinuclear ribosomal ribonucleic acid or many thymus nucleic acids, and it can be non-modified RNA or DNA, or the RNA or the DNA (for example, heterology DNA) that modify.For example, polynucleotide can consist of strand and double-stranded DNA, its mixture that is strand and double-stranded region; Strand and double-stranded RNA, its mixture that is strand and double-stranded region; The hybrid molecule that comprises DNA and RNA, it can be strand, or the mixture of two strands or strand and double-stranded region more typically." polynucleotide " have comprised chemically, the form of zymetology ground or metabolism ground modification.

As used herein, " polynucleotide of through engineering approaches " refer to the polynucleotide that changed from naturally occurring form, or as transformed, polynucleotide are introduced to host living beings by transgenosis.This type of modification comprises and for example connects two natural disjunct sequences, for example operatively connects encoding sequence and promotor that natural inoperation ground connects, or two encoding sequences are connected together to form chimeric encoding sequence.This type of modification also comprises the one or more Nucleotide that change naturally occurring polynucleotide, comprises Substitution, insertion or disappearance.

Polynucleotide sequence can mean " separation ", and wherein it removes out from natural environment.For example, be included in the heterology polynucleotide that coding in carrier has dihydroxyacid dehydratase active polypeptide or polypeptide fragment, can be considered to for purposes of the present invention separate.Another example of the polynucleotide that separate comprises recombination of polynucleotide that the heterologous host cell has or (partially or substantially) polynucleotide of the purifying in solution.The polynucleotide that separate according to the present invention or nucleic acid also comprise the molecule that this type of synthetic is produced.The polynucleotide passage of the separation of DNA polymer form can consist of one or more cDNA, genomic dna or synthetic DNA fragment.

As used herein, term " polypeptide " is intended to " polypeptide " of encompasses singular and " polypeptide " of plural number, and refers to the molecule connected to form by amido linkage (being also referred to as peptide bond) linearity by monomer (amino acid).Term " polypeptide " refers to any two or more amino acid whose chains, and does not relate to the length-specific of product.Therefore, peptide, dipeptides, tripeptides, oligopeptides, " protein ", " amino acid chain " or other an any chain being formed by two or more amino acid or term of many chains of being used in reference to, all be included in the definition of " polypeptide ", and term " polypeptide " can be used for replacing or with these terms in any one Alternate.Polypeptide can derive from the natural biological source or be produced by recombinant technology, but not necessarily by the nucleotide sequence of appointment, translated.It can be generated by any mode, comprises by chemosynthesis.

Take the variant of " separation " polypeptide or pieces and its derivative intends as the polypeptide under its physical environment not.Do not need special level of purification.For example, isolated polypeptide can be from it be removed primary or natural environment.The multiple polypeptides that restructuring produces in host cell is considered to be separated with described goal of the invention with protein, is polypeptide primary or restructuring, its by any suitable technical point from, fractionation or purifying partly or significantly.

As used herein, " recombinant microorganism " refers to that microorganism that use by recombinant DNA technology (for example, by the through engineering approaches host cell to comprise the biosynthetic pathway of biosynthetic pathway as butanols) transformed is as bacterium and yeast.

As used herein, term " codon degeneracy " refers to that nucleotide sequence allows the character changed in genetic code, and this variation does not affect the aminoacid sequence in coded polypeptide.The technician understands concrete host cell at " codon preference " that use the Nucleotide codon to be shown when determining given amino acid very much.Therefore, when synthetic gene, when improving the expression in host cell, expectation is designed gene, makes its codon usage frequency approach the preferred codon usage frequency of this host cell.

As it refers to gene for transforming various hosts or the nucleic acid molecule of coding region, term " codon optimized " refers to that the codon in the nucleic acid molecule of gene or coding region changes, and has reflected and has not changed the typical codon service condition of host living beings in the polypeptide situation of DNA encoding.This type of optimization comprises by least one or more than the codon of or significant number and replaces with the higher one or more codons of frequency of utilization in that organism.

Deviation in the nucleotide sequence of the amino acid whose codon that comprises any polypeptide chain of encoding, the modification in the sequence of this gene of permission coding.Because each codon is comprised of three Nucleotide, and the Nucleotide of composition DNA is limited to four species specificity bases, has 64 kinds of possible Nucleotide combinations, 61 kinds of coded amino acids wherein (signal of its excess-three kind codon end-of-encode translation).This paper will show which amino acid whose " genetic code " recasting which codon encode for table 1.Therefore, much amino acid has been assigned with more than a kind of codon.For example, amino acid alanine and proline(Pro) are encoded by four kinds of triplets, and Serine and arginine are by six kinds, and tryptophane and methionine(Met) are only by a kind of triplet coding.This degeneracy allows the DNA based composition change in wide region and can not change the aminoacid sequence by the protein of this DNA encoding.

table 1: standard genetic code

For the specific amino acids of encoding with specific cryptosystem, to the insertion in the peptide chain in extending, many biological performances go out preference.The difference that between codon preference or codon preference, organism, codon uses is that the degeneracy of genetic codon causes, and in a lot of organisms, very detailed record is arranged.Codon preference is normal relevant with the translation efficiency of messenger RNA(mRNA) (mRNA), and this it is believed that the characteristic that depends on the codon particularly be translated and the operability of specific transfer RNA (tRNA) molecule then.Generally speaking the advantage of selected tRNA in cell reflected the codon the most often used in peptide synthetic.Therefore, can to gene, be processed based on codon optimized, so that the expression optimization of gene in given biology.

Consider for diversified animal, plant and microbial species and have a large amount of gene orders, the relative frequency of calculating the codon use is possible.Codon use table is easy to get, " the codon usage data storehouse " for example provided at http://www.kazusa.or.jp/codon/ (access on March 20th, 2008), and these forms can be adjusted in many aspects.Referring to Nakamura, the people such as Y., Nucl.Acids Res.28:292 (2000).From GenBank Release 128.0[2002 February 15] to use table be table 2 hereinafter by recasting for the codon of the yeast that calculates.This table is used the mRNA name, and therefore, this table is used and is present in the uridylic (U) in RNA, rather than is present in the thymus pyrimidine (T) in DNA.Table 2 has passed through adjustment, has therefore calculated each amino acid whose frequency, but not whole 64 codons.

yuan 2: the codon of Saccharomyces cerevisiae (Saccharomyces cerevisiae) gene uses Yuan

By utilizing this table or similarly showing, those of ordinary skill in the art can be by these frequency application in any given peptide sequence, and produce the nucleic acid fragment of the coding region of codon optimized coding said polypeptide, but it uses the codon for given species optimization.

With frequency accidental assignment of password of the optimizing given peptide sequence of encoding, can be by calculating the codon frequency of every seed amino acid, then randomly for realizing to peptide sequence assignment of password artificially.In addition, many algorithms and computer software programs are that those of ordinary skill in the art easily obtains.For example, " EditSeq " function in the Lasergene bag, purchased from DNAstar, Inc., Madison, WI, the backtranslation function of VectorNTI external member, purchased from InforMax, Inc., Bethesda, MD, and " backtranslate " function in the GCG-Wisconsin bag, purchased from Accelrys, Inc., San Diego, CA.In addition, concerning codon optimized coding region sequence, various resources are for example openly to obtain, http://www.entelechon.com/bioinformatics/backtranslation.php? " backtranslation " function that lang=eng provides (on April 15th, 2008 accessed), and " backtranseq " function (on July 9th, 2002 accessed) of providing of http://bioinfo.pbi.nrc.ca:8090/EMBOSS/index.html.Building the frequency assignation codon of rudimentary algorithm based on given also can be realized with basic mathematical function by those of ordinary skill in the art.

Codon optimized coding region also can be designed by a plurality of methods known to those skilled in the art, comprise that software package is as " synthetic gene designer " (userpages.umbc.edu/～wug1/codon/sgd/ is published on March 19th, 2012).

When under the temperature suitable and solution ion strength condition, the nucleic acid fragment of single stranded form can be annealed to another nucleic acid fragment, polynucleotide or nucleic acid fragment " can be hybridized " to another nucleic acid fragment, for example cDNA, genomic dna or RNA molecule.Hybridization and wash conditions are well known, example is referring to Sambrook, J., Fritsch, E.F. and Maniatis, T.Molecular Cloning:A Laboratory Manual, the 2nd edition, Cold Spring Harbor Laboratory:Cold Spring Harbor, NY (1989) has especially carried out illustration in document Chapter 11 and table 11.1.Temperature and ionic strength conditions have been determined hybridization " severity ".For example can regulate stringency, with the similar fragment of screening moderate the homologous sequence of edge biology far away (from), the highly similar fragment to the screening gene of nearly edge bioautography functional enzyme (for example from).Stringency is determined in washing after hybridization.One group of preferred condition is used a series of washing steps, start is that 6X SSC, 0.5%SDS at room temperature wash 15 minutes, then repeat 30 minutes under 45 ℃, then use 0.2XSSC, 0.5%SDS 50 ℃ of lower repeated washings twice, each 30 minutes with 2X SSC, 0.5%SDS.Preferred one group of stringency adopts higher temperature, and wherein washing is identical with above-mentioned washing, and different is, and last temperature while washing 30 minutes twice in 0.2X SSC, 0.5%SDS is added to 60 ℃.The preferred highly strict condition of another group is that 0.1X SSC is used in last twice washing under 65 ℃, and 0.1%SDS carries out.For example, another group stringent condition is included in 0.1X SSC, 0.1%SDS hybridizes and uses 2X SSC, 0.1%SDS washing under 65 ℃, subsequently with 0.1X SSC, 0.1%SDS washing.

Hybridization needs two kinds of nucleic acid to contain complementary sequence, but depends on the severity of hybridization, between base, mispairing may occur.For making the suitable severity of nucleic acid hybridization depend on the length of nucleic acid and complementary degree, they are variable well known in the art.Article two, the similarity between nucleotide sequence or homology degree are higher, have the crossbred T of the nucleic acid of those sequences _mvalue just larger.The relative stability of nucleic acid hybridization is (to T _mshould be higher) reduce successively in the following order: RNA:RNA, DNA:RNA, DNA:DNA.With regard to the crossbred that is greater than 100 Nucleotide with regard to length, obtained the formula that calculates Tm (referring to people such as Sambrook, the same, 9.50-9.51).For the hybridization of shorter nucleic acid (being oligonucleotide), the position of mispairing becomes more important, and the length of oligonucleotide determined its specificity (referring to people such as Sambrook, the same, 11.7-11.8).In one embodiment, but the length of hybrid nucleic acid is at least about 10 Nucleotide.Preferably, but the minimum length of hybrid nucleic acid is at least about 15 Nucleotide; More preferably at least about 20 Nucleotide; And most preferably length is at least about 30 Nucleotide.In addition, the technician will recognize, can regulate according to the factor such as probe length the salt concn of temperature and washing soln in case of necessity.

" essential part " of amino acid or nucleotide sequence refers to such part, the nucleotide sequence presuming of the aminoacid sequence of the polypeptide that this part comprises or gene is identified described polypeptide or gene, described evaluation or can be completed by artificial evaluation sequence by those skilled in the art, or can utilize (the Basic Local Aligmnent Search Tool such as BLAST; Altschul, the people such as S.F., J.Mol.Biol., 215:403-410 (1993)) the sequence alignment of algorithm by computer automation and identifying carry out.In general, for infer identify polypeptide or nucleotide sequence whether with known protein or DNA homolog, need to have ten or more in abutting connection with amino acid or 30 or the sequence of polynucleotide more.In addition, for nucleotide sequence, comprise 20-30 and can be used in the method for the gene identification (as DNA hybridization) of sequence dependent and gene isolation (as the in situ hybridization of bacterial colony or plaque) in abutting connection with the gene specific oligonucleotide probe of Nucleotide.In addition, the short oligonucleotide of 12-15 base can be used as amplimer in PCR, in order to obtain the specific nucleic acid fragment that comprises this primer.Therefore, the sequence that " essential part " of nucleotide sequence comprises is enough to identify specifically and/or separate the nucleic acid fragment that comprises this sequence.This specification sheets has been instructed complete amino acid and the nucleotide sequence of encode specific protein matter.Have the beneficial effect of reporting sequence as this paper, the technician can use whole announcement sequences or their essential part for purpose well known by persons skilled in the art now.Therefore, the present invention includes sufficient sequence as herein provided, and the essential part of those above-mentioned sequences.

The relation of term " complementation " for hybridizing each other between the described nucleotide base.For example, for DNA, VITAMIN B4 and thymus pyrimidine complementation, and cytosine(Cyt) and guanine complementation.

Term " per-cent identity " refers to as known in the art by more a plurality of sequencings, relation between two or more peptide sequences or two or more polynucleotide sequences.In this area, " identity " also means the serial correlation degree between polypeptide or polynucleotide sequence, depends on the circumstances, as the coupling by this class sequence of characters string is measured." identity " and " similarity " can easily be calculated by currently known methods, described method include but not limited in following document disclosed those: 1.) Computational Molecular Biology (Lesk, A.M. edits) Oxford University:NY (1988); 2.) Biocomputing:Informatics and Genome Projects (Smith, D.W. edits) Academic:NY (1993); 3.) Computer Analysis of Sequence Data, Part I (H.G. edits for Griffin, A.M. and Griffin) Humania:NJ (1994); 4.) Sequence Analysis in Molecular Biology (von Heinje, G. edits) Academic (1987); And 5.) Sequence Analysis Primer (J. edits for Gribskov, M. and Devereux) Stockton:NY (1991).

Set the preferred method of definite identity for providing the optimum matching between sequence to be tested.The method of identity and similarity of determining has been weaved into code in the computer program that can openly obtain.Sequence alignment and % identity are calculated can use the MegAlign in LASERGENE information biology computation software package (LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, WI)) ^tMprogram is carried out.The multiple ratio of sequence is to being used " Clustal comparison method " to carry out, the method contains several different algorithms, comprise that " Clustal V comparison method " corresponding to the comparison method that is called Clustal V (is disclosed in Higgins and Sharp, CABIOS.5:151-153 (1989); Higgins, the people such as D.G., Comput.Appl.Biosci., 8:189-191 (1992)) in, and MegAlign that can be in LASERGENE information biology computation software package (DNASTAR Inc.) ^tMthe comparison method found in program.For multiple ratio pair, default value is corresponding to GAP PENALTY=10 and GAP LENGTH PENALTY=10.The default parameters that carries out the per-cent identity calculating of comparison in pairs and protein sequence by the Clustal method is KTUPLE=1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5.For nucleic acid, these parameters are KTUPLE=2, GAP PENALTY=5, WINDOW=4 and DIAGONALS SAVED=4.After using Clustal V program to carry out sequence alignment, by checking " sequence distance " form in same program, likely obtain " per-cent identity ".Described " Clustal W comparison method " is available and (Higgins and Sharp are shown in description, CABIOS.5:151-153 (1989) corresponding to the comparison method that is labeled as Clustal W in addition; Higgins, the people such as D.G., Comput.Appl.Biosci.8:189-191 (1992)) in, and MegAlign that can be in LASERGENE information biology computation software package (DNASTAR Inc.) ^tMthe comparison method found in the v6.1 program.Be used for the default parameters that multiple ratio is right (GAP PENALTY=10, GAP LENGTH PENALTY=0.2, Delay Divergen Seqs (%)=30, DNA Transition Weight=0.5, Protein Weight Matrix=Gonnet series, DNA weight Matrix=IUB).After using Clustal W program to carry out sequence alignment, by checking " sequence distance " table in same program, likely obtain " per-cent identity ".

Those skilled in the art is perfectly clear, and the sequence identity of multiple degree can be used for identifying polypeptide, comprises variant or polypeptide from other species, and wherein this class polypeptide has same or analogous function or activity.Useful consistence percentage includes but not limited to: 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, or any positive number per-cent of 55% to 100% can be useful when description is of the present invention, as 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.Suitable nucleic acid fragment not only has above-mentioned homology, and coding has at least 50 amino acid, preferably at least 100 amino acid, more preferably at least 150 amino acid, more preferably at least 200 amino acid, most preferably at least 250 amino acid whose polypeptide usually.

Term " sequence analysis software " refers to can be used for any computerized algorithm or the software program of analysis of nucleotide or aminoacid sequence." sequence analysis software " commercially available acquisition or stand-alone development.Typical sequence analysis software includes but not limited to: 1.) GCG suite of programs (Wisconsin Package Version 9.0, Genetics Computer Group (GCG), Madison, WI); 2.) BLASTP, BLASTN, BLASTX (people such as Altschul, J.Mol.Biol., 215:403-410 (1990)); 3.) DNASTAR (DNASTAR, Inc.Madison, WI); 4.) Sequencher (Gene Codes Corporation, Ann Arbor, MI); And 5.) described FASTA program combines Smith-Waterman algorithm (W.R.Pearson, Comput.Methods Genome Res., [Proc.Int.Symp.] (1994), Meeting Date 1992,111-20. editor: Suhai, Sandor, Plenum:New York, NY).Should be appreciated that in the context of present application for patent while using sequence analysis software to be analyzed, except as otherwise noted, otherwise analytical results is by " default value " based on institute's referral procedure.At this, " default value " used refers to initial any value or the parameter set loaded of software when initializers first.

Standard recombinant dna used herein and molecule clone technology are known in the art, and have in as Publication about Document more fully and describe: Sambrook, J., Fritsch, E.F. and Maniatis, T., Molecular Cloning:A Laboratory Manual, second edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989) (hereinafter " Maniatis "); And Silhavy, T.J., Bennan, M.L. and Enquist, L.W., Experiments with Gene Fusions, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1984); And Ausubel, the people such as F.M., Current Protocols in Molecular Biology, published by Greene Publishing Assoc.and Wiley-Interscience (1987).Additional method used herein is referring to Methods in Enzymology, the 194th volume, Guide to Yeast Genetics and Molecular and Cell Biology (part A, 2004, Christine Guthrie and Gerald R.Fink (editor), Elsevier Academic Press, San Diego, CA).

Genetic manipulation to recombinant host cell disclosed herein can utilize standard genetic technique and screening to carry out, and can in the host cell of any applicable genetic manipulation, complete (Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, the 201-202 page).In an embodiment, recombinant host cell is intestinal bacteria.In an embodiment, recombinant host cell disclosed herein can be any yeast or the fungal host useful to the expression of genetic modification and recombination.In other embodiments, recombinant host cell can be a member of following Pseudomonas: zygosaccharomyces belongs to that (Zygosaccharomyces), Schizosaccharomyces (Schizosaccharomyces), moral gram yeast belong (Dekkera), Issatchenkia (Issatchenkia), torulopsis (Torulopsis), Brettanomyces belong to (Brettanomyces), spore torulopsis (Torulaspora) is arranged, spore Hansenula (Hanseniaspora) is arranged, some bacterial classifications of genus kluyveromyces (Kluyveromyce) and mycocandida (Candida).In another embodiment, recombinant host cell can be Saccharomyces cerevisiae (Saccharomyces cerevisiae).

The applicant has been found that the recombinant host cell that can express and lipase is secreted in fermention medium produces catalyzer, and it is by the esterification of catalysis alcohol and carboxylic acid.This type of host cell has improved the host cell used in pure fermentative production, because the esterification of alcohol can allow cell to produce alcohol with higher efficiency, or the alcohol amount of producing is more than producing to host cell the alcohol amount of toxic effect.Use this type of recombinant microorganism also can reduce or eliminate to add Purification of Lipase to fermention medium to carry out the needs of methods described herein, this can provide the advantage of cost and operating aspect.

In addition, the fermentative production of alcohol utilizes reproducible biomass material so that carbon substrate to be provided usually, and recombinant microorganism changes into product alcohol by carbon substrate.This type of raw material can comprise a certain amount of triglyceride level.When implement extracting fermentation when from fermentation, removing product alcohol, triglyceride level can gather along with the time, reduced partition ratio and the recyclability of extraction agent.The lipase of recombinant host cell secretion provided herein can advantageously become free fatty acids by triglyceride hydrolysis, and it can be the substrate of esterification, and it also can produce less effect to the partition ratio of product alcohol extraction agent.

polypeptide with lipase activity

Recombinant host cell disclosed herein comprises the polynucleotide with polypeptide, and described polypeptide has lipase activity.The biology of originating in the example of lipase polynucleotide and polypeptide and they provides in table 3.

table 3: lipase polynucleotide and polypeptide example

The BLAST that the described lipase of use table 3 carries out the nonredundancy protein sequence database in NCBI as search sequence analyzes, thereby identifies the albumen with height (>90%) sequence similarity.Result is in (information on January 22nd, 2012 protein sequence of the nonredundancy from NCBI database retrieval obtain) shown in table 4.Although think that the albumen with the sequence similarity that is greater than 90% is to have the lipase of similarity with lipase described herein, will have with search sequence to be low to moderate～Sequence annotation of 30% similarity is that lipase and imagination are used together with method and composition as herein described.

table 4: other lipase polypeptide example

Except above and the lipase described in table 3 and 4, applicable lipase sequence can derive from any source, comprises for example mould genus of pears head (Absidia), achromobacter (Achromobacter), Aeromonas (Aeromonas), Alcaligenes (Alcaligenes), Alternaria (Alternaria), Aspergillus (Aspergillus), achromobacter (Achromobacter), Aureobasidium pullulans (Aureobasidium), bacillus (Bacillus), Beauveria (Beauveria), circumfili Pseudomonas (Brochothrix), mycocandida (Candida), chromobacterium (Chromobacter), Coprinus (Coprinus), fusarium (Fusarium), Geotrichum (Geotricum), Hansenula (Hansenula), Humicola (Humicola), Hyphozyma, Bacterium lacticum (Lactobacillus), Metarhizium (Metarhizium), Mucor (Mucor), Nectria (Nectria), Neurospora (Neurospora), paecilomyces (Paecilomyces), Penicillium (Penicillium), Rhodopseudomonas (Pseudomonas), Rhizoctonia (Rhizoctonia), Rhizomucor (Rhizomucor), head mold (Rhizopus), Rhodosporidium (Rhodosporidium), Rhodotorula (Rhodotorula), saccharomyces (Saccharomyces), Sus, Sporobolomyces (Sporobolomyces), thermophilic fungus belongs to (Thermomyces), Thiarosporella, Trichoderma (Trichoderma), Verticillium (Verticillium) and/or Ye Shi yeast belong (Yarrowia) bacterial strain.In an embodiment, the source of lipase is selected from Bu Shi colter mould (Absidia blakesleena), absidia corymbifera (Absidia corymbifera), Achromobacter iophagus (Achromobacter iophagus), Alcaligenes (Alcaligenes sp.), rape gives birth to chain lattice spores (Alternaria brassiciola), flavus (Aspergillus flavus), aspergillus niger (Aspergillus niger), Aspergillus albicans (Aspergillus kawachii), Tabin aspergillus (Aspergillus tubingensis), Aureobasidium pullulans (Aureobasidium pullulans), bacillus pumilus (Bacillus pumilus), bacstearothermophilus (Bacillus strearothermophilus), subtilis (Bacillus subtilis), Brochothrix thermosohata, Candida cylindracea (Candida cylindracea) (fold candida (Candida rugosa)), secondary Candida lipolytica (Candida paralipolytica), antarctic candida (Candida Antarctica) lipase A, antarctic candida (Candida Antarctica) lipase B, Ou Nuo is than candiyeast (Candida ernobii), deformity candiyeast (Candida deformans), thermophilic candiyeast (Candida thermophila), thickness look bacillus (Chromobacter viscosum), Coprinus cinereus (Coprinus cinerius), Fusarium oxysporum (Fusarium oxysporum), fusariun solani (Fusarium solani), fusariun solani (Fusarium solani pisi), the yellow sickle spore (Fusarium roseum culmorum) of wheat, geotrichum penicillatum (Geotricum penicillatum), Hansenula anomala (Hansenula anomala), short and small humicola lanuginosa (Humicola brevispora), short and small humicola lanuginosa high temperature mutation (Humicola brevis var.thermoidea), Humicola insolens (Humicola insolens), lactobacillus curvatus (Lactobacillus curvatus), Rhizopus oryzae (Rhizopus oryzae), penicillium cyclopium (Penicillium cyclopium), the skin mould (Penicillium crustosum) that falls, Penicilllum expansum (Penicillium expansum), Penicillium I (Penicillium sp.I), Penicillium II (Penicillium sp.II), Pseudomonas aeruginosa (Pseudomonas aerugmosa), Pseudomonas alcaligenes (Pseudomonas alcaligenes), pseudomonas cepacia (with Burkholderia (Burkholderia cepacia)), Pseudomonas fluorescens (Pseudomonas fluorescens), Pseudomonas fragi (Pseudomonas fragi), Pseudomonas Maltophilia (Pseudomonas maltophilia), pseudomonas mendocina (Pseudomonas mendocina), separate fat Pseudomonas mephitica (Pseudomonas mephitica lipolytica), Pseudomonas alcaligenes (Pseudomonas alcaligenes), Pseudomonas plantarii (Pseudomonas plantari), pseudomonas pseudoalcaligenes (Pseudomonas pseudoalcaligenes), pseudomonas putida (Pseudomonas putida), pseudomonas stanieri (Pseudomonas stutzeri) and Wisconsin pseudomonas (Pseudomonas wisconsinensis), dry thread Pyrenomycetes (Rhizoctonia solani), rhizomucor miehei (Rhizomucor miehei), Japan's head mold (Rhizopus japonicus), Rhizopus microsporus (Rhizopus microsporus), knotter head mold (Rhizopus nodosus), circle red winter spore yeast (Rhodosporidium toruloides), rhodotorula glutinis (Rhodotorula glutinis), Saccharomyces cerevisiae (Saccharomyces cerevisiae), sesame paddy shadow yeast (Sporobolomyces shibatanus), wild boar (Sus scrofa), thermophilic ankle joint bacterium (Talaromyces thermophiles), dredge the thermophilic hyphomycete of cotton shape (Thermomyces lanuginosus) (once being named as pubescence humicola lanuginosa (Humicola lanugmose)), Thiarosporella phaseolina, trichoderma harziarum (Trichoderma harzianum), Trichodermareesei (Trichoderma reesei) and Yarrowia lipolytica (Yarrowia lipolytica).In an embodiment, lipase is selected from and dredges the thermophilic hyphomycete of cotton shape (Thermomyces lanuginosus) lipase, Aspergillus (Aspergillus sp.) lipase, aspergillus niger (Aspergillus niger) lipase, Tabin aspergillus (Aspergillus tubingensis) lip3, antarctic candida (Candida antarctica) lipase B, Rhodopseudomonas (Pseudomonas sp.) lipase, penicillum requeforti (Penicillium roqueforti) lipase, penicillium cammenberti (Penicillium camembertii) lipase, mucor javanicus (Mucor javanicus) lipase, Burkholderia (Burkholderia cepacia) lipase, Alcaligenes (Alcaligenes sp.) lipase, candida rugosa (Candida rugosa) lipase, Candida parapsilosis (Candida parapsilosis) lipase, deformity candiyeast (Candida deformans) lipase, lipase A and B from geotrichum candidum (Geotrichum candidum), Neuraspora crassa (Neurospora crassa) lipase, the red shell bacterium of red sphere bundle (Nectria haematococca) lipase, different spore Fusariumsp (Fusarium heterosporum) lipase, De Shi head mold (Rhizopus delemar) lipase, rhizomucor miehei (Rhizomucor miehei) lipase, rhizopus arrhizus (Rhizopus arrhizus) lipase and Rhizopus oryzae (Rhizopus oryzae) lipase.

Those skilled in the art will know that coding has the polynucleotide sequence of polypeptide of lipase activity, and in upper table or polynucleotide sequence that derive from the indication source for example can be through codon optimized for recombinant host cell.In addition, one skilled in the art will appreciate that and can or guard replacement by given peptide sequence brachymemma, can not remove the lipase activity of this polypeptide simultaneously.Therefore, this paper provides to have with the sequence provided and active fragments thereof to have at least about 75%, at least about 80%, and at least about 90%, at least about 95%, at least about 97%, or at least about the polypeptide of 99% identity.The polynucleotide of this type of polypeptide of encoding also are provided.

For the embodiment of methods described herein and host cell, can be there is the polypeptide of lipase activity by microbial expression secretion, make at product alcohol production period lipase and there is activity in fermention medium.One skilled in the art will appreciate that the polypeptide of expressing on the microorganism surface, such as the cell wall protein of processing by Secretory Pathway, will be considered to secreted, because the polypeptide active of expressing can be used in extracellular on cell surface.Therefore, in an embodiment, the lipase of expression-secretion on the microorganism surface.The surface expression of albumen is known in the art, and its modified polypeptide is so that their target surface expressions.(Washida, M., S.Takahashi, M.Ueda and A.Tanaka (2001). " Spacer-mediated display of active lipase on the yeast cell surface. " Appl Microbiol Biotechnol 56 (5-6): 681-686, Matsumoto, T., H.Fukuda, M.Ueda, A.Tanaka and A.Kondo (2002). " Construction of yeast strains with high cell surface lipase activity by using novel display systems based on the Flolp flocculation functional domain. " Appl Environ Microbiol 68 (9): 4517-4522, Mormeneo, M., I.Andres, C.Bofill, P.Diaz and J.Zueco (2008). " Efficient secretion of Bacillus subtilis lipase A in Saccharomyces cerevisiae by translational fusion to the Pir4 cell wall protein. " Appl.Microbiol.Biotechnol.80 (3): 437-445, Liu, W., H.Zhao, B.Jia, L.Xu and Y.Yan (2010). " Surface display of active lipase in Saccharomyces cerevisiae using Cwp2 as an anchor protein. " Biotechnology Letters 32 (2): 255-260, Su, G.-d., X.Zhang and Y.Lin (2010). " Surface display of active lipase in Pichia pastoris using Sedl as an anchor protein. " Biotechnology Letters 32 (8): 1131-1136, Kuroda, K. and M.Ueda (2011). " Cell surface engineering of yeast for applications in white biotechnology. " Biotechnology Letters 33 (1): 1-9).In an embodiment, polypeptide provided herein is attached to polypeptide target on the albumen territory of cell surface.In an embodiment, polypeptide provided herein is attached on territory Flo1p, Pir4, Sed1, Sag1p, Cwp2 or Aga2.In an embodiment, polypeptide provided herein is attached on the albumen or protein fragments with GPI grappling motif.GPI grappling motif is known to those skilled in the art, and can be predicted by information biology, for example, for example, by using prediction engine (to be published on purpose on-line prediction engine March 19 in 2012, mendel.imp.ac.at/gpi/fungi_server.html).(the people such as Eisenhaber B., " A sensitive predictor for potential GPI lipid modification sites in fungal protein sequences and its application to genome-wide studies for Aspergillus nidulans; Candida albicans; Neurospora crassa; Saccharomyces cerevisiae, and Schizosaccharomyces pombe " J Mol Biol.2004 March 19; 337 (2): 243-53.) example of spendable peptide sequence territory (it is the cell surface to Saccharomyces cerevisiae (Saccharomyces cerevisiae) by polypeptide target) is shown in table 5.In table 5, the systematic naming method of albumen is according to saccharomyces genome database (Saccharomyces Genome Database, " SGD "; Online network address www.yeastgenome.org/; Information retrieval was on March 13rd, 2012).Being furnished with these disclosed those skilled in the art can use the peptide sequence of example and other this type of sequence construct known in the art by the polypeptide of lipase activity target recombinant microorganism cell surface.

table 5: for the cell surface grappling of Saccharomyces cerevisiae (S.cerevisiae) albumen of surface display the peptide sequence in territory.

coexpression Aga2 territory and Aga1 (SGD systematic naming method: YNR044W; SEQ ID NO:254)

In an embodiment, can modify lipase peptide sequence provided herein, make glycosylation, include but not limited to the N-glycosylation, be reduced or eliminate.This type of modification can, by the polynucleotide of sudden change coded polypeptide, make one or more glycosylation motifs be removed to carry out.In an embodiment, the glycosylation motif is N-glycosylation motif.In an embodiment, the glycosylation motif is NXS/T.In an embodiment, the polypeptide that has a lipase activity does not contain glycosylation motif NXS/T.

Can reduce or eliminate glycosylation by any method known in the art.For example, can use glycosylation inhibitor as tunicamycin, or can change the glycosylation mechanism in host cell.Also can use technology known in the art, by site-directed mutagenesis, remove the de-glycosylation motif.For example, can use the test kit (for example QuikChange II XL site-directed mutagenesis test kit, catalog number (Cat.No.) 200524, Stratagene, La Jolla, CA) of commercially available acquisition to carry out site-directed mutagenesis.Site-directed mutagenesis can be undertaken by the method for Kunkel, the method relates to mixes by uridylic template (the Kunkel TA (1985) that needs mutagenesis, Rapid and efficient site-specific mutagenesis without phenotypic selection.Proc.Natl.Acad.Sci.USA82:488-492), perhaps can carry out (Taylor JW by the method for mixing phosphorothioate, Qtt J and Eckstein F (1985), The rapid generation of oligonucleotide-directed mutations at high frequency using phosphorothioate-modified DNA.Nucleic Acids Res 13:8765-8785), perhaps by other method in vitro and in vivo known in the art, undertaken.Design of primers for the mutagenesis target site is well known in the art, and sequential analysis is known equally as the multiple sequences alignments for the identification of the mutagenesis target site.

In an embodiment, carrying out mutagenesis makes the N of motif by any other naturally occurring amino acid (A, R, D, C, E, Q, G, H, I, L, K, M, F, P, S, T, W, Y or V; Referring to table 1) replace.In an embodiment, the N of motif replaces with A.In an embodiment, carrying out mutagenesis makes the S/T of motif by any other naturally occurring amino acid (A, R, N, D, C, E, Q, G, H, I, L, K, M, F, P, w, Y or V; Referring to table 1) replace.In an embodiment, N and S/T all use any other naturally occurring amino acid (A on the N residue, R, J, D, C, E, Q, G, H, I, L, K, M, F, P, W, Y or V or S or T; A on the T residue, R, J, D, C, E, Q, G, H, I, L, K, M, F, P, W, Y or V, S or N; A on the S residue, R, J, D, C, E, Q, G, H, I, L, K, M, F, P, w, Y or V or T or N) replace.In an embodiment, glycosylation motif NXS/T replaces with motif AXS/T.

In a non-limitative example, lopsided candiyeast (C.deformans) comprises two glycosylation sequences, at the NIS of codon 146 with at 167 NNT.In an embodiment, in those glycosylation sites one or both of target replace and the glycosylation site of indication is replaced with AIS and ANT respectively.Antarctic candida (C.antarctica) has NDT 99, and the thin thermophilic hyphomycete of cotton shape (T.lanuginosus) has NIT 55.In an embodiment, the glycosylation site of sudden change indication, making calling sequence is ADT and AIT at indicating positions.

What in table 6, provide is the glycosylation site lipase open reading frame of prediction, they are from lopsided candiyeast (C.deformans), antarctic candida (C.antarctica) and dredge the thermophilic hyphomycete of cotton shape (T.lanuginosus), and the sudden change example in discarded those sites.First row has been listed the position that occurs glycosylation site in the polypeptide.Secondary series has provided the glycosylation sequences in this position, and in codon optimized polynucleotide its DNA sequence dna of coding.The 3rd row have provided after mutagenesis the peptide sequence in this position, and affect the required DNA sequence dna of this amino acid change.

table 6: the glycosylation site of prediction

In addition, the nucleic acid and the aminoacid sequence that have lopsided candiyeast (Candida deformans) lipase of two modifications (N146A and N167A) of listing in table 6 are given as SEQ ID NO:52 and 53.Nucleic acid and aminoacid sequence with Tabin aspergillus (A.tubingensis) lipase of two modifications (N59A and N269A) of listing in table 6 are given as SEQ ID NO:275 and 276.

As shown in example, use technology known in the art and/or provided herein, those skilled in the art can easily modify the glycosylation motif in lipase and measure the activity of this quasi-lipase with method and composition provided herein.

One skilled in the art will appreciate that this paper provides to have with the sequence provided and active fragments thereof and has at least about 75%, at least about 80%, at least about 90%, at least about 95%, at least about 97%, or at least about the polypeptide of 99% identity.The polynucleotide of this type of polypeptide of encoding also are provided.Those skilled in the art also will recognize that the active variant of sequence provided herein can be used known in the art and/or the techniques described herein to produce, for method and composition as herein described.

recombinant microorganism and butanols biosynthetic pathway

Not bound by theory, it is believed that improvement as herein described and method can be together with the microorganism of any generation alcohol useful, especially with the titre of the tolerance level higher than them, produce those recombinant microorganisms of alcohol.

It is known in the art producing pure microorganism.For example, methane for example, produces methyl alcohol by the fermentable oxidation of methane-oxidizing bacteria (, sending out spore Methylosimus trichosporium (Methylosinus trichosporium)), makes methyl alcohol (C ₁alkyl alcohol) contact the methyl alcohol ester that can make described carboxylic acid and methanol esterification form described carboxylic acid with catalyzer with carboxylic acid.Wild-type yeast bacterial strain CEN.PK113-7D (CBS8340, Centraal Buro voor Schimmelculture; The people such as van Dijken JP, 2000, An interlaboratory comparison of physiological and genetic properties of four Saccharomyces cerevisiae strains.Enzyme Microb.Technol.26:706-714) can produce ethanol; Make ethanol contact carboxylic acid and can the esterification carboxylic acid and the catalyzer formation ethyl ester of ethanol.

Produce pure recombinant microorganism and be also known in the art (such as people such as Ohta, 1991, Appl.Environ.Microbiol.57:893-900; The people such as Underwood, 2002, Appl.Environ.Microbiol.68:1071-1081; Shen and Liao, 2008, Metab.Eng.10:312-320; The people such as Hahnai, 2007, Appl.Environ.Microbiol.73:7814-7818; United States Patent (USP) 5,514,583, United States Patent (USP) 5,712,133; PCT Shen Qing Publication WO1995028476; The people such as Feldmann, 1992, Appl.Microbiol.Biotechnol.38:354-361; The people such as Zhang, 1995, Science267:240-243; 20070031918A1; United States Patent (USP) 7,223,575, United States Patent (USP) 7,741,119; US20090203099A1; U. S. application is announced 2009/0246846A1; With PCT Shen Qing Publication WO2010/075241, they are incorporated herein by reference).

The applicable recombinant microorganism that can produce butanols is known in the art, and this paper describes that some can produce the applicable microorganism of butanols.The recombinant microorganism that produces butanols by biosynthetic pathway can comprise the member of following genus: fusobacterium (Clostridium), zymomonas (Zymomonas), Escherichia (Escherichia), salmonella (Salmonella), serratia (Serratia), erwinia (Erwinia), Klebsiella (Klebsiella), Shigella (Shigella), Rhod (Rhodococcus), Rhodopseudomonas (Pseudomonas), bacillus (Bacillus), lactobacillus (Lactobacillus), enterococcus spp (Enterococcus), Alkaligenes (Alcaligenes), Klebsiella (Klebsiella), series bacillus belongs to (Paenibacillus), genus arthrobacter (Arthrobacter), corynebacterium (Corynebacterium), brevibacterium sp (Brevibacterium), Schizosaccharomyces (Schizosaccharomyces), kluyveromyces spp (Kluyveromyces), Ye Shi yeast belong (Yarrowia), Pichia (Pichia), mycocandida (Candida), Hansenula (Hansenula), Issatchenkia (Issatchenkia) or saccharomyces (Saccharomyces).In one embodiment, recombinant microorganism is optional from intestinal bacteria (Escherichia coli), plant lactobacillus (Lactobacillus plantarum) and Saccharomyces cerevisiae (Saccharomyces cerevisiae).In one embodiment, described recombinant microorganism is yeast.In one embodiment, described recombinant microorganism is the positive yeast of Crabtree, and it is selected from some bacterial classifications of saccharomyces (Saccharomyces), zygosaccharomyces genus (Zygosaccharomyces), Schizosaccharomyces (Schizosaccharomyces), moral gram yeast belong (Dekkera), torulopsis (Torulopsis), Brettanomyces genus (Brettanomyces) and mycocandida (Candida).The bacterial classification of the positive yeast of Crabtree includes but not limited to Saccharomyces cerevisiae (Saccharomyces cerevisiae), kluyveromyces (Saccharomyces kluyveri), schizosaccharomyces pombe (Schizosaccharomyces pombe), saccharomyces bayanus (Saccharomyces bayanus), budding yeast (Saccharomyces mikitae), Saccharomyces paradoxus (Saccharomyces paradoxus), Lu Shi zygosaccharomyces (Zygosaccharomyces rouxii) and Candida glabrata (Candida glabrata).In certain embodiments, described host cell is Saccharomyces cerevisiae (Saccharomyces cerevisiae).Saccharomyces cerevisiae (Saccharomyces cerevisiae) yeast is known in the art and can be purchased from multiple source, include but not limited to American type culture collection (Rockville, MD), Centraalbureau voor Schimmelcultures (CBS) fungal organism center of diversity, LeSaffre, Gert Strand AB, Ferm Solutions, North American Bioproducts, Martrex and Lallemand.Saccharomyces cerevisiae (Saccharomyces cerevisiae) includes but not limited to BY4741, CEN.PK113-7D, Ethanol yeast, Ferm Pro ^tMyeast,

xR yeast, Gert Strand Prestige Batch Turbo alcohol yeast, Gert Strand Pot Distillers yeast, Gert Strand Distillers Turbo yeast, FerMax ^tMgreen yeast, FerMax ^tMthe Gold yeast,

yeast, BG-1, PE-2, CAT-1, CBS7959, CBS7960 and CBS7961.

In addition, described and comprised n-butyl alcohol biosynthetic pathway (U.S. Patent Application Publication US20080182308A1, be incorporated herein by reference), the recombinant microorganism production host of 2-butanols biosynthetic pathway (United States Patent (USP) be incorporated herein by reference is openly announced US20070259410A1 and the US20070292927 that is incorporated herein by reference) and isopropylcarbinol biosynthetic pathway (United States Patent (USP) is openly announced US20070092957, is incorporated herein by reference).

Utilize the fermentative production butanols of the microorganism of microorganism and production butanols to disclose in for example United States Patent (USP) discloses 2009/0305370, the document is incorporated herein by reference.In certain embodiments, microorganism comprises the butanols biosynthetic pathway.In an embodiment, the heterology polynucleotide encoding in microorganism at least one, at least two, at least three or at least four polypeptide, in their catalysis approach, substrate is to the conversion of product.In an embodiment, all polypeptide of heterology polynucleotide encoding in microorganism, in their catalysis approach, substrate is to the conversion of product.In certain embodiments, described microorganism comprises the pyruvic carboxylase activity reduced or eliminated.The microorganism that is substantially free of the pyruvic carboxylase activity is described in U.S. Patent Application Publication 20090305363, and it is incorporated herein by reference.Being substantially free of enzyme with glyceraldehyde-3 phosphate dehydrogenase activity that NAD-relies on also is described in this patent as the microorganism of GPD2.

the butanols biosynthetic pathway

Some suitable isopropylcarbinol biosynthetic pathway is open in U.S. Patent Application Publication US20070092957, and this patent is incorporated herein by reference.The chart of disclosed isopropylcarbinol biosynthetic pathway is provided in Fig. 2.As, as described in the U.S. Patent Application Publication US20070092957A1 be incorporated herein by reference, the step in isopropylcarbinol biosynthetic pathway example comprises following conversion:

-pyruvic acid changes into acetylactis, and (referring to Fig. 2, wherein the step of approach a), for example is subject to acetolactate synthase catalysis;

-acetylactis changes into 2,3-dihydroxyl isovaleric acid (referring to Fig. 2, wherein the step b of approach), for example is subject to KARI catalysis;

-2,3-dihydroxyl isovaleric acid changes into 2-ketoisovaleric acid (referring to Fig. 2, wherein the step c of approach), for example is subject to the acetohydroxy acid dehydratase, also cries dihydroxyacid dehydratase (DHAD) catalysis:

-2-ketoisovaleric acid changes into isobutyric aldehyde (referring to Fig. 2, approach steps d wherein) and for example is subject to the catalysis of side chain 2-keto acid decarboxylase; And

-isobutyric aldehyde changes into isopropylcarbinol (referring to Fig. 2, wherein the step e of approach), for example is subject to the catalysis of branched-chain alcoho desaturase.

The substrate of the step f of alternative approach, g, h, i, j and k transforms in U.S. Patent Application Publication US2007/0092957A1 and describes to some extent to product, and it is incorporated herein by reference.

Can be used for above-mentioned substrate and describe to some extent in U.S. Patent Application Publication 20070092957 to gene and the polypeptide of product conversion and those additional isopropylcarbinol approach, it is incorporated herein by reference.U. S. application is announced 20070092957 and 20100081154 and has been described dihydroxylated acid dehydratase (DHAD), comprises from the DHAD of Streptococcus mutans (Streptococcus mutans) (SEQ ID NO:262) with from the DHAD of Lactococcus lactis (Lactococcus lactis) (SEQ ID NO:263).The U.S. Patent Application Publication 2009/0269823 and the 2011/0269199A1 that are incorporated herein by reference have described alcoholdehydrogenase, comprise from the alcoholdehydrogenase of Achromobacter xylosoxidans (Achromobacter xylosoxidans) (SEQ ID NO:258) with from the alcoholdehydrogenase of India Bai Yelinke Salmonella (Beijerinkia indica) (SEQ ID NO:259).The description of Ketol-acid Reductoisomerase (KARI) is referring to U.S. Patent Application Publication 20080261230A1,20090163376,20100197519 and PCT Shen Qing Publication WO/2011/041415, and they all are incorporated herein by reference.Keto acid decarboxylase comprises from those of Lactococcus lactis (Lactococcus lactis) (SEQ ID NO:260) and listeria grayi (Listeria grayi) (SEQ ID NO:261)

The mosaic gene that uses disclosed biosynthetic pathway to build bacterium and yeast and genetically engineered have been described in addition in the United States Patent (USP) 7,851,188 and 7,993,889 be incorporated herein by reference with the production isopropylcarbinol.

In another embodiment, the isopropylcarbinol biosynthetic pathway comprises the conversion of following substrate to product:

-pyruvic acid is to acetolactic conversion, and it can be by for example acetolactate synthase catalysis;

-acetylactis to 2, the conversion of 3-dihydroxyl isovaleric acid, it can be by for example Ketol-acid Reductoisomerase catalysis;

-2,3-dihydroxyl isovaleric acid is to the conversion of a-ketoisovaleric acid, and it can be by for example dihydroxylated acid dehydratase catalysis;

-alpha-ketoisocaproic acid is to the conversion of α-amino-isovaleric acid, and it can be by for example transaminase or valine dehydrogenase catalysis;

-α-amino-isovaleric acid is to the conversion of isobutylamine, and it can be by for example valine decarboxylase catalysis;

-isobutylamine is to the conversion of isobutyric aldehyde, and it can be by for example ω transaminase catalysis; And

-isobutyric aldehyde is to the conversion of isopropylcarbinol, and it can be by for example branched-chain alcoho desaturase catalysis.

-acetylactis to 2, the conversion of 3-dihydroxyl isovaleric acid, it can be by for example acetohydroxy acid catalysis;

-2,3-dihydroxyl isovaleric acid is to the conversion of alpha-ketoisocaproic acid, and it can be by for example acetohydroxy acid dehydratase catalysis;

-alpha-ketoisocaproic acid is to the conversion of isobutyryl-CoA, and it can be by for example branched-chain keto acids desaturase catalysis;

-isobutyryl-CoA is to the conversion of isobutyric aldehyde, and it can be by for example acetylize aldehyde dehydrogenase catalysis; And

In another embodiment, the isopropylcarbinol biosynthetic pathway comprises the conversion to product of substrate as shown in step k, g and e in Fig. 2.

Biosynthetic pathway for the production of spendable n-butyl alcohol is included in those that describe in U. S. application announcement 2008/0182308, and it is incorporated to this paper by reference.In one embodiment, the n-butyl alcohol biosynthetic pathway comprises the conversion of following substrate to product:

-a) acetyl-CoA is to the conversion of acetoacetyl-CoA, and it can be by for example acetyl-CoA Transacetylase catalysis;

-b) conversion of acetoacetyl-CoA to 3-hydroxybutyric acid-CoA, it can be by for example 3-hydroxybutyric acid-CoA desaturase catalysis;

-c) 3-hydroxybutyric acid-CoA is to the conversion of crotonoyl-CoA, and it can be by for example enoyl-CoA hydratase catalysis;

-d) crotonoyl-CoA is to the conversion of butyryl-CoA, and it can be by for example butyryl-CoA desaturase catalysis;

-e) butyryl-CoA is to the conversion of butyraldehyde, and it can be by for example butyraldehyde desaturase catalysis; And

-f) butyraldehyde is to the conversion of n-butyl alcohol, and it can be by for example butanols desaturase catalysis.

Biosynthetic pathway for the production of spendable 2-butanols is included in those that describe in U. S. application announcement 2007/0259410 and U. S. application announcement 2009/0155870, and they are incorporated to this paper by reference.In one embodiment, 2-butanols biosynthetic pathway comprises the conversion of following substrate to product:

-a) pyruvic acid is to α-acetolactic conversion, and it can be by for example acetolactate synthase catalysis;

-b) α-acetylactis is to the conversion of acetoin, and it can be by for example acetolactate decarboxylase catalysis;

-c) acetoin is to the conversion of 3-amino-2-butanols, and it can be by for example acetoin aminase catalysis;

-d) 3-amino-2-butanols is to the conversion of 3-amino-2-butanols phosphoric acid, and it can be kinase catalytic by for example amino butanol;

-e) 3-amino-2-butanols phosphoric acid is to the conversion of 2-butanone, and it can be by for example amino butanol phosphophosphorylase catalysis; And

-f) 2-butanone is to the 2-butanols, and it can be by for example butanols desaturase catalysis.

In another embodiment, 2-butanols biosynthetic pathway comprises the conversion of following substrate to product:

-c) acetoin is to the conversion of 2,3-butanediol, and it can be by for example butanediol dehydrogenation enzyme catalysis;

-d) 2,3-butanediol is to the conversion of 2-butanone, and it can be by for example diol dehydratase enzyme catalysis; And

-e) 2-butanone is to the 2-butanols, and it can be by for example butanols desaturase catalysis.

the method removed for situ product

As herein describedly improve microorganism and method can be combined with other situ product removing method, those that for example describe in PCT Shen Qing Publication WO2011/159998, this patent application is incorporated herein by reference.Fig. 1 has showed according to embodiments of the invention, the example process flow sheet for the production of product alcohol as ethanol or butanols.As shown in the figure, raw material 12 can be imported to the entrance of liquefaction vessel 10 and liquefied to produce raw slurry 16.Raw material 12 comprises the hydrolyzable polysaccharide that fermentable carbon substrate (for example, fermentable sugars is as glucose) is provided, and can be biomass, such as but not limited to rye, wheat, sugarcane or corn, or can derive from biomass.In certain embodiments, raw material 12 can be one or more components of the biomass of classification, and in other embodiments, raw material 12 can be biomass that grind, unassorted.In certain embodiments, raw material 12 can be corn, for example dry, grind, unassorted corn grain, and undissolved particle can comprise plumule, fiber and gluten.Undissolved solid be raw material 12 can not fermentation part.The purpose of discussing for this paper illustrated embodiment, raw material 12 will be described to the not classification corn of milling and forming usually, and wherein undissolved solid is not yet therefrom separated.Yet, be to be understood that illustrative methods as herein described and system can be through revising for different raw materials, no matter whether it passes through classification, and this will be readily apparent to persons skilled in the art.

The method of liquefaction raw material 12 relates to the polysaccharide hydrolysis in raw material 12 is become to comprise for example sugar of dextrin and oligose.Any known liquifying method and the corresponding liquefaction vessel of industrial common utilization be can use, sour method, acid-enzyme method or enzyme method included but not limited to.Can be used alone or in combination these class methods.In certain embodiments, can utilize described enzyme method and can be by applicable enzyme 14, α-amylase for example, import the entrance of liquefaction vessel 10.Also water can be imported to liquefaction vessel 10.In an embodiment, saccharifying enzyme (for example, glucoamylase) also can be introduced into liquefaction vessel 10.

Comprise the carbon substrate that can ferment (for example sugar) by the raw slurry 16 that makes of liquefaction raw material 12, and optionally root a tree name raw material packet containing the triglyceride level of oil form with derive from the undissolved solid of raw material.Raw slurry 16 can be discharged from the outlet of liquefaction vessel 10.In certain embodiments, raw material 12 is corn or corn kernel, and therefore raw slurry 16 is corn mash slurries.In certain embodiments, raw material 12 is lignocellulosic materials, so raw slurry 16 can be ligno-cellulose hydrolysate using.In certain embodiments, before raw slurry 16 is introduced to fermenting containers from wherein removing undissolved solid.

Raw slurry 16 is introduced to fermenting container 30 together with the microorganism of polynucleotide that comprises coding and have the polypeptide of lipase activity, and this lipase activity 32 provides according to the present invention.Fermenting container 30 is configured to make slurries 16 to ferment to produce alcohol.Particularly, the fermented carbon substrate in microorganism 32 contact slurries 16 is to produce product alcohol.Slurries can comprise fermentable carbon source, for example fermentable carbon source of oligose form, and water.

In certain embodiments, slurries 16 be provided to that saccharifying for example, is cracked into the carbohydrate (, oligose) by complicated in slurries 16 can be by the monose of easily metabolism of microorganism 32.Any known method for saccharifying of industrial common utilization all can be used, and includes but not limited to sour method, acid-enzyme method or enzyme method.In certain embodiments, synchronous saccharification and fermentation (SSF) can be as shown in Figure 1 in the interior generations of fermenting container 30.In certain embodiments, can by enzyme 38 as glucoamylase imports entrance in fermenting container 30 can be by the glucose of microorganism 32 metabolism so that starch or oligose are cracked into.

Carboxylic acid 28 and/or the natural oil that comprises triglyceride level 26 are introduced to fermenting container 30 together with optional catalyst 42.Optional catalyzer 42 can be before enzyme 38, be introduced into afterwards or simultaneously.Therefore, in certain embodiments, what the interpolation of enzyme 38 and optional catalyzer 42 can be substep (for example first adds catalyzer 42, then add enzyme 38, perhaps add on the contrary them) or basically synchronous (, just add the identical time of time of cost once with people or machine, or a kind of enzyme/catalyzer and then other catalyst/enzyme add, its time behaves or machine adds the time spent twice).Optional catalyzer 42 can esterification products alcohol with carboxylic acid 28 to form alcohol ester, and it is purified lipase in an embodiment.For example, with regard to the production of butanols, optional catalyzer 42 can make butanols and carboxylic acid 28 esterifications to form butyl ester.It is believed that catalyzer 42 is optionally for methods described herein because recombinant microorganism will express and show or secrete lipase in fermention medium with Catalytic Esterification.Yet, may expect to add purified lipase (optional catalyzer 42) and method provided herein and microorganism to allow to reduce the amount of the optional catalyst 42 added.

In the situation that will comprise during the natural oil of triglyceride level 26 adds to fermenting container 30; by oil 26 is contacted as the polypeptide of microorganism secretion provided herein or displaying and/or optional catalyzer 42 with the polypeptide with lipase activity, at least a portion acyl glyceride hydrolyzable becomes carboxylic acid 28 in oily 26.In certain embodiments, resulting acid/oil compositions comprises monoglyceride and/or the triglyceride from the partial hydrolysis of the acyl glyceride in described oil.In certain embodiments, resulting acid/oil compositions comprises the esterolytic by-product glycerin of acylglycerol.

In addition, root a tree name raw material, derive from the oil of raw material 12 and the acyl glyceride be present in slurries 16 also can be hydrolyzed to carboxylic acid 28.In certain embodiments, in liquid nutrient medium, the concentration of carboxylic acid is enough to form the two-phase fermenting mixture, and it comprises organic phase and water.

Carboxylic acid 28 can be any can with the carboxylic acid of product alcohol (for example butanols or ethanol) esterification with the alcohol ester that produces described carboxylic acid.For example, in certain embodiments, carboxylic acid 28 can be free fatty acids, and in certain embodiments, carboxylic acid or free fatty acids have the chain length of 4 to 28 carbon, 4 to 22 carbon in other embodiments, 8 to 22 carbon in other embodiments, 10 to 28 carbon, 10 to 22 carbon, 12 to 22 carbon in other embodiments in other embodiments in other embodiments, 4 to 18 carbon in other embodiments, 12 to 22 carbon in other embodiments, and 12 to 18 carbon in other embodiments, and 16 to 22 carbon in other embodiments.In certain embodiments, carboxylic acid 28 is one or more following lipid acid: lepargylic acid, capric acid, sad, castor-oil plant, coconut are (, as the combination of naturally occurring lipid acid, for example comprise lauric acid, tetradecanoic acid, palmitinic acid, sad, capric acid, stearic acid, caproic acid, eicosanoic acid, oleic acid and linolic acid), Unimac 5680, lauric acid, linseed oil, tetradecanoic acid, oleic acid, plam oil, palmitinic acid, palm-kernel, n-nonanoic acid, ricinolic acid, sebacic acid, soybean, stearic acid, Yatall MA, butter and #12 oxystearic acid.In certain embodiments, carboxylic acid 28 is one or more diacid, for example nonane diacid and sebacic acid.In certain embodiments, carboxylic acid 28 is one or more saturated monocarboxylic acids, carbon chain branches with restriction, the 2-alkyl that is called Guerbet alcohol that wherein said carboxylic acid or their mixture are known by oxidation-1-alkanol is prepared, and wherein carboxylic acid has 12 to 22 total carbon atom numbers.

Therefore, in certain embodiments, carboxylic acid 28 can be the mixture of two or more different lipid acid.In certain embodiments, carboxylic acid 28 comprises the free fatty acids of the hydrolysis that derives from acyl glyceride, and described hydrolysis is undertaken by any method known in the art, comprises hydrolysis chemistry or enzyme.In certain embodiments, as described above, use enzyme as catalyzer 42, carboxylic acid 28 can derive from by the enzymic hydrolysis of oily glyceryl ester natural oil 26.In certain embodiments, described lipid acid or their mixture comprise unsaturated fatty acids.The existence of unsaturated fatty acids reduces fusing point, and the advantage of processing aspect is provided.In unsaturated fatty acids, those monounsaturated fatty acids, have the lipid acid of single carbon-to-carbon double bond that is, and the advantage of the oxidative stability of considering with respect to the fusing point that does not carry out suitably heating and method can be provided.

In certain embodiments, natural oil 26 can be butter, corn, canola, the last of the ten Heavenly stems/sad triglyceride level, castor-oil plant, coconut, cottonseed, fish, Jojoba oil, lard, linseed oil, cow hoof, Ao Di, palm, peanut, Semen Brassicae campestris, paddy rice, safflower, soybean, Sunflower Receptacle, tung oil tree, manioca, pumpkin, palm, Semen Vitis viniferae and vegetables oil blend (maybe can be purified as the different chain length of higher concentration and unsaturated level (that is, oil) 18:1).In certain embodiments, natural oil 26 is mixtures of two or more natural oil, for example the mixture of palm and soybean oil.In certain embodiments, natural oil 26 is the oil of plant origin.In certain embodiments, the oil of plant origin can derive from available biomass in fermenting process, but this is dissimilar.Described biomass can be the identical or different sources that derive from from raw material 12.Therefore, for example, in certain embodiments, oil 26 can derive from corn, and raw material 12 can be sugarcane.For example, in certain embodiments, oil 26 can derive from corn, and the biomass of raw material 12 source is also corn.With regard to the 26 pairs of raw materials 12 of oil, any possible combination in different biomass source all can be used, as should be apparent for those skilled in the art.In certain embodiments, oil 26 derives from the biomass of using in described fermenting process.Therefore, oil 26 is directed to raw material 12 in certain embodiments.For example, when raw material 12 is corn, oil 26 is feed composition Semen Maydis oil and can be introduced in fermenting container 30 together with slurries 16 so.

In fermenting container 30, the alcohol produced by microorganism 32 and carboxylic acid 28 by the polypeptide with lipase activity by microorganism secretion (and optionally catalyzer 42) esterification to form alcohol ester.For example, with regard to the situation of production of butanol, make the butanols that produced by microorganism 32 and carboxylic acid 28 esterifications to form butyl ester.Situ product removes (ISPR) and can be used to alcohol ester is removed from fermentation broth.Utilize and express and secrete or show that the polypeptide with lipase activity can improve leavening property with the recombinant microorganism that forms ester in conjunction with ISPR.Not bound by theory, it is believed that lipase activity in fermention medium and during fermentation the esterification of product alcohol can improve the ability of microorganisms producing product alcohol, this ability is especially expected for producing the virose product alcohol of host cell tool.Therefore, this paper improves the method for microorganism to the product alcohol tolerance by the through engineering approaches microorganism to produce and to secrete the polypeptide with lipase activity, to provide.

In an embodiment, with using ISPR, do not use the effective titre in the similar fermentation of the microorganism that produces lipase to compare, use microorganisms lipase with form ester in conjunction with ISPR (for example, liquid-liquid extracts) can make effective titre improve at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or at least about 100%.Similarly, in an embodiment, with using ISPR, do not use the effective speed in the similar fermentation of the microorganism that produces lipase to compare, use microorganisms lipase with form ester in conjunction with ISPR (for example, liquid-liquid extracts) can make effective speed improve at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or at least about 100%.In an embodiment, effectively yield improved at least about 10, at least about 20%, at least about 30%, at least about 40% or at least about 50%.In certain embodiments, the fermentation broth obtained after pure esterification can comprise free (, nonesterified) alcohol, and in certain embodiments, the concentration of the free alcohol in the fermentation broth after pure esterification, when product alcohol is butanols for not higher than 1, 3, 6, 10, 15, 20, 25, 3025, 40, 45, 50, 55, or 60g/L, perhaps when product alcohol is ethanol, the concentration of the free alcohol in the fermentation broth after pure esterification is not for higher than 15, 20, 25, 3025, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100g/L.In certain embodiments, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or be converted to alcohol ester at least about the alcohol of effective titre of 90%.

In certain embodiments, fermentation broth contacts to form the two-phase mixture that comprises water and organic phase during the fermentation with extraction agent.Can carry out this type of liquid-liquid according to the described method of U.S. Patent Application Publication 2009/0305370 and extract, its disclosure is incorporated to this paper in full.U.S. Patent Application Publication 2009/0305370 has been described and has been produced butanols and adopt liquid-liquid to extract the method that reclaims butanols from fermentation broth, said method comprising the steps of: the immiscible extraction agent of fermentation broth and water is contacted, the two-phase mixture that comprises water and organic phase with formation.Usually, extraction agent can be to select self-saturating, monounsaturated, polyunsaturated (and they mix) C ₁₂-C ₂₂fatty alcohol, C ₁₂-C ₂₂lipid acid, C ₁₂-C ₂₂the ester of lipid acid, C ₁₂-C ₂₂alkanoic, C ₁₂-C ₂₂organic extraction agent of fatty amide and their mixture.Extraction agent also can be organic extraction agent, and it selects self-saturating, monounsaturated, polyunsaturated (and their mixture) C ₄-C ₂₂fatty alcohol, C ₄-C ₂₈lipid acid, C ₄-C ₂₈fatty acid ester, C ₄-C ₂₂alkanoic and their mixture.The example of suitable extraction agent comprises the extraction agent that comprises at least one solvent, and described solvent is selected from oleyl alcohol, behenyl alcohol, hexadecanol, lauryl alcohol, tetradecyl alcohol, stearyl alcohol, oleic acid, lauric acid, tetradecanoic acid, stearic acid, Myristicin acid methylester, Witconol 2301, lauryl aldehyde, 1 nonyl alcohol, 1-decanol, 1-hendecanol, 2-hendecanol, 1-aldehyde C-9,2-butyl octanol, 2-butyl-sad and their mixture.In an embodiment, described extraction agent comprises oleyl alcohol.In an embodiment, described extraction agent comprises the side chain saturated alcohol, for example can trade(brand)name the 2-butyl octanol that 12 (Sasol, Houston, TX) or Jarcol I-12 (Jarchem Industries, Inc., Newark, NJ) are commercially available.In an embodiment, described extraction agent comprises branched carboxylic acids, for example can be respectively with trade(brand)name

12, 16 know

the commercially available 2-butyl of 24 (Sasol, Houston, TX) is sad, 2-hexyldecanoic acid or 2-decyl TETRADECONIC ACID.For for method as herein described, for the extraction agent of ISPR, being the extraction agent of non-alcohol usually, (less like that carboxylic acid will be available for the esterification with product alcohol in order to avoid consuming the carboxylic acid 28 of fermenting container 30 by carboxylic acid 28 and the catalytic esterification of ethanol-extracted agent.For example, if oleyl alcohol is used as the ISPR extraction agent, owing to there being lipase activity, the carboxylicesters of oleyl alcohol can produce in fermenting container so.

According to the embodiment of Fig. 1, carboxylic acid 28 also can be used as ISPR extraction agent 28 or its component.As previously pointed out, carboxylic acid 28 can be provided and/or fermenting container 30 original positions form and/or in the situation that raw material 16 comprises that the oil form triglyceride level original position that can be hydrolyzed forms in the situation that natural oil 26 is provided to.In certain embodiments, ISPR extraction agent 28 comprises free fatty acids.In certain embodiments, ISPR extraction agent 28 comprises corn oil fatty acid (COFA).In certain embodiments, oil 26 is Semen Maydis oil, thereby ISPR extraction agent 28 is COFA.ISPR extraction agent (lipid acid) 28 contacts and forms the two-phase mixture that comprises water 34 and organic phase with fermentation broth.The product alcohol ester priority allocation formed in fermentation broth, to organic phase, forms the organic phase 36 that comprises ester.In fermentation broth any free product alcohol also priority allocation in the organic phase that comprises ester.Described two-phase mixture can be used as stream 39 and is moved out of fermenting container 30 and is introduced in container 35, and the organic phase 36 that comprises ester is separated with organic phase 34 therein.Two-phase mixture 39 is separated into the organic phase 36 that comprises ester and water 34 can be used any method as known in the art to realize, include but not limited to siphon, supply gas, decantation, centrifugal, use the auxiliary division mutually of gravity settling tank, film etc.The all or part of fermention medium that can be used as of water 34 is circulated to fermenting container 30 (as shown in the figure), or goes out of use and replaced by fresh culture, or processedly with the product alcohol that removes any remnants, then is circulated to fermenting container 30.

According to Fig. 1, the organic phase 36 that comprises ester is introduced in container 50, and alcohol ester reacts to reclaim product alcohol 54 with one or more substrates 52 therein.Can use any method be known in the art and/or describe in the PCT Shen Qing Publication WO2011/159998 be incorporated to way of reference to reclaim product alcohol 54, thereby obtain alcohol from alcohol ester.

example

As used herein, the abbreviation implication of use is as follows: " L " refers to rise, and " mL " refers to milliliter, and " μ L " refers to microlitre.

general method

the GC of the reaction product at water and extraction agent in mutually analyzes

Sample (approximately 5.0g) comprises corn oil fatty acid (COFA) as shifting out in the reaction mixture of extraction agent or fermentation broth from stirring, and carries out centrifugal with water phase separated and extraction agent phase.The sample of gained water or extraction agent phase (approximately 0.50g, record actual weight) is dissolved in the solution of 4.50mL5.5556mg/mL pentadecylic acid methyl esters (C15:0FAME, external standard) in Virahol.Centrifugal gained solution, to remove any suspended solids, then is added to about 1.25mL gained supernatant liquor in 2.0mL Agilent GC sample flasket, and PTFE film end-blocking for bottle.The isopropylcarbinol of analytic sample or lipid acid fat acid butyl ester on the Agilent6890GC that is furnished with 7683B sampler and automatic sampler.Post is Agilent DB-FFAP post (30m x0.25mm ID, 0.25 μ m film).Carrier gas is helium, and flow velocity is 1.8mL/ minute, at 80 ℃, with constant top, presses and measures; The injection splitting ratio is 20:1 at 250 ℃; Furnace temperature be 80 ℃ 2.0 minutes, be warming up to 250 ℃ with 10 ℃/minute from 80 ℃, then remain on 250 ℃ 20 minutes.Flame ionization detector is 250 ℃ of uses.Use following GC standard substance (Nu-Chek Prep; Elysian, MN) confirm the identity of lipid acid isobutyl ester product: isobutyl-cetylate, Unimac 5680 butyl ester, isooleic acid butyl ester, isobutyl-linoleate, isobutyl-linolenate, isobutyl-Arachidate.

the structure of bacterial strain

table 7:

the strain gene type used in example

table 8:

the characteristic information of the construct used in example

build PNY1500

Described strain BP 857 (" PNY1500 ") derives from CEN.PK113-7D, and (CBS 8340; Centraalbureau voor Schimmelcultures (CBS) Fungal Biodiversity Centre, Netherlands) and the disappearance that comprises following gene: URA3, HIS3.

the URA3 disappearance

For deleting endogenous URA3 coding region, the ura3::loxP-kanMX-loxP box by pcr amplification from pLA54 template DNA (SEQ ID NO:25).PLA54 comprises Kluyveromyces lactis (K.lactis) TEF1 promotor and kanMX mark, and flanking sequence is the loxP site, allows the Cre recombinase to participate in recombinating and removing mark.Use Phusion archaeal dna polymerase (New England BioLabs; Ipswich, MA) and primer BK505 and BK506 (SEQ ID NO:26 and 27) carry out PCR.The URA3 of described each primer partly derives from downstream 3 ' district, ，He coding region, URA3 promotor upstream 5 ' district, makes the integration of loxP-kanMX-loxP mark cause the replacement of URA3 coding region.The genetics technology of Application standard (Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, the 201-202 page) described PCR product is transformed in CEN.PK113-7D, and above transformant is selected at the YPD that comprises G418 (100 μ g/mL) in 30 ℃.The correct integration of the transformant of screening is verified by PCR, uses primer LA468 and LA492 (SEQ ID NO:28 and 29), and called after CEN.PK113-7D Δ ura3::kanMX.

the HIS3 disappearance

The amplification of four fragments of the PCR box lacked for seamless HIS3 is used Phusion High Fidelity PCR Master Mix (New England BioLabs) and be take the CEN.PK113-7D genomic dna as template, with Gentra Puregene Yeast/Bact kit (Qiagen; Valencia, CA) preparation.Primer oBP452 (SEQ ID NO:30) and primer oBP453 (SEQ ID NO:31) are used in the amplification of HIS3 Segment A, and it comprises 5 ' tail with 5 of HIS3 fragment B ' end homology.Primer oBP454 (SEQ ID NO:32) is used in the amplification of HIS3 fragment B, and it comprises 5 ' tail with 3 of HIS3 Segment A ' end homology, and primer oBP455 (SEQ ID NO:33), and it comprises 5 ' tail with 5 of HIS3 fragment U ' end homology.Primer oBP456 (SEQ ID NO:34) is used in the amplification of HIS3 fragment U, and it comprises 5 ' tail with 3 of HIS3 fragment B ' end homology, and primer oBP457 (SEQ ID NO:35), and it comprises 5 ' tail with 5 of HIS3 fragment C ' end homology.Primer oBP458 (SEQ ID NO:36) is used in the amplification of HIS3 fragment C, and it comprises 5 ' tail with 3 of HIS3 fragment U ' end homology, and primer oBP459 (SEQ ID NO:37).With PCR Purification kit (Qiagen) purified pcr product.The HIS3 fragments AB generates by overlapping PCR, by mixing HIS3 Segment A and HIS3 fragment B and being increased with primer oBP452 (SEQ ID NO:30) and oBP455 (SEQ ID NO:33).HIS3 fragment UC generates by overlapping PCR, by mixing HIS3 fragment U and HIS3 fragment C and being increased with primer oBP456 (SEQ ID NO:34) and oBP459 (SEQ ID NO:37).Gained PCR product electrophoresis on sepharose, then by Gel Extraction kit (Qiagen) purifying.The HIS3ABUC box generates by overlapping PCR, by mixing HIS3 fragments AB and HIS3 fragment UC and being increased with primer oBP452 (SEQ ID NO:30) and oBP459 (SEQ ID NO:37).With PCR Purification kit (Qiagen) purified pcr product.

The competent cell of preparation CEN.PK 113-7D Δ ura3::kanMX, and transform with the HIS3ABUCPCR box, Frozen-EZ Yeast Transformation II kit (Zymo Research used; Orange, CA).Transformation mixture is inoculated into and lacks uridylic and supplemented on the synthetic perfect medium of 2% glucose at 30 ℃.Having the transformant that his3 knocks out is screened with PCR, the primer is oBP460 (SEQ ID NO:38) and oBP461 (SEQ ID NO:39), uses the genomic dna prepared by Gentra Puregene Yeast/Bact kit (Qiagen).Correct transformant is chosen as bacterial strain CEN.PK113-7D Δ ura3::kanMX Δ his3::URA3.

remove the KanMX mark from Δ ura3 site and remove the URA3 mark from Δ his3 site

Described KanMX mark is removed by transform CEN.PK 113-7D Δ ura3::kanMX Δ his3::URA3 with pRS423::PGAL1-cre (SEQ ID NO:40), use Frozen-EZ Yeast Transformation II kit (Zymo Research) and be coated on 30 ℃ containing 2% glucose, not on the synthetic perfect medium containing Histidine and uridylic supplement.Make transformant 30 ℃ supplement in the YP of 1% semi-lactosi growth～6 hours to induce Cre recombinase and KanMX mark to shear, then be coated on YPD (2% glucose) flat board of 30 ℃ and recover.The isolate overnight growth is inoculated on the synthetic perfect medium that comprises the fluoro-vitamin B13 of 5-(0.1%) in YPD and at 30 ℃, has lost the isolate of the transformant of URA3 mark with selection.The isolate of the fluoro-vitamin B13 of anti-5-is grown and is inoculated in YPD to remove the pRS423::PGAL1-cre plasmid.The loss of KanMX mark and URA3 mark in the detection isolate, and detect the loss of detection pRS423::PGAL1-cre plasmid by the growth of the synthetic perfect medium flat board at YPD+G418 flat board, the synthetic perfect medium flat board that lacks uridylic and shortage Histidine.The G418 sensitivity also is chosen as to bacterial strain CEN.PK113-7D Δ ura3::loxP Δ his3 called after BP857 for the correct isolate of uridylic and Histidine auxotrophy.Described disappearance and mark remove by PCR and order-checking to be confirmed, the primer is that oBP450 (SEQ ID NO:41) and oBP451 (SEQ ID NO:42) are for Δ ura3, and primer oBP460 (SEQ ID NO:38) and oBP461 (SEQ ID NO:39) be for Δ his3, Gentra Puregene Yeast/Bact kit (Qiagen) preparation for genomic dna used.

build bacterial strain PNY2205

the PDC6 disappearance

The amplification of four fragments of the PCR box lacked for seamless PDC6 is used Phusion High Fidelity PCR Master Mix (New England BioLabs) and be take the CEN.PK113-7D genomic dna as template, with Gentra Puregene Yeast/Bact kit (Qiagen) preparation.Primer oBP440 (SEQ ID NO:18) and primer oBP441 (SEQ ID NO:19) are used in the amplification of PDC6 Segment A, and it comprises 5 ' tail with 5 of PDC6 fragment B ' end homology.Primer oBP442 (SEQ ID NO:20) is used in the amplification of PDC6 fragment B, and it comprises 5 ' tail with 3 of PDC6 Segment A ' end homology, and primer oBP443 (SEQ ID NO:21), and it comprises 5 ' tail with 5 of PDC6 fragment U ' end homology.Primer oBP444 (SEQ ID NO:22) is used in the amplification of PDC6 fragment U, and it comprises 5 ' tail with 3 of PDC6 fragment B ' end homology, and primer oBP445 (SEQ ID NO:23), and it comprises 5 ' tail with 5 of PDC6 fragment C ' end homology.Primer oBP446 (SEQ ID NO:24) is used in the amplification of PDC6 fragment C, and it comprises 5 ' tail with 3 of PDC6 fragment U ' end homology, and primer oBP447 (SEQ ID NO:56).With PCR Purification kit (Qiagen) purified pcr product.The PDC6 fragments AB generates by overlapping PCR, by mixing PDC6 Segment A and PDC6 fragment B and being increased with primer oBP440 (SEQ ID NO:18) and oBP443 (SEQ ID NO:21).PDC6 fragment UC generates by overlapping PCR, by mixing PDC6 fragment U and PDC6 fragment C and (SEQ ID NO:56 is increased with primer oBP444 (SEQ ID NO:22) and oBP447.Gained PCR product electrophoresis on sepharose, then by GelExtraction kit (Qiagen) purifying.Described PDC6ABUC box generates by overlapping PCR, by mixing PDC6 fragments AB and PDC6 fragment UC and being increased with primer oBP440 (SEQ ID NO:18) and oBP447 (SEQ ID NO:56).With PCR Purification kit (Qiagen) purified pcr product.

The competent cell of preparation CEN.PK113-7D Δ ura3::loxP Δ his3, and transform with PDC6 ABUC PCR box, Frozen-EZ Yeast Transformation II kit (Zymo Research) used.Transformation mixture is inoculated into and lacks on the synthetic perfect medium that uridylic is aided with 2% ethanol at 30 ℃.The transformant knocked out with pdc6 is screened by PCR, the primer is oBP448 (SEQ ID NO:57) and oBP449 (SEQ ID NO:58), uses the genomic dna prepared by Gentra Puregene Yeast/Bact kit (Qiagen).Correct transformant is chosen as bacterial strain CEN.PK 113-7D Δ ura3::loxP Δ his3 Δ pdc6::URA3.

CEN.PK 113-7D Δ ura3::loxP Δ his3 Δ pdc6::URA3 isolate overnight growth is inoculated on the synthetic perfect medium that comprises the fluoro-vitamin B13 of 5-(0.1%) in YPD and at 30 ℃, has lost the isolate of the transformant of URA3 mark with selection.Described disappearance and mark remove by PCR and order-checking to be confirmed, the primer is oBP448 (SEQ ID NO:57) and oBP449 (SEQ ID NO:58), uses the genomic dna prepared by Gentra Puregene Yeast/Bact kit (Qiagen).Shortage from the PDC6 gene of isolate proves by the PCR negative findings, and the Auele Specific Primer of the coding region of PDC6 used is oBP554 (SEQ ID NO:59) and oBP555 (SEQ ID NO:60).Correct isolate is chosen as bacterial strain CEN.PK113-7D Δ ura3::loxP Δ his3 Δ pdc6 and is named as BP891.

pDC1 disappearance ilvDSm integrates

Described PDC1 gene is deleted and replaced by ilvD, and its coding region is from Streptococcus mutans (Streptococcus mutans) ATCC700610.Described A fragment is followed the ilvD coding region from Streptococcus mutans (Streptococcus mutans), for the PCR box of integrating for PDC1 disappearance-ilvDSm, use Phusion High Fidelity PCR Master Mix (New England BioLabs) increased and use the NYLA83 genomic dna as template, Gentra Puregene Yeast/Bact kit (Qiagen) preparation for described genomic dna.NYLA83 is the bacterial strain (structure is described in U.S. Patent Application Publication 20110124060, and this full patent texts is incorporated herein by reference) that PDC1 disappearance-ilvDSm integrates that carrying of describing in U.S. Patent Application Publication 2009/0305363 (this full patent texts is incorporated herein by reference).Primer oBP513 (SEQ ID NO:61) and primer oBP515 (SEQ ID NO:62) are used in the amplification of PDC1 Segment A, and it comprises 5 ' tail with 5 of PDC1 fragment B ' end homology.Fragment B, the U of the described PCR box of integrating for PDC1 disappearance-ilvDSm and C are used Phusion High Fidelity PCR Master Mix (New England BioLabs) increased and use the CEN.PK113-7D genomic dna as template, with Gentra Puregene Yeast/Bact kit (Qiagen) preparation.Primer oBP516 (SEQ ID NO:63) is used in the amplification of PDC1 fragment B, it comprises 5 ' tail with 3 of PDC1 Segment A-ilvDSm ' end homology, with primer oBP517 (SEQ ID NO:64), it comprises 5 ' tail with 5 of PDC1 fragment U ' end homology.Primer oBP518 (SEQ ID NO:65) is used in the amplification of PDC1 fragment U, and it comprises 5 ' tail with 3 of PDC1 fragment B ' end homology, and primer oBP519 (SEQ ID NO:66), and it comprises 5 ' tail with 5 of PDC1 fragment C ' end homology.Primer oBP520 (SEQ ID NO:67) is used in the amplification of PDC1 fragment C, and it comprises 5 ' tail with 3 of PDC1 fragment U ' end homology, and primer oBP521 (SEQ ID NO:68).With PCR Purification kit (Qiagen) purified pcr product.PDC1 Segment A-ilvDSm-B generates by overlapping PCR, by mixing PDC1 Segment A-ilvDSm (SEQ ID NO:171) and PDC1 fragment B and using primer oBP513 (SEQ ID NO:61) and oBP517 (SEQ ID NO:64) is increased.PDC1 fragment UC generates by overlapping PCR, by mixing PDC1 fragment U and PDC1 fragment C and being increased with primer oBP518 (SEQ ID NO:65) and oBP521 (SEQ ID NO:68).Gained PCR product electrophoresis on sepharose, then by Gel Extraction kit (Qiagen) purifying.Described PDC1A-ilvDSm-BUC box (SEQ ID NO:172) generates by overlapping PCR, by mixing PDC1 Segment A-ilvDSm-B and PDC1 fragment UC and using primer oBP513 (SEQ ID NO:61) and oBP521 (SEQ ID NO:68) is increased.With PCRPurification kit (Qiagen) purified pcr product.

The competent cell of preparation CEN.PK113-7D Δ ura3::loxP Δ his3 Δ pdc6, and transform with PDC1 A-ilvDSm-BUC PCR box, Frozen-EZ Yeast Transformation II kit (Zymo Research) used.Transformation mixture is inoculated into and lacks on the synthetic perfect medium that uridylic is aided with 2% ethanol at 30 ℃.Transformant with knock out-ilvDSm of pdc1 integration is screened with PCR, the primer is oBP511 (SEQ ID NO:69) and oBP512 (SEQ ID NO:70), uses the genomic dna prepared by Gentra Puregene Yeast/Bact kit (Qiagen).Shortage from the PDC1 gene of isolate proves by the PCR negative findings, and the Auele Specific Primer of PDC1 coding region used is oBP550 (SEQ ID NO:71) and oBP551 (SEQ ID NO:72).Correct transformant is chosen as bacterial strain CEN.PK 113-7D Δ ura3::loxP Δ his3 Δ pdc6 Δ pdcl::ilvDSm-URA3.

CEN.PK113-7D Δ ura3::loxP Δ his3 Δ pdc6 Δ pdc1::ilvDSm-URA3 overnight growth is inoculated on the synthetic perfect medium that comprises the fluoro-vitamin B13 of 5-(0.1%) in YPD and at 30 ℃, has lost the isolate of the transformant of URA3 mark with selection.Removing by PCR and order-checking of the disappearance of described PDC1, the integration of ilvDSm and mark confirmed, the primer is oBP511 (SEQ ID NO:69) and oBP512 (SEQ ID NO:70), uses the genomic dna prepared by Gentra Puregene Yeast/Bact kit (Qiagen).Correct isolate is chosen as bacterial strain CEN.PK113-7D Δ ura3::loxP Δ his3 Δ pdc6 Δ pdc1::ilvDSm called after BP907.

pDC5 disappearance sadB integrates

Described PDC5 gene is deleted and replaced by the sadB coding region, described coding region is from Achromobacter xylosoxidans (Achromobacter xylosoxidans) (the sadB gene is described to some extent in U.S. Patent application 2009/0269823, and the document is incorporated herein by reference in full).At first a fragment of the PCR box of integrating for PDC5 disappearance-sadB is cloned in plasmid pUC19-URA3MCS.

PUC19-URA3MCS is based on (SEQ ID NO:94) and the sequence that comprise the URA3 gene in multiple clone site (MCS) of pUC19, and this gene is from Saccharomyces cerevisiae Saccharomyces cerevisiae (S.cerevisiae).The gene that pUC19 comprises pMB1 replicon and a coding β-lactamase, this gene is responsible for copying and selecting in intestinal bacteria.Except the encoding sequence of URA3, the upstream of this gene to downstream sequence all is included at yeast, expressing URA3.Described carrier can be used for clone's purpose, and can be used as yeast integration plasmid.

Described DNA has been contained the URA3 coding region, together with URA3 upstream of coding region 250bp and downstream 150bp, described sequence is from Saccharomyces cerevisiae (Saccharomyces cerevisiae), primer oBP438 (SEQ ID NO:89) is used in the amplification of CEN.PK113-7D genomic dna, it comprises BamHI, AscI, PmeI and FseI restriction site, and oBP439 (SEQ ID NO:90), it comprises XbaI, PacI and NotI restriction site, uses Phusion High-Fidehity PCR Master Mix (New England BioLabs).Genomic dna is used Gentra Puregene Yeast/Bact kit (Qiagen) to prepare.Described PCR product and pUC19, after BamHI and XbaI digestion, connect and generate carrier pUC19-URA3MCS with the T4 ligase enzyme.Described carrier is by PCR and order-checking confirmation, and the primer is oBP264 (SEQ ID NO:91) and oBP265 (SEQ ID NO:92).

Described sadB encoding sequence and PDC5 fragment B are cloned into the sadB-BU part that pUC19-URA3MCS generates PDC5A-sadB-BUC PCR box.Described sadB encoding sequence amplification is usingd pLH468-sadB (SEQ ID NO:93) as template, use primer oBP530 (SEQ ID NO:73), it comprises the AscI restriction site, with primer oBP531 (SEQ ID NO:74), it comprises 5 ' tail with 5 of PDC5 fragment B ' end homology.Primer oBP532 (SEQ ID NO:75) is used in the amplification of PDC5 fragment B, and it comprises 5 ' tail with 3 of sadB ' end homology, and primer oBP533 (SEQ ID NO:76), and it comprises the Pme1 restriction site.With PCR Purification kit (Qiagen) purified pcr product.SadB-PDC5 fragment B generates by overlapping PCR, by mixing sadB fragment U and PDC5 fragment B and being increased with primer oBP530 (SEQ ID NO:73) and oBP533 (SEQ ID NO:76).After the enzymic digestion with suitable, gained PCR product is connected on the site that pUC19-URA3MCS is corresponding with the T4DNA ligase enzyme through AscI and PmeI digestion.The gained plasmid is as template with amplification sadB-fragment B-fragment U, and the primer is oBP536 (SEQ ID NO:77) and oBP546 (SEQ ID NO:78), and it comprises 5 ' tail with 5 of PDC5 fragment C ' end homology.Primer oBP547 (SEQ ID NO:79) is used in the amplification of PDC5 fragment C, and it comprises 5 ' tail with 3 of PDC5sadB-fragment B-fragment U ' end homology, and primer oBP539 (SEQ ID NO:80).With PCR Purification kit (Qiagen) purified pcr product.PDC5sadB-fragment B-fragment U-fragment C generates by overlapping PCR, by mixing PDC5sadB-fragment B-fragment U and PDC5 fragment C and being increased with primer oBP536 (SEQ ID NO:77) and oBP539 (SEQ ID NO:80).Gained PCR product purifying on sepharose is then used Gel Extraction kit (Qiagen).PDC5 A-sadB-BUC box (SEQ ID NO:173) generates by amplification PDC5sadB-fragment B-fragment U-fragment C, the primer is oBP542 (SEQ ID NO:81), it comprises 5 ' tail with 50 nucleotide homologies in primary PDC5 encoding sequence upstream, and oBP539 (SEQ ID NO:80).With PCR Purification kit (Qiagen) purified pcr product.

The competent cell of preparation CEN.PK113-7D Δ ura3::loxP Δ his3 Δ pdc6 Δ pdc1::ilvDSm, and transform with PDC5 A-sadB-BUC PCR box, use Frozen-EZ Yeast Transformation II kit (Zymo Research).Transformation mixture is seeded to and lacks on the synthetic perfect medium of glucose that uridylic is aided with 1% ethanol (without glucose) at 30 ℃.Having pdc5 knocks out the transformant that sadB integrates and is screened with PCR, the primer is oBP540 (SEQ ID NO:82) and oBP541 (SEQ ID NO:83), uses the genomic dna prepared by Gentra Puregene Yeast/Bact kit (Qiagen).Shortage from the PDC5 gene of isolate proves by the PCR negative findings, and the Auele Specific Primer of PDC5 coding region used is oBP552 (SEQ ID NO:84) and oBP553 (SEQ ID NO:85).Correct transformant is chosen as bacterial strain CEN.PK113-7D Δ ura3::loxP Δ his3 Δ pdc6 Δ pdc1::ilvDSm Δ pdc5::sadB-URA3.

CEN.PK 113-7D Δ ura3::loxP Δ his3 Δ pdc6 Δ pdc1::ilvDSm Δ pdc5::sadB-URA3 overnight growth is inoculated on synthetic perfect medium in YPD (0.1% ethanol) and at 30 ℃, be aided with ethanol (without glucose) and comprise the fluoro-vitamin B13 of 5-(0.1%), to select to have lost the isolate of URA3 mark.Described PDC5 disappearance, sadB integration and mark remove by PCR to be confirmed, the primer is oBP540 (SEQ ID NO:82) and oBP541 (SEQ ID NO:83), uses the genomic dna prepared by Gentra Puregene Yeast/Bact kit (Qiagen).Described correct isolate is chosen as bacterial strain CEN.PK113-7D Δ ura3::loxP Δ his3 Δ pdc6 Δ pdc1::ilvDSm Δ pdc5::sadB called after BP913.

the GPD2 disappearance

For deleting endogenous GPD2 coding region, gpd2::loxP-URA3-loxP box (SEQ ID NO:174) carries out pcr amplification, uses loxP-URA3-loxP PCR as template DNA.LoxP-URA3-loxP (SEQ ID NO:170) comprises the mark of the URA3 from pRS426 that both wings are the loxP recombinase site.PCR is used Phusion archaeal dna polymerase and primer LA512 (SEQ ID NO:95) and LA513 (SEQ ID NO:96).The GPD2 of each primer partly derives from GPD2 upstream of coding region 5 ' district and downstream 3 ' district, coding region, makes the integration of loxP-URA3-loxP mark cause the replacement of GPD2 coding region.Described PCR product is transformed into BP913 and transformant screens on the synthetic perfect medium that is aided with 1% ethanol (without glucose) that lacks uridylic.The correct integration of transformant of screening verifies by PCR, and the primer is oBP582 and AA270.

The circulation of described URA3 mark is by transforming pRS423::PGAL1-cre and being inoculated into the shortage Histidine at 30 ℃, is aided with on the synthetic perfect medium of 1% ethanol.Be aided with 1% ethanol, and, on the synthetic perfect medium that comprises the fluoro-vitamin B13 of 5-(0.1%), transformant is to have bar mosasic, and at 30 ℃ of isolates of hatching to have selected to lose the transformant of URA3 mark.It is upper to remove the pRS423::PGAL1-cre plasmid that the isolate of the fluoro-vitamin B13 of anti-5-is grown in YPE (1% ethanol).Described disappearance and mark remove by PCR to be confirmed, the primer is oBP582 (SEQ ID NO:97) and oBP591 (SEQ ID NO:98).Correct isolate is chosen as bacterial strain CEN.PK 113-7D Δ ura3::loxP Δ his3 Δ pdc6 Δ pdc1::ilvDSm Δ pdc5::sadB Δ gpd2::loxP called after PNY1503 (BP1064).

the FRA2 disappearance

The FRA2 disappearance is designed to delete 250 Nucleotide from encoding sequence 3 ' end, and initial 113 Nucleotide that retain the FRA2 encoding sequence are motionless.A reading frame terminator codon appears at the position of removing 7 Nucleotide in downstream.The amplification of four fragments of the PCR box lacked for seamless FRA2 is used Phusion High Fidelity PCR Master Mix (New England BioLabs) and be take the CEN.PK113-7D genomic dna as template, with Gentra Puregene Yeast/Bact kit (Qiagen) preparation.Primer oBP594 (SEQ ID NO:99) and primer oBP595 (SEQ ID NO:102) are used in the amplification of FRA2 Segment A, and it comprises 5 ' tail with 5 of FRA2 fragment B ' end homology.Primer oBP596 (SEQ ID NO:103) is used in the amplification of FRA2 fragment B, and it comprises 5 ' tail with 3 of FRA2 Segment A ' end homology, and primer oBP597 (SEQ ID NO:104), and it comprises 5 ' tail with 5 of FRA2 fragment U ' end homology.Primer oBP598 (SEQ ID NO:105) is used in the amplification of FRA2 fragment U, and it comprises 5 ' tail with 3 of FRA2 fragment B ' end homology, and primer oBP599 (SEQ ID NO:106), and it comprises 5 ' tail with 5 of FRA2 fragment C ' end homology.Primer oBP600 (SEQ ID NO:107) is used in the amplification of FRA2 fragment C, and it comprises 5 ' tail with 3 of FRA2 fragment U ' end homology, and primer oBP601 (SEQ ID NO:108).With PCR Purification kit (Qiagen) purified pcr product.The FRA2 fragments AB generates by overlapping PCR, by mixing FRA2 Segment A and FRA2 fragment B and being increased with primer oBP594 (SEQ ID NO:99) and oBP597 (SEQ ID NO:104).FRA2 fragment UC generates by overlapping PCR, by mixing FRA2 fragment U and FRA2 fragment C and being increased with primer oBP598 (SEQ ID NO:105) and oBP601 (SEQ ID NO:108).Gained PCR product electrophoresis on sepharose, then by Gel Extraction kit (Qiagen) purifying.Described FRA2ABUC box generates by overlapping PCR, by mixing FRA2 fragments AB and FRA2 fragment UC and being increased with primer oBP594 (SEQ ID NO:99) and oBP601 (SEQ ID NO:108).With PCR Purification kit (Qiagen) purified pcr product.

The competent cell of preparation PNY1503 also transforms with FRA2ABUC polymerase chain reaction box, uses Frozen-EZ Yeast Transformation II kit (Zymo Research).Transformation mixture is inoculated into and lacks on the synthetic perfect medium that uridylic is aided with 1% ethanol at 30 ℃.The transformant knocked out with fra2 screens by PCR, the use primer is oBP602 (SEQ ID NO:109) and oBP603 (SEQ ID NO:110), uses the genomic dna prepared by Gentra Puregene Yeast/Bact kit (Qiagen).Correct transformant grows in YPE (yeast extract, protein peptone, 1% ethanol), and 30 ℃ of coated plates on the synthetic perfect medium that comprises the fluoro-vitamin B13 of 5-(0.1%) to select to have lost the isolate of URA3 mark.Confirm that by PCR disappearance and mark remove, described PCR has primer oBP602 (SEQ ID NO:109) and oBP603 (SEQ ID NO:110), uses the genomic dna prepared by Gentra Puregene Yeast/Bact kit (Qiagen).Shortage from the FRA2 gene of isolate proves by the PCR negative findings, uses the specific primer in FRA2 coding region, oBP605 (SEQ ID NO:111) and oBP606 (SEQ ID NO:112).Described correct isolate is selected as bacterial strain CEN.PK113-7D MATa ura3 Δ:: loxP his3 Δ pdc6 Δ pdc1 Δ:: P[PDC1]-the DHAD|ilvD_Sm-PDCltpdc5 Δ:: P[PDC5]-ADH|sadB_Ax-PDC5t gpd2 Δ:: loxP fra2 Δ called after PNY1505 (BP1135).

aDH1 disappearance and kivDLl (y) integrate

The ADH1 gene is deleted, and is replaced by the kivD coding region from Lactococcus lactis (Lactococcus lactis), and described kivD coding region is to optimize for the expression codon in Saccharomyces cerevisiae (S.cerevisiae).At first ADH1 disappearance-kivD_Ll (y) is integrated to seamless box clones in plasmid pUC19-URA3MCS.

Through codon optimized, using in Saccharomyces cerevisiae (S.cerevisiae) the kivD coding region of expressing and use pLH468 (SEQ ID NO:129) as template from Lactococcus lactis (Lactococcus lactis), use primer oBP562 (SEQ ID NO:113), it comprises the PmeI restriction site, with primer oBP563 (SEQ ID NO:114), it comprises 5 ' tail with 5 of ADH1 fragment B ' end homology.ADH1 fragment B amplification is from genomic dna, preparation as mentioned above, the primer is oBP564 (SEQ ID NO:115), it comprises 5 ' tail with 3 of kivD_L1 (y) ' end homology, with primer oBP565 (SEQ ID NO:116), it comprises the FseI restriction site.With PCR Purification kit (Qiagen) purified pcr product.KivD_L1 (y)-ADH1 fragment B generates by overlapping PCR, by mixing kivD_L1 (y) and ADH1 fragment B PCR product and using primer oBP562 (SEQ ID NO:113) and oBP565 (SEQ ID NO:116).PmeI and FseI digestion for resulting PCR product, link on the site by the correspondence of the pUC19-URA3MCS of applicable enzymic digestion with the T4DNA ligase enzyme.The amplification of ADH1 Segment A is from genomic dna, and the primer is oBP505 (SEQ ID NO:117), and it comprises the SacI restriction site, and primer oBP506 (SEQ ID NO:118), and it comprises the AscI restriction site.With SacI and AscI digestion ADH1 Segment A PCR product, and connect into the corresponding site of the plasmid that comprises kivD_L1 (y)-ADH1 fragment B with the T4DNA ligase enzyme.ADH1 fragment C amplification is from genomic dna, and the primer is oBP507 (SEQ ID NO:119), and it comprises the PacI restriction site, and primer oBP508 (SEQ ID NO:120), and it comprises the SalI restriction site.Digest ADH1 fragment C PCR product with PacI and SalI, and be connected to the corresponding site of the plasmid that comprises ADH1 Segment A-kivD_L1 (y)-ADH1 fragment B with the T4DNA ligase enzyme.From carrier pRS316-UAS (PGK1)-P _fBA1-GUS's mixed promoter UAS (PGK1)-P (SEQ ID NO:130) has increased _fBA1, primer oBP674 (SEQ ID NO:121) is used in amplification, and it comprises the AscI restriction site, and primer oBP675 (SEQ ID NO:122), and it comprises the Pmel restriction site.Digested UAS (PGK1)-P with PacI and SalI _fBA1the PCR product, and be connected to the corresponding site of the plasmid that comprises kivD_L1 (y)-ADH1 fragments AB C with the T4DNA ligase enzyme.From resulting plasmid amplification whole integration box carried out purifying with PCR Purification kit (Qiagen), amplification use primer oBP505 (SEQ ID NO:117) and oBP508 (SEQ ID NO:120).

The competent cell that has prepared PNY1505, and use Frozen-EZ Yeast Transformation II kit (Zymo Research), transformed competent cell with ADH1-kivD_L1 (y) the PCR box of above structure.Transformation mixture is inoculated into and lacks on the synthetic perfect medium that uridylic is aided with 1% ethanol at 30 ℃.Transformant is grown and is inoculated on synthetic perfect medium in YPE (1% ethanol), and described substratum comprises the fluoro-vitamin B13 of 5-(0.1%), has lost the isolate of URA3 mark 30 ℃ of selections.The integration of the disappearance of ADH1 and kivD_L1 (y) is confirmed by PCR, outside primer used is oBP495 (SEQ ID NO:123) and oBP496 (SEQ ID NO:124), with used kivD_Ll (y) Auele Specific Primer oBP562 (SEQ ID NO:113) and outside primer oBP496 (SEQ ID NO:124), use the genomic dna prepared by Gentra Puregene Yeast/Bact kit (Qiagen).Correct isolate is selected as bacterial strain CEN.PK 113-7D MATa ura3 Δ:: loxP his3 Δ pdc6 Δ pdc1 Δ:: P[PDC1]-the DHAD|ilvD_Sm-PDC1tpdc5 Δ:: P[PDC5]-ADH|sadB_Ax-PDC5t gpd2 Δ:: loxP fra2 Δ adh1 Δ:: UAS (PGK1) P[FBA1]-kivD_L1 (y)-ADHlt, and be named as PNY1507 (BP1201).

build integrative vector pUC19-kan::pdc1::FBA-alsS::TRX1

The FBA-alsS-CYCt box passes through from pRS426::GPD::alsS::CYC (as United States Patent (USP) 7,851,188 is described, the document is incorporated herein by reference in full) in 1.7kb BbvCI/PacI fragment is moved on to pRS426::FBA::ILV5::CYC (as United States Patent (USP) 7,851,188 is described, and the document is incorporated herein by reference in full) in built, with BbvCI/PacI, carried out digestion to discharge the ILV5 gene before it.Ligation is transformed to intestinal bacteria (E.coli) TOP10 cell, and has screened transformant by PCR, and the primer used is N98SeqF1 (SEQ ID NO:125) and N99SeqR2 (SEQ ID NO:126).Separated FBA-alsS-CYCt box with NotI from described carrier with BglII, for cloning the pUC19-URA3::ilvD-TRX1 (the Klenow fragment is used to prepare the end that is applicable to connection) into the AflII site.Obtained and comprised the transformant that is arranged in the alsS box of described carrier with whole two kinds of orientations, and confirmed by PCR, the primer used is N98SeqF4 (SEQ ID NO:125) and N1111 (SEQ ID NO:128) for structure " A ", for structure " B ", is N98SeqF4 (SEQ ID NO:125) and N1110 (SEQ ID NO:153).Then, the Geneticin selection type of the carrier of " A " configuration is by removing URA3 gene (1.2kb NotI/Nael fragment) and adding the Geneticin box.The Klenow fragment is used to prepare whole ends connected that is suitable for, and having screened transformant to select to have with the identical orientation of URA3 mark with before the clone of Geneticin resistant gene by PCR, the primer used is BK468 (SEQ ID NO:131) and N160SeqF5 (SEQ ID NO:154).Resulting clone is called as pUC19-kan::pdc1::FBA-alsS::TRX1 (clone A) (SEQ ID NO:155).

build alsS and integrate bacterial strain

Make pUC19-kan::pdc1::FBA-alsS integrative vector linearizing mentioned above with PmeI, and proceeded to PNY1507.Therefore PmeI inscribe carrier in clone's pdc1-TRX1 intergenic region also causes the targeted integration (Rodney Rothstein, Methods in Enzymology, 1991, the 194 volumes, 281-301 page) in this site.Selected transformant on the YPE that has added 50 μ g/mL G418.By PCR, with regard to the integration event, the transformant that forms patch is screened, the primer used is N160SeqF5 (SEQ ID NO:154) and oBP512 (SEQ ID NO:70).Manufacture the ability of isopropylcarbinol by the assessment bacterial strain, for the acetolactate synthase function, two transformants have been carried out to indirectly testing.In order to carry out this test, at E. coli-Yeast shuttle vectors (pYZ090 Δ alsS and pBP915; Respectively SEQ ID NO:43 and 44) on additional isopropylcarbinol pathway gene is provided.A clone is called PNY2205.Containing the parent strain of plasmid be named as PNY2204 (MATa ura3 Δ:: loxP his3 Δ pdc6 Δ pdc1 Δ:: P[PDC1]-DHAD|ilvD_Sm-PDClt-pUC19-loxP-kanMX-loxP-P[FBA1]-ALS|alsS_Bs-CYClt pdc5 Δ:: P[PDC5]-ADH|sadB_Ax-PDC5t gpd2 Δ:: loxP fra2 Δ adh1 Δ:: UAS (PGK1) P[FBA1]-kivD_L1 (y)-ADHlt).

build bacterial strain PNY2211

Build PNY2211 with multi-step from Saccharomyces cerevisiae (S.cerevisiae) bacterial strain PNY1507, as described in following paragraph.At first described bacterial strain is modified to comprise the phosphoketolase gene.Next, use the integrative vector of the contiguous phosphoketolase gene of target that acetolactic acid sy nthase gene (alsS) is added in bacterial strain.Finally, use homologous recombination to remove phosphoketolase gene and integrative vector sequence, cause that alsS is seamless is inserted into the Δ at pdc1:: the intergenic region between the primary TRX1 gene of ilvD and chromosome x II.Gained genotype PNY2211 is MATa ura3 Δ:: 1oxPhis3 Δ pdc6 Δ pdc1 Δ:: P[PDC11-DHAD|ilvD_Sm-PDClt-P[FBA1]-ALS|alsS_Bs-CYClt pdc5 Δ:: P[PDC5]-ADH|sadB_Ax-PDC5t gpd2 Δ:: loxP fra2 Δ adh1 Δ:: UAS (PGK1) P[FBA1]-kivD_L1 (y)-ADHlt.

By homologous recombination, the phosphoketolase box gene is imported in PNY1507.The following construction and integration body that generates.Plasmid pRS423::CUP1-alsS+FBA-budA (describes to some extent before in US2009/0305363, the document is incorporated herein by reference in full) digest to remove the FBA-budA sequence of 1.8kb with NotI and XmaI, and after processing by the Klenow fragment connection carrier again.Next, replace CUP1 promoter variants (M4 variant with the TEF1 promoter variants, as before by people such as Nevoigt, the described variant of Appl.Environ.Microbiol.72:5266-5273 (2006), the document is incorporated herein by reference in full), this is replaced via the DNA from DNA2.0 (Menlo Park, CA) is synthetic and completes with the vector construction service.Gained plasmid pRS423::TEF (M4)-alsS cuts (part of removing the 1.6kb that comprises part alsS gene and CYC1 terminator) with StuI and MluI enzyme, then with the PCR product of 4kb, mix, described PCR product generates with primer N1176 (SEQ ID NO:164) and N1177 (SEQ ID NO:165) from pRS426::GPD-xpk1+ADH-eutD (SEQ ID NO:175), and mix with the PCR product D NA of 0.8kb, described PCR product is used primer N822 (SEQ ID NO:160) and N1178 (SEQ ID NO:166) to generate from pastoris genomic dna (ENO1 promoter region), and use breach reparation cloning process to be transformed in Saccharomyces cerevisiae (S.cerevisiae) bacterial strain BY4741 (ATCC#201388), referring to people such as Ma, Gene 58:201-216 (1987).Obtain transformant by seeding cells on the synthetic perfect medium without Histidine.The expectation plasmid (pRS423::TEF1 (M4)-xpk1+ENO1-eutD, SEQ ID NO:156) of correct assembling is by being used PCR primer N821 and N1115 (being respectively SEQ ID NO:159 and 163) and being confirmed by restrictive diges-tion (BglI).Subsequently to two cloning and sequencings.TEF (M4) by separating 3.1kb with SacI and NotI digestion-xpk1 gene also is cloned into (Clone A, excise with AflII) in the pUC19-URA3::ilvD-TRX1 carrier by it.Cloned sequence processes to generate flat end for connecting by the Klenow fragment.The ligation product is transformed in intestinal bacteria Stb13 cell, selects amicillin resistance.The PCR (primer N1110 (SEQ ID NO:153) and N1114 (SEQ ID NO:162)) that is inserted through of TEF1 (M4)-xpk1 is confirmed.Described carrier is processed with linearizing AflII and by the Klenow fragment.The 1.8kb KpnI-HincII Geneticin resistance box of describing in WO2011159853A1 (being incorporated to by reference this paper) by connecting and cloned after processing by the Klenow fragment.The ligation product is transformed in intestinal bacteria Stb13 cell, selects amicillin resistance.The PCR (primer N160SeqF5 (SEQ ID NO:154) and BK468 (SEQ ID NO:131)) that is inserted through of Geneticin box is confirmed.This paper provides plasmid sequence (pUC19-URA3::pdc1::TEF (M4)-xpk1::kan, SEQ ID NO:157).

Separating obtained integration box (pdc1::TEF1 (M4)-xpk1::KanMX::TRX1) (the 5.3kb band that AscI and NaeI digestion produce, it carries out gel-purified) and it is transformed in PNY1507, Zymo Research Frozen-EZ Yeast Transformation Kit (catalog number (Cat.No.) T2001) is used in described conversion.Add selection transformant on 50 μ g/ml G418 by being inoculated into YPE.Integration on the expectation locus is confirmed by PCR (primer N886 and N1214 are respectively SEQ ID NO:161 and 167).Next, the plasmid pRS423::GALlp-Cre (SEQ ID NO:169) of coding Cre recombinase is for removing the KanMX box of loxP-flank.Correct box removes by PCR (primer oBP512 and N160SeqF5 (being respectively SEQ ID NO:168 and 154)) to be confirmed.Finally, the Geneticin selective marker that use comprises is by alsS integrated plasmid as herein described (SEQ ID NO:155; PUC19-kan::pdc1::FBA-alsS::TRX1, clone A) be transformed in this bacterial strain.The acetolactate synthase of two intasomies of test, this test is by transforming and carry out with plasmid pYZ090 Δ alsS (SEQ ID NO:43) and pBP915 (SEQ ID NO:44), use Amberg, Protocol#2 in Burke and Strathem " Methods in Yeast Genetics " (2005), and (growth and isopropylcarbinol measuring method are as follows: all bacterial strains are on the synthetic perfect medium that lacks Histidine and uridylic for the growth in the substratum that assessment comprises glucose and isopropylcarbinol production, its ethanol that comprises 0.3% glucose and 0.3% is as carbon source (the 10mL substratum (VWR catalog number (Cat.No.) 89095-260) in 125mL ventilation Erlemmeye flask.After incubated overnight (30 ℃, 250rpm's

in 40New Brunswick Scientific Shaker), in the synthetic perfect medium that comprises 2% glucose and 0.05% ethanol, (the 20mL substratum (VWR catalog number (Cat.No.) 89095-260) in the Erlenmeyer flask closely sealed at 125mL) is diluted to 0.2OD (Eppendorf BioPhotometer measurement) by culture.After cultivating 48 hours (30 ℃, 250rpm's

in 40New Brunswick Scientific Shaker), culture supernatants (using the Spin-X centrifuge tube to filter unit, Costar catalog number (Cat.No.) 8169) is used HPLC, announces 20070092957 described methods according to U. S. application and is analyzed).In two clones one is positive and is called PNY2218.

PNY2218 processes with the Cre recombinase, and by PCR (primer N886 and N160SeqR5; Respectively SEQ ID NO:161 and 158) screening loses institute's DCRP of xpk1 gene and pUC19 integrative vector sequence.During integrative vector inserts, after the homologous dna of the DNA upstream of restructuring xpk1 and importing, this only stays the alsS gene (for " seamless " inserts, because carrier, marker gene and loxP sequence are lost) that is incorporated into the pdc1-TRX1 intergenic region.Although this restructuring can occur on any point, as if carrier is integrated in the situation that to select without hereditary mycin be stable, and only after importing Cre recombinase, observe described recombination event.A clone is called PNY2211.

build Saccharomyces cerevisiae bacterial strain (Saccharomyces cerevisiae) PNY2242

Built bacterial strain PNY2242 with a plurality of steps from (mentioned above) PNY1507.At first, the mosaic gene that will comprise FBA1 promotor, alsS coding region and CYC1 terminator has been integrated into chromosome x II, is positioned at the TRX1 upstream region of gene.The locus sequence of described modification is SEQ ID No:176.Next step, two copies of coding horse liver alcohol dehydrogenase gene are integrated in dyeing VII and XVI.On karyomit(e) VII, comprise the PDC1 promotor, the mosaic gene of hADH coding region and ADH1 terminator is placed on fra2 Δ locus (disappearance of original FRA2 is as mentioned above).The locus sequence of described modification is SEQ ID No:177.On chromosome x VI, the zone occupied by long-term repeat element YPRC Δ 15 before the mosaic gene that comprises PDC5 promotor, hADH coding region and ADH1 terminator is integrated into.The locus sequence of described modification is SEQ ID No:178.Then natural gene YMR226c and ALD6 have been deleted.The elimination of YMR226c is seamless its coding region of having removed only.The locus sequence of described modification is SEQ ID No:179.Use the mark of CRE-lox mediation to remove (method mentioned above), deleted the upstream sequence of ALD6 coding region and 700bp, therefore resulting locus comprises a loxP site.The locus sequence of described modification is SEQ ID No:180.Finally, plasmid is introduced to bacterial strain to express Anaerostipes caccae variant KARI (pLH702, SEQ ID.No.181) and DHAD (pYZ067DkivDDhADH, SEQ ID.No.182), obtain bacterial strain PNY2242.

example 1

the expression of deformity candiyeast (Candida deformans) LIP1 lipase in yeast

DNA sequence dna from the primary LIP1 lipase of lopsided candiyeast (C.deformans) is obtained from GenBank (accession number AJ428393), and open reading frame (ORF) is through optimizing to express (DNA2.0) in yeast.Gained DNA sequence dna and wild-type sequence have 76% sequence identity, and the identical albumen of encoding.

The synthetic DNA (DNA2.0) that expresses the ORF sequence of optimizing that comprises, and the clone advances (Oldenburg KR in yeast-shuttle vehicle by the gained cloned dna molecule by the breach reparation, Vo KT, Michaelis S and Paddon C (1997) Recombination-mediated PCR-directed plasmid construction in vivo in yeast.Nucleic Acids Res 25:451-452).In brief, LIP1 lipase ORF is used primer AK10-33_CdL5 and AK10-34_CdL3 (being respectively SEQ ID NO:10 and 11) to be increased, they comprise with plasmid pNAK34 (SEQ ID NO:x) in 5th ' district of regional homology.By gained PCR product with together with PacI restriction endonuclease linearizing pNAK34, cotransformation is to Saccharomyces cerevisiae (S.cerevisiae) bacterial strain PNY1500 in, this conversions is by basically carrying out as (Gietz RD and Woods RA (2006) Yeast transformation by the LiAc/SS Carrier DNA/PEG method.Methods Mol Biol 313:107-120) described lithium acetate/PEG conversion.Conversion reaction is applied on synthetic complete nutrient agar (Sherman F (2002) Getting started with yeast.Methods in Enzymology 350:3-41), this substratum comprises 2% glucose and does not contain the auxotrophy mixture (Formedium of Histidine, UK, catalog number (Cat.No.) DSCK-042; The SCD-His substratum).30 ℃ hatch 3 days after, picking His ⁺bacterium colony is for further analyzing.

The positive isolate of LIP1 lipase is coated on the SC-His substratum that comprises tributyrin and at 30 ℃ and hatches 3 days.The positive isolate of LIP1 lipase has the clear area around them, indicates their secretions can be hydrolyzed the feed fat powder enzyme of tributyrin; In contrast, the contrast yeast strain does not cause that clear area appears in the tributyrin in nutrient agar.Plasmid from 3 isolates reclaims by plasmid rescue (Robzyk K and Kassir Y (1992) A simple and highly efficient procedure for rescuing autonomous plasmids from yeast.Nucleic Acids Res.20:3790), and use M13-is reverse and T7-promoter primer (being respectively SEQ ID NO:16 and 17), on ABI Prism 3730xl DNA Analyzer, use BigDye Terminator Cycle order-checking chemistry to be checked order.The prediction plasmid product that this sequence and breach are repaired cloning process (data are not shown) mates fully.The gained plasmid is pNAK10 (SEQ ID NO:45; Fig. 3).

example 2

in yeast, Yuan Da dredges the thermophilic hyphomycete of cotton shape (Thermomyces lanuginosus) lipase

DNA sequence dna from the primary lipase (Tlan lipase) of dredging the thermophilic hyphomycete of cotton shape (Thermomyces lanuginosus) is obtained from GenBank (accession number AF054513), and this sequence is through optimizing to express (DNA2.0) in yeast.Gained DNA sequence dna and wild-type sequence have 76% sequence identity, and the identical albumen of encoding.

The synthetic DNA (DNA2.0) that expresses the ORF sequence of optimizing that comprises, and by the clone of the breach reparation as example 1, the gained cloned dna molecule is advanced in yeast-shuttle vehicle.In brief, the synthetic thermophilic hyphomycete of thin cotton shape (T.lanuginosus) Tlan lipase ORF is used primer AK10-42_T15-1 and AK10-43_T13 (being respectively SEQ ID NO:12 and 13) to be increased, and they comprise (the SEQ ID NO:45 with plasmid pNAK10; 5th ' district of the regional homology Fig. 3).By gained PCR product with together with SpeI restriction endonuclease linearizing pNAK10 cotransformation in Saccharomyces cerevisiae (S.cerevisiae) bacterial strain PNY1500.Conversion reaction is applied on the SCD-His substratum).30 ℃ hatch 3 days after, by the plasmid that comprises Tlan lipase sequence of bacterium colony pcr analysis bacterium colony, this bacterium colony PCR is used primer AK10-41_T15-1 and AK10-42_T13 (SEQ ID NO:12 and 13).

The positive isolate of Tlan lipase is coated on the SCD-His substratum that comprises tributyrin and at 30 ℃ and hatches 3 days.The positive isolate of Tlan lipase has the clear area around them, indicates their secreting function lipase activitys; In contrast, the contrast yeast strain does not cause that clear area appears in the tributyrin in nutrient agar.Retrieve the plasmid from three isolates by plasmid rescue, and use M13-is reverse and T7-promoter primer (SEQ ID NO:16 and 17) is checked order.The prediction plasmid product that this sequence and breach are repaired cloning process (data are not shown) mates fully.A plasmid is called pTVAN2 (SEQ ID NO:100).

example 3

dredge the extensive expression of the thermophilic hyphomycete of cotton shape (T.lanuginosus) lipase in yeast

From a positive isolate of example 2, PNY1020, in the SCD-His substratum, preculture is spent the night, and this is used to inoculate four 500mL cultures of SCD-His substratum; Two asparaginyl glycosylation inhibitor tunicamycin (5 μ g/mL for culture; Sigma-Aldrich, St.LouisMO) process.Flask is hatched with the 250rpm condition with 30 ℃ in the shaking culture case.After 8 hours, by 50mL YPD substratum (yeast extract, 10g/L; Peptone, 20g/L; Glucose, 20g/L) be added in each flask, and by the culture overnight incubation.Morning, after exhausting glucose, culture under 4 ℃ with 8000rpm centrifugal 10 minutes.Hold back strainer by concentrated about 500 times of supernatant liquor pressurization by 10,000 daltonian molecular weight.Measure the protein concentration of retention, and, by SDS-Polyacrylamide Gel Electrophoresis 20 μ g albumen, this electrophoresis is used the acrylamide Bis-Tris gel (1nvitrogen, Carlsbad CA) of 4-12%, carries out according to the specification sheets of manufacturers.Gel dyes and decolours with Coomassie Blue R-250.The albumen that both tunicamycin treatment is crossed has lower molecular weight, shows as its higher movability (not shown) in gel.The characteristic of Tlan lipase band is confirmed by amino-end sequencing.

Tlan lipase albumen in the prediction retention (through or without both tunicamycin treatment after express) concentration for analyzing total solubilized albumen of 25% based on SDS-PAGE, and these two kinds of retentions that comprise Tlan lipase albumen (through or without both tunicamycin treatment after express) be used as the catalyzer (example 5) of external esterification isopropylcarbinol and corn oil fatty acid.

example 4

the production of corn oil fatty acid

750g crude corn oil, 2112g water and 285g50% sodium hydroxide solution are packed into and be equipped with 5 liters of round-bottomed flasks of mechanical stirrer, thermopair, heating mantles, condenser and nitrogen threeway.Mixture is heated to 90 ℃ and keep two hours, and in during this period of time, it is single-phase that it has become dense emulsion form.Tlc indication in latter stage during this period of time in mixture without remaining Semen Maydis oil.Then mixture is cooled to 74 ℃ and add 900g25% sulfuric acid with acidifying mixture, then it is cooled to 50 ℃ and separate water layer.The water washing twice of 1500mL40 ℃ for oil reservoir, then with the washing of 1L saturated brine once, and subsequently dry on sal epsom and pass through diatomite filtration.Product is the oil of the redness that 610g is limpid.Fatty acid content by AOCS method Ca5a-40 to the titration demonstration 95% of free fatty acids, it is expressed with oleic acid.By will in the dry pyridine of 1mL, sample (104mg) being reacted and sample will be carried out to silanization with N-methyl-N-(TMS) trifluoroacetamide of 100uL.Gas chromatography-mass spectrography (GCMS) analysis of the product of silanization has been shown to the existence of TMS (trimethyl silicon based) derivative of 16:0,18:2,18:1,18:0 and 20:0 carboxylic acid.

example 5

by the reaction by the lipase-catalyzed isopropylcarbinol of secretion and corn oil fatty acid produce isobutyl-- the COFA ester

Reaction mixture comprises 3.6g isopropylcarbinol (2-methyl isophthalic acid-propyl alcohol), 14.7g the corn oil fatty acid prepared by Semen Maydis oil (COFA) (example 4), 45.1g moisture 2-(N-morpholino) ethyl sulfonic acid damping fluid (0.20M, and 0.487mg (10ppm in water pH5.4); Table 7) or 0.974mg (20ppm in water; Table 9) Tlan lipase albumen (through or without both tunicamycin treatment after express; Example 3), stir this mixture at 30 ℃, and take out sample in predetermined point of time from each reaction mixture, centrifugal immediately, and separate and analyze isopropylcarbinol (iBuOH) and the corn oil fatty acid isobutyl ester (iBuO-COFA) (table 10) of water layer and organic layer.The reaction that comprises Tlan lipase produces than the significantly more iBuO-COFA of contrast reaction; In the situation that tunicamycin exists the lipase sample of yeast secretary to exist and produce significantly more iBuO-COFA than unrestraint agent.

table 9: isopropylcarbinol (iBuOH) is being changed into to corn oil fatty acid isobutyl ester (iBuO- cOFA) the Tlan concentration in reaction.

table 10: for the reaction described in table 9 moisture level part (AQ) and organic grade of part (ORG) the weight of the isobutyl ester (iBuO-COFA) of the isopropylcarbinol of middle existence (iBuOH) and corn oil fatty acid amount.

example 6

produce fatty acid butyl ester during yeast culture

Tlan lipase isolate PNY1020 and control strain PNY908 preculture in the SCD-His substratum, and the flask that comprises 25mL SC-His substratum for inoculation (the Packed top cover of tool).The aseptic COFA of 8.25g (33%w/w), isopropylcarbinol (0.50g adds after 8 hours in growth) and tunicamycin (Tnm, ultimate density 5 μ g/ml) improvement following (table 11) for flask:

table 11:

Flask is hatched under 30 ℃ and 250rpm condition, and samples after hatching 24 hours and 96 hours.Glucose, ethanol, isopropylcarbinol and the alkyl fatty esters in water by HPLC and GC analytic sample, and analyze isopropylcarbinol and the alkyl fatty esters (table 12 and 13) in organic phase by GC.When being added in culture by isopropylcarbinol and COFA, the bacterial strain (flask F8 and F9) of expressing lipase produces than the more iBuO-COFA of contrast bacterial strain (flask F4).The cell of crossing by both tunicamycin treatment produces more ester than the cell of processing without inhibitor.

yuan 12: the HPLC of moisture F1-F10 sample analyzes.

Sample	Time	Glucose	Glycerine	Acetate	Ethanol	iBuOH
							?	(hour)	(mM)	(mM)	(mM)	(mM)	(mM)
?	?	?	?	?	?	?
							F1	24	0.1	25	8.2	170.4	0.8
F2	24	0.1	3.3	7.3	179.4	1.2
							F3	24	50.9	1.6	3.5	106.4	266.6
F4	24	0.0	1.5	5.5	182.0	144.3
							F5	24	0.1	3.9	10.5	171.1	0.5
F6	24	0.1	5.0	7.9	184.7	0.1
							F7	24	89.9	1.1	2.4	39.0	267.2

F8	24	0.1	1.8	1.7	189.9	144.6
							F9	24	65.9	1.7	2.6	80.7	143.5
F10	24	10.8	16.6	5.4	141.6	0.4
							?	?	?	?	?	?	?
F1	96	0.0	?	0.7	91.7	0.3
							F2	96	0.0	3.1	7.0	179.3	0.1
F3	96	50.6	1.2	3.0	108.6	247.9
							F4	96	0.0	1.6	7.2	183.8	130.0
F5	96	0.0	3.3	9.2	163.9	0.1
							F6	96	0.0	4.9	9.7	182.6	0.1
F7	96	90.3	0.9	1.5	38.6	248.6
							F8	96	0.0	1.9	2.0	190.7	128.1
F9	96	0.7	2.1	1.2	184.2	128.5
							F10	96	0.0	17.8	2.2	153.2	0.1

table 13: for the diastatochromogenes described in table 11 moisture level part (AQ) and organic grade of part (ORG) isopropylcarbinol (iBuOH) existed in and the isobutyl ester (iBuO-of corn oil fatty acid cOFA) weight.

example 7

antarctic candida (c andida antarctica) expression of lipase B in yeast

The DNA sequence dna of antarctic candida (Candida antarctica) lipase B (CalB lipase) is obtained from GenBank (accession number Z30645), and this sequence is through optimizing to express (DNA2.0 in yeast, Menlo Park, CA).Gained DNA sequence dna and wild-type sequence have 72% sequence identity, and the identical albumen of encoding.

The synthetic DNA (DNA2.0) that comprises CalB open reading frame (ORF) sequence of expressing optimization, and the breach reparation of passing through clone advances the gained cloned dna molecule in yeast-shuttle vehicle.In brief, CalB lipase ORF is used primer CALBL_gap_for and CALBL_gap_rev (SEQ ID NO:14 and 15) to be increased, they comprise with plasmid pNAK34 (SEQ ID NO:232) in 5th ' district of regional homology.By lithium acetate/PEG, transform, by gained PCR product with use the linearizing plasmid pNAK33 of HpaI restriction endonuclease (SEQ ID NO:231), pNAK34 (SEQ ID NO:232) or pNAK35 (SEQ ID NO:233) together with cotransformation in Saccharomyces cerevisiae (S.cerevisiae) bacterial strain PNY1500.Conversion reaction is applied on the SCD-His substratum.30 ℃ hatch 3 days after, by the plasmid that comprises CalB lipase sequence of bacterium colony pcr analysis bacterium colony, this bacterium colony PCR is used primer CALBL_gap_for and CALBL_gap_rev (SEQ ID NO:14 and 15).

The positive isolate of CalB lipase is coated on the SCD-His substratum that comprises tributyrin and at 30 ℃ and hatches 3 days.The positive isolate of CalB lipase has the clear area around them, indicates their secreting function lipase activitys; In contrast, the contrast yeast strain does not cause that clear area appears in the tributyrin in nutrient agar.Retrieve the plasmid from 3 isolates by plasmid rescue, and use M13-is reverse and T7-promoter primer (SEQ ID NO:16 and 17).The prediction plasmid product that this sequence and breach are repaired cloning process mates fully.The gained plasmid is called pTVAN7 (TEF1 (M2) promotor), pTVAN3 (TEF1 (M4) promotor) and pTVAN8 (TEF1 (M6) promotor) (being respectively SEQ ID NO:278,277 and 240).

example 8

the surface display of Tlan lipase

Following introducing is fixed to the territory on yeast cell surface by the thermophilic hyphomycete of thin cotton shape (T.lanuginosus) lipase of secretion.Pastoris genomic dna (PNY1500) is as the template in the PCR reaction, the primer is AK11-46 and AK11-47 (being respectively SEQ ID NO:215 and 216), their amplifications are by 320 amino acid whose codons of C-end of the yeast α-agglutinant protein of SAG1 coding, and a sequence of 5 ' end interpolation that is comprising the joining region of being rich in glycine and Serine.Use Phusion archaeal dna polymerase (New England Biolabs), according to the specification sheets of manufacturers, complete amplification.

This GS-SAG1DNA clones into pCR-BluntII-TOPO (InVitrogen) and is transformed into the TOPO of DH5 α.PGS-SAG1 plasmid (SEQ ID NO:217) reclaims and confirms correct sequence by DNA sequencing by mini-prep (Qiagen).DNA is used primer Sagtgap1 and Sagtgap2 (being respectively SEQ ID NO:218 and 219) to be increased, and they comprise that the homology zone clones into lipase expression vector pTVAN11, pTVAN12 and pTVAN13 (being respectively SEQ ID NO:220,221 and 222) with the breach reparation.Together with the pTVAN11 that purified PCR product digested with PacI (TEF1 (M2) promotor), pTVAN12 (TEF1 (M4) promotor) or pTVAN13 (TEF1 (M6) promotor), be transformed in yeast strain PNY1500.Conversion reaction is applied to the SCD-His substratum; Show the positive bacterium colony of test and express lipase activity on the tributyrin flat board.Save plasmid and be transformed in bacillus coli DH 5 alpha and carry out purifying from these isolates (Yeast Plasmid Miniprep Kit, Zymo Research).Sequential analysis illustrates the chimeric expectation nucleotide sequence of lipase-SAG1.

Express grow overnight in the 50mL SCD-His substratum that 250mL that the bacterial strain (PNY1052, PNY1053 and PNY1054) of lipase and control strain (PNY1500) hatch under 30 ℃ and 250rpm condition ventilates in the top cover flask.Morning, transfer to the 21.5mL culture in 125mL flask (top pressure closure), adds 1.75mL glucose (500g/L), 2.5mL10XYEP (100g/L yeast extract, 200g/L peptone), and 0.313mL isopropylcarbinol.Take out sample, add subsequently 10.3mL COFA and aseptic stirring rod, send flask back to and hatched.Take out sample (5mL) and carry out HPLC and GC analysis after 24 hours, and add 1.75mL glucose and 0.313mL isopropylcarbinol.Took out the second sample after 72 hours.Analytic sample (table 14) as mentioned above.Express the chimeric bacterial strain of SAG1-lipase and produce more fatty acid butyl ester (FABE) than the contrast bacterial strain.Bacterial strain PNY1054 has the strong promoter that drives that mosaic transcribes, and its produces than the FABE of many 6 times of contrast bacterial strain, and have than the bacterial strain of weak promoter only produce than contrast bacterial strain many～30% FABE.

yuan 14: at water and the isopropylcarbinol in organic phase (iBuOH) of indicator strain diastatochromogenes the amount of recording with lipid acid isobutyl ester (FABE).

example 9

cell surface display: cell association test

Carry out this test to determine the lipase activity of being expressed by SAG1-lipase mosaic be in fact whether cell association or be secreted in nutrient solution.

Express 250mL that the bacterial strain (PNY1052, PNY1053 and PNY1054) of lipase and control strain (PNY1500) hatch under 30 ℃ and 250rpm condition 25mL SCD-His substratum in the top cover flask (the 6.7g/L yeast nitrogen-without amino acid of ventilating, 1926mg does not contain the auxotrophy mixture of Histidine, 20g/L glucose) the middle growth 24 hours.Centrifugal separating cell and nutrient solution subsequently; Twice of washed cell throw out also is resuspended in the 25mL50mM MES damping fluid of pH5.5.With the 1M MES damping fluid of pH5.5, acellular substratum used is adjusted to 50mM.Isopropylcarbinol (ultimate density 20g/L) and COFA (33%wt/wt) are added in substratum and cell suspending liquid used.Two reactions are hatched 72 hours under 30 ℃ and 250rpm condition.As mentioned above by GC analytic sample (table 15).After with COFA and isopropylcarbinol, hatching 72 hours, the suspension of expressing the cell of lipase form than compared with control cells suspension many～FABE of 2.5 times.In contrast, the FABE in acellular media samples gathers indifference, shows that Sag1-lipase chimeric protein is that specific cell is associated under these conditions.

table 15: at water and the isopropylcarbinol in organic phase (iBuOH) of indicator strain diastatochromogenes the amount of recording with lipid acid isobutyl ester (FABE).

example 10

the through engineering approaches isopropylcarbinol is produced yeast and is dredged the thermophilic hyphomycete of cotton shape (T.lanuginosus) fat to secrete the fat enzyme

The amplification of Tlan lipase transgenosis is from plasmid pTVAN6 (SEQ ID NO:183), oligonucleotide AK11-24 (SEQ ID NO:132) and AK11-25 (SEQ ID NO:133) are used in this amplification, and they comprise the AscI site at their 5 ' end.The PCR product digests with AscI and is connected in the pBP1236 (SEQ ID NO:185) of AscI digestion.This plasmid is for implementing the people such as the Akada (people such as Akada R, (2006), PCR-mediated seamless gene deletion and marker recycling in Saccharomyces cerevisiae.Yeast23:399-405) technology, integrate for the transgenosis of the fra2 Δ locus at yeast.To connect mixture is transformed in competence bacillus coli DH 5 alpha (Invitrogen, Carlsbad CA) and is applied on LB-penbritin agar plate.Dull and stereotyped bacterium colony grow overnight in the LB-penbritin from this, and use Qiaprep Spin Miniprep test kit isolated plasmid dna.Recombinant plasmid digests by AscI and agarose gel electrophoresis is identified.DNA sequencing is for the identification of the genetically modified direction of the lipase in construct.Plasmid pNAK15 (SEQ ID NO:186) comprises reverse wild-type lipase transgenosis, and pNAK16 (SEQ ID NO:187) the wild-type lipase transgenosis that comprises forward.

Amplification lipase transgenosis and the flanking DNA (and it comprises that the URA3 gene is as selected marker) that makes them be oriented in the fra2 Δ and integrate from these plasmids, the amplification the primer is oBP691 (SEQ ID NO:136) and oBP696 (SEQ ID NO:137).Use QIAQuick PCR Purification kit purifying concentrated PCR product.Yeast strain PNY2211 (as mentioned above build) is under 30 ℃ and 250rpm condition, at YPE substratum (10g/L yeast extract, the 20g/L peptone, 20mL/l95% ethanol) grow overnight in, and transform with the PCR product, be applied to subsequently SCE-Ura nutrient agar (6.7g/L yeast nitrogen-without amino acid (YNB; Difco 291940, BD, Franklin Lakes NJ), 1926mg/L does not contain the auxotrophy mixture (DSCK102, Formedium, Norfolk UK) of Ura, 20mL/L95% ethanol) on.By Ura ⁺bacterium colony is applied on the new system substratum, is applied to subsequently FOA substratum (6.7g/L YNB, 1g/L 5-fluororotic acid, 200mg/L uridylic, 20mL/l 95% ethanol) upper to select isolate again, and this isolate has been lost the URA3 selective marker.

Check the whether correct integration transgenosis of anti-FOA transformant and check by bacterium colony PCR whether selective marker loses, this bacterium colony PCR is used the primer pair of following each flank of integration box: for having the genetically modified construct of forward (from pNAK16), use primer pair AK11-26 (SEQ ID NO:134) and oBP730 (SEQ ID NO:138) and AK11-27 (SEQ ID NO:135) and oBP731 (SEQ ID NO:139).For thering is reverse genetically modified construct (from pNAK15), use primer pair AK11-27 (SEQ ID NO:135) and oBP730 (SEQ ID NO:138) and AK11-26 (SEQ ID NO:134) and oBP731 (SEQ ID NO:139).Select the isolate that produces correct PCR product further to study, and called after PNY931 (oppositely) and PNY932 (forward).

Lipase integrates yeast strain and their parent strain PNY2211 transforms to import the isopropylcarbinol pathways metabolism with plasmid pBP915 (SEQ ID NO:44) and pYZ090 Δ alsS (SEQ ID NO:43).Bacterial strain is overnight incubation in the YPE substratum, transform by plasmid DNA as mentioned above subsequently, and be applied to SCE-His-Ura nutrient agar (6.7g/L YNB, 1850mg/L auxotrophy mixture-His-Ura (DSCK162, Formedium), 20mL/l95% ethanol).Bacterium colony is applied on the SCE-His-Ura nutrient agar again, and called after PNY934 and PNY935.

example 11

produce isopropylcarbinol and lipid acid isobutyl ester by allos lipase Yuan Da

Bacterial strain PNY934 and PNY935 are applied on SC-His-Ura DE nutrient agar (6.7g/L YNB, 1850mg/L auxotrophy mixture-His-Ura, 3g/L glucose, 3mL/195% ethanol); These cells are used from the preculture thing of inoculation 3mL SC-His-Ura DE substratum together with the cell (it produces isopropylcarbinol, still not secretion heterogenous lipase) of control strain PNY2242.These growths～6 hour.2mL is for being seeded in the 50mL same medium of the 250mL flask of being furnished with the ventilation top cover; These grow overnight are to optical density(OD) (OD ₆₀₀)～1.Morning, add glucose, yeast extract and peptone, makes their concentration be respectively 35,10 and 20g/L, and final volume is 75mL.By in this culture evenly distribute to three identical 125mL shaking flask (top pressure closure comprises stirring rod), add subsequently 10.3mL corn oil fatty acid (COFA) and flask is hatched under 30 ℃ and 250rpm condition.In 0 hour (0.6mL, before adding COFA) and 24 hours and 72 hours (each 5mL, after mixing water and COFA phase fully) sampling.As mentioned before, by HPLC and GC analytic sample.Added 1.5mL500g/L glucose in the time of 24 hours.

The isopropylcarbinol produced in these fermentations is distributing between 3 level parts: the free isopropylcarbinol at water and COFA in mutually, and lipid acid isobutyl ester (FABE) level part of producing of the esterification by isopropylcarbinol and lipid acid.Shown in table 16, the bacterial strain of secretion lipase produces considerable FABE, and control strain only produces a small amount of FABE, the FABE that its bacterial strain that only accounts for secretion lipase produces～12%.Lipase-catalyzed isopropylcarbinol esterification becomes FABE to cause water isopropylcarbinol concentration to reduce, and by isopropylcarbinol total amount per-cent in system, is about 10%, is 53% to 43% in the time of 24 hours, and is 45% to～31% in the time of 72 hours.

yuan 16: at water and the isopropylcarbinol in organic phase (iBuOH) of indicator strain diastatochromogenes the amount of recording with lipid acid isobutyl ester (FABE).Show three same flask average ± standard is inclined to one side poor.According to the volumetric wear caused due to sampling, amount is revised.

example 12

Produce isopropylcarbinol and lipid acid isobutyl ester-more glycosylated and nonglycosylated lipase by the secretion of allos lipase

Repeat the experiment of example above, comprise bacterial strain PNY936, the N55A mutant of its secretion Tlan lipase.In this experiment, added glucose (glucose of 1.5mL500g/L) for twice after 24 hours and 48 hours.

The bacterial strain of secretion lipase produces more FABE (many～5-6 doubly in this case) than the contrast bacterial strain.As the result of FABE formation, the ratio of the total isopropylcarbinol in moisture level part reduces, reduction～10% and reduction～15% in the time of 72 hours in the time of 24 hours.Secrete therein significantly more FABE level part of culture ratio contrast deposits yields of the product isopropylcarbinol bacteria growing of lipase.The FABE amount produced in the fermentation with PNY936 bacterial strain (secretion glycosylation-variant lipase) significantly is not different from the FABE amount by the bacterial strain generation of secretion wild-type lipase.

table 17: at water and the isopropylcarbinol in organic phase (iBuOH) of indicator strain diastatochromogenes the amount of recording with lipid acid isobutyl ester (FABE).Show three same flask average ± standard is inclined to one side poor.According to the volumetric wear caused due to sampling, amount is revised.

example 13

the through engineering approaches isopropylcarbinol is produced yeast to express lopsided candiyeast (C.deformans) and South Pole vacation silk yeast (C.antarctica) lipase

Coding is increased from plasmid pNAK10 (SEQ ID NO:45), pNAK31 (SEQ ID NO:238) and pTVAN8 (SEQ ID NO:240) from LIP1 and the CalB lipase transgenosis of the wild-type lipase of lopsided candiyeast (C.deformans) and antarctic candida (C.antarctica) respectively, oligonucleotide AK11-24 (SEQ ID NO:132) and AK11-25 (SEQ ID NO:133) are used in this amplification, and they comprise the AscI site at their 5 ' end.The PCR product digests and is connected to the pNAK36 (SEQ ID NO:223) of AscI digestion with AscI.To connect mixture is transformed in competence bacillus coli DH 5 alpha (Invitrogen) and is applied on LB-penbritin agar plate.Dull and stereotyped bacterium colony grow overnight in the LB-penbritin from this, and use Qiaprep Spin Miniprep test kit isolated plasmid dna.Recombinant plasmid digests by AscI and agarose gel electrophoresis is identified.Plasmid pNAK38 (SEQ ID NO:224) is included in the CalB lipase under the control of TEF1 (M6) promotor, pNAK37 (SEQ ID NO:225) is included in the LIP1 lipase under the control of TEF1 (M4) promotor, and pNAK39 (SEQ ID NO:226) is included in the LIP1 lipase under the control of TEF1 (M6) promotor.

Amplification lipase transgenosis and the flanking DNA (and it comprises that the URA3 gene is as selected marker) that makes them be oriented in the gpd2 Δ and integrate from these plasmids, the amplification the primer is oBP691 (SEQ ID NO:136) and oBP696 (SEQ ID NO:137).Use QIAQuick PCRPurification kit purifying concentrated PCR product.Yeast strain PNY1556 under 30 ℃ and 250rpm condition, grow overnight in YPE substratum (10g/L yeast extract, 20g/L peptone, 20mL/l 95% ethanol), and, with the conversion of PCR product, being applied to subsequently on the SCE-Ura nutrient agar.By Ura ⁺bacterium colony is applied on the new system substratum, is applied to subsequently on the FOA substratum to select to have lost the isolate of selected marker again.

Check the whether correct integration transgenosis of anti-FOA transformant and check by bacterium colony PCR whether selective marker loses, this bacterium colony PCR is used the primer pair of following each flank of integration box: genomic dna is used PureGene test kit (Qiagen), basically as described in manufacturers, carries out purifying.This is as the template of PCR reaction, and oligomer HY48 (SEQ ID NO:227) and HY49 (SEQ ID NO:228) are used in this reaction.Positive intasome is applied on the FOA substratum, and reclaims the isolate of anti-FOA.The isolate of from gpd2 Δ locus, having lost the URA3 mark is identified by PCR, uses as mentioned above oligomer HY48 and HY49.

Lipase is integrated yeast strain and control strain, PNY1556, transformed to import the isopropylcarbinol pathways metabolism with plasmid pBP2092 (SEQID NO:237), as described below: bacterial strain is overnight incubation in the YPE substratum, transform by plasmid DNA as mentioned above subsequently, and be applied on the SCE-His nutrient agar.Bacterium colony is applied on the SCE-Ura nutrient agar again, and called after PNY1022 (TEF1 (M4)-LIP1), PNY1023 (TEF1 (M6)-LIP1) and PNY1024 (TEF1 (M4)-CalB).

example 14

by heterogenous expression deformity candiyeast (C.deformans) and South Pole vacation in producing the isopropylcarbinol bacterium silk yeast (C.antarct ica) lipase is produced isopropylcarbinol and lipid acid isobutyl ester

Bacterial strain PNY1022, PNY1023 and PNY1024 are applied on SC-Ura DE nutrient agar again; These cells are for inoculating the preculture thing of 3mL SC-His-Ura DE substratum, and its is grown～6 hours.2ml is for being seeded in the 50mL same medium of the 250mL flask of being furnished with the ventilation top cover; These grow overnight are to optical density(OD) (OD ₆₀₀)～1.Morning, add glucose, yeast extract and peptone, makes their concentration be respectively 35,10 and 20g/l, and final volume is 75mL.By in this culture evenly distribute to three identical 125mL shaking flask (top pressure closure comprises stirring rod), add 10.3mL corn oil fatty acid (COFA) and flask is hatched under 30 ℃ and 250rpm condition.0 hour (before adding COFA), and 24 hours, 48 hours and 94 hours (after mixing water and COFA phase fully) sampling.By HPLC and GC analytic sample.At 24 hours, add 1.5mL500g/L glucose (in 1.2mL to PNY1556 culture); At 48 hours, 1.6mL glucose is added in each flask.

The isopropylcarbinol produced in these fermentations distributes between mutually 3: the free isopropylcarbinol at water and COFA in mutually, and the lipid acid isobutyl ester (FABE) of the esterification generation by isopropylcarbinol and lipid acid.Shown in table 18, express the bacterial strain of lopsided candiyeast (C.deformans) lipase and produced considerable FABE at 24 and 94 hours.PNY1023 have drive lopsided candiyeast (C.deformans) lipase transgene expression than strong promoter, the FABE that it prepares is approximately the twice of FABE prepared by PNY1022.Interestingly, produce significantly more total isopropylcarbinol to 94 hours PNY1023 than other bacterial strain.

The bacterial strain of expressing CalB lipase produces than the FABE of the bacterial strain much less of expressing lopsided candiyeast (C.deformans) enzyme, but in its flask, has than the remarkable more FABE of control fermentation.Control strain (fat-free enzyme transgenosis) only became FABE by the esterification of 10mg isopropylcarbinol by 94 hours, and this may be due to endogenic lipase activity.The fermentation of being undertaken by the product isopropylcarbinol bacterium of expressing lipase all shows than the remarkable lower water isopropylcarbinol concentration of control fermentation.In the situation that ferment with lopsided candiyeast (C.deformans) expression strain (PNY1022 and PNY1023), this significantly gathers corresponding to FABE's; The bacterial strain of expressing antarctic candida (C.antarctica) gathers the ester of much less.For lopsided candiyeast (C.deformans) expression strain, when using weak promoter to express lipase gene, total isopropylcarbinol output is equivalent to fat-free enzyme contrast; When using strong promoter, total isopropylcarbinol yield increased is high by 24%.

table 18: at water and the isopropylcarbinol in organic phase (iBuOH) of indicator strain diastatochromogenes the amount of recording with lipid acid isobutyl ester (FABE).

example 15

express Tabin aspergillus (Aspergillus tubingensis) LIP3 lipase in yeast

The DNA (DNA2.0) of composite coding Tabin aspergillus (Aspergillus tubingensis) LIP3 lipase, this synthetic use through optimizing the codon for expressing at Saccharomyces cerevisiae (S.cerevisiae).Use primer Atublip1 and AtubLip2 (being respectively SEQ ID NO:229 and 230), this DNA increases to utilize Phusion archaeal dna polymerase (New England Biolabs).The PCR product is transformed in yeast strain PNY1500 together with breach plasmid pNAK33 (TEF1 (M2) promotor), pNAK34 (TEF1 (M4) promotor) and pNAK35 (TEF1 (M6) promotor) (being respectively SEQID NO:231,232 and 233).Conversion reaction is applied to the SCD-His substratum; Show the positive bacterium colony of test and express lipase activity on the tributyrin flat board.Save plasmid and be transformed in bacillus coli DH 5 alpha and carry out purifying from these isolates (Yeast Plasmid Miniprep Kit, Zymo Research).Sequential analysis illustrates the genetically modified expectation nucleotide sequence of Tabin aspergillus (A.tubingensis) lipase.They are respectively for TEF1 (M2), TEF1 (M4) and TEF1 (M6) promoter variants ((being respectively SEQ ID NO:234,235 and 236)) difference called after pTVAN9, pTVAN4 and pTVAN10.

Express grow overnight in the 50mL SCD-His substratum that 250mL that bacterial strain PNY1055 (pTVAN9), the PNY1056 (pTVAN4) of lipase and PNY1057 (pTVAN10) and wild-type control strain (PNY827) hatch under 30 ℃ and 250rpm condition ventilates in the top cover flask.Morning, transfer to the 22mL culture in 125mL flask (top pressure closure), adds 1.75mL glucose (500g/L), 2.5mL10X YEP (100g/L yeast extract, 200g/L peptone), and 0.313mL isopropylcarbinol.Take out sample, add subsequently 10.3mL COFA and aseptic stirring rod, send flask back to and hatched.Take out sample (5mL) and carry out HPLC and GC analysis after 24 hours, and add 1.75mL glucose and 0.313mL isopropylcarbinol.Took out the second sample after 96 hours.Analytic sample as mentioned above.The bacterial strain of expressing lipase can become FABE by the isopropylcarbinol esterification; At 96 hours, the FABE amount formed by these bacterial strains was measured many ten times than the FABE formed by control strain.As implied above, the bacterial strain of the promotor of transcribing by the driving lipase with medium tenacity forms the FABE of maximum.

table 19: at water and the isopropylcarbinol in organic phase (iBuOH) of indicator strain diastatochromogenes the amount of recording with lipid acid isobutyl ester (FABE).

example 16

the glycosylated gene of the lipase of expressing in yeast is removed

The glycosylated conserved site of N-glycosylation sequences coupling asparaginyl, N-X-S/T (Drickamer K and Taylor ME (2006) Introduction to Glycobiology (the 2nd edition) Oxford University Press, USA), they identify in LIP1 and CalB.LIP1 has two glycosylation sites (at the NIS of residue 146 with at the NNT of residue 167), and CalB has a glycosylation site (at the NDT of residue 99).As described below by changing these with the site-directed mutagenesis (and at LIP1, forming double-mutant) of A replacement N in all cases.

Use QuikChange Site-Directed Mutagenesis test kit (Strategene, La Jolla CA), according to the specification sheets of manufacturers, carry out mutagenesis, combine following plasmid and primer:

Using the heat-stabilised poly synthase provided together with test kit, after utilizing mutagenic primer amplification plasmid main chain, with DpnI restriction endonuclease DNA digestion.In intestinal bacteria XL1-Blue competent cell, and use Qiaprep Spin Miniprep Kit (Qiagen) to reclaim the Plasmid Transformation after processing.Identify the clone of sudden change by the DNA sequence analysis to the mutagenesis plasmid.Plasmid is called after pTVAN20, pTVAN25, pTVAN26 and pTVAN27 respectively.Plasmid (with the control plasmid with wild-type lipase gene) is transformed in the PNY1500 yeast strain.

Claims

1. method comprises:

A) provide fermention medium, described fermention medium comprises the fermented carbon substrate that derives from biomass material, the alcohol that originates from the fermented carbon substrate that derives from biomass material and alcohol and produces yeast microorganism, wherein said alcohol is produced microorganism and is comprised the through engineering approaches polynucleotide that coding has the polypeptide of lipase activity, and described microbial expression is also showed or secretes described polypeptide, making in described fermention medium and have described lipase activity;

B) described fermention medium is contacted with carboxylic acid;

During wherein said lipase activity is present in described fermention medium with the q.s that will be changed into by least a portion of the alcohol of described microorganisms alcohol ester in extracellular.

2. method according to claim 1, also comprise and make described fermention medium contact to form the two-phase mixture that comprises water and organic phase with extraction agent.

3. method according to claim 2, wherein said extraction agent comprises described carboxylic acid.

4. according to method in any one of the preceding claims wherein, wherein said product alcohol is C ₂-C ₈alkyl alcohol.

5. according to method in any one of the preceding claims wherein, wherein said product alcohol is ethanol.

6. method according to claim 5, wherein said alcohol ester comprises fatty-acid ethyl ester.

7. according to the described method of any one in claim 1 to 4, wherein said product alcohol is butanols.

8. method according to claim 7, wherein said alcohol ester comprises fatty acid butyl ester.

9. method according to claim 8, wherein said alcohol ester also comprises fatty-acid ethyl ester.

10. according to method in any one of the preceding claims wherein, the wherein said polypeptide with lipase activity is showed on the surface of described yeast microorganism.

11. method according to claim 10, the wherein said polypeptide with lipase activity comprise with SEQ ID NO:249,250,251,252,253 or their fragment in any one there is the sequence at least about 70% identity.

12. according to method in any one of the preceding claims wherein, the polynucleotide that wherein said coding has a polypeptide of lipase activity comprise with there is SEQ ID NO:1,3,5,7,8,9,46,48,50,52,54,255,271 or 273 polynucleotide have the sequence at least about 70% identity.

13. according to method in any one of the preceding claims wherein, the wherein said polypeptide with lipase activity comprise with have SEQ ID NO:2,4,6,256,47,49,51,53,55,241,242,243,244,245,246,247,248,272 or 274 or their polypeptide of active fragments there is the sequence at least about 70% identity.

14. method according to claim 13, the wherein said polypeptide with lipase activity does not comprise the glycosylation motif.

15. according to method in any one of the preceding claims wherein, the wherein said polypeptide with lipase activity be do not have glycosylated.

16., according to method in any one of the preceding claims wherein, wherein said carboxylic acid comprises free fatty acids, described free fatty acids derives from Semen Maydis oil, Tower rape oil, plam oil, linseed oil, curcas oil or soybean oil.

17., according to method in any one of the preceding claims wherein, wherein said carboxylic acid derives from the biomass material identical with the described carbon substrate that ferments.

18., according to method in any one of the preceding claims wherein, wherein said carboxylic acid comprises having C ₁₂-C ₂₂the carboxylic acid of the aliphatic chain of straight chain or branching.

19., according to the described method of any one in claim 2-18, wherein saidly occur with contacting simultaneously of carboxylic acid with contacting with described of extraction agent.

20., according to method in any one of the preceding claims wherein, wherein by the alcohol of described microorganisms, at least about 60% effective titre, be converted to alcohol ester.

21. according to method in any one of the preceding claims wherein, wherein said fermention medium also comprises triglyceride level, triglyceride, monoglyceride and phosphatide or their combination, and wherein said lipase activity makes at least a portion of described triglyceride level, triglyceride, monoglyceride and phosphatide or their combination be hydrolyzed to form free fatty acids.

22. according to method in any one of the preceding claims wherein, effective titre of the wherein said alcohol during fermentation produced is greater than effective titre of being produced the alcohol that microorganism during fermentation produces by the alcohol of polynucleotide that does not comprise coding and have the polypeptide of lipase activity, and described microbial expression is also secreted or shows described polypeptide, makes in described fermention medium and has described lipase activity.

23. according to method in any one of the preceding claims wherein, the effective speed of the wherein said alcohol during fermentation produced is greater than by the alcohol of polynucleotide that does not comprise coding and have the polypeptide of lipase activity and produces the microorganism speed that during fermentation alcohol produces, and described microbial expression is also secreted or shows described polypeptide, makes in described fermention medium and has described lipase activity.

24. recombinant host cell comprises:

(a) the pure production ways of through engineering approaches; With

(b) polynucleotide of through engineering approaches, its coding has the polypeptide of lipase activity.

25. recombinant host cell according to claim 24, the wherein said polypeptide with lipase activity comprise with SEQ ID NO:2,4,6,256,47,49,51,53,55,241,242,243,244,245,246,247,248,272 or 274 or their active fragments there is the sequence at least about 70% identity.

26. according to the described recombinant host cell of claim 24 or 25, the wherein said polypeptide with lipase activity comprise with SEQ ID NO:249,250,251,252,253 or their fragment in any one there is the sequence at least about 70% identity.

27. recombinant host cell according to claim 26, the wherein said polypeptide with lipase activity does not comprise the glycosylation motif.

28. according to the described recombinant host cell of any one in claim 24-27, the wherein said polypeptide with lipase activity be do not have glycosylated.

29. recombinant host cell according to claim 24, the through engineering approaches polynucleotide that wherein said coding has the polypeptide of lipase activity comprise and SEQ ID NO:1,3,5,7,8,9,46,48,50,52,54,255,271 or 273 sequences that have at least about 70% identity.

30. recombinant host cell comprises:

(a) pure production ways; With

(b) polynucleotide of through engineering approaches, its coding has the polypeptide of lipase activity, the wherein said polypeptide with lipase activity comprise with SEQ ID NO:2,4,6,256,47,49,51,53,55,241,242,243,244,245,246,247,248,272 or 274 or their active fragments there is the sequence at least about 70% identity.

31. recombinant host cell according to claim 30, the wherein said polypeptide with lipase activity also comprise with SEQ ID NO:249,250,251,252,253 or their fragment in any one there is the sequence at least about 70% identity.

32., according to the described recombinant host cell of any one in claim 24-31, wherein said pure production ways is the production of butanol approach.

33. recombinant host cell according to claim 32, wherein said production of butanol approach is the isopropylcarbinol production ways.

34., according to the described recombinant host cell of any one in claim 24-33, wherein said host cell also comprises pyruvic carboxylase activity minimizing or that eliminate.

Produce the method for microorganism to produced pure tolerance 35. improve alcohol, described method comprises:

(a) the through engineering approaches microorganism is to express and secrete or show the polypeptide with lipase activity;

(b), therein under the condition of described microorganisms alcohol, the microorganism of described through engineering approaches is contacted with following:

I. triglyceride level, triglyceride, monoglyceride, phosphatide, free fatty acids or their mixture; And

Ii. carbon substrate.

36. method according to claim 35, wherein make the microorganism of described through engineering approaches contact with triglyceride level, triglyceride, monoglyceride and phosphatide or their combination, and lipase wherein said secretion or that show change into free fatty acids by least a portion of described triglyceride level, triglyceride, monoglyceride and phosphatide or their combination.

37. according to the described method of claim 35 or 36, the formation of wherein said lipase-catalyzed alcohol ester.

38. method according to claim 35, effective titre of the alcohol of wherein said microorganisms be greater than by not yet through through engineering approaches to express and to secrete effective titre of alcohol of the microorganisms of the polypeptide with lipase activity.

39., according to the described method of any one in claim 35-38, wherein said pure biosynthetic pathway is the pure biosynthetic pathway of through engineering approaches.

40., according to the described method of claim 39, the pure biosynthetic pathway of wherein said through engineering approaches is the isopropylcarbinol biosynthetic pathway.

41., according to the described method of claim 40, wherein said isopropylcarbinol biosynthetic pathway comprises the conversion of following substrate to product:

(a) pyruvic acid is to acetylactis;

(b) acetylactis to 2,3-dihydroxyl isovaleric acid;

(c) 2,3-dihydroxyl isovaleric acid is to the 2-ketoisovaleric acid;

(d) the 2-ketoisovaleric acid is to isobutyric aldehyde; And

(e) isobutyric aldehyde is to isopropylcarbinol.

42. during fermentation produce the method for butyl ester, comprising:

(a) provide fermention medium, described fermention medium comprises carbon substrate and triglyceride level, triglyceride, monoglyceride and phosphatide or their mixture; And

(b) making described fermention medium and the alcohol that comprises the butanols biosynthetic pathway produce microorganism contacts, wherein said microorganism also comprises the through engineering approaches polynucleotide that coding has the polypeptide of lipase activity, and described microbial expression is also secreted or shows described polypeptide, makes in described fermention medium and has described lipase activity.

43., according to the described method of claim 42, wherein said fermention medium also comprises one or more carboxylic acids.

44., according to the described method of claim 42, wherein said carbon substrate derives from biomass.

45., according to the described method of claim 42, wherein said biomass are corn or sugarcane.

46., according to the described method of claim 42, wherein said carbon substrate and described triglyceride level, triglyceride, monoglyceride and phosphatide all derive from described identical biomass.

47. fermention medium, comprise alcohol and produce microorganism, butyl ester and butanols, described alcohol is produced microorganism and is comprised the butanols biosynthetic pathway and comprise the through engineering approaches polynucleotide that coding has the polypeptide of lipase activity, and described polypeptide is expressed and secretes or show.

48. animal food prods, the microorganism that comprises any one in claim 24-34.