CN101652482A - The production of butanol of being undertaken by the metabolic engineering yeast - Google Patents

Abstract

The method that discloses the metabolic engineering yeast and produced propyl carbinol.In one embodiment, the metabolic engineering yeast can the metabolism carbon source to generate propyl carbinol, at least a approach generates the kytoplasm acetyl-CoA that increases with respect to the kytoplasm acetyl that wild-type yeast generated-CoA, and at least a heterologous gene is encoded and expression can utilize NADH acetyl-CoA to be changed at least a enzyme of the pathways metabolism of propyl carbinol.In another embodiment, the method of producing propyl carbinol comprises that (a) provides following metabolic engineering yeast, its can the metabolism carbon source to generate propyl carbinol, at least a approach generates the kytoplasm acetyl-CoA that increases with respect to the kytoplasm acetyl that wild-type yeast generated-CoA, and at least a heterologous gene is encoded and expression can utilize NADH acetyl-CoA to be changed at least a enzyme of the pathways metabolism of propyl carbinol; And (b) cultivate this yeast to generate propyl carbinol.Other embodiment is also disclosed.

Description

The production of butanol of being undertaken by the metabolic engineering yeast

Cause the relevant personnel:

State as followsly, we (hereinafter having listed name, address and nationality) invented the invention of describing in the following specification sheets, be entitled as " production of butanol of being undertaken by the metabolic engineering yeast ".

Uvini?Gunawardena

Address: Pasadena, California

Nationality: Sri Lanka

Peter?Meinhold

Address: Pasadena, California

Nationality: Germany

Matthew?W.Peters

Address: Pasadena, California

Nationality: the U.S.

Jun?Urano

Address: CALVER city, California

Nationality: the U.S.

Reid?M.Renny?Feldman

Address: Los Angeles, California

Nationality: the U.S.

The U.S. Provisional Patent Application serial number 60/871 that people such as the application's requirement (1) Jun Urano submitted on December 21st, 2006,427, " production of butanol of being undertaken by the metabolic engineering yeast " (BUTANOLPRODUCTION BY METABOLICALLY ENGINEERED YEAST); (2) the U.S. Provisional Patent Application serial number 60/888 submitted on February 2nd, 2007 of people such as JunUrano, 016, " the propyl carbinol production of being undertaken by the metabolic engineering yeast " (N-BUTANOL PRODUCTION BYMETABOLICALLY ENGINEERED YEAST); (3) the U.S. Provisional Patent Application serial number 60/928 submitted on May 8th, 2007 of people such as Uvini P.Gunawardena, 283, the rights and interests of " production of butanol of being undertaken by the metabolic engineering yeast " (BUTANOL PRODUCTION BY METABOLICALLYENGINEERED YEAST).By addressing this paper is taken in above-mentioned each piece application at this.

Invention field

The present invention relates to the yeast cell of metabolic engineering, be used for producing propyl carbinol with high yield, as an alternative and reproducible transport fuel, and be used for other application.But yeast of the present invention is comprised and will change into the pathways metabolism of propyl carbinol such as the carbon source of glucose and/or other metabolize carbohydrates and biomass or the like by engineered one-tenth.

Background of invention

Current, the about 1,400 hundred million gallons of gasoline of the annual consumption of the U.S., and the about 3,400 hundred million gallons of gasoline of the annual consumption in the whole world.These consumption quantity are also in increase.2005 EPACT (Energy PolicyAct of 2005) stipulated will in gasoline, use 7,500,000,000 gallons of recyclable fuels in 2012.In his State of the Union Message in 2007 (2007 State of the Union address), president's requirement improves recyclable fuel standard (renewable fuel standard) size (RFS) and enlarges the scope of recyclable fuel standard, requires in 35,000,000,000 gallons of reproducible alternative fuel of use in 2017.Ministry of Energy has set the year two thousand thirty and has replaced 30% target (i.e. " 30X30 " motion) of the current petrol consumption of the U.S. with biofuel.In May, 2007, the Brazil and the U.S. have signed " ethanol agreement " (the Ethanol Agreement), to promote the exploitation of America biofuel, unite biofuel manufacturer the biggest in the world-current 70% of world's alcohol production that accounts for.

Biofuel not only has the potentiality of the dependency (this is vital for Homeland Security) that reduces the oil input of United States Foreign Trade Zones Board state, and has the potentiality of the remarkable reduction greenhouse gas emission relevant with Global warming.Obtain biofuel from conversion energy based on the raw material of carbon.It is reproducible that agricultural raw material is considered to, and reason is that though their release of carbon dioxide when burning, they catch the carbonic acid gas of almost equal amount by photosynthesis.

In the U.S., as the oxidation additive of normal benzene, as the substitute of methyl tertiary butyl ether (MTBE), a kind of chemical in back is difficult to regain in underground water and soil pollution ethanol day by day.At 10% mixture, by boosting of octane rating, ethanol reduces the possibility of engine knock (engine knock).The use of 10% ethanol petrol is enforceable in some city, and the possibility of the auto exhaust of harmful level is possible in these cities, in the some months especially in the winter time.The North America vehicle need not repacking and just can move with 10% ethanol/90% gasoline (being E10) from about 1980.

Yet, in order to use ethanol with greater concn, must be ad hoc engineered or the engine and the fuel system of repacking vehicle.Designed variable fuel vehicle (flexible fuel vehicle) (FFV), with gasoline or with the operation of the mixture of height to 85% ethanol (E85).Yet, because the energy that one gallon of ethanol contains lacks than one gallon of gasoline, so the FFV mileage that common per gallon obtains when acting as a fuel with E85 is few greater than 20-30%.Can obtain to transform bag, be used for conventional vehicles is changed into FFV, it generally includes electronic installation and avoids corroding with the protection engine with the volume of fuel (because the alcoholic acid intrinsic energy is lower) that improves each circulation and injected and the chemical treatment of some situation.Current American has and surpasses 4,000,000 variable fuel vehicles and on the way travels, though 2002 discover that E85 is less than 1% in the fuel that these vehicles consume.

Butanols acts as a fuel and has several better than the alcoholic acid advantage.Though it can prepare from the raw material identical with ethanol, different with ethanol, it is compatible at arbitrary proportion with diesel oil with gasoline.Butanols can also need not repacking as pure fuel in existing automobile, and the Richard Branson jazz group of Virgin airline has proposed butanols as jet fuel.Different with ethanol, butanols does not absorb water, and so can store and dispensing in existing petrochemical industry Infrastructure.Because its higher intrinsic energy, fuel economy (per gallon mileage) is better than ethanol.Also have, butanols-gasoline mixture has the vapour pressure lower than alcohol-gasoline mixture, and this is important in reducing the steam hydrocarbon emission.These characteristics provide potentiality to use with the identical mode of gasoline for butanols, and need not to reequip the do not have to burden of more frequent postcombustion of vehicle and human consumer.

Fusobacterium (Clostridum) bacterial strain of natural generation propyl carbinol can generate propyl carbinol via leading to the approach of propyl carbinol from butyryl-CoA in use.A shortcoming of fusobacterium bacterial strain is that the process that propyl carbinol generates with two steps takes place, and it involves, and acid generates vegetative period and solvent afterwards generates the phase.Also have, generate a large amount of by products in this process, such as hydrogen, ethanol and acetone, so the stoichiometry productive rate with propyl carbinol is restricted to the glucose that the every mol of about 0.6mol propyl carbinol consumes.In addition, the fusobacterium bacterial strain has been lost them generated the ability (Cornillot etc., J.Bacteriol.179:5442-5447,1997) of solvent under the cultured continuously condition.Show the clostridium approach among Fig. 1, shown that conversion of glucose becomes acid and solvent in the clostridium acetobutylicum (Clostridum acetobutylicum), comprised the approach that generates propyl carbinol from acetyl-CoA.

Summary of the invention

In one embodiment, following metabolic engineering yeast is provided, its can the metabolism carbon source to generate propyl carbinol, at least a approach is configured to generate the kytoplasm acetyl that another kytoplasm acetyl-the CoA amount increases-CoA amount that generates with respect to wild-type yeast, and at least a heterologous gene is encoded and expression can utilize NADH acetyl-CoA to be changed at least a enzyme of the pathways metabolism of propyl carbinol.

In another embodiment, the method of producing propyl carbinol is provided, this method comprises that (a) provides following metabolic engineering yeast, its can the metabolism carbon source to generate propyl carbinol, at least a approach is configured to generate the kytoplasm acetyl that another kytoplasm acetyl-the CoA amount increases-CoA amount that generates with respect to wild-type yeast, and at least a heterologous gene is encoded and expression can utilize NADH acetyl-CoA to be changed at least a enzyme of the pathways metabolism of propyl carbinol; And (b) cultivate this metabolic engineering yeast, the time of cultivation and condition are in order to generate propyl carbinol.

In another embodiment, provide and used yeast to produce the method for propyl carbinol, this method comprises that (a) metabolic engineering yeast generates to improve kytoplasm acetyl-CoA; (b) the metabolic engineering yeast to be to express the pathways metabolism that carbon source is changed into propyl carbinol, wherein this approach need at least a for this yeast non-natural enzyme, wherein step (a) and (b) can be with arbitrary order enforcement; And (c) cultivate this yeast, the time of cultivation and condition are in order to generate the propyl carbinol of recyclable amount.

In also having an embodiment, provide and used yeast to produce the method for propyl carbinol, this method comprises that (a) cultivates the metabolic engineering yeast, the time of cultivation and condition are in order to generate the yeast cell biomass but do not activate propyl carbinol and generate; And (b) at another section period change culture condition, the time of cultivation and condition are in order to generate the propyl carbinol of recyclable amount.

In another embodiment, provide following metabolic engineering yeast, it can the metabolism carbon source also generate acetyl-CoA amount of measuring increase with respect to acetyl-CoA that wild-type yeast generated.

In another embodiment, the method that improves the zymic metabolic activity is provided, this method comprises that yeast generates the kytoplasm acetyl that another kytoplasm acetyl-the CoA amount increases-CoA amount with respect to wild-type yeast generated.

In also having an embodiment, following metabolic engineering yeast is provided, it has at least a approach and is configured to generate the kytoplasm acetyl that another kytoplasm acetyl-the CoA amount increases-CoA amount that generates with respect to wild-type yeast.

Other embodiment is also disclosed.

The accompanying drawing summary

Illustrate exemplary embodiment of the present invention in the accompanying drawing, wherein:

Fig. 1 illustrates glucose in the clostridium acetobutylicum, pentose and starch small grain (granulose) and changes into the pathways metabolism that is involved in acid and the solvent.Hexose (for example glucose) and pentose are converted to pyruvic acid, ATP and NADH.Subsequently, pyruvic acid is become acetyl-CoA by pyruvic acid-ferredoxin oxide-reductase oxidative decarboxylation.The reducing equivalent quilt that is generated in this step only ferruginous hydrogenase (iron-onlyhydrogenase) changes into hydrogen.Acetyl-CoA is the tapping point intermediate product, leads to the generation of organic acid (acetate and butyric acid) and solvent (acetone, butanols and ethanol).

Fig. 2 illustrates the chemistry route that generates butanols in yeast.

Fig. 3 illustrates yeast saccharomyces cerevisiae (Sacchromyces cerevisiae) and generates the employed approach of acetyl-CoA.

Figure 4 and 5 illustrate the various exemplary plasmid that can be used for expressing according to present disclosure various enzymes.

Fig. 4 illustrates and can be used for as expressing the exemplary plasmid of various enzymes according to present disclosure described in the table 1.

Fig. 5 illustrates and can be used for as expressing the exemplary plasmid of various enzymes according to present disclosure described in the table 2.

Fig. 6 illustrates Gevo 1099 and Gevo 1103 propyl carbinol in time generates, and compares with the contrast conivium that only contains carrier, Gevo 1110 and Gevo 1111, and is as follows:

Gevo?1099；

Gevo?1103；

Gevo 1110; With

Gevo?1111。

Fig. 7 illustrates the bcd, the etfb that contain from clostridium acetobutylicum and the pGV1090 plasmid of etfa gene, and described gene is inserted in the phage LacO-1 promotor (P of EcoR1 and BamHI site and improvement _L-lac) the downstream.This plasmid also carries replication orgin gene and the chloramphenicol resistance gene of pBR322.

Fig. 8 illustrates the pGV1095 plasmid that is used to express from the butyraldehyde desaturase (bdhB) of clostridium acetobutylicum, and this butyraldehyde desaturase is inserted in EcoRI and BamHI site and improvement phagemid λ LacO-1 promotor (P _L-lac) the downstream.This plasmid also carries CoIEI replication orgin gene and chloramphenicol resistance gene.

Fig. 9 illustrates the pGV1094 plasmid of the enoyl-CoA hydratase (crt) that is used to express from clostridium acetobutylicum, and this enoyl-CoA hydratase is inserted in EcoRI and BamHI site and improvement phage LacO-1 promotor (P _L-lac) the downstream.This plasmid also carries ori gene and chloramphenicol resistance gene.

Figure 10 illustrates the pGV1037 plasmid that is used to express the hydroxyl butyryl-CoA desaturase (hbd) from clostridium acetobutylicum, and this hydroxyl butyryl-CoA desaturase is inserted in EcoRI and BamHI site and improvement phage LacO-1 promotor (P _L-lac) the downstream.This plasmid also carries ori gene and chloramphenicol resistance gene.

Figure 11 illustrates the pGV1031 plasmid of the thiolase (thl) that is used to express from clostridium acetobutylicum, and this thiolase is inserted in the downstream of EcoRI and BamHI site and LacZ gene.This plasmid also carries replication orgin gene and the ampicillin resistance gene of pBR322.

Figure 12 illustrates the pGV1049 plasmid that is used for expressing from the enoyl-CoA hydratase (crt) of Bai Shi clostridium (Clostridium beijerinckii), and this enoyl-CoA hydratase is inserted in EcoRI and BamHI site and improvement phage LacO-1 promotor (P _L-lac) the downstream.This plasmid also carries ori gene and chloramphenicol resistance gene.

Figure 13 illustrates the pGV1050 plasmid that is used for expressing from Bai Shi clostridial hydroxyl butyryl-CoA desaturase (hbd), and this hydroxyl butyryl-CoA desaturase is inserted in EcoRI and BamHI site and improvement phage LacO-1 promotor (P _L-lac) the downstream.This plasmid also carries ori gene and chloramphenicol resistance gene.

Figure 14 illustrates the pGV1091 plasmid that is used for expressing from Bai Shi clostridial alcoholdehydrogenase (adhA), and this alcoholdehydrogenase is inserted in HindIII and BamHI site and improvement phage LacO-1 promotor (P _L-lac) the downstream.This plasmid also carries chloramphenicol resistance gene.

Figure 15 illustrates the pGV1096 plasmid that is used for expressing from Bai Shi clostridial alcoholdehydrogenase (aldh), and this alcoholdehydrogenase is inserted in EcoRI and BamHI site and improvement phage LacO-1 promotor (P _L-lac) the downstream.This plasmid also carries ori gene and chloramphenicol resistance gene.

Detailed Description Of The Invention

The recombination yeast microorganism that with high yield carbon source is changed into n-butanol by engineered one-tenth has been described. Particularly, described and can the metabolism carbon source have generated the recombination yeast microorganism of n-butanol with 50% the productive rate that surpasses theoretical value at least 5% productive rate of theoretical value and some situation. As used herein, term " productive rate " refers to molar yield. For example, when 1 mole of glucose was converted to 1 mole of n-butanol, productive rate equaled 100%. Particularly, term " productive rate " is defined as the product molal quantity that every mole of carbon source monomer obtains, and can be expressed as percentage. Except as otherwise noted, productive rate is expressed as the percentage of theoretical yield. " theoretical yield " is defined as the stoichiometric regulation according to the metabolic pathway that is used for the generation product, every mole of maximum molal quantity of the product of specifying substrate to generate. For example, to become the theoretical yield that a kind of typical case of n-butanol transforms be 100% to glucose. Therefore, to become the productive rate of n-butanol be 95% can be expressed as 95% or 95% theoretical yield of theoretical yield to glucose.

Microorganism disclosed herein is to use genetic engineering technology by engineered, to provide the enzyme that utilizes heterogenous expression the next microorganism that generates n-butanol with high yield. The butanols productive rate depends on that carbon source becomes the high yield conversion of acetyl-CoA and the high yield conversion that follow-up acetyl-CoA becomes butanols. The present invention relates to the combination of these two aspects, cause generating with high yield the microorganism of n-butanol.

As used herein, term " microorganism " comprises protokaryon and microbial species eucaryon, it is from bacterium and eucaryote category (Domains Bacteria and Eukaryote), and the latter comprises yeast and filamentous fungi, protozoan, algae or high protist. Term " cell " and " microbial cell " can with term " microorganism " Alternate. In a preferred embodiment, microorganism is yeast (for example Saccharomyces cerevisiae/saccharomyces cerevisiae (Saccharomyces cerevisiae) or Kluyveromyces lactis (Kluyveromyce lactis)) or ETEC/Escherichia coli (E.coli).

" yeast " refers to a category of most eukaryotes, is arranged in mycota in phylogenetics, under sac fungus and Basidiomycota (phyla Ascomycota and Basidiomycota). About 1500 primary yeast species have been described so far. Yeast mainly is unicellular microorganism, mainly breeds by asexual budding, although the yeast of having described some many cells yeast and having bred by the binary fragmentation. Most of species are classified as aerobic bacteria, but facultative and yeast anaerobism also are well-known. Relevant with culture propagation physiology, yeast is classified as two groups: the Crabtree positive with the Crabtree feminine gender.

In brief, the Crabtree effect is defined as when under aerobic conditions cultivating because high glucose concentration that (for example the oxygen consumption that has microorganism of 50 gram glucose/L) is suppressed. So, because the existence of glucose, the yeast cells with Crabtree positive phenotypes continues fermentation, no matter the availability of oxygen, and the yeast cells with the negative phenotype of Crabtree does not represent the inhibition to oxygen consumption of glucose mediation. Usually the example that has the yeast cells of Crabtree positive phenotypes includes but not limited to that saccharomyces (Saccharomyces), Zygosaccharomyces belong to (Zygosaccharomyces), have spore torulopsis (Torulaspora) and moral to restrain the yeast cells of saccharomyces (Dekkera). Usually the example that has the yeast cells of the negative phenotype of Crabtree includes but not limited to the yeast cells of Kluyveromyces (Kluyveromyces), pichia (Pichis), Hansenula (Hansenula) and candida (Candida).

In this application after the definition of employed some term, hereinafter illustration some detailed aspect of the present invention and embodiment. Term " carbon source " refers generally to be suitable as substrate or the compound of the carbon source of yeast cell growth. Carbon source can be various forms, includes, but are not limited to polymer such as xylan and pectin, carbohydrate, acid, alcohol, aldehyde, ketone, amino acid, peptide etc. This type of carbon source more specifically comprises, for example, various monose such as glucose and fructose, oligosaccharides such as lactose or sucrose, polysaccharide, cellulosic material, saturated or unsaturated aliphatic acid, butanedioic acid, lactic acid, acetic acid, ethanol, or its mixture and from the not purified mixture of renewable raw materials, such as cheese whey penetrant (cheese whey permeate), corn steep liquor (corn steep liquor), beet molasses (sugar beet molasses) and fructus hordei germinatus.

The carbon source of serving as for the suitable starting material that generates the n-butanol product comprises, but be not limited to, biomass water hydrolysis products, glucose, starch, cellulose, hemicellulose, wood sugar, lignin, dextrose, fructose, galactolipin, corn, liquefaction corn flour, corn steep liquor (byproduct of corn wet milling technique, it contains the nutrients that leaches from corn between soak period), molasses, lignocellulosic and maltose. The photosynthetic organism physical efficiency generates carbon source in addition, as photosynthetic product. In a preferred embodiment, the optional authigenic material hydrolysate of carbon source and glucose. Glucose, dextrorotation sugar and starch can be from sources endogenous or external source.

Should be noted that can be with other carbon source place of glucose that more be easy to get and/or cheap, and this change to host microorganism is comparatively small. For example, in certain embodiments, it may be preferred using other reproducible and economically feasible substrate. These comprise: agricultural wastes, the packaging material based on starch, corn fiber hydrolysate product, soy molasses, fruit processing industry refuse and whey permeate etc.

Five-carbon sugar only can be processed these sugared microorganism strains as carbon source, for example intestinal bacteria B.In some embodiments, can use the carbon source of glycerine (glycerol) (a kind of three carbon carbohydrate) as bio-transformation.The impure glycerine that in other embodiments, can use glycerine (glycerin) or obtain from the tri-glyceride of plant and animal fat and oil by hydrolysis is as carbon source, as long as any impurity can not have a negative impact to host microorganism.

Term " enzyme ", as used herein, refer to any catalysis or promote the material of one or more chemistry or biochemical reaction, it generally includes the enzyme that is made of polypeptide wholly or in part, but can comprise the enzyme that is made of differing molecular (comprising polynucleotide).

Term " polynucleotide " can exchange with term " nucleic acid " in this article and use, the organic polymer that finger is made of two or more monomers (comprising Nucleotide, nucleosides or its analogue), include but not limited to strand or double-stranded, any length thymus nucleic acid (DNA) justice or antisense is arranged and strand or double-stranded when suitable, any length Yeast Nucleic Acid (RNA) justice or antisense is arranged, comprise siRNA.Term " Nucleotide " index kind is connected by ribose or ribodesose and purine or pyrimidine bases and with phosphate group and the compound formed arbitrary, and it is the basic structural unit of nucleic acid.Term " nucleosides " refers to compound (as guanosine or adenosine) combined by purine or pyrimidine bases and ribodesose or ribose and that form, and it especially sees nucleic acid.Term " nucleotide analog " or " nucleoside analog " refer to that respectively wherein one or more independent atoms are by different atoms or by the Nucleotide or the nucleosides of different functional group's replacements.Thereby the term polynucleotide comprise nucleic acid, DNA, RNA, its analogue and the fragment of any length.The polynucleotide of three or more Nucleotide are also referred to as nucleotide oligomer or oligonucleotide.

Term " protein " or " polypeptide " as used herein, refer to the organic polymer that is made of two or more amino acid monomers and/or its analogue.As used herein, term " amino acid " or " amino acid monomer " refer to any natural and/or synthetic amino acid, comprise two kinds of optical isomers of glycine and D or L.Term " amino acid analogue " refers to the amino acid that wherein one or more independent atoms are replaced by different atoms or different functional group.Thereby the term polypeptide comprises the aminoacid polymers of any length, comprise full length protein and peptide with and analogue and fragment.Three or more amino acid whose polypeptide are also referred to as protein oligomer or oligopeptides.

Term " allos " or " external source ", as employed about molecule (particularly enzyme and polynucleotide) herein, the molecule that finger is expressed in organism their origins or beyond their organism of occurring in nature discovery, do not have and concern expression level, it can be lower than, be equal to or higher than the expression level of this molecule in natural microbial.

On the other hand, term " natural " or " endogenous ", as employed about molecule (particularly enzyme and polynucleotide) herein, finger is at molecule their origins or that express in their organism of occurring in nature discovery, do not have and concern expression level, it can be lower than, be equal to or higher than the expression level of this molecule in natural microbial.

In certain embodiments, natural, not engineered microorganism can not change into carbon source propyl carbinol or one or more its metabolic intermediates, because for example, this type of wild-type host lacks needed one or more enzymes in the propyl carbinol generation approach.

In certain embodiments, natural, not engineered microorganism is merely able to less than 0.1% productive rate of theoretical yield the carbon source of trace is changed into propyl carbinol.

For example, microorganism such as intestinal bacteria or yeast belong bacterial classification (Saccharomyces sp.) generally do not have the pathways metabolism that sugar such as conversion of glucose is become propyl carbinol, but the propyl carbinol generation approach of the bacterial strain (for example clostridium) of the propyl carbinol of self-generating in the future is transferred in heterologous host bacterium or eucaryon, such as intestinal bacteria or yeast belong bacterial classification, and use the gained recombinant microorganism to produce propyl carbinol.

Generally speaking microorganism, is suitable as the host, if they have intrinsiccharacteristic, such as solvent resistance, this can allow their metabolism carbon sources in containing the environment of solvent.

Term " host ", " host cell " and " recombinant host cell " can exchange use in this article, not only refer to specific subject cell but also refer to the offspring or the potential offspring of such cell.Because in the follow-up generation,,, but still be included in the scope of this term as used herein so such offspring may not be same with parental cell in fact because some may take place for sudden change or environmental influence modifies.

For producing the useful host of propyl carbinol can be microorganism eucaryon or protokaryon.Yeast cell is preferred host, such as, but be not limited to yeast saccharomyces cerevisiae or Kluyveromyces lactis.In certain embodiments, other suitable yeast host microorganism includes, but are not limited to Pichia (Pichia), the West mould genus of alpine yarrow (Yarrowia), Aspergillus (Aspergillus), genus kluyveromyces (Kluyveromyces), pipe capsule yeast belong (Pachysolen), Rhodotorula (Rhodotorula), zygosaccharomyces belongs to (Zygosaccharomyces), the mould genus of semi-lactosi (Galactomyces), Schizosaccharomyces (Schizosaccharomyces), Penicillium (Penicillium), spore torulopsis (Torulaspora) is arranged, Debaryomyces (Debaryomyces), intend Weir yeast belong (Williopsis), moral gram yeast belong (Dekkera), Kloeckera (Kloeckera), strange yeast belong of plum (Metschnikowia) and mycocandida (Candida) species.

Particularly, recombinant microorganism disclosed herein is by the engineered isodynamic enzyme that can use in propyl carbinol is produced with activation (particularly expressing).Particularly, in certain embodiments, this recombinant microorganism is by the engineered isodynamic enzyme that changes into propyl carbinol with activating catalytic acetyl-CoA.

Term " activation " as employed about biologically active molecules (such as enzyme), refers to improve in the genome of microorganism and/or the protein group any modification of the biologic activity of the biologically active molecules in this microorganism herein.Exemplary activation includes but not limited to cause molecule to become biology from the biology inactive form to be had activity form and has activity form to become the modification that biology more has the conversion of activity form from biology, and the modification that causes biologically active molecules in microorganism, to be expressed, wherein this biologically active molecules was not before expressed.For example, the activation of biologically active molecules can be as the enforcement of getting off, promptly in microorganism, express the natural or allogenic polynucleotide that encoding human is learned bioactive molecule, in microorganism, express the natural or allogenic polynucleotide that encoding human is learned the enzyme that is involved in the route of synthesis of bioactive molecule, in microorganism, express the natural or allogenic molecule of the expression that strengthens biologically active molecules.

If gene or dna sequence dna are not the genomic parts of microorganism as its normal presence form, promptly it is not the genomic part of wild-type microorganisms in natural situation, and then this gene or dna sequence dna are " allogenic " for this microorganism.For example and not add restriction,, think that each DNA is allogenic below the coding for yeast saccharomyces cerevisiae.Escherichia coli protein or enzyme, from the control sequence of the protein of any other microorganism beyond the yeast saccharomyces cerevisiae or enzyme, non-transcribed and translation, and yeast saccharomyces cerevisiae protein or RNA sudden change or that alternate manner is modified, no matter mutant produces by selection, or is gone in the yeast saccharomyces cerevisiae by engineered.In addition, think that the proteinic construct of wild-type yeast saccharomyces cerevisiae of transcribing and/or translating under the control that has at allos regulatory element (inducible promoter, enhanser etc.) also is an allogeneic dna sequence DNA.

When the NADH that is generated during the oxidizing reaction of carbon source equals to be utilized to that acetyl-CoA changed into the NADH of metabolic end product, say that the metabolism of carbon source is " equilibrated ".Have only under these conditions, all NADH are only round-robin.In not having the round-robin situation, the NADH/NAD+ ratio becomes unbalance (promptly raising), and this can guide organism into final death, unless there is the alternate pathways metabolism to keep equilibrated NADH/NAD ⁺Ratio.

In certain embodiments, if microorganism is not used aerobic or anaerobic respiration, the propyl carbinol productive rate is the highest so, because carbon is with the form loss (lost) of carbonic acid gas in these situations.

In certain embodiments, microorganism under anaerobic generates propyl carbinol in fermentable ground, makes carbon not run off with the form of carbonic acid gas.

Term " aerobic respiration " refers to that oxygen wherein is the respiratory pathways that final electron acceptor(EA) and energy generate with the form of ATP molecule usually.Term " aerobic respiration approach " is used interchangeably with word " aerobic metabolism ", " oxidative metabolism " or " cellular respiration " in this article.

On the other hand, term " anaerobic respiration " refers to that oxygen wherein is not the respiratory pathways that final electron acceptor(EA) and energy generate with the form of ATP molecule usually.This comprises that wherein oxygen organic or inorganic molecule (for example nitric acid, fumaric acid, methyl-sulphoxide, sulfocompound such as sulfuric acid and metal oxide) in addition is the respiratory pathways of final electron acceptor(EA).Word " anaerobic respiration approach " is used interchangeably with word " anaerobic metabolism " and " anaerobic respiration " in this article.

" anaerobic respiration " must distinguish with " fermentation ".In " fermentation ", NADH contributes its electronics to the molecule that same pathways metabolism generated by the electronics that carries among the generation NADH.For example, in one of colibacillary fermentable approach, the NADH that generates by glycolysis-gives pyruvic acid with its transfer transport, produces lactic acid.

Can only be metabolism carbon source in the situation of " balance " in fermentation in the microorganism that moves under the fermentable condition.When the NADH that is generated during the oxidizing reaction of carbon source equaled acetyl-CoA and changes into fermentation NADH that end product utilized, saying ferments was " balance ".Have only under these conditions, all NADH are only recirculation.If there is not recirculation, NADH/NAD ⁺Than becoming uneven, this causes organism finally dead, unless there is the alternative pathways metabolism of available to keep balance NADH/NAD ⁺Ratio.When the hydrogen that is generated between oxidation period equaled to be transferred to the hydrogen of fermentation end product, then monologue story-telling with gestures face fermentation (written fermentation) was " balance ".Have only under these conditions, recirculation becomes oxidised form to all NADH with reduced form ferredoxin.Will know importantly whether fermentation is equilibrated, because if be not like this, so whole written reaction is incorrect.

Anaerobic condition is that the Institute of Micro-biology of high productivity generation propyl carbinol is preferred.

Fig. 2 illustrates the approach that according to embodiment of the present invention carbon source is changed into propyl carbinol in the yeast.This approach can be considered and has two differentiated parts, and it comprises that (1) carbon source changes into acetyl-CoA and (2) acetyl-CoA changes into propyl carbinol.Owing to the compartmentation of metabolic reaction in the yeast (with other eukaryote) with in order to ensure generating the acetyl-CoA of capacity to drive the second section of this approach from glucose, the generation of acetyl-CoA is necessary in the cytosol, and therefore raises in disclosed in this article some through engineering approaches variant.

About part (1) is that carbon source changes into butanols, can engineered yeast microorganism to improve the flux that pyruvic acid in the cytosol becomes acetyl-CoA.

As shown in Figure 3, yeast saccharomyces cerevisiae generates acetyl-CoA in plastosome and in cytosol.Because becoming the conversion of propyl carbinol, acetyl-CoA occurs in the cytosol, so the acetyl in the cytosol-CoA generation has raise in the through engineering approaches cell.Optional is to reduce or to contain that the acetyl-CoA in the plastosome generates.

In one embodiment, by raising pass kytoplasm " pyruvic oxidase bypass " (Prank etc., (1996) .Yeast 12 (16): flux 1607), can generate acetyl-CoA from pyruvic acid, 1-3 is illustrated as Fig. 3 step.In order to improve the flux that passes this path, can cross one or more enzymes in expression pyruvic carboxylase (PDC), aldehyde dehydrogenase (ALD) and acetyl-CoA synthase (ACS).

This raising " PDH bypass " activity in path or the operation of flux can cause surpassing the realization of 5% butanols productive rate of theoretical maximum.

Because this acetyl-the CoA generation pass generates acetaldehyde as intermediate product, reduces to minimumly away from the shunting of acetyl-CoA synthetic approach so preferably acetaldehyde is entered, and mainly is that the activity by alcoholdehydrogenase (ADH) further is reduced into ethanol with acetaldehyde.Therefore, reduce or eliminate acetyl-CoA generation that the ADH activity can further improve the pyruvic oxidase alternative pathway.

For example, the genome of the positive yeast yeast saccharomyces cerevisiae of Crabtree contains 7 kinds of known ADH genes.Wherein, ADH1 is the active main source of kytoplasm ADH, and deleted the cell of ADH1 can not anaerobic growth (Drewke etc., (1990) .J.Bacteriology 172 (7): 3909).So, possible preferably deletion ADH1 with acetaldehyde is become alcoholic acid transform reduce to minimum.Yet, but other ADH isoform catalysis acetaldehyde becomes the alcoholic acid reduction, and their reduction or deletion are also contained in the present invention.

This reduce acetaldehyde become operation that alcoholic acid transforms individually or with the raise realization of 10% the butanols productive rate that can cause surpassing theoretical maximum in combination of " PDH bypass " mentioned above flux.

In addition, pyruvic oxidase catalysis pyruvic acid becomes acetyl-CoA and CO ₂Direct conversion, simultaneously with NAD ⁺Be reduced into NADH.So in certain embodiments, in the yeast cytosol, cross the expression pyruvic oxidase.Perhaps, pyruvic acid is changed into formic acid and acetyl-CoA, and (it is also with NAD by hydrogenlyase ⁺Be reduced into NADH) activity gained formic acid further is metabolized to CO ₂

Because above-mentioned acetyl-CoA generation pass utilizes pyruvic acid as substrate, reduce to minimum so preferably pyruvic acid is entered the shunting of other pathways metabolism.Pyruvic carboxylase (PDC) activity has been represented the metabolic main tenuigenin of pyruvic acid path.Therefore, reduce or eliminate acetyl-CoA generation that the PDC activity can further improve above-mentioned path.

With eliminate PDC activity (so eliminating " PDH bypass " path) in combination, can realize surpassing 50% butanols productive rate of theoretical maximum to the operation of the pathways metabolism that pyruvic acid changed into acetyl-CoA.This improvement is the result to three important operations of the natural pathways metabolism of yeast cell: (1) is eliminated the carbon that generates via ethanol and is run off; (2) eliminate in the cell in the Acetyl-CoA synthetase activity that consumes big energy; (3) balance glucose becomes the cofactor (NAD for example of the whole approach that is involved in the conversion of butanols ⁺/ NADH) generation and consumption (glucose becomes acetyl-CoA and generates 4 NADH, and acetyl-CoA becomes the conversion of butanols and consumes 4 NADH).By improving the whole metabolism fitness of host's yeast cell, there is ATP can be utilized and reduce NAD in the cell by biosynthetic process by making thus ⁺/ NADH is than the unbalance butanols approach function that promotes, back two kinds of operations can raise to productive rate and contribute maximum.

About part (2), promptly carbon source becomes the conversion of butanols, can engineered yeast so that acetyl-CoA is changed into butanols.

In an illustrated embodiment, acetyl-CoA-Transacetylase changes into acetoacetyl-CoA with acetyl-CoA, hydroxyl butyryl-CoA desaturase changes into hydroxyl butyryl-CoA with acetoacetyl-CoA, enoyl-CoA hydratase changes into crotonoyl-CoA with hydroxyl butyryl-CoA, and butyryl-CoA desaturase (bcd) changes into butyryl-CoA with crotonoyl-CoA.For the reduction of coupling crotonoyl-CoA and the oxidation of NADH, Bcd needs existence and the activity of electron transfer protein (etfA and etfB).Butyraldehyde desaturase/butanols desaturase changes into butyryl-CoA butyraldehyde and butyraldehyde is changed into butanols then.This enzyme can be from clostridium acetobutylicum.

Put down in writing the approach that uses heterogenous expression in the U.S. Patent application serial number of submitting on December 3rd, 2,007 11/949,724 (at this by addressing income this paper) and come the gene of self-produced solvent bacterium (for example fusobacterium species) acetyl-CoA to be changed into the example of second section of the approach of propyl carbinol.

In some embodiments, recombinant microorganism can be expressed one or more heterologous genes, and its coding is given the enzyme of the ability that generates propyl carbinol.For example, recombinant microorganism can be expressed one or more the heterologous gene in coding anaerobism 2-'s desaturase (Pdh), pyruvic acid formic acid lyase (Pfl), NADH dependency hydrogenlyase (Fdh), acetyl-CoA-Transacetylase (thiolase), hydroxyl butyryl-CoA desaturase, enoyl-CoA hydratase, butyryl-CoA desaturase, butyraldehyde desaturase, propyl carbinol desaturase, the difunctional butyraldehyde/propyl carbinol desaturase.This type of allogeneic dna sequence preferably obtains from allos microorganism (such as clostridium acetobutylicum or Bai Shi clostridium), and can use conventional Protocols in Molecular Biology with the suitable host of one or more importings in these heterologous genes.These allogeneic dna sequences make recombinant microorganism can generate propyl carbinol, generate propyl carbinol or its metabolic intermediate with the big amount of amount that generates than the corresponding Institute of Micro-biology of wild-type at least.

In certain embodiments, recombinant microorganisms express allos thiolase disclosed herein or acetyl-CoA-Transacetylase is such as by from the thl coded by said gene of fusobacterium.

Thiolase (E.C.2.3.1.19) or acetyl-CoA Transacetylase is that the catalysis ethanoyl is condensed to the enzyme on acetyl-CoA molecule.This enzyme in clostridium acetobutylicum by gene thl coding (GenBank accession number U08465, protein ID AAA82724.1), in other enzyme, this enzyme descended expression (Bermejo etc. at the natural promoter that it is used for the acetone generation in intestinal bacteria, Appl.Environ.Mirobiol.64:1079-1085,1998).The homology enzyme also identifies, and can easily identify at the blast search of protein sequence above by implementing.These homologues (homolog) also can serve as the suitable thiolase in the propyl carbinol approach of heterogenous expression.Only lift several examples, these homology enzymes comprise, but be not limited to those from and the following: clostridium acetobutylicum (for example, protein ID AAC26026.1), Clostridium baratii (C.pasteurianum) (for example, protein ID ABA18857.1), the Bai Shi clostridium (for example, protein IDEAP59904.1 or EAP59331.1), clostridium perfringens (Clostridium perfringens) (for example, protein ID ABG86544.1, ABG83108.1), clostridium difficile (Clostridium difficile) (for example, protein ID CAJ67900.1 or ZP_01231975.1), the pyrolysis hot anaerobic bacillus(cillus anaerobicus) of sugar (Thermoanaerobacterium thermosaccharolyticum) (for example, protein IDCAB07500.1), the hot anaerobic bacillus(cillus anaerobicus) of Tengchong (Thermoanaerobacter tengcongensis) (for example, AAM23825.1), (for example give birth to hydroxide carbon thermophile bacteria (Carboxydothermus hydrogenoformans), protein ID ABB 13995.1), Desulfotomaculum reducens MI-1 (for example, protein ID EAR45123.1), candida tropicalis (Candida tropicalis) (for example, protein IDBAA02716.1 or BAA02715.1), yeast saccharomyces cerevisiae (for example, protein ID AAA62378.1 or CAA30788.1), the bacillus bacterial classification, Megasphaera elsdenii (Megasphaera elsdenii), and Butyrivibrio fibrisolvens (Butyrivibrio fibrisolvens).In addition, endogenous yeast saccharomyces cerevisiae thiolase also can be activated in the propyl carbinol approach (ScERGIO) of heterogenous expression.

Calculating according to the BLAST of NCBI, share at least about 55%, 60%, 65%, 70%, 75% or 80% sequence identity, or be the suitable thiolase homologue that can in recombinant microorganism of the present invention, use at least about the homologue of 65%, 70%, 80% or 90% sequence homology.This type of homologue includes, but is not limited to: Bai Shi clostridium NCIMB 8052 (ZP_00909576.1 or ZP_00909989.1), clostridium acetobutylicum ATCC 824 (NP_149242.1), clostridium tetani (Clostridium tetani) E88 (NP_781017.1), clostridium perfringens bacterial strain 13 (NP_563111.1), clostridium perfringens SM101 (YP_699470.1), Clostridium baratii (ABA18857.1), pyrolysis sugar hot anaerobic bacillus(cillus anaerobicus) (CAB04793.1), clostridium difficile QCD-32g58 (ZP_01231975.1) and clostridium difficile 630 (CAJ67900.1).

In certain embodiments, recombinant microorganisms express allos 3-maloyl group of the present invention-CoA desaturase is such as by from the hbd coded by said gene of fusobacterium.

3-hydroxyl butyryl-CoA desaturase (BHBD) is the enzyme that catalysis acetoacetyl-CoA changes into 3-maloyl group-CoA.This enzyme exists (S) that generate 3-maloyl group-CoA or (R) the different variants of isomer.Those skilled in the art are by for example implementing easily to identify the homology enzyme at the blast search of clostridium acetobutylicum BHBD above.All these homology enzymes can serve as the BHBD in the propyl carbinol approach of heterogenous expression.These homology enzymes include, but are not limited to and the following: Ke Shi clostridium (Clostridium kluyveri), and it expresses two kinds multi-form (Miller etc., J.Bacteriol.138:99-104,1979) of this enzyme; Conciliate fiber butyric acid vibrios (Butyrivibrio fibrisolvens), it contains the bhbd gene, and it is organized in homologous genes seat interior (Asanuma etc., Current Microbiology 51:91-94,2005 of its butyric acid approach remainder; Asanuma etc., Current Microbiology 47:203-207,2003).The gene clone of coding short chain acyl-CoA desaturase (SCAD) is from Megasphaera elsdenii and at expression in escherichia coli.Can measure external activity (Becker etc., Biochemistry 32:10736-10742,1993).In other fusobacterium bacterial strain, identified other homologue, such as Ke Shi clostridium (Hillmer etc., FEBS Lett.21:351-354,1972; Madan etc., Eur.J.Biochem.32:51-56,1973), Bai Shi clostridium, clostridium thermosaccharolyticum (C.thermosaccharolyticum), clostridium tetani.

In certain embodiments, wherein express BHBD, it may be useful selecting the enzyme of the organism identical with upstream thiolase or downstream enoyl-CoA hydratase origin.The destruction of the potential protein-protein interaction during this can be avoided when the enzyme of expressing from different organisms this approach between the adjacent eggs white matter.

In certain embodiments, recombinant microorganisms express allos enoyl-CoA hydratase disclosed herein is such as by from the crt coded by said gene of fusobacterium.

Enoyl-CoA hydratase or alkene acyl-CoA hydratase are the catalysis cis becomes corresponding β-hydroxyl acyl CoA derivative with trans alkene acyl-reversible hydration of CoA substrate enzymes.In clostridium acetobutylicum, metabolic this step of butyric acid is come catalysis (GenBank protein numbering AAA95967, Kanehisa, Kanehisa, Novartis Found Symp.247:91-101,2002 by the EC 4.2.1.55 of crt coded by said gene; 01-3 is discussed, 19-28,244-52).Enoyl-CoA hydratase (Crt) from clostridium acetobutylicum has been purified to homogeneous and has obtained sign (Waterson etc., J.Biol.Chem.247:5266-5271,1972).It all shows as the protein of homogeneous at natural and state sex change.This enzyme shows as tetramer performance function, and molecular weight subunit is 28.2kDa and 261 residues (Waterson etc. have reported that molecular weight is that 40kDa and length are 370 residues).The enzyme of purifying in buffered soln when 4 ℃ are preserved or when freezing loss of activity (Waterson etc., J.Biol.Chem.247:5266-5271,1972).The optimal pH of this enzyme is pH 8.4 (Schomburg etc., NucleicAcids Res.32:D431-433,2004).Different with Mammals enoyl-CoA hydratase with extensive substrate specificity, the enzyme of bacterium hydration crotonoyl-CoA and hexenoyl-CoA.For crotonoyl-CoA has obtained V _MaxAnd K _mValue is 6.5x10 ⁶Every mole of mole per minute and 3x 10 ^-5M.This enzyme is higher than 7x10 in crotonoyl-CoA concentration ⁵Be suppressed (Waterson etc., J.Biol.Chem.247:5252-5257,1972 during M; Waterson etc., J.Biol.Chem.247:5258-5265,1972).

Parsed the structure (Engel etc., J.Mol.Biol.275:847-859,1998) of many enzymes of enoyl-CoA hydratase family.Crt gene high expression level in intestinal bacteria, and show than the higher ratio of in clostridium acetobutylicum, being seen (187.5U/mg surpasses 128.6U/mg) (Boynton etc., J.Bacteriol.178:3015-3024,1996) alive.Encoded in eukaryote and the prokaryotic organism many different homologues of enoyl-CoA hydratase, they are synthetic as butyric acid metabolism, lipid acid, the part of β-Yang Hua and other relational approach plays a role (Kanehisa, Novartis Found Symp.247:91-101,2002; 01-3 is discussed, 19-28,244-52; Schomburg etc., Nucleic Acids Res.32:D431-433,2003).Manyly in these enzymes furtherd investigate.Alkene acyl-CoA hydratase from beef liver has obtained extremely deep research and has characterized (Waterson etc., J.Biol.Chem.247:5252-5257,1972) completely.Get close to most directly to the ClustalW of homologue comparison for 20 kinds that have generated from the enoyl-CoA hydratase of bacterium.From 40% to 85% of the sequence identity of homologue does not wait.

Calculating according to the BLAST of NCBI, share at least about 45%, 50%, 55%, 60%, 65% or 70% sequence identity, or be the suitable Crt homologue that can in recombinant microorganism of the present invention, use at least about the homologue of 55%, 65%, 75% or 85% sequence homology.This type of homologue comprises, but be not limited to: clostridium tetani E88 (NP_782956.1), clostridium perfringens SM101 (YP_699562.1), clostridium perfringens bacterial strain 13 (NP_563217.1), Bai Shi clostridium NCIMB 8052 (ZP_00909698.1 or ZP_00910124.1), Wo Shi supports Zymomonas mobilis Wo Shi subspecies brother Dettingen bacterial strain (Syntrophomonas wolfeisubsp.wolfei str.Goettingen) (YP_754604.1) altogether, Desulfotomaculum reducens MI-1 (ZP_01147473.1 or ZP_01149651.1), pyrolysis sugar hot anaerobic bacillus(cillus anaerobicus) (CAB07495.1) and living hydroxide carbon thermophile bacteria Z-2901 (YP_360429.1).

The crt gene that studies have shown that the coding enoyl-CoA hydratase that carries out in clostridium is to encode as the part of bigger BCS operon.Yet, to the slightly different arrangement that studies show that of Butyrivibrio fibrisolvens (B.fibriosolvens) (the butyro-bacterium of a kind of generation) from cud.Bunch collection and thl, crt, hbd, bcd, etfA and the etfB gene arranged though I type Butyrivibrio fibrisolvens has as the part of operon, but II type bacterial strain has similar bunch but lack crt gene (Asanuma etc., Curr.Microbiol.51:91-94,2005; Asanuma etc., Curr.Microbiol.47:203-207,2003).Since this protein gives full expression in intestinal bacteria and thoroughly characterize, the clostridium acetobutylicum enzyme is the preferred enzyme of propyl carbinol approach institute of heterogenous expression so.Other possible target thing come autohemagglutination nuclear fusobacterium Wen's subspecies (Fusobacterium nucleatum subsp.Vincentii) (Q7P3U9-Q7P3U9_FUSNV), clostridium difficile (P45361-CRT CLODI), Clostridium baratii (P81357-CRT_CLOPA) and Bacterium melitense (Brucella melitensis) homologous gene (Q8YDG2-Q8YDG2_BRUME).

In certain embodiments, recombinant microorganisms express allos butyryl-CoA desaturase disclosed herein and corresponding in case of necessity electron transfer protein are such as by from bcd, the etfA of fusobacterium and etfB coded by said gene.

Clostridium acetobutylicum butyryl-CoA desaturase (Bcd) is that the carbon-to-carbon double bond among catalysis crotonoyl-CoA reduces to produce the enzyme of butyryl-CoA.The oxidation of this reductive coupling NADH.Yet, two kinds of electron transfer proteins of this enzyme require, i.e. etf and etfB (Bennett etc., Ferns Microbiology Reviews 17:241-249,1995).

The gene of codase beta-hydroxy butyryl-coenzyme A (CoA) desaturase, enoyl-CoA hydratase and the butyryl-CoA desaturase of clostridium acetobutylicum ATCC 824 is at the BCS operon collection of putting on an arrow, and its GenBank accession number is U17110.

Butyryl-CoA desaturase (Bcd) protein sequence (GenBank accession number AAA95968.1) is shown in SEQ ID NO:3.

Calculating according to the BLAST of NCBI, share at least about 55%, 60%, 65%, 70%, 75% or 80% sequence identity, or be the suitable Bcd homologue that can in recombinant microorganism of the present invention, use at least about the homologue of 70%, 80%, 85% or 90% sequence homology.This type of homologue comprises, but be not limited to: clostridium tetani E88 (NP_782955.1 or NP_781376.1), clostridium perfringens bacterial strain 13 (NP_563216.1), Bai Shi clostridium (AF494018_2), Bai Shi clostridium NCIMB 8052 (ZP_00910125.1 or ZP_00909697.1), with pyrolysis sugar hot anaerobic bacillus(cillus anaerobicus) (CAB07496.1), the hot anaerobic bacillus(cillus anaerobicus) MB4 of Tengchong (NP_622217.1).

Calculating according to the BLAST of NCBI, share at least about 45%, 50%, 55%, 60%, 65% or 70% sequence identity, or be the suitable Hbd homologue that uses in can described in this article recombinant microorganism at least about the homologue of 60%, 70%, 80% or 90% sequence homology.This type of homologue comprises, but be not limited to: clostridium acetobutylicum ATCC 824 (NP_349314.1), clostridium tetani E88 (NP_782952.1), clostridium perfringens SM101 (YP_699558.1), clostridium perfringens bacterial strain 13 (NP_563213.1), clostridium saccharobutyricum (Clostridium saccharobutylicum) (AAA23208.1), Bai Shi clostridium NCIMB 8052 (ZP_00910128.1), Bai Shi clostridium (AF494018_5), the hot anaerobic bacillus(cillus anaerobicus) MB4 of Tengchong (NP_622220.1), pyrolysis sugar hot anaerobic bacillus(cillus anaerobicus) (CAB04792.1) and Alkaliphilusmetalliredigenes QYMF (ZP_00802337.1).

Bcd is to the K of butyryl-CoA _mBe 5.The gene of clostridium acetobutylicum bcd and the corresponding ETF of coding has been cloned in intestinal bacteria-clostridium acetobutylicum shuttle vectors.In the clostridium acetobutylicum ATCC 824 that transforms with this plasmid, detect the Bcd activity (Boynton etc., Journal of Bacteriology178:3015-3024,1996) of rising.Clostridium acetobutylicum P26 2Bcd is to the K of butyryl-CoA _mBe about 6 μ M (DiezGonzalez etc., Current Microbiology 34:162-166,1997).The homologue of Bcd (homologues) is generating butyro-anaerobe Megasphaera elsdenii (Williamson etc. with relevant ETF, Biochemical Journal 218:521-529,1984), peptostreptococcus elsdenii (Peptostreptococcus elsdenii) (Engel etc., Biochemical Journal 125:879,1971), Bu Shi is health spore bacterium (Syntrophospora bryanti) (Dong etc. altogether, Antonie Van LeeuwenhoekInternational Journal of General and Molecular Microbiology 67:345-350,1995), with usur treponema (Treponema phagedemes) (George etc., Journal ofBacteriology 152:1049-1059,1982) the middle evaluation.The structure of Megasphaera elsdenii Bcd is resolved (Djordjevic etc., Biochemistry 34:2163-2171,1995).The blast search of clostridium acetobutylicum ATCC 824 Bcd has been identified a large amount of homologous sequences in extremely a plurality of species, above enumerated some in the homologue herein.The gene of any these homologues of coding all can be used for the present invention.Notice, in a kind of microorganism (such as intestinal bacteria), can produce expression problem and/or electron transport problem during these genes of heterogenous expression, but quite different in another kind of microorganism.In addition, a kind of homology enzyme can have in given microorganism expresses and/or the electron transport problem, but not so other homology enzyme then can be.The availability of gene different, that be equal to substantially provides more design alternatives when engineered recombinant microorganism.

A kind of promising bcd that clones in intestinal bacteria already and express is from Megasphaera elsdenii, and the external activity of expressed enzyme can be measured (Becker etc., Biochemistry 32:10736-10742,1993).O ' Neill etc. have reported that etfA and eftB gene clone and the heterogenous expression in intestinal bacteria reaches the coded proteinic function sign (O ' Neill etc., J.Biol.Chem.273:21015-21024,1998) from Megasphaera elsdenii.Measured activity with the ETF assay method, this assay method is got up the reduction of NADH oxidation and crotonoyl-CoA through the Bcd coupling.The activity of reorganization ETF in the ETF assay method that contains Bcd is with similar as natural enzyme activity that Whitfield and Mayhew reported.Therefore, utilize Megasphaera elsdenii Bcd and ETF albumen thereof that the solution of synthetic butyryl-CoA is provided.The K of Megasphaera elsdenii Bcd when recombinant expressed _mBe measured as 5 μ M, and be 14 μ M (DuPlessis etc., Biochemistry 37:10469-77,1998) when in natural host, expressing.Megasphaera elsdenii Bcd shows at extremely low concentration and is subjected to etheric acid inhibition (Ki is 0.1uM) (Vanberkel etc., Eur.J.Biochem.178:197-207,1988).Generate in the butyro-bacterial strain of separating fiber butyric acid vibrios at two and to have identified the gene cluster that contains thl, crt, hbd, bcd, etfA and etfB.Compare with clostridium acetobutylicum, these proteinic amino acid sequence similarities are high (Asanuma etc., Current Microbiology 51:91-94,2005; Asanuma etc., CurrentMicrobiology 47:203-207,2003).In mammlian system, in plastosome, found the similar enzyme that involves the short-chain fat acid oxidase.

In certain embodiments, recombinant microorganisms express allos " trans-2-alkene acyl-CoA reductase enzyme " or " TER " disclosed herein.

Trans-2-alkene acyl-CoA reductase enzyme or TER are the protein that can catalysis crotonoyl-CoA changes into butyryl-CoA.In certain embodiments, recombinant microorganisms express and TER from the Bcd/EtfA/EtfB catalysis same reaction of fusobacterium and other bacterial species.Describe from the plastosome TER of very thin eye worm (E.gracilis) is existing, and derived from many TER albumen of many species with have the active protein of TER and identify, form TER protein families (U.S. Patent application 2007/0022497, Cirpus etc.; Hoffmeister etc., J.Biol.Chem., 280:4329-4338,2005, state they complete this paper of incorporating into by carrying at this).The brachymemma cDNA of very thin eye worm gene is functional expression in intestinal bacteria.This cDNA or can express with propyl carbinol pathway gene thl, crt, adhE2 and hbd from the gene of the homologue of other microorganism is in order to generate propyl carbinol in intestinal bacteria, yeast saccharomyces cerevisiae or other host.

TER albumen also can be identified by the bioinformatics method of common general knowledge, such as BLAST.The proteic example of TER includes, but not limited to from the TER such as following species: Euglena (Euglena spp.) includes but not limited to very thin eye worm; Aeromonas (Aeromonas spp.) includes but not limited to Aeromonas hydrophila (A.hydrophila); Cold zygosaccharomyces (Psychromonas spp.) includes but not limited to the cold Zymomonas mobilis in deep-sea (P.ingrahamii); Photobacterium (Photobacterium spp.) includes but not limited to deep-sea luminous bacillus (P.profundum); Vibrio (Vibrio spp.) includes but not limited to Vangustum, vibrio cholerae (V.cholerae), separates alginic acid vibrios (V.alginolyticus), Vibrio parahaemolyticus (V.parahaemolyticus), Vibrio vulnificus (V.vulnificus), Fei Shi vibrios (V.fischeri), Vibrio splindidus (V.splendidus); Shiva Bordetella (Shewanella spp.) includes but not limited to S.amazonensis, S.woodyi, S.frigidimarina, S.paeleana, S.baltica, denitrification Shiva Salmonella (S.denitrificans); Oceanospirillum (Oceanospirillum spp.); Xanthomonas (Xanthomonasspp.) includes but not limited to rice Xanthomonas campestris (X.oryzae), Xanthomonas campestris (X.campestris); Look Halobacterium (Chromohalobacter spp.) need to include but not limited to salt look salt bacillus (C.salexigens); Idiomarina spp. includes but not limited to I.baltica; Pseudoalteromonas belongs to (Pseudoalteromonasspp.), includes but not limited to Atlantic Ocean pseudoalteromonas (Patlantica); Replace zygosaccharomyces (Alteromonas spp.); Saccharophagus spp., include but not limited to S.degradans, S.marinegamma proteobacterium, S.alpha proteobacterium, Rhodopseudomonas (Pseudomonasspp.) includes but not limited to Pseudomonas aeruginosa (P.aeruginosa), pseudomonas putida (P.putida), Pseudomonas fluorescens (P.fluorescens); Burkholder Pseudomonas (Burkholderia spp.), include but not limited to B.phytofirmans, new onion bulkholderia cepasea (B.cenocepacia), onion bulkholderia cepasea (B.cepacia), B.ambifaria, Vietnam's bulkholderia cepasea (B.vietnamensis), B.multivorans, B.dolosa; Methyl Bacillaceae (Methylbacillus spp.) includes but not limited to M.flageliatus; Oligotrophy zygosaccharomyces (Stenotrophomonas spp.) includes but not limited to have a liking for maltose oligotrophy Zymomonas mobilis (S.maltophilia); Assemble Bacillaceae (Congregibacter spp.), include but not limited to C.litoralis; Serratia (Serratia spp.) includes but not limited to be out of shape spot Serratia (S.proteamaadans); Ocean Zymomonas mobilis (Marinomonas spp.); Xytella spp. includes but not limited to X.fastidiosa; Reinekea spp.; Colwell Bordetella (Colwellia spp.) includes but not limited to C.psychrerythraea; Yersinia (Yersinia spp.) includes but not limited to Yersinia pestis (Y.pestis), artificial tuberculosis yersinia genus (Y.pseudotuberculosis); Methyl Pseudomonas (Methylobacillus spp.) includes but not limited to M.flageliatus; Cytophaga Pseudomonas (Cytophagaspp.) includes but not limited to Cytophaga hutchinsonii (C.hutchinsonii); Flavobacterium (Flavobacteriumspp.) includes but not limited to F.johnsoniae; Little Pseudomonas that quivers (Microscilla spp.) includes but not limited to M.marina; Polar region Bacillaceae (Polaribacter spp.) includes but not limited to P.irgensii; Fusobacterium includes but not limited to clostridium acetobutylicum, Bai Shi clostridium, separates the fiber clostridium; Coxiella (Coxiellaspp.) includes but not limited to Bei Shi Ke Kesi body (C.burnetii).

Outside aforementioned, term " trans-2-alkene acyl-CoA reductase enzyme " or " TER " refer to can catalysis crotonoyl-CoA to change into butyryl-CoA and according to the calculating of the NCBI BLAST that uses default parameter, share at least about 40% with the arbitrary of the Aeromonas hydrophila TER of the very thin eye worm TER of brachymemma or total length or the two, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or bigger sequence identity, or at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or the protein of bigger sequence similarity.

As used herein, identical Nucleotide or occurrence of amino acid in the same position in " sequence identity " sequence of referring to be compared." sequence similarity " considered approximate match, and only measure according to some of " difference " or " homogeny " when this type of is substituted marking meaningful, conservative or highly possible alternative the tax with than the nonconservative or unlikely more favourable score that substitutes in described tolerance.

Using TER to replace another advantage of Bcd/EtfA/EtfB is that TER is activated at monomeric form, and protein expression and enzyme self are all insensitive to oxygen.

As used herein, " trans-2-alkene acyl-CoA reductase enzyme (TER) homologue " from other organism (for example refers to, belong to a worm or Aeromonas class (phylum)) the enzyme homeopeptide, it has and defined identical TER essential characteristic above, but share less than 40% sequence identity and 50% sequence similarity standard, as discussed above.The substituting of one or more amino-acid residues, interpolation, deletion, inversion or insertion are contained in sudden change.This allows the expression of enzymes during the expression phase of aerobic growth and propyl carbinol process, can potentially allow biofuel generative process more efficiently.

In certain embodiments, recombinant microorganisms express allos butyraldehyde desaturase/propyl carbinol desaturase disclosed herein is such as by from bdhA/bdhB, the aad of fusobacterium or adhE2 coded by said gene.

Butyraldehyde desaturase (BYDH) is that catalysis butyryl-CoA NADH dependency is reduced into the enzyme of butyraldehyde.Butyraldehyde further is reduced into propyl carbinol by propyl carbinol desaturase (BDH).This reduction is also with the NADH oxidation.Clostridium acetobutylicum contains and demonstrates the gene that butyryl-CoA is changed into several enzymes of propyl carbinol.

One of these enzymes are by aad coding (Nair etc., J.Bacteriol.176:871-885,1994).This gene is called adhE in clostridium acetobutylicum strain DSM 792.This enzyme is the part of sol operon, and its encode difunctional BYDH/BDH (Fischer etc., Journal of Bacteriology 175:6959-6969,1993; Nair etc., J.Bacteriol.176:871-885,1994).

The gene product of aad functional expression in intestinal bacteria.Yet under aerobic conditions, gained is active to keep very low, the indication oxygen sensitivity.According to the activity of butyraldehyde with respect to the activity of acetaldehyde for surpassing 100 times high, in the formation that mainly acts on propyl carbinol of Aad but not ethanol (Hair etc., Journal ofBacteriology 176:5843-5846,1994).

According to the calculating of the BLAST of NCBI, share at least about 50%, 55%, 60% or 65% sequence identity, or be the suitable homologue that uses in can disclosed in this article recombinant microorganism at least about the homologue of 70%, 75% or 80% sequence homology.This type of homologue includes, but is not limited to: clostridium tetani E88 (NP_781989.1), clostridium perfringens bacterial strain 13 (NP_563447.1), clostridium perfringens ATCC13124 (YP_697219.1), clostridium perfringens SM101 (YP_699787.1), Bai Shi clostridium NCIMB8052 (ZP_00910108.1), clostridium acetobutylicum ATCC 824 (NP_149199.1), clostridium difficile 630 (CAJ69859.1), clostridium difficile QCD-32g58 (ZP_01229976.1) and thermal fiber clostridium (Clostridium thermocellum) ATCC 27405 (ZP_00504828.1).

Other two kinds of NADH dependency propyl carbinol desaturases (BDH I, BDH II) are purified, and their gene (bdhA bdhB) clones.The GenBank accession number of BDH I is AAA23206.1, and protein sequence is shown in SEQ ID NO:10.

The GenBank accession number of BDH II is AAA23207.1, and protein sequence is shown in SEQ IDNO:11.

These genes are adjacent on karyomit(e), but by their promoter transcription (Walter etc., Gene 134:107-111,1993).BDH I utilizes NADPH as cofactor, and BDH II utilizes NADH.Yet, notice that relative cofactor preference is that pH is dependent.From in the intestinal bacteria lysate, observing BDH I activity (Petersen etc., Journal of Bacteriology173:1831-1834,1991) behind the plasmid expression bdhA.BDH II it is reported that the activity to butyraldehyde that has is active 46 times high to acetaldehyde, and low 50 times of backward activity.BDH I only is active about 2 times high (Welch etc., Archives of Biochemistry and Biophysics 273:309-318,1989) to acetaldehyde to the activity of butyraldehyde.So, in one embodiment, in the propyl carbinol approach of heterogenous expression, use BDH II or BDH II homologue.In addition, these enzymes have activity most at 5.5 relatively low pH, can consider this proterties when selecting appropriate host and/or processing condition.

Though gene mentioned above is transcribed under the condition of producing solvent, a kind of different gene, adhE2 transcribes (Fontaine etc., J.Bacteriol.184:821-830,2002, GenBank accession number AF321779) under the condition of producing alcohol.These conditions are in neutral pH existence relatively.This enzyme has been crossed in colibacillary anaerobism is cultivated and has been expressed, and has high NADH dependency BYDH and BDH activity.In certain embodiments, this enzyme is preferred enzyme.The protein sequence of this enzyme (GenBank accession number AAK09379.1) is shown in SEQID NO:1.

According to the calculating of the BLAST of NCBI, share at least about 50%, 55%, 60% or 65% sequence identity, or be the suitable homologue that uses in can disclosed in this article recombinant microorganism at least about the homologue of 70%, 75% or 80% sequence homology.This type of homologue comprises, but be not limited to: clostridium perfringens SM101 (YP_699787.1), clostridium perfringens bacterial strain 13 (NP_563447.1), clostridium perfringens ATCC 13124 (YP_697219.1), clostridium tetani E88 (NP_781989.1), Bai Shi clostridium NCIMB8052 (ZP_00910108.1), clostridium difficile QCD-32g58 (ZP_01229976.1), clostridium difficile 630 (CAJ69859.1), clostridium acetobutylicum ATCC 824 (NP_149325.1) and thermal fiber clostridium ATCC27405 (ZP_00504828.1).

In certain embodiments, can use with any aforementioned polypeptides samely at least about 70%, 80%, 90%, 95%, 99%, or any homology enzyme of sharing at least about 60%, 70%, 80%, 90%, 95% sequence homology (similar) replaces these wild type peptides.These enzymes of sharing essential sequence identity or similarity can be the wild-type enzymes from different organisms, perhaps can be the enzymes that artificial is recombinated.

In certain embodiments, can use coding to have the gene that replaces the above-mentioned enzyme of coding with any gene of the identical active enzyme of any above-mentioned enzyme.These enzymes can be the wild-type enzymes from different organisms, perhaps can be enzymes artificial, reorganization or engineered.

In addition because the inherent degeneracy of genetic code, also can use coding substantially the same or function on other nucleotide sequence of the aminoacid sequence that is equal to clone and express these zymoid polynucleotide of coding.Skilled person in the art will appreciate that modifying encoding sequence can be favourable to strengthen its expression in specific host.The most normal codon that is utilized is called best codon (optimalcodon) in a kind of species, and the codon that not too often utilizes is classified as rare or low rate of utilization codon.Can substitute codon and select, promptly be called the process of " codon optimized " or " control species codon preference " sometimes with the preferred codon of reflection host.The methodology of optimizing nucleotide sequence for the expression in the plant is provided, and for example, in U.S. Patent No. 6,015,891 reach in the reference of wherein quoting.

In certain embodiments, recombinant microorganism disclosed herein has one or more fusobacteriums that comes self-produced solvent, such as clostridium acetobutylicum or Bai Shi clostridial allogeneic dna sequence.Exemplary clostridium acetobutylicum is strains A TCC824, and exemplary Bai Shi clostridium is bacterial strain NCIMB 8052.

Expression of gene can realize by conventional molecular biology method.For example, heterologous gene can be under the control of inducible promoter or composition promotor.Heterologous gene can be integrated in the karyomit(e) of host microorganism, perhaps as stable delivery (" heredity ") existing for the extrachromosomal inheritance element of daughter cell.This type of extrachromosomal inheritance element (such as plasmid, BAC, YAC etc.) can contain the selection marker of guaranteeing the existence of this type of genetic elements in daughter cell in addition.

In certain embodiments, recombinant microorganism disclosed herein also can generate one or more metabolic intermediates that propyl carbinol generates approach, such as acetoacetyl-CoA, maloyl group-CoA, crotonoyl-CoA, butyryl-CoA or butyraldehyde, and/or its derivative, such as butyric acid.

In some embodiments, described hereinly generated propyl carbinol with the recombinant microorganism that activates one or more isodynamic enzymes mentioned above through the allos approach by engineered for generating propyl carbinol.

As used herein, term " approach " refers to comprise that one or more are subjected to biological procedures enzyme control, that substrate conversion become the chemical reaction of product.Thereby being used for the approach that carbon source changes into propyl carbinol is to comprise that one or more are subjected to biological procedures enzyme control, that carbon source changed into the reaction of propyl carbinol." allos approach " refers at least a by the catalytic approach of at least a isodynamic enzyme of wherein at least a or number of chemical reaction.On the other hand, " natural approach " refers to that wherein one or more chemical reactions are by the catalytic approach of natural enzyme.

In certain embodiments, recombinant microorganism disclosed herein is by the engineered allos approach (being also referred to as the propyl carbinol approach in this article) that generates propyl carbinol with activation, it comprises: (1) 2 acetyl-CoA changes into acetoacetyl-CoA, (2) acetoacetyl-CoA changes into maloyl group-CoA, (3) maloyl group-CoA changes into crotonoyl-CoA, (4) crotonoyl-CoA changes into butyryl-CoA, and (5) butyraldehyde changes into propyl carbinol (seeing the exemplary diagram of Fig. 2).

2 acetyl-CoA change into acetoacetyl-CoA and can implement by the natural or heterologous gene of expressing coding acetyl-CoA-Transacetylase (thiolase) or Thl in recombinant microorganism.Suitable exemplary thiolase is by following genes encoding in the disclosed in this article recombinant microorganism: from the thl of clostridium acetobutylicum (particularly from strains A TCC 824), or from Clostridium baratii, Bai Shi clostridium (particularly from bacterial strain NCIMB 8052 or bacterial strain BA101), candida tropicalis, bacillus, Megasphaera elsdenii or separate the gene of the coding homology enzyme of fiber butyric acid vibrios, or be selected from the intestinal bacteria thiolase gene of fadA or atoB.

Acetoacetyl-CoA changes into maloyl group-CoA and can implement by the natural or heterologous gene of expressing coding maloyl group-CoA desaturase Hbd in recombinant microorganism.Suitable exemplary Hbd is by following genes encoding in the disclosed in this article recombinant microorganism: from the hbd of clostridium acetobutylicum (particularly from strains A TCC 824), or from Ke Shi clostridium, Bai Shi clostridium (particularly from bacterial strain NCIMB8052 or bacterial strain BA101), clostridium thermosaccharolyticum, clostridium tetani, separate the gene of the coding homology enzyme of fiber butyric acid vibrios, Megasphaera elsdenii or intestinal bacteria (fadB).

Maloyl group-CoA changes into crotonoyl-CoA and can implement by the natural or heterologous gene of expressing coding enoyl-CoA hydratase or Crt in recombinant microorganism.Exemplary crt suitable in the disclosed in this article recombinant microorganism is by the crt from clostridium acetobutylicum (particularly from strains A TCC 824), or from the genes encoding of the coding homology enzyme of Butyrivibrio fibrisolvens, poly-nuclear fusobacterium Wen subspecies, clostridium difficile, Clostridium baratii or Bacterium melitense.

Crotonoyl-CoA changes into butyryl-CoA and can implement by the natural or heterologous gene of expressing coding butyryl-CoA desaturase in recombinant microorganism.Exemplary butyryl-CoA desaturase suitable in the disclosed in this article recombinant microorganism is by the bcd/etfA/etfB from clostridium acetobutylicum (particularly from strains A TCC 824), or from Megasphaera elsdenii, peptostreptococcus elsdenii, Bu Shi altogether health spore bacterium, usur treponema, separate the gene of the coding homology enzyme of fiber butyric acid vibrios or Mammals plastosome Bcd homologue coding.

Butyraldehyde changes into propyl carbinol and can implement by the natural or heterologous gene of expressing coding butyraldehyde desaturase or propyl carbinol desaturase in recombinant microorganism.Exemplary butyraldehyde desaturase/propyl carbinol desaturase suitable in the disclosed in this article recombinant microorganism is by bdhA, bdhB, aad or adhE2 from clostridium acetobutylicum (particularly from strains A TCC824), or from coding ADH-1, ADH-2 of Bai Shi clostridium (particularly from bacterial strain NCIMB8052 or bacterial strain BA101) or the genes encoding of ADH-3.

In certain embodiments, the enzyme of the pathways metabolism from acetyl-CoA to propyl carbinol is (i) thiolase (Thl), (ii) maloyl group-CoA desaturase (Hbd), (iii) enoyl-CoA hydratase (Crt), (iv) alcoholdehydrogenase (AdhE2), or at least a in propyl carbinol desaturase (Aad) or butyraldehyde desaturase (Ald) the single in addition function propyl carbinol desaturase (BdhA/BdhB) and (v) trans-2-alkene acyl-CoA reductase enzyme (TER) (Fig. 2).In certain embodiments, Thl, Hbd, Crt, AdhE2, Ald, BdhA/BdhB and Aad are from fusobacterium.In certain embodiments, fusobacterium is a clostridium acetobutylicum.In certain embodiments, TER is from very thin eye worm or from Aeromonas hydrophila.

In certain embodiments, one or more in one or more heterologous gene coding acetyl-CoA-Transacetylases (thiolase), hydroxyl butyryl-CoA desaturase (hbd), enoyl-CoA hydratase (crt) and alcoholdehydrogenase (adhE2), butyryl-CoA desaturase (bcd), butyraldehyde desaturase (bdhA/bdhB)/butanols desaturase (aad) and the trans-2-alkene acyl-CoA reductase enzyme (TER).

For example, acetyl-CoA-Transacetylase (thiolase) can be the thl from clostridium acetobutylicum, or from the homology enzyme of Clostridium baratii, Bai Shi clostridium, candida tropicalis, bacillus bacterial classification, Megasphaera elsdenii or Butyrivibrio fibrisolvens, or be selected from the intestinal bacteria thiolase of fadA or atoB.

Hydroxyl butyryl-CoA desaturase can be the hbd from clostridium acetobutylicum, or from Ke Shi clostridium, Bai Shi clostridium, clostridium thermosaccharolyticum, clostridium tetani, separate the homology enzymes of fiber butyric acid vibrios, Megasphaera elsdenii or intestinal bacteria (fadB).

Enoyl-CoA hydratase can be the crt from clostridium acetobutylicum, or from Butyrivibrio fibrisolvens, the poly-homology enzyme of examining fusobacterium Wen subspecies, clostridium difficile, Clostridium baratii or Bacterium melitense.

Butyryl-CoA desaturase can be the bcd/etfA/etfB from clostridium acetobutylicum, or from Megasphaera elsdenii, peptostreptococcus elsdenii, Bu Shi altogether health spore bacterium, usur treponema, separate the homology enzyme of fiber butyric acid vibrios or eucaryon plastosome bcd homologue.

Butyraldehyde desaturase/butanols desaturase can be bdhA, bdhB, aad or the adhE2 from clostridium acetobutylicum, or from Bai Shi clostridial ADH-1, ADH-2 or ADH-3.

Trans-2-alkene acyl-CoA reductase enzyme (TER) can be from very thin eye worm or Aeromonas hydrophila.

One or more allogeneic dna sequences can produce the solvent clostridium from being selected from clostridium acetobutylicum or Bai Shi clostridial, or are total to health spore bacterium, usur treponema or intestinal bacteria from clostridium difficile, Clostridium baratii, Ke Shi clostridium, clostridium thermosaccharolyticum, clostridium tetani, candida tropicalis, bacillus bacterial classification, Bacterium melitense, Megasphaera elsdenii, Butyrivibrio fibrisolvens, poly-nuclear fusobacterium Wen subspecies, peptostreptococcus elsdenii, Bu Shi.

In certain embodiments, clostridium acetobutylicum is strains A TCC824, and the Bai Shi clostridium is bacterial strain NCIMB 8052 or bacterial strain BA101.In certain embodiments, share at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% sequence identity, or be suitable for the present invention at least about the homologue of 50%, 60%, 70%, 80%, 90% sequence identity (according to the calculating of NCBI BLAST, use default parameter).

Partly (1): engineered pyruvic acid becomes the conversion of acetyl-CoA

As mentioned above, the pyruvic acid conversion that becomes acetyl-CoA can occur in the through engineering approaches cell by two kinds of general paths: (A) aforesaid " PDH bypass " path or (B) in the cytosol by PDH or become the direct conversion of acetyl-CoA by the pyruvic acid that PFL carries out.

(A) generate acetyl-CoA through " PDH bypass " path

About generating the path (A) of acetyl-CoA from pyruvic acid, kytoplasm acetyl-CoA generation approach be by three kinds enzymatic, shown in step 1,2 and 3 among Fig. 3.The activity of the enzyme by improving those speed limits has realized the more effective way of generation acetyl-CoA.For example, in yeast saccharomyces cerevisiae, if the ALD activity is restrictive in approach, crossing of ALD6 expressed the overall flux that can improve thus by this approach so.Realized that through one of following mechanism or its combination the acetyl-CoA that raises in the cytosol forms:

In one embodiment, by Pyruvate Decarboxylase Gene (for example, yeast saccharomyces cerevisiae PDC1, PDC5 and/or PDC6; Acetyl-the CoA that can generate rising is expressed in crossing of step 1).

In another embodiment, by acetaldehyde dehydrogenase gene (for example, yeast saccharomyces cerevisiae ALD6; Acetyl-the CoA that can generate rising is expressed in crossing step 2).

In another embodiment, by acetyl-CoA synthase gene (for example, yeast saccharomyces cerevisiae ACS1 or ACS2 or the two; Acetyl-the CoA that can generate rising is expressed in crossing of step 3).

In a different embodiment, ALD and ACS (yeast saccharomyces cerevisiae ALD6; Step 2) the two the time cross acetyl-CoA (step 2 and 3) that expression can generate rising.

In another embodiment, cross acetyl-CoA that expression can generate rising PDC, ALD and ACS gene the time and generate (step 1-3).

Generate in order further to improve acetyl-CoA, the main kytoplasm ethanol that can reduce or eliminate in the yeast generates approach.In the positive yeast saccharomyces cerevisiae of Crabtree, this realizes that by deletion ADH1 ADH1 is the active main source of kytoplasm ADH.The cell of having deleted ADH1 can not anaerobic growth (Drewke etc., (1990) .J.Bacteriology 172 (7): 3909), and so may reduce to minimum acetaldehyde is become the alcoholic acid conversion by preferably deletion ADH1.Eliminating this approach selectivity drives acetaldehyde and leads to acetate and lead to acetyl-CoA subsequently and generate (Fig. 3, step 5).Therefore, can implement crossing of gene mentioned above in having the active cell of ADH reduction or that eliminate expresses.

Similarly, can reduce or eliminate kytoplasm ADH activity by deletion ADHI or ADHII in such as Kluyveromyces lactis, in order to improve through " PDH bypass " path the flux to acetyl-CoA from pyruvic acid at the negative yeast of Crabtree.Therefore, in this organism,, can improve the flux in process " PDH bypass " path via the expression of crossing of KIALD6, KIACS1 independent or combination or KIACS2 with above proposed similar to yeast saccharomyces cerevisiae.

(B) directly generate acetyl-CoA from pyruvic acid

About generate the path (B) of acetyl-CoA from pyruvic acid, can improve acetyl-CoA generation by the expressing excessively of gene that forms complete PDH mixture.For example, crossing the gene of expressing can be from intestinal bacteria (aceE, aceF and lpdA), zymomonas mobilis (Zymomonas mobilis) (pdhAa, pdhA β, pdhB and Ipd), streptococcus aureus (Staphylococcus aureus) (pdhA, pdhB, pdhC and Ipd), subtilis (Bacillus subtilis), Corynebacterium glutamicum (Corynebacteriumglutamicum) or Pseudomonas aeruginosa (Pseudomonas aeruginosa) (step 4).

Pyruvate dehydrogenase complex catalysis pyruvic acid becomes the conversion of acetyl-CoA.In yeast saccharomyces cerevisiae, this mixture is positioned in the mitochondrial inner membrane space.Therefore, the another kind of method that in the tenuigenin of yeast saccharomyces cerevisiae, obtains higher level acetyl-CoA be engineered cell with cross express can be in tenuigenin pyruvate dehydrogenase complex (step 4) eucaryon or protokaryon of performance function.In certain embodiments, recombinant microorganism disclosed herein is included in activated pyruvic oxidase (Pdh) under anaerobism or the little aerobic condition.Pyruvic oxidase or NADH dependency hydrogenlyase can be allogenic for recombinant microorganism, the encoding sequence that is encoding such enzymes is allogenic, or the transcriptional regulatory district is allogenic (comprising artificial), or encoded polypeptide comprises sequence variation, and it makes this enzyme to the feedback inhibition that some metabolic intermediate or substrate bring resistance be arranged.

Up to date, just accepted extensively Pdh and under anaerobic do not brought into play function, but several parts of nearest reports have proved situation really not so (de Graef, M. etc., 1999, Journal of Bacteriology, 181,2351-57; Vernuri, G.N. etc., 2002, Applied and Environmental Microbiology, 68,1715-27).In addition, other microorganism such as enterococcus faecalis (Enterococcus faecalis) is even under anaerobic also show the high in-vivo activity of Pdh mixture, and prerequisite is that growth conditions makes stable state NADH/NAD ⁺Lower than enough (Snoep, J.L. etc., 1991, Ferns Microbiology Letters, 81,63-66).Replace regulating the oxygen of Pdh expression and function, shown that Pdh is subjected to NADH/NAD ⁺The adjusting of ratio (de Graef, M. etc., 1999, Journal of Bacteriology, 181,2351-57).If consuming NADH, the propyl carbinol approach of expressing in host cell must be enough to soon keep low NADH/NAD at cell interior ⁺Level, Pdh so endogenous or heterogenous expression can keep active and NADH is provided, and it is enough to this approach of balance.

These Pdh enzymes can disclosed in this article recombinant microorganism in balance propyl carbinol approach.

The expection of the expression of functional Pdh under anaerobic improves the NADH mole number that every mole of glucose obtains.Kim etc. put down in writing Pdh make in intestinal bacteria consume every mole of glucose can obtain nearly 4 moles of NADH (Kim, Y. etc. (2007) .Appl.Environm.Microbiol., 73,1766-1771).Also can engineered yeast, to express PDH mixture from bacterial origin of all kinds.For example, from the Pdh of enterococcus faecalis with similar from colibacillary Pdh, but at much lower NADH/NAD ⁺Level is inactivated.In addition, some organism such as subtilis and nearly all lactic-acid-bacterium bacterial strain use Pdh in anaerobic metabolism.If propyl carbinol generate approach can with endogenous fermentable approach competition, in the microorganism of expressing the active Pdh of anaerobism, express the expection of propyl carbinol generation approach so and cause propyl carbinol productive rate greater than 1.4%.

Perhaps, can express two kinds of bacterial enzymes by crossing, promptly pyruvic acid formic acid lyase (for example intestinal bacteria pflB) and hydrogenlyase (for example Candida boidinii (Candida boidnii) fdh1) generate acetyl-CoA in cytosol.Use this approach, pyruvic acid is converted to acetyl-CoA and formic acid.Hydrogenlyase catalysis formic acid becomes the NADH dependency conversion of carbonic acid gas then.The net result of these reactions is identical with the situation that pyruvic acid changes into acetyl-CoA with pyruvate dehydrogenase complex:

Pyruvic acid+NAD ⁺→ acetyl-CoA+NADH+CO ₂

NADH dependency hydrogenlyase (Fdh; EC 1.2.1.2) catalysis formic acid becomes CO ₂Oxidation and NAD simultaneously ⁺Become the reduction of NADH.Fdh can be used to improve availability in the NADH born of the same parents in the host microorganism according to the present invention, and is used in NADH aspect balance propyl carbinol and generates approach.Particularly, can in host microorganism, activate the dependent Fdh of (particularly being expression) biologic activity NADH.When having the hydrogenlyase approach of this new introducing, 1 mole of formic acid can form 1 mole of NADH when changing into carbonic acid gas.In certain embodiments, in natural microbial, hydrogenlyase changes into CO with formic acid ₂And H ₂, do not involve cofactor.

In addition, can use method known to those skilled in the art that the gene of the external enzyme of any coding (or mentioned herein any other) (or any control or regulate and control the regulatory element of its expression) is carried out orthogenesis.This type of action allows that those skilled in the art are expression and the active enzyme of optimizing in the yeast.

In addition, in order to regulate and control this approach, can express pyruvic carboxylase, Acetyl-CoA synthetase and acetaldehyde dehydrogenase gene from other fungi and bacterial species.Multiple biological physical efficiency is served as the source of these enzymes, includes, but are not limited to: the yeast belong bacterial classification comprises yeast saccharomyces cerevisiae mutant strain and saccharomyces uvarum (S.uvarum); Genus kluyveromyces comprises heat-resisting kluyveromyces (K.thermotolerans), Kluyveromyces lactis and Kluyveromyces marxianus (K.marxianus); Pichia; Hansenula comprises multiple-shaped nuohan inferior yeast (H.polymorpha); Mycocandida; Trichosporon (Trichosporon), Yamadazyma comprise Y.stipitis; Torulaspora pretoriensis; Schizosaccharomyces pombe (Schizosaccharomyce pombe); Genera cryptococcus bacterial classification (Cryptococcus sp.); Aspergillus bacterium; Neurospora bacterial classification (Neurospora sp.) or Ustilago bacterial classification (Ustilago sp.).The example of useful pyruvic carboxylase be those from saccharomyces bayanus (Saccharomyces bayanus) (1PYD), Candidaglabrata, Kluyveromyces lactis (KIPDC1), or Aspergillus nidulans (Aspergillus nidulans) (PdcA), with from white candiyeast (Candida albicans), Neuraspora crassa (Neurosporacrassa), Aspergillus nidulans, or the acetyl of Kluyveromyces lactis (ACS1)-CoA synthase and (ALDDH) from aspergillus niger (Aspergillus niger), the white candiyeast, cryptococcus neoformans (Cryptococcusneoformans) acetaldehyde dehydrogenase (alddh).The source of useful former ribozyme includes, but are not limited to intestinal bacteria, zymomonas mobilis, bacillus bacterial classification, clostridium species, Rhodopseudomonas bacterial classification, lactococcus bacterial classification (Lactococcus sp.), enterobacter bacterial classification (Enterobacter sp.) and salmonella bacterial classification (Salmonella sp.).To realize the enhanced activity, can obtain the further enhancing of this approach by engineered these enzymes, it is realized by site-directed mutagenesis and other evolvement method (this comprises technology well known by persons skilled in the art).

Prokaryotic organism, such as, but be not limited to, intestinal bacteria, zymomonas mobilis, streptococcus aureus, bacillus bacterial classification, clostridium species, Clavibacter, Rhodopseudomonas bacterial classification, lactococcus bacterial classification, enterobacter bacterial classification and salmonella bacterial classification, the source that can serve as this kind of enzyme mixture.For example, the pyruvate dehydrogenase complex from intestinal bacteria (aceE, aceF and lpdA), zymomonas mobilis (pdhA α, pdhA β, pdhB and Ipd), streptococcus aureus (pdhA, pdhB, pdhC and pdhC), subtilis, Corynebacterium glutamicum and Pseudomonas aeruginosa can be used for this purpose.

It is well known in the art cultivating and operating yeast method.In yeast cell, cross expressing gene, be well known in the art, and the present invention is contained any these class methods and is used to make up yeast strain of the present invention with the method for various lower horizontal expression genes, containment genetic expression or deletion genes.

Can use any method that exogenous nucleic acid molecule is imported in the yeast, and many these class methods are well known to a person skilled in the art.For example, it is the common method that is used for nucleic acid is imported yeast cell that conversion, electroporation, joint and protoplastis merge.Referring to for example Ito etc., J.Bacterol.153:163-168 (1983); Durrens etc., Curr.Genet.18:7-12 (1990); And Becker and Guarente, Methodsin Enzymology 194:182-187 (1991).

In one embodiment, the gene of interest integration that enters dna fragmentation or target gene takes place according to principle of homologous recombination.According to this embodiment, contain the module that comprises at least a yeast marker gene, have or do not have the arbitrary flank of integration box for the treatment of integrator gene (internal module) and be the terminal homologous dna fragmentation (causing recombination sequence) of deciding integration site with target.By proper method with this box transformed yeast after, cause homologous recombination between the recombination sequence and can cause internal module to be replaced in the genome and the chromosomal region between two corresponding sites of recombination sequence of causing of integrating box.

In one embodiment, in order to delete gene, integrate box and can comprise suitable yeast selection marker, its flank is for causing recombination sequence.In one embodiment, for heterologous gene is integrated in the yeast chromosomal, the integration box is included in the heterologous gene under suitable promotor and the terminator control, and with selection marker, flank is for causing recombination sequence.In one embodiment, heterologous gene comprises suitable natural gene, and expectation improves the copy number of natural gene.Selected marker gene can be any marker gene of using in yeast, includes, but not limited to URA3 gene or homologous gene from yeast saccharomyces cerevisiae; Or hygromycin gene, they are respectively applied for carries out selection based on auxotroph complementation or antibiotics resistance to transformant.Can choose at random and cause recombination sequence, this depends on and is suitable for expecting the expectation integration site used.

In addition, in one embodiment, use the technology that well known to a person skilled in the art in genome, to remove the marker gene that some is imported into.For example,, and select FOA resistance bacterium colony by cell coated plate in the substratum that contains FOA (5-fluoro-vitamin B13) that will contain URA3, can obtain the URA3 sign lose (Boeke, J. etc., 1984, Mol.Gen.Genet.197,345-47).

The exogenous nucleic acid molecule that is included in the present disclosure yeast cell can be maintained in this cell in any form.For example, exogenous nucleic acid molecule can be integrated in the genome of cell, or maintain the episome state, it can stably transmit (" heredity ") and give daughter cell.This type of extrachromosomal inheritance element (such as plasmid etc.) can contain the selection marker of guaranteeing the existence of this type of genetic elements in daughter cell in addition.In addition, can be stably or instantaneous ground transformed yeast cell.In addition, yeast cell described herein can contain the specific as mentioned above exogenous nucleic acid molecule of single copy or multiple copied.

The method that is used for from the exogenous nucleic acid molecule express polypeptide is well known to a person skilled in the art.These class methods include but not limited to make up nucleic acid, make regulatory element promote the expression of the nucleotide sequence of coding expectation polypeptide.Be typically, regulatory element is to regulate the dna sequence dna that other dna sequence dna is expressed at transcriptional level.So, regulatory element includes but not limited to promotor, enhanser or the like.For example, foreign gene can be under the control of induction type or composition promotor.In addition, being used at yeast is well known to a person skilled in the art from the method for exogenous nucleic acid molecule express polypeptide.For example, can be at genus kluyveromyces (referring to for example U.S. Patent No. 4,859,596 and 4,943,529, by addressing with the complete income this paper of each piece) and yeast belong (referring to for example Gelissen etc., Gene 190 (1): the nucleic acid construct of expressing allogenic polypeptide 87-97 (1997)) is known.In another embodiment, the allos controlling elements can be used for activating or containing the expression of native gene.In addition, in the time will containing or eliminate expression, can eliminate the gene of relevant enzyme, protein or RNA by known deleting technique.

As described in this article, the yeast in the present disclosure scope can be by identifying the specific selection technology of certain enzyme expressed, that express excessively or containment.The method that evaluation has the bacterial strain of expectation phenotype is well known to a person skilled in the art.These class methods include but not limited to that PCR and nucleic acid hybridization technique such as Northern and Southern analyze, the energy for growth of going up or changing when having specific substrates (substrate), chemical, selective agent etc. in particular substrate (substrate).In some situation, can use immunohistochemistry and Measurement for Biochemistry to measure cell and whether contain specific nucleic acid by detecting coded polypeptide expression.For example, can use and coded enzyme is had specific antibody measure specific yeast cell and whether contain this coded enzyme.In addition, can use Measurement for Biochemistry to measure the specific nucleic acid molecule whether cell contains the codase polypeptide by the product that detects the result that expresses as enzyme polypeptide and generate.For example, with the carrier transformant of coding Acetyl-CoA synthetase and detect that the kytoplasm acetyl of rising-CoA concentration indication carrier exists and gene product has activity.The method that is used to detect the existence of certain enzyme activity or specific product is well known to a person skilled in the art.For example, can be as Dalluge etc., the existence of measuring acetyl-CoA that Anal.Bioanal.Chem.374 (5): 835-840 (2002) is put down in writing.

Yeast cell of the present invention has the enzymic activity of reduction, such as the alcoholdehydrogenase activity that reduces.Term " reduction " as employed about cell and certain enzyme activity, refers to the enzymic activity lower than level measured in the compared yeast cell of same species herein.So, lack the active yeast cell of alcoholdehydrogenase and be considered as having the alcoholdehydrogenase activity of reduction, because great majority (if not all words) comparable yeast strain has at least some alcoholdehydrogenase activity.The reason of the enzymic activity of this type of reduction can be that lower enzyme concn, lower enzyme ratio are lived or its combination.Many different methods can be used for making that yeast has the enzymic activity of reduction.For example, can engineered yeast cell, to have the locus that suffers the destructive codase, it uses mutagenesis commonly used or the technology of knocking out to realize.Referring to for example Methods inYeast Genetics (version in 1997), Adams, Gottschling, Kaiser, and Stems, Cold SpringHarbor Press (1998).

Perhaps, can use antisense technology to reduce enzymic activity.For example, can engineered yeast, to contain the cDNA of encoding antisense molecule, this antisense molecule stops enzyme to generate.Term " antisense molecule " as used herein, is contained any nucleic acid molecule that contains the sequence corresponding with endogenous peptide coding chain.Antisense molecule also can have flanking sequence (for example regulating sequence).So, antisense molecule can be ribozyme or antisense oligonucleotide.Ribozyme can have any universal architecture, includes, but not limited to hair clip, tup or axe (axhead) structure, and prerequisite is this molecule cutting RNA.

Yeast with enzymic activity of reduction can use any method to identify.For example, can use common method easily to identify the active yeast of the alcoholdehydrogenase with reduction, for example form by measuring ethanol through gas chromatography.

In one embodiment, can use two step processes to generate propyl carbinol from one of metabolic engineering bacterial strain of present disclosure.Because high-caliber butanols (for example is 1.5% in substratum, this generally changes with yeast and bacterial classification) can be virose for cell, a kind of strategy that obtains a large amount of propyl carbinols is to cultivate can not generate butanols therein or only generate the bacterial strain that generates propyl carbinol under the condition of butanols of inapparent, avirulent amount.This step is allowed the accumulation of a large amount of viable cell, and promptly the biomass of significant quantity can convert thereof into the growth conditions that wherein generates propyl carbinol then.Such strategy is allowed a large amount of propyl carbinols of generation before toxicity problem becomes important and slows down the cell growth.For example, culturing cell (wherein propyl carbinol generates and to be contained or lack) under aerobic conditions converts anaerobism or little aerobic condition to then to generate propyl carbinol (for example by activation according to the present invention by the engineered pathways metabolism that suits of going in the bacterial classification).Perhaps, the expression of relevant enzyme can be under induction type control, for example heat sensitivity promotor or other heat sensitivity step (such as the thermostability of enzyme self), make and close (non-activity) with relevant approach or enzyme when first step takes place, induce generation (for example temperature transition) and propyl carbinol to generate.The method that is used for gene is carried out induction type control is known.Heat-staple enzyme is known, perhaps can select by means known in the art.As in other process of present disclosure,, just can reclaim it according to an embodiment in case generate propyl carbinol.

Be used for being disclosed in the U.S. Provisional Application serial number of submitting on December 3rd, 2,007 11/949,724, by addressing income this paper from the process of microorganism (comprising yeast) recovery propyl carbinol.

Those skilled in the art can understand, and can carry out various omissions, interpolation and modification to invention as described above, and not depart from scope of the present invention, and all these type of modifications and variations intentions drop in the invention scope of appended claims qualification.Take in this paper at this by addressing all reference, patent, patent application and other file that to be quoted.

Embodiment

Table 1 has been enumerated one group of gene described in the embodiment 1-38.The relevant primer (forward with reverse) of each gene that can be used for increasing and the sequence of each primer have been provided.Gene is according to the suitable naming rule of each species is enumerated; In some cited gene front two letters are arranged, they have represented the genus of given gene origin and first letter of kind.For some gene title, with suffix " co ", indication uses bacteria Escherichia coli or the preferred codon rate of utilization of yeast yeast saccharomyces cerevisiae to make up the synthetic gene that codon is optimized, as specified in the text.

Table 1

Gene	Gene SEQ?? ID?? NO：	?? The primer title	?? SEQ?? ID?? NO：	?? Primer sequence
					??Cb-hbd	??155	??Gevo-311	??42	??GAGGTTGTCGACATGAAAAAGATTTTTGTACTTGGAG
		??Gevo-175	??43	??AATTGGATCCTTATTTAGAATAATCATAGAATCCT
					??Cb-crt	??156	??Gevo-312	??44	??GTTCTTGTCGACATGGAATTAAAAAATGTTATTCTTG
		??Gevo-171	??45	??AATTGGATCCTTATTTATTTTGAAAATTCTTTTCTGC
					??Cb-bcd	??157	??Gevo-313	??46	??CAAGAGGTCGACATGAATTTCCAATTAACTAGAGAAC
		??Gevo-314	??47	??GCGTCCGGATCCCTATCTTAAAATGCTTCCTGCG
					??Cb-etfA	??158	??Gevo-315	??48	??CGGAAAGTCGACATGAATATAGCAGATTACAAAGGC
		??Gevo-173	??49	??AATTGGATCCTTATTCAGCGCTCTTTATTTCTTTA
					??Cb-etfB	??159	??Gevo-316	??50	??CAAAATGTCGACATGAATATAGTAGTTTGTGTAAAAC
		??Gevo-317	??51	??TAATTTGGATCCTTAGATGTAGTGTTTTTCTTTTAAT
					??Cb-??adhA	??160	??Gevo-319	??52	??GAACCAGTCGACATGGCACGTTTTACTTTACCAAG
		??Gevo-177	??53	??AATTGGATCCTTACAAATTAACTTTAGTTCCATAG
					??Cb-aldh	??161	??Gevo-318	??54	??TCCATAGTCGACATGAATAAAGACACACTAATACCT
		??Gevo-249	??55	??AATTGGATCCTTAGCCGGCAAGTACACATCTTCTTTGTC??T
					??Ca-thl	??162	??Gevo-308	??56	??GATCGAGTCGACATGAAAGAAGTTGTAATAGCTAG
		??Gevo-309	??57	??GTTATAGGATCCCTAGCACTTTTCTAGCAATATTG
					??Ca-hbd	??163	??Gevo-281	??58	??GTGGATGTCGACATGAAAAAGGTATGTGTTATAGGTG
		??Gevo-161	??59	??AATTGGATCCTTATTTTGAATAATCGTAGAAACCT
					??Ca-crt	??164	??Gevo-282	??60	??TCCTACGTCGACATGGAACTAAACAATGTCATCCT
		??Gevo-283	??61	??TAACTTGGATCCCTATCTATTTTTGAAGCCTTCAAT
					??Ca-bcd	??165	??Gevo-284	??62	??CAAGAGGTCGACATGGATTTTAATTTAACAAGAGAAC
		??Gevo-285	??63	??CAATAAGGATCCTTATCTAAAAATTTTTCCTGAAATAAC
					??Ca-etfA	??166	??Gevo-286	??64	??CGGGAAGTCGACATGAATAAAGCAGATTACAAGGGC
		??Gevo-287	??65	??GTTCAAGGATCCTTAATTATTAGCAGCTTTAACTTG
					??Ca-etfB	??167	??Gevo-288	??66	??CAAAATTGTCGACATGAATATAGTTGTTTGTTTAAAAC
		??Gevo-289	??67	??GTTTTAGGATCCTTAAATATAGTGTTCTTCTTTTAATTTT

?? Gene	?? Gene?? SEQ?? ID?? NO：	?? The primer title	?? SEQ?? ID?? NO：	?? Primer sequence
									??G
??Ca-??adhE2	??168	??Gevo-292	??68	??CAAGAAGTCGACATGAAAGTTACAAATCAAAAAGAAC
							??Gevo-293	??69	??TCCTATGCGGCCGCTTAAAATGATTTTATATAGATATCC??T
??Ca-aad	??169	??Gevo-290	??70	??AGGAAAGTCGACATGAAAGTCACAACAGTAAAGGA
							??Gevo-291	??71	??ATTTAAGCGGCCGCTTAAGTTGTTTTTTAAAACAATTT??A
??Ca-??bdhA	??170	??Gevo-294	??72	??CATAACGTCGACATGCTAAGTTTTGATTATTCAATAC
							??Gevo-247	??73	??AAT?TGGATCCTTAATAAGATTTTTTAAATATCTCAA
??Ca-??bdhB	??171	??Gevo-295	??74	??CATAACGTCGACATGGTTGATTTCGAATATTCAATAC
							??Gevo-159	??75	??AATTGGATCCTTACACAGATTTTTTGAATATTTGTA
??Ca-thl-??co	??1	??Gevo-310	??76	??GATCGAGAATTCATGAAAGAAGTTGTAATAGCTAG
							??Gevo-309	??77	??GTTATAGGATCCCTAGCACTTTTCTAGCAATATTG
??Ca-hbd-??co	??2	??Gevo-296	??78	??CGGATAGTCGACATGAAAAAGGTATGTGTTATAGGC
							??Gevo-297	??79	??TCCCAAGGATCCTTATTTTGAATAATCGTAGAAACCCT
??Ca-crt-??co	??3	??Gevo-282	??80	??TCCTACGTCGACATGGAACTAAACAATGTCATCCT
							??Gevo-283	??81	??TAACTTGGATCCCTATCTATTTTTGAAGCCTTCAAT
??Ca-bcd-??co	??4	??Gevo-284	??82	??CAAGAGGTCGACATGGATTTTAATTTAACAAGAGAAC
							??Gevo-298	??83	??GTAAAGGGATCCTTAACTAAAAATTTTTCCTGAAATG
??Ca-eftA-??co	??5	??Gevo-286	??84	??CGGGAAGTCGACATGAATAAAGCAGATTACAAGGGC
							??Gevo-299	??85	??GTTCAAGGATCCTTAATTATTAGCAGCTTTAACCTG
??Ca-eftB-??co	??6	??Gevo-288	??86	??CAAAATTGTCGACATGAATATAGTTGTTTGTTTAAAAC
							??Gevo-300	??87	??GACTTTGGATCCTTAAATATAGTGTTCTTCTTTCAG
??Ca-??adhE2-??co	??7	??Gevo-292	??88	??CAAGAAGTCGACATGAAAGTTACAAATCAAAAAGAAC
							??Gevo-301	??89	??ATTTTCGGATCCTTAAAATGATTTTATATAGATATCTTTT??A
??Me-bcd-??co	??8	??Gevo-302	??90	??CTTATAGTCGACATGGATTTTAACTTAACAGATATTC
							??Gevo-303	??91	??CCGCCAGGATCCTTAACGTAACAGAGCACCGCCGGT
??Me-effA-??co	??9	??Gevo-304	??92	??CGGAAAGTCGACATGGATTTAGCAGAATACAAAGGC
							??Gevo-305	??93	??CTTTGTGGATCCTTATGCAATGCCTTTCTGTTTC
??Me-eftB-??co	??10	??Gevo-306	??94	??CAAACTGAATTCATGGAAATATTGGTATGTGTCAAAC
							??Gevo-307	??95	??ACCAACGGATCCTTAAATGATTTTCTGGGCAACCA
??ERG10	??154	??Gevo-273	??96	??GTTACAGTCGACATGTCTCAGAACGTTTACATTG
							??Gevo-274	??97	??GATAACGGATCCTCATATCTTTTCAATGACAATAG
??IpdA	??20	??Gevo-610	??119	??ttttGTCGACACTAGTatgagtactgaaatcaaaactcagggtcgtg
							??Gevo-611	??120	??ttttCTCGAGttacttcttcttcgctttcgggttcgg

?? Gene	Gene SEQ?? ID?? NO：	?? The primer title	?? SEQ?? ID?? NO：	?? Primer sequence
					??aceE	??21	??Gevo-606	??116	??ttttGTCGACACTAGTatgtcagaacgtttcccaaatgacgtgg
		??Gevo-607	??117	??ttttCTCGAGttacgccagacgcgggttaactttatctg
					??aceF	??22	??Gevo-653	??136	??ttttGTCGACACTAGTatggctatcgaaatcaaagtaccggacatcggg
		??Gevo-609	??118	??ttttCTCGAGttacatcaccagacggcgaatgtcagacag
					??PDA1	??23	??Gevo-660	??143	??ttttCTCGAGactagtATGgcaactttaaaaacaactgataagaagg
		??Gevo-661	??144	??ttttagatctTTAATCCCTAGAGGCAAAACCTTGC
					??PDB1	??24	??Gevo-662	??145	??ttttCTCGAGactagtATGgcggaagaattggaccgtgatgatg
		??Gevo-663	??146	??tttGGATCCTTATTCAATTGACAAGACTTCTTTGACAG
					??PDX1	??25	??Gevo-664	??147	??TtttCTCGAGactagtATGttacttgctgtaaagacattttcaatgcc
		??Gevo-665	??148	??ttttggatccTCAAAATGATTCTAACTCCCTTACGTAATC
					??LAT1	??26	??Gevo-656	??139	??ttttCTCGAGgctagcATGGCATCGTACCCAGAGCACACCAT??TATTGG
		??Gevo-657	??140	??ttttGGATCCTCACAATAGCATTTCCAAAGGATTTTCAAT
					??LPD1	??27	??Gevo-658	??141	??ttttCTCGAGactagtATGGTCATCATCGGTGGTGGCCCTGC??TGG
		??Gevo-659	??142	??ttttGGATCCTCAACAATGAATAGCTTTATCATAGG
					??PDC1	??28	??Gevo-639	??129	??ttttctcgagactagtATGTCTGAAATTACTTTGGG
		??Gevo-640	??130	??ttttggatccTTATTGCTTAGCGTTGGTAGCAGCAG
					??CUP1??prom	??178	??Gevo-637	??127	??ttttGAGCTCgccgatcccattaccgacatttggg
		??Gevo-638	??128	??aaaGTCGACaccgatatacctgtatgtgtcaccaccaatgtatctataagtatc??catGCTAGCCCTAGGtttatgtgatgattgattgattgattg
					??pflA	??36	??PflA_forw	??98	??cattgaattcatgtcagttattggtcgcattcac
		??PflA_Rev	??99	??cattgtcgacttagaacattaccttatgaccgtactg
					??pflB	??37	??PflB_forw	??100	??cattgaattcatgtccgagcttaatgaaaagttagcc
		??PflB_Rev	??101	??cattgtcgacttacatagattgagtgaaggtacgag
					??Cb-??FDH1	??38	??fdh1_forw	??102	??cattgaattcatgaagatcgttttagtcttatatggtgc
		??fdh1_rev	??103	??cattgtcgacttatttcttatcgtgtttaccgtaagc
					??KIALD6	??39	??KIALD6_rig??ht3	??104	??gttaggatccttaatccaacttgatcctgacggccttg
		??KIALD6_Lef??t5	??105	??ccaagtcgacatgtcctctacaattgctgagaaattgaacctc
					??KIACS1	??40	??KIACS1_Ri??ght3	??106	??gttagcggccgcttataatttcacggaatcgatcaagtgc
		??KIACS1_Lef??t5	??107	??ccaagctagcatgtctcctgctgttgataccgcttcc
					??KIACS2	??41	??KIACS2_rig??ht3	??108	??ggttggatccttatttcttctgctgactgaaaaattgattttctactgc
		??KIACS2_Lef??t5	??109	??ccaagaattcatgtcgtcggataaattgcataagg
					??ACS1	??30	??Gevo-479	??112	??catgccgtcgacatgtcgccctctgccgtac
		??Gevo-480	??113	??gattaagcggccgcttacaacttgaccgaatcaattag
					??ACS2	??31	??Gevo-483	??114	??gatgaagtcgacatgacaatcaaggaacataaagtag
		??Gevo-484	??115	??gttaaaggatccttatttctttttttgagagaaaaattg
					??ALD6	??29	??Gevo-643	??133	??ccaagtcgacatgactaagctacactttgacac
		??Gevo-644	??134	??gtcggtaagagtgttgctgtggactcg
					??Ca-ter	??179	??Gevo-345	??183	??atgtttgtcgacatgatagtaaaagcaaagtttgta
		??Gevo-346	??184	??cttaatgcggccgcttaaggttctaattttcttaataattc
					??Ah-ter	??180	??Gevo-343	??185	??Gcttgagtcgacatgatcattaaaccgaaagttcg

?? Gene	Gene SEQ?? ID?? NO：	?? The primer title	?? SEQ?? ID?? NO：	?? Primer sequence
							??Gevo-344	??186	??atttaaggatcctcacagttcgacaacatcaaattta
??Eg-ter	??181	??Gevo-347	??187	??catcacgtcgacatggccatgttcaccactac
							??Gevo-348	??188	??ctcgcgggatccttactgctgagctgcgctc
??Sc-ccr	??182	??Gevo-341	??189	??gtcttagtcgacatgaccgtgaaagacattctg
							??Gevo-342	??190	??attggcggatcctcacacattacggaaacggtta

Table 2 has been enumerated one group of plasmid construction body and features relevant thereof, as described in the embodiment.Comprise related plasmid title (pGV) in the table; The prototroph sign that exists, its for plasmid in appropriate nutrition defective type bacterial classification selection and to keep be useful; Promoter sequence (from given genes of brewing yeast zone); Gene under the aforementioned promotor control; Other promotor+assortment of genes, if present.

Table 2: the summary sheet of the features relevant of the plasmid among the embodiment

Title	The prototroph sign	Promotor 1	Gene 1	Promotor 2	Gene 2
						??pGV1099	??HIS3	??TEF1	??(AU1?tag)
??pGV1100	??TRP1	??TEF1	??(HA?tag)
						??pGV1101	??LEU2	??TEF1	??(AU1?tag)
??pGV1102	??URA3	??TEF1	??(HA?tag)
						??pGV1103	??HIS3	??TDH3	??(myc?tag)
??pGV1104	??TRP1	??TDH3	??(myc?tag)
						??pGV1105	??LEU2	??TDH3	??(myc?tag)
??pGV1106	??URA3	??TDH3	??(myc?tag)
						??pGV1208	??TRP1	??TEF1	??Ca-hbd-co
??pGV1209	??LEU2	??TEF1	??Ca-crt-co
						??pGV1213	??URA3	??TEF1	??Ca-adhE2-co
??pGV1214	??HIS3	??TDH3	??Me-bcd-co
						??pGV1217	??TRP1	??TEF1	??Ca-hbd-co	??TDH3	??Ca-eftA-co
??pGV1218	??LEU2	??TEF1	??Ca-crt-co	??TDH3	??Ca-eftB-co
						??pGV1219	??HIS3	??TEF1	??ScERG10	??TDH3	??Me-bcd-co
??pGV1220	??HIS3	??TEF1	??Ca-thl-co	??TDH3	??Ca-bcd-co
						??pGV1221	??TRP1	??TEF1	??Ca-hbd-co	??TDH3	??Me-eftA-co
??pGV1222	??LEU2	??TEF1	??Ca-crt-co	??TDH3	??Me-eftB-co
						??pGV1223	??HIS3	??TEF1	??ScERG10	??TDH3	??Ca-bcd-co
??pGV1224	??HIS3	??TEF1	??Ca-thl-co	??TDH3	??Me-bcd-co
						??pGV1225	??HIS3	??TEF1	??Ca-thl-co	??TDH3	??Ca-ter
??pGV1226	??HIS3	??TEF1	??Ca-thl-co	??TDH3	??Ah-ter

Title	The prototroph sign	Promotor 1	Gene 1	Promotor 2	Gene 2
						??pGV1227	??HIS3	??TEF1	??Ca-thl-co	??TDH3	??Eg-ter
??pGV1228	??HIS3	??TEF1	??Ca-thl-co	??TDH3	??Sc-ccr
						??pGV1262	??LEU2	??TEF1	??ScACS1
??pGV1263	??URA3	??TEF1	??ScACS2
						??pGV1319	??URA3	??TDH3	??Ca-AdhE2_co	??TEF1	??ACS1
??pGV1320	??URA3	??TDH3	??Ca-AdhE2_co	??TEF1	??ACS2
						??pGV1321	??LEU2	??TDH3	??ALD6
??pGV1326	??LEU2	??TEF1	??ALD6
						??pGV1334	??HIS3	??TDH3	??/pdA
??pGV1339	??LEU2	??TEF1	??Ca_Crt_co	??TDH3	??ALD6
						??pGV1379	??HIS3	??TDH3	??aceE
??pGV1380	??HIS3	??TDH3	??aceF
						??pGV1381	??HIS3	??TDH3	??LAT1
??pGV1383	??HIS3	??TDH3	??PDA1
						??pGV1384	??HIS3	??TDH3	??PDB1
??pGV1385	??HIS3	??TDH3	??PDX1
						??pGV1388	??URA3	??CUP1	??n/a
??pGV1389	??URA3	??TDH3	??PDC1
						??pGV1399	??LEU2	??TEF1	??Ca-hbd-co	??TDH3	??ALD6
??pGV1414	??URA3	??MET3	??n/a
						??pGV1428	??HIS3	??TDH3	??n/a
??pGV1429	??TRP1	??TDH3	??n/a
						??pGV1430	??LEU2	??TDH3	??n/a
??pGV1483	??URA3	??MEt3	??n/a
						??pGV1603	??TRP1	??TDH3	??aceE
??pGV1604	??LEU2	??TDH3	??aceF
						??pGV1605	??URA3	??TEF1	??adhE2	??TDH3	??PDC1
??1102Fdh1	??URA3	??TEF1	??Cb-FDH1
						??1103PflA	??HIS3	??TDH3	??pflA
??1104PflB	??TRP1	??TDH3	??pflB
						??1208_PflA	??TRP1	??TEF1	??Ca_hbd_co	??TDH3	??pflA
??1208KI	??HIS3	??TEF1	??Ca_hbd_co
						??1208KIALD6	??HIS3	??TEF1	??Ca_hbd_co	??TDH3	??KIALD6
??1208KIPflA	??HIS3	??TEF1	??Ca_hbd_co	??TDH3	??pflA
						??1208KIPflA	??TRP1	??TEF1	??Ca_Crt_co	??TDH3	??pflB
??1208-IpdA	??TRP1	??TEF1	??thl	??TDH3	??-IpdA
						??1209_PflB	??LEU2	??TEF1	??Ca_Crt_co	??TDH3	??pflB
??1209-aceE	??LEU2	??TEF1	??crt	??TDH3	??aceE
						??1209KI	??TRP1	??TEF1	??Ca_Crt_co

Title	The prototroph sign	Promotor 1	Gene 1	Promotor 2	Gene 2
						??1209kIACS1	??LEU2	??TEF1	??Ca_Crt_co	??TDH3	??KIACS1
??1209kIACS2	??LEU2	??TEF1	??Ca_Crt_co	??TDH3	??KIACS2
						??1213_Fdh1	??URA3	??TDH3	??Ca_AdhE2_co	??TEF1	??Cb-FDH1
??1213-aceF	??URA3	??TEF1	??adhE2	??TDH3	??aceF
						??1213KI	??URA3	??TDH3	??Ca_AdhE2_co
??1213KIPflA	??LEU2	??TEF1	??Ca_thl_co	??TDH3	??Cb-FDH1
						??1227KI	??LEU2	??TEF1	??Ca_thl_co	??TDH3	??Eg-TER-co
??1388-PDC1	??URA3	??CUP1	??PDC1
						??1428_PflA	??HIS3	??TDH3	??pflA
??1428ALD6	??HIS3	??TDH3	??KIALD6
						??1428-IpdA	??HIS3	??TDH3	??IpdA
??1429_PflB	??TRP1	??TDH3	??pflB
						??1429-aceE	??TRP1	??TDH3	??aceE
??1429ACS1	??TRP1	??TDH3	??KIACS1
						??1430_Fdh1	??LEU2	??TDH3	??Cb-FDH1
??1430-aceF	??LEU2	??TDH3	??aceF
						??1431ACS2	??URA3	??TDH3	??KIACS2
??pGV1103-??Ipd1	??HIS3	??TDH3	??LPD1

Table 3 has been described the butanols that is generated in yeast wine brewing (bacterial strain W303a) yeast that carries various plasmids and express one group of gene that is imported (as enumerating) thus

Table 3: the butanols of yeast saccharomyces cerevisiae transformant generates

The conivium title	The plasmid combination	The gene that is imported	Butanols amount 72h p.i (μ M)
				??Gevo?1094；??Gevo?1095	??pGV1208；??pGV1209；??pGV1225；??pGV1213	??Ca-hbd-co；Ca-Crt-co；??Ca-thl-co+Ca-ter；Ca-??adhE2-co	??129；145
??Gevo?1096；??Gevo?1097	??pGV1208；??pGV1209；??pGV1226；??pGV1213	??Ca-hbd；Ca-Crt；Ca-thl-??co+Ah-ter；Ca-adhE2-??co	??207；216
				??Gevo?1098；??Gevo?1099	??pGV1208；??pGV1209；??pGV1227；??pGV1213	??Ca-hbd；Ca-Crt；Ca-thl-??co+Eg-ter；Ca-adhE2-??co	??251；313
??Gevo?1100，??Gevo?1101	??pGV1208；??pGV1209；	??Ca-hbd；Ca-Crt；Ca-thl-??co+Sc-ter；Ca-adhE2-	??109；109

All gene clones and combination rules are to use method (Miller, J.H., 1992 of having set up at first; Sambrook, J. etc., 2001) in intestinal bacteria, implement.

For at one group of useful carrier of yeast Expression in Saccharomyces Cerevisiae (Mumberg, D. etc. (1995) before on the books Gene156:119-122; Sikorski and Heiter (1989) Genetics122:19-27).Particularly, these publications have been put down in writing a group selection sign (HIS3, LEU2, TRP1, URA3) and yeast saccharomyces cerevisiae replication orgin, and they also have use in cited many carriers in table 2.

Embodiment 1: the plasmid construction that is used for expressing at the yeast yeast saccharomyces cerevisiae butanols pathway gene

Just use and introduce the SalI site and just after terminator codon, introduce the primer in BamHI site at upstream from start codon, by PCR from the beginning home-brewed wine yeast strain W303a genomic dna cloning yeast saccharomyces cerevisiae thiolase gene ERG10.With this PCR product with SalI and BamHI digestion and be cloned into pUC19 (Yanisch-Perron, C, Vieira, J., 1985, Gene, 33, same loci 103-19) is to generate pGV1120.

Use plasmid pGV1031, pGV1037, pGV1094 and pGV1095 template respectively as pcr amplification acetone-butanol clostridium gene (Ca-) Ca-thl, Ca-hbd, Ca-crt and Ca-bdhB.Use the template of pGV1090 as pcr amplification Ca-bcd, Ca-etfA and Ca-etfB.Use the genomic dna of the clostridium ATCC824 Ca-bdhA that increases.Amplified fragments is digested and is cloned into the same loci of pUC19 with SalI and BamHI.This scheme generates plasmid pGV1121, pGV1122, pGV1123, pGV1124, pGV1125, pGV1126, pGV1127, pGV1128, and they contain gene C a-thl, Ca-hbd, Ca-crt, Ca-bcd, Ca-etfA, Ca-etfB, Ca-bdhA and Ca-bdhB respectively.

Use is designed to just introduce the SalI site and just introduce the primer in BamHI site in the terminator codon downstream at upstream from start codon, by pcr amplification Bai Shi clostridium (Cb-) gene C b-hbd, Cb-crt, Cb-bcd, Cb-etfA, Cb-etfB, Cb-aldh and Cb-adhA.Use plasmid pGV1050, pGV1049, pGV1096 and pGV1091 template respectively as pcr amplification Cb-hbd, Cb-crt, Cb-aldh and Cb-adhA.Use the template of the genomic dna of Bai Shi clostridium ATCC 51743 as Cb-bcd, Cb-etfA and Cb-etfB.Pcr amplified fragment is digested and is cloned into the same loci of pUC19 with SalI and BamHI.These rules generate plasmid pGV1129, pGV1130, pGV1131, pGV1132, pGV1133, pGV1134 and pGV1135, and they contain gene C b-hbd, Cb-crt, Cb-bcd, Cb-etfA, Cb-etfB, Cb-aldh and Cb-adhA respectively.

Also cloned to carry out codon optimized (co) clostridium acetobutylicum and Megasphaera elsdenii (Me-) gene at expression in escherichia coli.These genes comprise Ca-thl-co, Ca-hbd-co, Ca-crt-co, Ca-bcd-co, Ca-etfA-co, Ca-etfB-co, Ca-adhE2-co, Me-bcd-co, Me-etfA-co and Me-etfB-co.Use is designed to just introduce the SalI site and just introduce the primer in BamHI site in the terminator codon downstream in the upstream of initiator codon, amplification these genes except Ca-thl-co and Me-etfB-co.In the situation of Ca-thl-co and Me-etfB-co, design of primers becomes just to introduce in the upstream of initiator codon the EcoRI site and just introduces the BamHI site in the downstream of terminator codon.The same loci that gained PCR product is digested (SalI and BamHI or EcoRI and BamHI) with suitable restriction enzyme and is cloned into pUC19 is to generate plasmid pGV1197, pGV1198, pGV1199, pGV1200, pGV1201, pGV1202, pGV1203, pGV1205, pGV1206, and they contain gene C a-thl-co, Ca-hbd-co, Ca-crt-co, Ca-bcd-co, Ca-etfA-co, Ca-etfB-co, Ca-adhE2-co, Me-etfA-co and Me-etfB-co respectively.The Me-bcd-co gene directly is cloned among the pGV1103 to generate pGV1214 as the SalI-BamHI fragment.

Said gene is cloned into high copy Yeast expression carrier pGV1099, pGV1100, pGV1101, pGV1102, pGV1103, pGV1104, pGV1105 and pGV1106.The characteristic that is used for gene cloning carrier and gained plasmid construction body has been described in the table 2.

Use SalI and BamHI to discharge thiolase gene ERG10 and Ca-thl and be cloned into pGV1099 (carrying the HIS3 sign) from pGV1120 and pGV1121 respectively to generate pGV1138 and pGV1139.Use EcoRI and BamHI to take out the thiolase gene Ca-thl-co of codon optimization and be cloned into pGV1099 to generate pGV1207 from pGV1197.So, these gene clones become to meet two copies AU1 label (SEQ ID NO:172) reading frame and use yeast saccharomyces cerevisiae TEF1 promoter region (SEQ IDNO:175) to express.Use SalI and BamHI respectively hydroxyl butyryl-CoA-dehydrogenase gene Ca-hbd (from pGV1122), Cb-hbd (from pGV1129) and Ca-hbd-co (from pGV1198) to be cloned into pGV1100 (carrying the LEU2 sign) to generate pGV1140, pGV1141 and pGV1208.This causes these gene clones to become to meet the reading frame of HA label (SEQ ID NO:173) and use the TEF1 promoter expression.Use SalI and BamHI respectively enoyl-CoA hydratase gene C a-crt (from pGV1123), Cb-crt (from pGV1130), Ca-crt-co (from pGV1199) to be cloned into pGV1101 (carrying the TRP1 mark) to generate pGV1142, pGV1143 and pGV1209.So, these gene clones become to meet two copies the AU1 label reading frame and use the TEF1 promoter expression.

Butyryl-CoA desaturase is cloned into myc label (SEQ ID NO:174) back with corresponding electron transport gene etfA and etfB, uses from the TDH3 promoter region (SEQ ID NO:176) of yeast saccharomyces cerevisiae and express.Ca-bcd (from pGV1124), Cb-bcd (from pGV1131), Ca-bcd-co (from pGV1200) and Me-bcd-co gene clone are gone into pGV1103 (carrying the HIS3 sign) to generate pGV1144, pGV1145, pGV1210 and pGV1214.Respectively Ca-etfA (from pGV1125), Ca-etfB (from pGV1126), Cb-etfA (from pGV1132), Cb-etfB (from pGV1133), Ca-etfB-co (from pGV1202) and Me-etfA-co (from pGV1205) gene clone are gone into pGV1104 (carrying the LEU2 sign) to generate pGV1146, pGV1147, pGV1148, pGV1149, pGV1212 and pGV1215.Respectively Ca-etfA-co (from pGV1201) and Me-etfB-co (from pGV1206) are cloned into pGV1104 (carrying the TRP1 sign) to generate pGV1211 and pGV1216.

Aldehyde dehydrogenase gene Cb-aldh (from pGV1134) is cloned into pGV1102 (carrying the URA3 sign) to generate pGV1150.The Cb-aldh gene is placed to the reading frame that meets HA label (SEQ ID NO:173), uses the TEF1 promoter expression.Difunctional aldehyde/alcoholdehydrogenase Ca-aad, Ca-adhE2 and Ca-adhE2-co and certain alcohols desaturase Ca-bdhA, Ca-bdhB and Cb-adhA are cloned into myc label back, under the control of TDH3 promotor, express.Use is designed to just introduce the SalI site and just introduce the primer in NotI site in the terminator codon downstream at upstream from start codon, by pcr amplification Ca-aad and Ca-adhE2.Use the template of plasmid pGV1089, and use the template of acetone-butanol clostridium gene group DNA as Ca-adhE2 as Ca-aad.Use SalI and NotI that these PCR product cloning are gone into pGV1106 (carrying the URA3 sign) to generate pGV1136 (Ca-aad) and pGV1137 (Ca-adhE2).The Ca-adhE2-co (from pGV1203) that uses SalI and BamHI that codon is optimized is cloned into pGV1106 to generate pGV1213.Use SalI and BamHI respectively alcoholdehydrogenase Ca-bdhA (from pGV1127), Ca-bdhB (from pGV1128) and Cb-adhA (from pGV1135) to be cloned into pGV1106 to generate pGV1151, pGV1152 and pGV1153.

Therefore, the yeast expression gene mentioned above of butyryl-coA desaturase, electron transfer protein A, electron transfer protein B and certain alcohols desaturase and thiolase, hydroxyl butyryl-CoA desaturase, enoyl-CoA hydratase or the aldehyde dehydrogenase of TEF1 promoters driven are made up in paired mode, as gathering in the table 2.

For this purpose, the NotI (filling and leading up) that will go into pGV1138 from pGV1144 (TDH3 promotor and Ca-bcd) with from EcoICRI to the XhoI fragment cloning of pGV1145 (TDH3 promotor and Cb-bcd) respectively with Klenow to the XhoI site to generate pGV1167 (ERG10+Ca-bcd) and pGV1168 (ERG10+Cb-bcd).Also respectively that these are identical EcoICRI to XhoI fragment is cloned into pGV1139 similarly to generate pGV1169 (Ca-thl+Ca-bcd) and pGV1170 (Ca-thl+Cb-bcd).Use identical strategy, respectively will be from pGV1146 (TDH3 promotor and Ca-etfA), pGV1148 (TDH3 promotor and Ca-etfB), pGV1147 (TDH3 promotor and Cb-etfA), and EcoICRI to the XhoI fragment cloning of pGV1149 (TDH3 promotor and Cb-etfB) is gone into pGV1140, pGV1141, pGV1142, the NotI of pGV1143 (filling and leading up) with Klenow to the XhoI site to generate pGV1171 (Ca-hbd+Ca-etfA), pGV1172 (Ca-crt+Ca-etfB), pGV1173 (Cb-hbd+Cb-etfA), and pGV1174 (Cb-crt+Cb-etfB).Similarly (the filling and leading up) by will going into pGV1150 from EcoICRI to the XhoI fragment cloning of pGV1151 (TDH3 promotor and Ca-bdhA), pGV1152 (TDH3 promotor and Ca-bdhB) and pGV1153 (TDH3 promotor and Cb-adhA) respectively with Klenow to the XhoI site to generate pGV1175 (Cb-aldh+Ca-bdhA), pGV1176 (Cb-aldh+Ca-bdhB) and pGV1177 (Cb-aldh+Cb-adhA), with aldehyde dehydrogenase and alcoholdehydrogenase combination.

In the situation of the gene that codon is optimized, respectively will from EcoICRI to the XhoI fragment cloning of pGV1210 (TDH3 promotor and Ca-bcd-co), pGV1211 (TDH3 promotor and Ca-etfA-co), pGV1212 (TDH3 promotor and Ca-etfB-co) go into the BamHI of pGV1207, pGV1208 and pGV1209 (mend with Klenow flat) to the XhoI site to generate pGV 1220 (Ca-thl-co+Ca-bcd-co), pGV1217 (Ca-hbd-co+Ca-etfA-co) and pGV1218 (Ca-crt-co+Ca-etfB-co).To go into same group of carrier to generate pGV1224 (Ca-thl-co+Me-bcd-co), pGV 1221 (Ca-hbd-co+Me-etfA-co) and pGV1222 (Ca-crt-co+Me-etfB-co) from EcoICRI to the XhoI fragment cloning of pGV1214 (TDH3 promotor and Me-bcd-co), pGV1215 (TDH3 promotor and Me-etfA-co), pGV1216 (TDH3 promotor and Me-etfB-co) respectively.In addition, the BamHI (mend with Klenow flat) that will go into pGV1138 from pGV1210 (TDH3 promotor and Ca-bcd-co) with from EcoICRI to the XhoI fragment cloning of pGV1214 (TDH3 promotor and Me-bcd-co) to the XhoI site to generate pGV1223 (ERG10+Ca-bcd-co) and pGV1219 (ERG10+Me-bcd-co).

Outside above-mentioned approach, generated the surrogate that utilizes the bcd/etfA/etfB mixture, the construct of promptly trans-enoyl reductase and crotonoyl-CoA reductase enzyme.Cloned trans-enoyl reductase gene from clostridium acetobutylicum (Ca-ter), Aeromonas hydrophila (Ah-ter) and very thin eye worm (Eg-ter), and (Sc-ccr) cloned crotonoyl-coA reductase enzyme from massif streptomycete (Streptomyces collinus).Use just is designed to introduce the SalI site and just at the primer in introducing NotI site, terminator codon downstream at upstream from start codon, from acetone-butanol clostridium gene group DNA pcr amplification Ca-fer.Use just is designed to introduce in the upstream of initiator codon the SalI site and just at the primer in the downstream of terminator codon introducing BamHI site, respectively from pGV1114, pGV1115 and pGV1166PCR increased Ah-ter, Eg-ter and Sc-ccr.The sequence of these three kinds of genes has been carried out codon optimized for the expression in the intestinal bacteria.Also have, Eg-ter sequence encoding disappearance may involve the protein of the localized N-stub area of plastosome.Use suitable restriction enzyme that corresponding PCR product cloning is gone into pGV1103 to generate pGV1155 (Ca-ter), pGV1156 (Ah-ter), pGV1157 (Eg-ter) and pGV1158 (Sc-ccr).

In order to be used for expressing the butanols approach, each and thiolase gene in these surrogates of bcd/etfA/etfB mixture are made up on a plasmid at yeast.With Ca-ter, Ah-ter, Eg-ter and Sc-ccr gene and the Ca-thl-co assortment of genes, the BamHI (mend with Klenow flat) by will going into pGV1207 from EcoICRI to the XhoI fragment cloning of pGV1155, pGV1156, pGV1157 and pGV1158 respectively to the XhoI site to generate pGV1225 (Ca-thl-co+Ca-ter), pGV 1226 (Ca-thl-co+Ah-ter), pGV1227 (Ca-thl-co+Eg-ter) and pGV1228 (Ca-thl-co+Sc-ccr).

Embodiment 2: yeast extract/Western engram analysis

Express for analysing protein, prepare rough yeast protein extract by quick TCA precipitation scheme.Collect 1 normal cell of OD600 and handling 10 minutes with 200 μ L 1.85N NaOH/7.4%2-mercaptoethanols on ice.Add 200 μ L 50%TCA and with sample at incubation 10 minutes more on ice.By with 25,000rcf collected in centrifugal 2 minutes sedimentary protein and clean with the ice-cold acetone of 1ml.Pass through with 25 once more, 000rcf collected protein in centrifugal 2 minutes.Then throw out is resuspended in the SDS sample buffer and boiled (99 ℃) 10 minutes.With sample in whizzer with centrifugal 30 seconds of maximum speed to remove insolubles.

Nitrocellulose is opened and be transferred to sample by the SDS-PAGE branch.Using TMB Western trace test kit (KPL) to carry out Western analyzes.HA.11, myc (9E10) and AU1 antibody derive from Covance.As manufacturers is described, implement Westerns, just when using myc antibody, use the 0.3x to 0.5x that is supplemented with 1% detection piece powder (detector block powder) to detect piece solution (detectorblock solution).Utilize this method to check all expression of gene described in the embodiment 1.

Embodiment 3: yeast conversion

Use lithium acetate method (Gietz, R.D.a.R.A.W., 2002, Methods in Enzymology, 350,87-96) carried out yeast saccharomyces cerevisiae (W303a) and transformed.In brief, 1ml is spent the night yeast culture dilutes the fresh YPD substratum of 50mL and in 30 ℃ of shaking tables incubation 5-6 hour.Collecting cell is used the 50mL sterile water wash, uses the 25mL sterile water wash again.With 1mL 100mM lithium acetate re-suspended cell and be transferred to Eppendorf tube.By coming sedimentation cell in centrifugal 10 seconds.Abandon supernatant liquor and cell is resuspended in the 100mM lithium acetate of 4 times of volumes.15 μ L cells are added into DNA mixture (plasmid DNA of 72 μ L 50%PEG, 10 μ L 1M lithium acetates, 3uL 10mg/ml sex change salmon sperm dna, every kind of expectation of 2 μ L and sterilized water are to cumulative volume 100 μ L).With sample in 30 ℃ of incubations 30 minutes and in 42 ℃ of heat shocks 22 minutes.Come collecting cell by centrifugal 10 seconds then, be resuspended in 100 μ L SOS substratum (Sambrook, J., Fritsch, E.F., Maniatis, T., 1989), and do not containing suitable SC option board (Kaiser C, M., the S. and the Mitchel of uridylic, tryptophane, leucine or Histidine, A, 1994) upward coating.

Embodiment 4: the generation of propyl carbinol

To expressing transformant (above table 1) the assessment propyl carbinol generation that generates the various combination of the relevant enzyme of approach with the butanols of being advised.Be prepared as follows the pre-culture of conivium, soon go into 3ml SC substratum (Kaiser C, M., S. and Mitchel, A, 1994), under aerobic conditions shook 16 hours with 250rpm in 30 ℃ from the minority colony inoculation of SC agar plate.The gained cell is precipitated 5 minutes and is resuspended in 500 μ lSC substratum with 4000xg.Assess the cell growth by suitable dilution 600nm absorbancy.For each conivium of being tested, it is previous with the saturated anaerobism balch pipe with the SC anaerobic culture medium of eliminating dissolved oxygen of N2 gas that the injection cell (200 μ l) that produces 15 OD is gone into to be equipped with 5ml.Pipe in 30 ℃ of incubations, is shaken with 250rpm and to prevent cell settlement.

After inoculation 10,26,44 and 70 hours to pipe sampling, promptly take out 500 μ l cultures with asepsis injector.Afterwards, 250 μ l, 40% glucose solution is injected into each pipe to keep carbon an amount of in the substratum.At each time point, the sample that is reclaimed is centrifugal with sedimentation cell, and freezing immediately supernatant liquor is until having collected all samples.

Propyl carbinol by gas chromatography (GC) assay determination transformant generates.All freezing samples are filtered by 0.2 μ m filter in the room temperature thawing and with 400 each sample of μ l and as the 80 μ l 10mM amylalcohols that internal contrast adds.200 μ l gained filtrates are placed the GC phial and carry out the GC analysis.On the serial II Plus gas chromatograph of the band flame ionization detector (FID) that is equipped with the HP-7673 automatic sampling system, move sample.Retention time based on the trusted standard product is come identification of analytes, and uses 5 working curves to come quantitatively.With 1 μ l volume injection all samples.Go up the direct analysis of implementing the propyl carbinol product at the DB-FFAP capillary column (30m length, 0.32mm ID, 0.25 μ m film thickness) that connects the FID detector.The temperature program(me) that is used for that pure product is separated is 225 ℃ of syringes, 225 ℃ of detectors, 50 ℃ of baking boxs 0 minute, then 8 ℃ of/minute gradients to 80 ℃, 13 ℃ of/minute gradients to 170 ℃, 50 ℃ of/minute gradients to 220 ℃, then 220 ℃ 3 minutes.

Generate in order to assess butanols, tested two independent transformant of each plasmid combination.Above table 3 has been summed up the result.Two Gevo titles under " conivium title " refer to make up two independent transformant being assessed for each plasmid.

Hereinafter (Fig. 6) shown that two kinds of best producers are that transformant Gevo1099 and Gevo1102 change the butanols amount that is generated in time with respect to being the conivium that Gevo1110 and Gevo1111 transform with empty carrier only.Gevo1099 and Gevo1102 show butanols generation rising in time, and 24-72 hour butanol concentration is increased to 313 μ M and is increased to 317 μ M from 57 μ M from 123 μ M respectively after inoculation.

Embodiment 5: clone and the expression of intestinal bacteria pyruvic oxidase subunit in yeast saccharomyces cerevisiae

The purpose of this embodiment is to describe escherichia coli cloning aceE, aceF and the lpdA gene that is included in three kinds of subunits of the pyruvic oxidase (PDH) that finds in the intestinal bacteria how certainly together.Use PCR from genomic dna this three kinds of genes that increase.This embodiment also illustration the protein of these three kinds of genes be to express in the yeast saccharomyces cerevisiae how at host organisms.

Use bacillus coli gene group DNA as template, by pcr amplification from colibacillary lpdA gene.For specific amplification lpdA, use primer Gevo-610 and Gevo-611; Supplied other pcr amplification reagent in the test kit of manufacturers, for example, KOD warm start polysaccharase (Novagen, Inc., products catalogue #71086-5), and use according to the scheme of manufacturers.Forward and reverse primer mix the Nucleotide in coding SalI and XhoI restriction endonuclease site respectively.Gained PCR product with SalI and XhoI digestion and be cloned into pGV1103, is produced pGV1334.To the complete order-checking of lpdA DNA of being inserted.

Use and similar way mentioned above, will insert pGV1334 from aceE and the aceF gene of intestinal bacteria.Use primer Gevo-606 and Gevo-607 from intestinal bacteria genomic dna amplification aceE gene,, and be cloned into the carrier pGV1334 that cuts through SalI+XhoI, produce pGV1379 with SalI+XhoI digestion.To the complete order-checking of aceE inset.In order to obtain to have the plasmid of the different prototroph selection markers that are suitable for the yeast saccharomyces cerevisiae expression, clone the aceE inset and be cloned into the pGV1104 that cuts through SalI+XhoI from pGV1379 as the SalI+XhoI fragment, produce pGV1603.

Use primer Gevo-653 and Gevo-609 from intestinal bacteria genomic dna amplification aceF gene.Gained 1.9kb product with SalI+XhoI digestion and be cloned into carrier pGV1334 through the same enzyme cutting, is produced pGV1380.To the complete order-checking of aceF inset.In order to obtain to have the plasmid of the different choice sign that is suitable for the yeast saccharomyces cerevisiae expression, clone the aceF inset and be cloned into pGV1105 from pGV1380, produce pGV1604.

In order in yeast saccharomyces cerevisiae, to express these protein, arbitrary combination transformed saccharomyces cerevisiae bacterial strain Gevo1187 (CEN.PK) with pGV1334, pGV1603 and pGV1604, and go up and select transformant at the suitable substratum (dropout media) of eliminating, as described in the embodiment 3.In contrast, be pGV1103, pGV1104 and pGV1105 difference transformant with corresponding empty carrier.It is following that the culture that obtains is measured LpdA, AceE or AceF expresses to cultivating from transformant, promptly prepare rough yeast protein extract and by Western engram analysis their (based on detecting existing Myc epi-position in each protein), as described in the embodiment 2.

Embodiment 6: from genomic dna cloning yeast saccharomyces cerevisiae PDH subunit, eliminate modification and their expression in brewing yeast cell of endogenous mitochondrial targeting sequence

In most of eukaryotes, pyruvic oxidase (PDH) mixture is positioned at plastosome inside.Rely on them to contain about 20-40 amino acid of so-called mitochondrial targeting sequence in their N-stub area, the range protein that comprises PDH is drawn towards and enters plastosome.The existence of such sequence can be by experiment or calculate (for example by program MitoProt: Http:// mips.qsf.de/cqi-bin/proi/medqen/mitofilter) measure.Behind the protein success input line plastochondria specific proteins hydrolysis cutting takes place, the target sequence is eliminated, and causes " through what cut " input form.As everyone knows, eliminating such sequence by the proteinic encoding sequence of hereditary change from this protein causes that this protein becomes and can not divide a word with a hyphen at the end of a line into plastosome.So, a kind of be used for the tempting strategy that the protein changed course of normal circumstances in plastosome enters cytosol involved only express coded protein remaining " through what cut " that part of gene partly after plastosome input and the cutting of follow-up proteolytic enzyme.

The purpose of this embodiment is to describe the clone of several genes that constitute the yeast saccharomyces cerevisiae pyruvate dehydrogenase complex, and expression and the detection of these genes in the brewing yeast cell culture.

By several genes of each subunit of PCR clones coding PDH, it uses the rules described in the embodiment 5 basically, and just template is genes of brewing yeast group DNA.The genes of brewing yeast and the employed corresponding primer that will increase have been shown in the table 1.

Be positioned at proteinic gene in the cytosol in order to generate coded prediction, each becomes the zone in that part of downstream of predictive coding mitochondrial targeting sequence in each gene of amplification to first kind of cited in the primer (cited in the table 1) design of primers.Use be used for the increasing coded unique restriction enzyme sites of primer of each gene is gone into carrier pGV1103 with gained PCR product cloning, produces cited plasmid in the table 2.To the complete order-checking of each inset.In order to test each expression of gene, only use each transformed saccharomyces cerevisiae bacterial strain Gevo1187 (CEN.PK) among pGV1381, pGV1383, pGV1384 or the pGV1385, it follows the rules described in the embodiment 3 basically, and selects the HIS+ bacterium colony on the superseded substratum that SC-his limits.By the preparation of molten born of the same parents' thing and and Western trace (in order to detect existing Myc label in each protein) measure protein expression, as described in the embodiment 2.

Embodiment 7: the clone of yeast saccharomyces cerevisiae subunit LPD1 and expression and the expression in brewing yeast cell thereof

This embodiment has described how to pass through PCR home-brewed wine pastoris genomic dna clone gene LPD1, and how to detect the expression of LPD1 in host's brewing yeast cell.

In PCR reaction, use primer Gevo-658 to add the Lpd1 open reading-frame (ORF) that the Gevo-659 amplification lacks the Nucleotide of those predictive coding mitochondrial targeting sequences, basically as described in the embodiment 5.With the 1.5kb product with XhoI+BamHI digestion and be cloned into pGV1103 through the cutting of same restrictions enzyme.The DCRP pGV1103-1pd1 of institute is transformed into Gevo1187 and selects the gained bacterium colony by HIS+ prototroph, basically as described in the embodiment 3.Cultivation contains the culture and following the detections LPD1 expression of the cell of pGV1103-1pd1, and promptly harvested cell then is Western trace (at an existing Myc label on the protein), basically as described in the embodiment 2.

Embodiment 8: the clone of intestinal bacteria PDH subunit and the expression in Kluyveromyces lactis thereof

Some yeast, especially those known be " Crabtree feminine gender ", provide as the distinct advantages of producing the host.Alcoholic acid Crabtree positive strain (for example yeast saccharomyces cerevisiae) is different with under aerobic conditions excessive glucose being fermented into, and Crabtree negative strain (such as those of genus kluyveromyces) can circulate metabolizable glucose to generate biomass through TCA with replacing.Therefore, the negative yeast of Crabtree tolerates the deactivation (for example by deletion KIPDC1 gene) (at aerobic growing period) of so-called glucose alienation PDH bypass path.

The gene clone that following examples have been described three kinds of subunits of the intestinal bacteria PDH that how will encode goes into to be suitable for the carrier of expressing in the yeast Kluyveromyces lactis, and how to detect those expression of gene in addition.

Pcr amplification bacillus coli gene lpdA, aceE and aceF as described in example 5 above.With gained PCR product with SalI+XhoI digestion and be cloned into carrier pGV1428, pGV1429 and the pGV1430 that all cuts respectively through SalI+XhoI.These steps have produced plasmid pGV1428-lpdA, pGV1429-aceE and pGV1430-aceF.To the complete order-checking of each inset.According to currently known methods (Kooistra R for example, Hooykaas PJ, Steensma HY. (2004) Yeast.15; 21 (9): 781-92) transform lactic acid yeast kluyveromyces kind (for example Gevo1287), and select the gained bacterium colony by suitable prototroph with one of these plasmids or arbitrary combination.Use rough yeast protein extract and Western engram analysis (based on detecting existing Myc epi-position in each protein) to measure LpdA, AceE or AceF expression as described in example 2 above to cultivate the culture that obtains from transformant.

Embodiment 9: cross the active measurement of PDH in the cell of expressing the PDH subunit

The purpose of this embodiment is to describe how to measure the PDH activity by the means of external test method.

Put down in writing method ((1992) .Eur J Biochem 209 (2) such as Wenzel TJ: 697-705) of PDH active level in the molten born of the same parents' thing of pair cell in the document.This method utilization is from being rich in the molten born of the same parents' thing of mitochondrial cell fraction deutero-.A different embodiments utilization of this method is originated as PDH from the molten born of the same parents' thing of cell that full cell obtains.This type of molten born of the same parents' thing is as preparation as described in previous (embodiment 2).Another embodiment of this measuring method is used from highly being rich in the molten born of the same parents' thing of kytoplasm (non-plastosome) proteinic cell fraction deutero-cell.This biological chemistry classification meeting reduces the contribution of endogenous plastosome PDH in assay method.The method for preparing the molten born of the same parents' thing of this type of enrichment be commercial and well known to a person skilled in the art (plastosome/cytosol classification test kit for example, Bio Vision, Inc., Mountain View, CA).

In another embodiment, by the existence of Myc epi-position label coded in one or more expression plasmids from cellular immunization purifying PDH activity.The method of protein of immunity purifying band epi-position label is to well known to a person skilled in the art (for example Harlow and Lane, Antibodies:A Laboratory Manual, (1988) CSHL Press).So be different from endogenous mixture and serve as activity source in the aforementioned PDH external test method through the PDH mixture of immune purifying.

Embodiment 10: cross the measurement of expressing acetyl-CoA in the born of the same parents that increase in the cell of PDH

The purpose of this embodiment is to describe level in the born of the same parents that how can measure acetyl-CoA (being the product of PDH) in the yeast cell that a group is cultivated.

In order to measure acetyl-CoA in the born of the same parents, those are carried yeast conversion body assessment cell acetyl-CoA level of expressing the necessary suitable plasmid combination of a whole set of PHD gene (for example pGV1334, pGV1603 and pGV1604), and (for example pGV1103, pGV1104 and pGV1105) compares with the contrast transformant that only contains carrier.In shaking bottle the superseded substratum that suitably limits (for example SC-His ,-Leu ,-yeast cell is cultured in Trp) saturated.Measure the optical density(OD) (OD600) of culture and by coming sedimentation cell in centrifugal 5 minutes with 2800xg.Use broken instrument (bead beater) lysing cell of pearl formula and molten born of the same parents' thing is used to use establishment method (Zhang etc., Connection of Propionyl-CoA Metabolism to PolyketideBiosynthesis in Aspergillus nidulans.Genetics, 168:785-794) protein determination that carries out and acetyl-CoA determination and analysis.

Embodiment 11: intestinal bacteria PDH subunit gene and butanols generate the coexpression of approach in yeast saccharomyces cerevisiae

The purpose of this embodiment is to describe how to constitute the gene that butanols generates the gene coexpression coding intestinal bacteria PDH subunit of approach with those in host's yeast saccharomyces cerevisiae.PDH can not have the functional PDH of heterogenous expression to improve the productive rate of the butanols that is generated with respect to only expressing the butanols approach in the cytosol with butanols generation approach coexpression.

The cloned genes lpdA of institute, aceE and aceF (seeing embodiment 5) subclone are gone into butanols pathway gene plasmid, specifically are pGV1208, pGV1209 and pGV1213 (table 2).For this reason, pGV1334, pGV1603 and pGV1604 are all digested with restriction enzyme EcoICRI+XhoI, and the inset of gained release is connected into pGV1208, pGV1209 and pGV1213, these plasmids digest through BamHI, mend flat dangling with the Klenow archaeal dna polymerase, through XhoI digestion, standard molecular biology method (Sambrook, J.Fritsch are all used in all these operations then, E.F., Maniatis, T., 1989).These steps have produced pGV1208-lpdA, pGV1209-aceE and pGV1213-aceF respectively.The gained plasmid is transformed into Gevo1187 with pGV1227 and selects HIS, LEU, TRP and URA prototroph, and all operations is basically as described in the embodiment 3.Use adds pGV1209 through the pGV1208 of parental plasmid and adds pVG1213 and add bacterial strain that pGV1227 transforms in contrast, the influence that butanols is generated in order to assessment PDH coexpression.Butanols generates and implements as described in example 4 above.The propyl carbinol productive rate is greater than 10%.

Embodiment 12: the generation of functional a kind of PDH form under anaerobic or under excessive NADH condition

The purpose of this embodiment be described under the anaerobism activated, or exist existing with respect to normal aerobic growing period than high [NADH]/[NAD ⁺] than the time activated mutant PDH separation.Such mutant PDH wants, because its tolerable even under little aerobic or anaerobic condition lasting PDH enzymic activity is arranged all.

Before (Kim, Y. etc. (2007) .Appl.Environm.Microbiol., 73,1766-1771; U.S. Patent application No.11/949,724, complete income this paper) put down in writing and obtained and identified the method that allows the little aerobic or active change pattern of anaerobism PDH.

Embodiment 13: it is active or do not have intestinal bacteria PDH subunit gene and butanols in the active Wine brewing yeast strain of pyruvic carboxylase to generate the coexpression of approach to have a pyruvic carboxylase of reduction

The purpose of this embodiment is how to describe in that to have a pyruvic carboxylase of reduction (PDC) active or do not have in host's Wine brewing yeast strain of pyruvic carboxylase (PDC) and constitute the gene that butanols generates the gene co-expressing coding intestinal bacteria PDH subunit of approach.The two utilizes and therefore competes available pyruvic acid pond PDC and PDH.Although product acetyl-CoA of PDH can directly be utilized by the butanols approach, but the product acetaldehyde of PDC can further be reduced into ethanol (through alcoholdehydrogenase), a kind of unwanted by-products of butylic fermentation perhaps can be converted to acetyl-CoA through the synergy that acetaldehyde dehydrogenase adds acetyl-CoA synthase.So, reduce or eliminate that PDC is active can to improve the productive rate that pyruvic acid in the cell of also crossing expressive function PDH in cytosol becomes butanols.

The generation of pdc-Wine brewing yeast strain

Document (Flikweert for example, M.T. etc., (1996) .Yeast 15; 12 (3): 247-57; Flikweert MT etc., (1999) .FEMS Microbiol Lett.1; 174 (1): 73-9; Van Maris AJ etc., (2004) ApplEnviron Microbiol.70 (1): put down in writing PDC activity 159-66) or do not had the active Wine brewing yeast strain of PDC, and they are well known to a person skilled in the art with reduction.In one embodiment, lack the active Wine brewing yeast strain of all PDC and have genotype pdc1 Δ pdc5 Δ pdc6 Δ.This type of bacterial strain lacks can detected PDC activity, and can not grow on the glucose as sole carbon source, but can survive when growth medium is supplemented with the ethanol of carbon source as an alternative or acetate.In another embodiment, the derivative of this bacterial strain has been evolved on glucose grows, and glucose is convenient and commonly used carbon source.The 3rd bacterial strain that embodiment is a related gene type pdc2 Δ with the active bacterial strain of PDC that reduces greatly is at document (Flikweert MT etc., (1999) .Biotechnol Bioeng.66 (1): also on the books 42-50).Any of these bacterial strain all can serve as PDH and add the useful host that the butanols approach is expressed.Where necessary, can come engineered any pdc-mutant strain by standard molecular biology means and yeast genetic technique, make those auxotroph signs to utilize, make and to select plasmid pGV1208-lpdA, pGV1209-aceE and pGV1213-aceF and stable maintenance in host cell.This type of genetic engineering meeting takes place by destroying relevant native gene, and it is by based on the destruction box of URA3, and follow-uply selects to eliminate URA3 and indicate and implement by FOA is anti-.

The butanols of crossing in the pdc-bacterial strain of expressing PDH generates

The cloned genes lpdA of institute, aceE and aceF (seeing embodiment 5) subclone are gone into butanols pathway gene plasmid, specifically are pGV1208, pGV1209 and pGV1213 (table 2), basically as described in the embodiment 11.

Plasmid group pGV1208-lpdA is added pGV1209-aceE to be added pGV1213-aceF and adds pGV1227 or group pGV1208 in contrast and add pGV1209 and add pGV1213 and add that pGV1227 is transformed into suitable pdc-yeast mutant and cultivate the gained bacterium colony in liquid culture.Implementing butanols as described in example 4 above generates.The propyl carbinol productive rate is greater than 50%.

Possiblely be that it is active or do not have the active bacterial strain of PDC can show significant growth defect to have a PDC of reduction, and the other carbon source (for example acetate or ethanol) of therefore may having to replenish.Because the growth defect in the pdc-yeast saccharomyces cerevisiae is derived from the disappearance in their kytoplasms acetyl-CoA pond,, the successful expression of expection PDH in cytosol save this growth defect so can generating enough acetyl-CoA.This type of growth recovery can serve as in the cytosol to be read in the active useful body of PDH.

Embodiment 14: pfl in the yeast saccharomyces cerevisiae (pyruvic acid formic acid lyase) and FDH1 (hydrogenlyase) express

The clone of intestinal bacteria pflB (the pyruvic acid formic acid lyase of non-activity) and pflA (pyruvic acid formic acid lyase activating enzymes)

In order to clone intestinal bacteria pflB and pflA, use bacillus coli gene group DNA and pflB_forw, PflB_rev and PflA_forw, PflA_rev primer amplification gene respectively.In order to clone Candida boidinii FDH1 (Cb-FDH1) gene, use the genomic dna of Candida boidinii with fdh_forw and fdh_rev primer.Utilize the restriction site SalI and the EcoRI that mix forward and cdna reverse amplimer respectively, the DNA that is increased is connected on pGV1103, the pGV1104 and pGV1102 of SalI and EcoRI digestion, produces pGV1103pflA, pGV1104pflB and pGV1002fdh1.Be with myc, myc and HA label respectively from the protein of gained plasmid expression.

Utilize gained plasmid (pGV1103pflA, pGV1104pflB and pGV1002fdh1) and carrier (pGV1103, pGV1104 and pGV1102) to come transformed yeast bacterial strain Gevo1187, pointed as embodiment 3, to generate transformant and contrast (PFL-) transformant of expressing PflA, PflB, Fdh1 (PFL+).Select this two groups of transformant by HIS, TRP and URA prototroph.

Use rough yeast protein extract and Western engram analysis that gained transformant assessment PflA, PflB and Cb-Fdh1 are expressed as described in example 2 above.

Those are proved all three kinds proteinic yeast conversion bodies assessment cell acetyl-CoA levels of expression, compare with the contrast transformant that only contains carrier.For this reason, to shake the doleiform formula at Sc-ura, his cultivates PFL+ and PFL-cell in the trp substratum.Measure the optical density(OD) (OD600) of culture and by coming sedimentation cell in centrifugal 5 minutes with 2800xrcf.Use the broken instrument lysing cell of pearl formula and molten born of the same parents' thing is used to use establishment method (Zhang etc., Connection of Propionyl-CoA Metabolism toPolyketide Biosynthesis in Aspergillus nidulans.Genetics, 168:785-794) protein determination that carries out and acetyl-CoA determination and analysis.Assess the acetyl-CoA amount of every mg total cellular protein.

Express the influence that propyl carbinol is generated in order to assess PflA, PflB and Fdh1, pflA, pflB and Cb-FDH1 subclone are gone into to contain the butanols pathway gene (table 1) of pGV1208, pGV1209 and pGV1213.For this reason, use standard molecular biology method (Sambrook, J.Fritsch, E.F., Maniatis, T., 1989), with pGV1103pflA, pGV1104pflB and pGV1002fdh1 with the digestion of EcoICRI+XhoI restriction enzyme and connect into through pGV1208, the pGV1209 of BamHI (mending flat terminal subsequently with Klenow)+XhoI digestion and pGV1213 to generate pGV1208PflA, pGV1209PflB and pGV1213Fdh1.The gained plasmid is transformed into Gevo1187 with pGV1227 and selects His, Leu, Trp and Ura prototroph.Use Gevo1110 and Gevo1111 conivium (table 1) in contrast.Implementing butanols as described in example 4 above generates.The propyl carbinol productive rate is greater than 10%.

Embodiment 15: have the pyruvic carboxylase activity of reduction or do not have PflA, PflB and Fdh1 in the active yeast saccharomyces cerevisiae of pyruvic carboxylase to express

Carry out the clone of intestinal bacteria pflB (the pyruvic acid formic acid lyase of non-activity) and pflA (pyruvic acid formic acid lyase activating enzymes) and Cb-FDH1 as described in example 14 above.

Utilize gained plasmid (pGV1103pflA, pGV1104pflB and pGV1002fdh1) and carrier (pGV1103, pGV1104 and pGV1102) to come transformed saccharomyces cerevisiae (related gene type: ura3, trp1, his3, leu2, pdc1, pdc5, pdc6) yeast strain, pointed as embodiment 3, to generate (PFL+) transformant and contrast (PFL-) transformant of expressing PflA, PflB, Cb-Fdh1.Select this two groups of transformant by HIS, TRP and URA prototroph.

Use rough yeast protein extract and Western engram analysis that gained transformant assessment PflA, PflB and Fdh1 are expressed as described in example 2 above.

As described in example 14 above, those are proved all three kinds proteinic yeast conversion bodies assessment cell acetyl-CoA levels of expression, compare with the contrast transformant that only contains carrier.

In order to assess expression PflA, PflB and Fdh1, pGV1208PflA1, pGV1209PflB and pGV1213Fdh1 are transformed into yeast saccharomyces cerevisiae (MAT A, ura3, trp1, his3, leu2, pdc1, pdc5, pdc6) and select His, Leu, Trp and Ura prototroph with pGV1227 the influence that propyl carbinol generates.Use Gevo1110 and 1111 conivium (table 1) in contrast.Implementing butanols as described in example 4 above generates.The propyl carbinol productive rate is greater than 50%.

Embodiment 16: have the ADH1 activity of reduction or do not have Pfl and Fdh1 in the active yeast saccharomyces cerevisiae of ADH1 to express

The clone of intestinal bacteria pflB (the pyruvic acid formic acid lyase of non-activity) and pflA (pyruvic acid formic acid lyase activating enzymes)

Carry out the clone of intestinal bacteria pflB (the pyruvic acid formic acid lyase of non-activity) and pflA (pyruvic acid formic acid lyase activating enzymes) and Cb-FDHI as described in example 14 above.

As described in example 3 above, utilize gained plasmid (pGV1103pflA, pGV1104pflB and pGV1002fdh1) and carrier (pGV1103, pGV1104 and pGV1102) to come transformed yeast bacterial strain Gevo1253 (adh1 Δ) to generate (PFL+) transformant and contrast (PFL-) transformant of expressing PflA, PflB, Fdh1.Select this two groups of transformant by HIS, TRP and URA prototroph.

Use rough yeast protein extract and Western engram analysis that gained transformant assessment PflA, PflB and Fdh1 are expressed as described in example 2 above.

As described in example 14 above those are proved and express all three kinds proteinic yeast conversion body assessment cell acetyl-CoA levels, compare with the contrast transformant that only contains carrier.

In order to assess expression PflA, PflB and Fdh1, pGV1208PflA, pGV1209PflB and pGV1213Fdh1 are transformed into Gevo1253 with pGV1227 and select His, Leu, Trp and Ura prototroph the influence that propyl carbinol generates.Use Gevo1110 and 1111 conivium (table 1) in contrast.Implementing butanols as described in example 4 above generates.The propyl carbinol productive rate is greater than 10%.

Embodiment 17: home-brewed wine yeast clone PDC1 gene and crossing in yeast saccharomyces cerevisiae thereof are expressed

The purpose of this embodiment is to be described in the composition promoters active control gene of clones coding pyruvic carboxylase down, and describes the such expression of gene in the Saccharomyces cerevisiae host cell.

In the PCR reaction of implementing as described in example 5 above basically, use primer Gevo-639 to add the Gevo-640 home-brewed wine pastoris genomic dna complete PDC1 ORF that increases.Gained 1.7kb product with XhoI+BamHI digestion and connect into carrier pGV1106 through the SalI+BamHI cutting, is produced pGV1389 (seeing Table 2).With the complete order-checking of inset.

In order in yeast saccharomyces cerevisiae, to cross expression Pdc1, eliminate on the substratum with pGV1389 transformed saccharomyces cerevisiae bacterial strain Gevo1187 (CEN.PK) and at SC-ura and select transformant, described in embodiment 3.Use rough yeast protein extract and Western engram analysis (based on detection) to measure the Pdc1 expression, described in embodiment 2 to cultivate the culture that obtains from transformant to existing Myc epi-position in the recombinant expression protein.

Embodiment 18: the clone is to allow the inducible expression of Pyruvate Decarboxylase Gene

The constructive expression of gene (for example pyruvic carboxylase) may be undesired at some point of culture growing period, maybe may apply unexpected metabolism or selective pressure to those overexpressing cells.So, the system that needs employing genetic expression to be regulated, gene of interest can mainly be expressed so that culture growth and the maximizing performance in follow-up fermentation at Best Times thus.

The purpose of this embodiment is the gene that is described in clones coding pyruvic carboxylase under the promotor control that can induce adjusting, and describes the such expression of gene in the Saccharomyces cerevisiae host cell.

Discharge existing PDC1 ORF among the pGV1389 (seeing embodiment 19) and be cloned into carrier pGV1414 as the XbaI+BamHI fragment, produce carrier pGV1483 through AvrII+BamHI digestion.So, carrier pGV1483 (table 2) is characterised in that yeast saccharomyces cerevisiae MET3 gene promoter (SEQ ID NO:177) drives the PDC1 expression of gene.The MET3 promotor is reticent aspect transcribing when having methionine(Met), but falls certain threshold value activity that become when following in methionine level.Plasmid pGV1483 is transformed into Gevo1187 and identifies transformant by the selection on the SC-ura substratum, described in embodiment 3.Cultivation carry pGV1483 Gevo1187 culture and measure PDC1 and express, basically as described in the embodiment 2.

In another embodiment of this embodiment, the PDC1 gene is expressed under the CUP1 of yeast saccharomyces cerevisiae copper inducible gene promoter (SEQ ID NO:178) control.At first, using primer by PCR home-brewed wine pastoris genomic dna amplification CUP1 gene promoter in as described in example 5 above the reaction basically.PGV1106 with the PCR product cuts through SacI+SalI with SacI+SalI digestion and insertion produces pGV1388.To the complete order-checking of CUP1 promoter sequence of being inserted.Then, will insert pGV1388, produce pGV1388-PDC1 from pGV1389, the XbaI+BamHI fragment that contains the PDC1 gene through AvrII+BamHI digestion.PGV1388-PDC1 is transformed into Gevo1187 with plasmid, described in embodiment 3, and identifies transformant lacking on the SC-ura defined medium of copper.In not having the SC-ura substratum of supplementation with copper, cultivate the culture of transformant, reach OD600＞0.5, add copper sulfate at that time to final concentration 0.5mM until them.Culture was cultivated 24 hours to 48 hours as required again, measured Pdc1 by the Western trace then and express, basically as described in the embodiment 2.

Embodiment 19: measured the active external test method of the PDC that is generated in the culture of the yeast cell of expressing pyruvic carboxylase

The purpose of this embodiment is to describe for measuring the active useful external test method of existing total pyruvic carboxylase in the cell (particularly from crossing a group cell of expressing the PDC enzyme).

Be used to measure on the books and be to well known to a person skilled in the art that (Maitra PK and Lobo be Biol Chem.25 Z.1971.J from the active assay method of PDC of the molten born of the same parents' thing of total cell; 246 (2): 475-88; Schmitt HD and Zimmermann FK.1982.J Bacterid.151 (3): 1146-52; Eberhardt etc., (1999) Eur.J.Biochem.262 (1), 191-201).

In another embodiment of this embodiment, following measurement promptly at first uses specific antibody or use at PDC to precipitate PDC at the antibody mediated immunity as existing Myc epi-position label among (but not being endogenous) PDC that crosses expression expressed among embodiment RF20 and the RF21 by express the PDC activity that PDC generated described in embodiment 17 and 18.Be used for the existing method of protein of specific immunity sediment composite mixture and be well known to a person skilled in the art (for example Harlow and Lane, 1988, Antibodies:A Laboratory Manual, CSHL Press).The PDC mixture of immunoprecipitation serves as the source of the material that will use aforementioned assay method mensuration then.That this method is so allowed is allogenic, cross the specific assay of the PDC that expresses.

Embodiment 20: contain also that PDC crosses the production of butanol rate of expressing the rising that causes in the yeast saccharomyces cerevisiae that functional butanols generates approach

The purpose of this embodiment is that illustration PDC crosses expression and how to improve and also express butanols and generate production of butanol rate in the yeast saccharomyces cerevisiae culture of approach.

Cross the Wine brewing yeast strain of expressing the PDC gene (van Hoek etc., (1998) .ApplEnviron Microbiol.64 (6): 2133-40) before on the books.These experiments disclosed (1) though the endogenous PDC level in the yeast saccharomyces cerevisiae account for total cell protein matter up to 3.4%, still can pass through the existence of expression construct and further improve; (2) mistake that the is in high growth rates fermentation capacity (the high specific speed that ethanol generates) of expressing the culture of PDC raises with respect to control strain.The flux that the butanols that provides via allos generates approach can be provided in the expression excessively of these results suggest PDC under some growth conditions.

In order in the situation that has the butanols approach, to cross expression PDC gene, cut out the PDC1 gene by SpeI digestion from pGV1389, with Klenow archaeal dna polymerase fragment the DNA that cuts out is dangled to mend and put down, use the XhoI digested vector then.Fragment is inserted pGV1213 (this carrier digests through BamHI, mends the end of truncation with the Klenow enzyme, digests with XhoI then), produce plasmid pGV1605.With plasmid pGV1605 or pGV1057 (Mumberg, D., Deng (1995) Gene 156:119-122) be transformed into Gevo1187 with plasmid pGV1208, pGV1209 and pGV1213, basically as described in example 3 above, and select His, Leu, Trp and Ura prototroph.Implement fermentation to generate butanols, measure as described in example 4 above.Comprising pGV1605 causes than comprising the higher production of butanol rate of pGV1057 and plasmid pGV1208, pGV1209 and pGV1213 (the butanols amount that time per unit generated) in the aforementioned fermentation.The propyl carbinol productive rate is greater than 5%.

Embodiment 21: it is active and contain also that PDC crosses the production of butanol rate of expressing the rising that causes in the brewing yeast cell that functional butanols generates approach to have an alcoholdehydrogenase of reduction

The purpose of this embodiment is to demonstrate how to obtain enhanced production of butanol rate by crossing expression PDC gene in the situation that has butanols generation approach in the yeast strain of the active defective of alcoholdehydrogenase (ADH).

From the acetaldehyde that pyruvic acid generates two kinds of main destiny are arranged by PDC: it can further be metabolized to acetyl-CoA by the effect of acetaldehyde dehydrogenase and acetyl-CoA synthase, and it can be used as the useful substrate of butanols route of synthesis then; Perhaps, it can further be metabolized to ethanol by reduction process by the effect of alcoholdehydrogenase (ADH).Therefore, reduction or elimination ADH (the especially ADH enzyme of those preference acetaldehyde) can reduce or eliminate this undesired acetaldehyde alienation path and improve the available acetyl of butanols approach-CoA pond.

Simultaneously plasmid pGV1208, pGV1209, pGV1213 and pGV1605 corotation are dissolved bacterial strain Gevo1187 (it has related gene type ADH1+) or bacterial strain Gevo1266 (it has related gene type adh1 Δ).To unelected His, Leu, Trp and the Ura prototroph of selecting of transformed bacteria, basically as described in the embodiment 3.Implement fermentation to generate butanols, measure as described in example 4 above.The propyl carbinol productive rate is greater than 10%.Bacterial strain Gevo1266 (adh1 Δ) shows the butanols productive rate that improves than the parallel fermentation of implementing in bacterial strain Gevo1187 (ADH1+).

Embodiment 22: have the alcoholdehydrogenase activity of reduction and expressive function butanols and generate that PDC crosses the butanols productive rate of expressing the rising that causes in the Kluyveromyces lactis cell of approach

The purpose of this embodiment is to describe to have the ADH activity that reduces greatly or do not have the butanols in the active lactic acid yeast kluyveromyces strain of ADH to generate.Predict that expressing the butanols approach in such bacterial strain can produce than express the significantly bigger every input glucose of butanols productive rate of butanols approach in having the active bacterial strain of ADH.

Generation with active lactic acid yeast kluyveromyces strain of alcoholdehydrogenase of reduction.

Transform the cell of Kluyveromyces lactis and the method for the gene in the destruction Kluyveromyces lactis and (promptly replace functional open reading-frame (ORF) with selection marker, follow follow-up elimination sign) (Kooistra R before on the books, Hooykaas PJ, Steensma HY. (2004) Yeast.15; 21 (9): 781-92).Kluyveromyces lactis has the gene of four kinds of coding ADH enzymes, and wherein two kinds (being KIADH1 and KIADH2) are positioned kytoplasm.Document (Saliola, M., etc., (1994) Yeast 10 (9): put down in writing the Kluyveromyces lactis sudden change derivative of having deleted these all four kinds of genes 1133-40) and (be called Kluyveromyces lactis adh ⁰), and cultivate the desired culture condition of this bacterial strain ideally.A kind of alternative pattern of this way adopts the sign of giving the resistance of medicine G418/ Geneticin (geneticin), for example provides as the kan gene.Such way is useful, and sign can be for use as the selection marker in the follow-up conversion because it stays URA3.

Kluyveromyces lactis adh ⁰Butanols is expressed the expression of approach in the bacterial strain

Simultaneously plasmid pGV1208, pGV1209, pGV1213 and pGV1605 corotation are dissolved bacterial strain Gevo1287 (it is ADH+) or adh ⁰Bacterial strain.To unelected His, Leu, Trp and the Ura prototroph of selecting of transformed bacteria.Implement fermentation to generate butanols, measure as described in example 4 above.The propyl carbinol productive rate is greater than 10%.Bacterial strain Gevo1287 generates than the significantly more butanols of the parallel fermentation of implementing in the isogenic adh0 bacterial strain of others.

Embodiment 23: the ALD6 in the yeast saccharomyces cerevisiae crosses expression

In order to clone the ALD6 gene of yeast saccharomyces cerevisiae, adopted for two steps merged the PCR method, it eliminates inner SalI restriction enzyme sites so that the operation of follow-up molecular biology.Use primer to generate two kinds of overlapping PCR products crossing over yeast saccharomyces cerevisiae ALD6 gene order to Gevo-643+Gevo-644 and Gevo-645+Gevo-646 and as the genes of brewing yeast group DNA of template.With SalI+BamHI digestion gained PCR fragment and connect into through the pGV1105 of similar restrictive diges-tion and pGV1101 to generate pGV1321 and pGV1326.Subsequently, subclone ALD6, promptly with EcoICRI+XhoI digestion pGV1321 and pGV1326 and connect into the pGV1209 of BamHI (and mending flat terminal subsequently with Klenow)+XhoI digestion respectively and pGV1208 to generate pGV1339 and pGV1399.

Utilize gained plasmid (pGV1339 and pGV1399) and carrier (pGV1105 and pGV1101) to come transformed yeast bacterial strain Gevo1187 respectively, described in embodiment 3, to generate (" Ald6+ ") transformant or the contrast transformant of expressing ALD6.By selecting TRP and LEU prototroph to select this two groups of transformant in the substratum suitable eliminating.

Use rough yeast protein extract and Western engram analysis that gained transformant assessment Ald6 is expressed, described in embodiment 2.

Those are proved the proteic yeast conversion body assessment of expression Ald6 enhanced acetaldehyde dehydrogenase activity, compare with the contrast transformant that only contains carrier.For this reason, in shaking bottle, in suitable superseded substratum, cultivate Ald6+ and control cells.Measure the optical density(OD) (OD600) of culture and by coming sedimentation cell in centrifugal 5 minutes with 2800xg.Use the broken instrument lysing cell of pearl formula and molten born of the same parents' thing is used to use establishment method (Van Urk etc. for example, Biochim.Biophys.Acta.191:769) protein determination that carries out and aldehyde dehydrogenase activation analysis.

To express Ald6 to the influence that propyl carbinol generates in order assessing, pGV1339 to be transformed into Gevo1187 and to select His, Leu, Trp and Ura prototroph with pGV1208, pGV1227 and pGV1213.Use Gevo1110 and 1111 conivium (table 1) in contrast.Implementing butanols as described in example 4 above generates.The propyl carbinol productive rate is greater than 5%.

Embodiment 24: do not have the Ald6 in the yeast saccharomyces cerevisiae of alcoholdehydrogenase I activity (adh1 Δ) to cross expression

Implement the clone of ALD6 gene as described in example 23 above.

Utilize gained plasmid (pGV1339 and pGV1399) and carrier (pGV1100 and pGV1101) to come transformed yeast bacterial strain Gevo1253 as described in example 3 above respectively to generate transformant and the contrast transformant of expressing Ald6+.On suitable superseded substratum, select these two groups of transformant.

Use rough yeast protein extract and Western engram analysis that gained transformant assessment Ald6 is expressed as described in example 2 above.

As described in example 23 above those are proved and express the proteic yeast conversion body assessment of Ald6 enhanced acetaldehyde dehydrogenase activity.

To express the consequence that propyl carbinol is generated in order assessing, pGV1339 to be transformed into Gevo1253 and to select His, Leu, Trp and Ura prototroph with pGV1209, pGV1227 and pGV1213.Use Gevo1110 and 1111 conivium (table 1) in contrast.Implementing butanols as described in example 4 above generates.The propyl carbinol productive rate is greater than 10%.

Embodiment 25: acetyl-CoA synthase gene crossing in yeast saccharomyces cerevisiae expressed

The purpose of this embodiment is to describe the clone of the gene of coding acetyl-CoA synthase activity, and the such expression of gene in host's brewing yeast cell.Particularly, arbitrary or the two coding acetyl-CoA synthase activity of genes of brewing yeast ACS1 or ACS2.

In order to clone ACS1 and ACS2 gene, utilization is as the genes of brewing yeast group DNA of template, and primer Gevo-479+Gevo-480 (ACS1) and Gevo-483+Gevo-484 (ACS2), each group contains SalI and BamHI restriction site respectively in forward and reverse primer.With gained PCR fragment with SalI+BamHI digestion and connect into through the pGV1101 of similar restrictive diges-tion and pGV1102 to generate pGV1262 and pGV1263.Subsequently, subclone ACS1 and ACS2 are promptly with EcoICRI+XhoI digestion pGV1262 and pGV1263 and connect into pGV1213 through BamHI (and mending flat terminal subsequently with Klenwo)+XhoI digestion to generate pGV1319 and pGV1320.

Utilize gained plasmid pGV1262 and pGV1263 and carrier pGV1101 and pGV1102 to come transformed yeast bacterial strain Gevo1187 as described in example 3 above respectively to generate transformant and the contrast transformant of expressing ACS1+, ACS2+.Select this two groups of transformant by LEU, URA prototroph.Use rough yeast protein extract and Western engram analysis that transformant assessment Acs1 or Acs2 are expressed as described in example 2 above.

Those are proved expression Acs1 or the proteic yeast conversion body assessment of Acs2 enhanced acetyl-CoA synthase activity, compare with the contrast transformant that only contains carrier.For this reason, in SC-LEU, URA substratum, cultivate ACS1+ or ACS2+ and control cells to shake bottle pattern.Measure the optical density(OD) (OD600) of culture and by coming sedimentation cell in centrifugal 5 minutes with 2800xrcf.Use the broken instrument lysing cell of pearl formula and molten born of the same parents' thing is used to use establishment method (Van Urk etc., Biochim.Biophys.Acta.191:769) protein determination that carries out and acetyl-CoA synthase activity analysis.

Cross expression to the influence that propyl carbinol generates in order to assess Acs1 or Acs2, pGV1319 and 1320 is transformed into Gevo1187 and selects His, Leu, Trp and Ura prototroph with pGV1208, pGV1209 and pGV1227.Use Gevo1110 and 1111 conivium (table 1) in contrast.Implementing butanols as described in example 4 above generates.The propyl carbinol productive rate is greater than 5%.

Embodiment 26: do not have in the brewing yeast cell of alcoholdehydrogenase I activity (adh1 Δ) crossing of acetyl-CoA synthase express

Clone the ACS1 and the ACS2 gene of yeast saccharomyces cerevisiae as described in example 25 above.

Utilize gained plasmid pGV1262 and pGV1263 and carrier pGV1101 and pGV1102 to come transformed yeast bacterial strain Gevo1253 as described in example 3 above respectively to generate transformant and the contrast transformant of expressing ACS1+, ACS2+.Select this two groups of transformant by LEU, URA prototroph.Use rough yeast protein extract and Western engram analysis that transformant assessment Acs1 or Acs2 are expressed as described in example 25 above.

As described in example 26 above those are proved and express Acs1 or the proteic yeast conversion body assessment of Acs2 enhanced acetyl-CoA synthase activity.

In order to assess expression Acs1 or Acs2, pGV1319 and 1320 is transformed into Gevo1253 and selects His, Leu, Trp and Ura prototroph with pGV1208, pGV1209 and pGV1227 the influence that butanols generates.Use Gevo1110 and 1111 conivium (table 1) in contrast.Implementing butanols as described in example 4 above generates.The propyl carbinol productive rate is greater than 5%.

Embodiment 27:ALD6, ACS1 and ACS2 crossing in yeast saccharomyces cerevisiae expressed

Described in embodiment 23 and 25, clone ALD6, ACS1 and ACS2 gene.

Utilize gained plasmid pGV1321 and pGV1262 or pGV1263 and carrier pGV1105 and pGV1102 to come transformed yeast bacterial strain Gevo1187 as described in example 3 above respectively to generate transformant and the contrast transformant of expressing ALD6+ACS1+, ALD6+ACS2+.Select this two groups of transformant by LEU and URA prototroph.

To transformant ALD6+ACS1+ and ALD6+ACS2+ assessment enhanced acetyl-CoA synthase activity, compare with the contrast transformant that only contains carrier.For this reason, in SC-LEU, URA substratum, cultivate ALD6+ACS1+, ALD6+ACS2+ and control cells to shake bottle pattern as described in example 25 above.

Add cross expressing of Acs1 or Acs2 and how to cause higher butanols to generate in order to assess Ald6, Gevo1187 is transformed and select His, Leu, Trp and Ura prototroph with pGV1208, pGV1339, pGV1227 and pGV1319 or 1320.Use Gevo1110 and 1111 conivium (table 1) in contrast.Assessing butanols as described in example 4 above generates.The propyl carbinol productive rate is greater than 5%.

Embodiment 28: do not have ALD6 in the yeast saccharomyces cerevisiae of alcoholdehydrogenase I activity (adhl Δ) to add ACS1 or ACS2 crosses expression

Described in embodiment 23 and 25, clone ALD6, ACS1 and ACS2 gene.

Utilize gained plasmid pGV1321 and pGV1262 or pGV1263 and carrier pGV1105 and pGV1102 to come transformed yeast bacterial strain Gevo1253 (Δ ADH1) as described in example 3 above respectively to generate bacterial strain or the contrast transformant of expressing ALD6+ACS1+ or ALD6+ACS2+.Select this two groups of transformant by LEU and URA prototroph.

To transformant ALD6+ACS1+ or ALD6+ACS2+ assessment enhanced acetyl-CoA synthase activity, compare with the contrast transformant that only contains carrier.For this reason, in SC-LEU, URA substratum, cultivate ALD6+ACS 1+ or ALD6+ACS2+ and control cells to shake bottle pattern as described in example 25 above.

To express the influence that SALD6 and ACS1 or ACS2 generate butanols in order assessing, Gevo1253 to be transformed and selected HIS, LEU, TRP and URA prototroph with pGV1208, pGV1339, pGV1227 and pGV1319 or 1320.Use Gevo1110 and 1111 conivium (table 1) in contrast.Implementing butanols as described in example 4 above generates.The propyl carbinol productive rate is greater than 10%.

Embodiment 29: the butanols approach is cloned into is used for the carrier of expressing at the yeast of genus kluyveromyces

Be suitable for the carrier of in the bacterial strain Kluyveromyces lactis, expressing for the butanols pathway gene is cloned into, discharge hbd, Crt, Thl+TER and be cloned into pGV1428,1429 and 1430 from pGV1208, pGV1209 and pGV1227 to generate pGV1208KI, pGV1209KI and pGV1227KI through similar digestion by SacI and NotI restrictive diges-tion.For ADHE2 is cloned into Kluyveromyces lactis, with pGV1213 with MluI and SacI digestion and connect into pGV1431 through similar digestion to generate pGV1213KI.Gained plasmid pGV1208KI, pGV1209KI, pGV1227KI and pGV1213KI are transformed into Kluyveromyces lactis (bacterial strain Gevo1287; Related gene type: MATa, trp1, his3, leu2, ura3) and to transformant selection TRP, HIS, LEU and URA prototroph (Kooistra R, Hooykaas PJ, Steensma HY. (2004) Yeast.15; 21 (9): 781-92).Implementing butanols as described in example 4 above generates.

Embodiment 30: pyruvic acid formic acid lyase and the expression of hydrogenlyase I in Kluyveromyces lactis

The clone of intestinal bacteria pflB (the pyruvic acid formic acid lyase of non-activity) and pflA (pyruvic acid formic acid lyase activating enzymes)

In order to clone intestinal bacteria pflB and pflA, use bacillus coli gene group DNA and pflB_forw, PflB_rev and PflA_forw, PflA_rev primer amplification gene respectively.In order to clone Candida boidinii FDH1 gene, in the PCR reaction, use genomic dna and fdh_forw and fdh_rev primer as the Candida boidinii of template.Utilize the restriction site SalI and the EcoRI that mix forward and cdna reverse amplimer respectively, the DNA that is increased is connected on pGV1428, the pGV1429 and pGV1430 of SalI and EcoRI digestion, generates pGV1428pflA, pGV1429pflB and pGV1430fdh1.From the protein belt myc of gained plasmid expression label, in order to carry out protein expression research.

By currently known methods (Kooistra R, Hooykaas PJ, Steensma HY. (2004) Yeast.15; 21 (9): 781-92) utilize gained plasmid (pGV1428pflA, pGV1429pflB and pGV1430fdh1) and carrier (pGV1428, pGV1429 and pGV1430) to come transformed yeast bacterial strain Kluyveromyces lactis (Gevo1287; Related gene type: MatA, trp1, his3, leu2 and ura3) to generate (PFL+) transformant and contrast (PFL-) transformant of expressing PflA, PflB, Cb-Fdh1.Select this two groups of transformant by HIS, TRP and LEU prototroph.

Use rough yeast protein extract and Western engram analysis that gained transformant assessment PflA, PflB and Fdh1 are expressed as described in example 2 above.

Those are proved all three kinds proteinic yeast conversion bodies assessment cell acetyl-CoA levels of expression, compare with the contrast transformant that only contains carrier.For this reason, in SC-LEU, HIS, TRP substratum, cultivate PFL+ and PFL-cell to shake bottle pattern.Measure the optical density(OD) (OD600) of culture and with centrifugal 5 minutes of 2800xrcf with sedimentation cell.Use the broken instrument lysing cell of pearl formula and molten born of the same parents' thing is used to use establishment method (Zhang etc., Connection of Propionyl-CoA Metabolism toPolyketide Biosynthesis in Aspergillus nidulans.Genetics, 168:785-794) protein determination that carries out and acetyl-CoA determination and analysis.Assess the acetyl-CoA amount of every mg total cellular protein.

Express the influence that butanols is generated in order to assess PflA, PflB and Fdh1, pflA, pflB and Cb-FDHI subclone are gone into to contain the butanols pathway gene (table 1) of pGV1208KI, pGV1209KI, pGV1227KI and pGV1213KI.For this reason, use standard molecular biology method (Sambrook, J.Fritsch, E.F., Maniatis, T., 1989), with pGV1428pflA, pGV1429pflB and pGV1002fdh1 with the digestion of EcoICRI+XhoI restriction enzyme and connect into through pGV1208KI, the pGV1209KI of BamHI (mending flat terminal subsequently with Klenow)+XhoI digestion and pGV1213KI to generate pGV1208KIPflA, pGV1209KIPflB and pGV1213KIFdh1.The gained plasmid is transformed into lactic acid yeast kluyveromyces strain (MATa, pdc1, trp1, his3, leu2 ura3) and selects His, Leu, Trp and Ura prototroph with pGV1227KI.The Kluyveromyces lactis transformant that use comprises pGV1428, pGV1429, pGV1430 and pGV1431 is conivium in contrast.Implementing butanols as described in example 4 above generates.The propyl carbinol productive rate is greater than 10%.

Embodiment 31: pyruvic acid formic acid lyase and the hydrogenlyase I expression in lacking the active Kluyveromyces lactis of pyruvic carboxylase

Clone intestinal bacteria pflB (the pyruvic acid formic acid lyase of non-activity) and pflA (pyruvic acid formic acid lyase activating enzymes) Cb-FDHI as described in example 30 above.

By currently known methods (Kooistra R, Hooykaas PJ, Steensma HY. (2004) Yeast.15; 21 (9): 781-92) utilize gained plasmid (pGV1428pflA, pGV1429pflB and pGV1430fdh1) and carrier (pGV1428, pGV1429 and pGV1430) to come transformed yeast bacterial strain Kluyveromyces lactis (MatA, pdc1, trp1, his3, leu2 and ura3) to generate (PFL+) transformant and contrast (PFL-) transformant of PflA, PflB, Cb-Fdh1.Select this two groups of transformant by HIS, TRP and LEU prototroph.

Use rough yeast protein extract and Western engram analysis that gained transformant assessment PflA, PflB and Cb-Fdh1 are expressed as described in example 2 above.

Those are proved all three kinds proteinic yeast conversion bodies assessment cell acetyl-CoA levels of expression, compare with the contrast transformant that only contains carrier.For this reason, in SC-LEU, HIS, TRP substratum, cultivate PFL+ and PFL-cell and assessment to shake bottle pattern as described in example 30 above.

For how the expression of assessing PflA, PflB and Fdh1 causes higher butanols generate, pGV1208KIPflA, pGV1209KIPflB and pGV1213KIFdh 1 are transformed into Kluyveromyces lactis (MATa, pdd Δ, trp1, his3, leu2, ura3) and select His, Leu, Trp and Ura prototroph with pGV1227KI.The Kluyveromyces lactis transformant that use comprises pGV1428, pGV1429, pGV1430 and pGV1431 is conivium in contrast.Implementing butanols as described in example 4 above generates.The propyl carbinol productive rate is greater than 50%.

Embodiment 32:Pf1 (pyruvic acid formic acid lyase) and Fdh1 (hydrogenlyase I) expression in lacking the active Kluyveromyces lactis of Adh1

Clone intestinal bacteria pflB (the pyruvic acid formic acid lyase of non-activity), pflA (pyruvic acid formic acid lyase activating enzymes) and Cb-FDH1 as described in example 30 above.

By currently known methods (Kooistra R, Hooykaas PJ, Steensma HY. (2004) Yeast.15; 21 (9): 781-92) utilize gained plasmid (pGV1428pflA, pGV1429pflB and pGV1430fdh1) and carrier (pGV1428, pGV1429 and pGV1430) to come transformed yeast bacterial strain Kluyveromyces lactis (MATa, trp1, his3, Leu2, ura3) to generate (EcPFL+) transformant and contrast (EcPFL-) transformant of expressing PflA, PflB1, Fdh1.Select this two groups of transformant by HIS, TRP and LEU prototroph.

Use rough yeast protein extract and Western engram analysis that gained transformant assessment PflA, PflB and Fdh1 are expressed as described in example 2 above.

Those are proved all three kinds proteinic yeast conversion bodies assessment cell acetyl-CoA levels of expression, compare with the contrast transformant that only contains carrier.For this reason, in SC-LEU, HIS, TRP substratum, cultivate EcPFL+ and EcPFL-cell and assessment to shake bottle pattern as described in example 30 above.

For how the expression of assessing PflA, PflB and Fdh1 causes higher butanols generate, pGV1208KIPflA, pGV1209KIPflB and pGV1213KIFdh1 are transformed into Kluyveromyces lactis (MATa, adh1 Δ, trp1, his3, leu2, ura3) and select His, Leu, Trp and Ura prototroph with pGV1227KI.The Kluyveromyces lactis transformant that use comprises pGV1428, pGV1429, pGV1430 and pGV1431 is conivium in contrast.Implementing butanols as described in example 4 above generates.The propyl carbinol productive rate is greater than 20%.

Embodiment 33:KIALD6 crossing in Kluyveromyces lactis expressed

In order to clone KIALD6, in the PCR reaction, use Kluyveromyces lactis genomic dna and primer KIALD6_left5 and KIALD6_right3 (seeing Table 1) as template, similar described in others and the embodiment 5.Aforementioned primer comprises SalI and BamHI restriction site respectively, with gained PCR fragment with SalI+BamHI digestion and connect into pGV1428 through similar restrictive diges-tion to generate pGV1428KLALD6.Subsequently, subclone KIALD6, soon pGV1428ALD6 digests with EcoICRI+XhoI and connects into through the BamHI pGV1208KI that (mending flat terminal subsequently with Klenow)+XhoI digests with generation pGV1208KIALD6.

By currently known methods (Kooistra R, Hooykaas PJ, Steensma HY. (2004) Yeast.15; 21 (9): 781-92) utilize gained plasmid pGV1428ALD6KI and carrier pGV1428 to come transformant and the contrast transformant of transformed yeast bacterial strain Kluyveromyces lactis (MATa, trp1, his3, leu2, ura3) respectively to generate expressing K IALD6+ and KIALD6-.Select this two groups of transformant by HIS prototroph.

Use rough yeast protein extract and Western engram analysis that gained transformant KIALD6+ and KIALD6-assessment KIAld6 are expressed as described in example 2 above.

Those were proved the proteic Kluyveromyces lactis transformant assessment of expressing K IAld6 enhanced acetaldehyde dehydrogenase activity, compared with the contrast transformant that only contains carrier.For this reason, in the SC-HIS substratum, cultivate KIALD6+ and KIALD6-cell and assessment to shake bottle pattern as described in example 23 above.

Express how to cause higher butanols generation in order to assess crossing of KIALD6, pGV1208KIALD6 is transformed into Kluyveromyces lactis (MATa, trp1, his3, leu2, ura3) and selects HIS, LEU, TRP and URA prototroph with pGV1209KI, pGV1227KI and pGV1213KI.The transformant that use pGV1428, pGV1429, pGV1430 and pGV1431 conversion Kluyveromyces lactis produce is conivium in contrast.Implementing butanols as described in example 4 above generates.The propyl carbinol productive rate is greater than 5%.

Embodiment 34: aldehyde dehydrogenase crossing in lacking the active Kluyveromyces lactis of Adh1 expressed

Put down in writing the clone of kluyveromyces KIALD6 gene among the embodiment 33.

By currently known methods (Kooistra R, Hooykaas PJ, Steensma HY. (2004) Yeast.15; 21 (9): 781-92) utilize gained plasmid pGV1428ALD6 and carrier pGV1428 to come transformant and the contrast transformant of transformed yeast bacterial strain Kluyveromyces lactis (MATa, adh1 Δ, trp1, his3, leu2, ura3) respectively to generate expressing K IALD6+ and KIALD6-.Select this two groups of transformant by HIS prototroph.

Use rough yeast protein extract and Western engram analysis that gained transformant assessment KIAld6 is expressed as described in example 2 above.

As described in example 30 above those are proved the proteinic Kluyveromyces lactis transformant assessment of expressing K IAld6 enhanced acetaldehyde dehydrogenase activity.

Express how to cause higher butanols generation in order to assess crossing of KIAld6, pGV1208KIALD6 is transformed into Kluyveromyces lactis (MATa, adh1 Δ, trp1, his3, leu2, ura3) and selects HIS, LEU, TRP and URA prototroph with pGV1209KI, pGV1227KI and pGV1213KI.The transformant that use pGV1428, pGV1429, pGV1430 and pGV1431 conversion Kluyveromyces lactis produce is conivium in contrast.Implementing butanols as described in example 4 above generates.The propyl carbinol productive rate is greater than 10%.

Embodiment 35: acetyl-CoA synthase gene crossing in the yeast Kluyveromyces lactis expressed

Two kinds of side direction homologous genes in the yeast Kluyveromyces lactis genome (being KIACS1 and KIACS2) coding acetyl-CoA activity.In order to clone KIACS1 and KIACS2, utilization is as Kluyveromyces lactis genomic dna and the primer KIACS1_left5+KIACS2_Right3 (ACS1) and the KIACS2_Left5+KIACS2_Right3 (ACS2) (seeing Table 1) of template, and it contains NotI+SalI and SalI+BamHI restriction site respectively in forward and reverse primer.With gained PCR fragment with suitable enzymic digestion and connect into through the pGV1429 of similar restrictive diges-tion and pGV1431 to generate pGV1429ACS1 and pGV1431ACS2.Subsequently, subclone KIACS1 and KIACS2 are about to pGV1429ACS1 and pGV1431ACS2 with SacId and NotI digestion and connect into through the pGV1209KI of similar digestion and pGV1213KI with generation pGV1209KIACS1 and pGVKIACS2.

Utilize gained plasmid pGV1429ACS1 and pGV1431ACS2 and empty carrier pGV1429 and pGV1431 to transform Kluyveromyces lactis (MATa, trp1, his3, leu2, ura3) respectively by currently known methods to generate proteic transformant of expressing K IACS1+, KIACS2+ and KIACS-and contrast transformant.Select this two groups of transformant by TRP, URA prototroph.Use rough yeast protein extract and Western engram analysis that transformant assessment KIAcs1 and KIAcs2 are expressed as described in example 2 above.

Those are proved expressing K IAcs1 and the proteic yeast conversion body assessment of KIAcs2 enhanced acetyl-CoA synthase activity, compare with the contrast transformant that only contains carrier.For this reason, in SC-TRP, URA substratum, cultivate KIACS1+, KIACS2+ and KIACS-cell and assessment to shake bottle pattern as described in example 25 above.

Express how to cause higher butanols generation in order to assess crossing of KIACS1 and KIACS2, pGV1209KIACS1 and pGV1209KIACS2 are transformed into bacterial strain Gevo1287 and transformant is selected His, Leu, Trp and Ura prototroph with pGV1208KI and pGV1227KI.The transformant that use pGV1428, pGV1429, pGV1430 and pGV1431 conversion Kluyveromyces lactis (MATa, trp1, his3, leu2, ura3) produce is conivium in contrast.Implementing butanols as described in example 4 above generates.The propyl carbinol productive rate is greater than 5%.

Embodiment 36: acetyl-CoA synthase gene crossing in lacking the active yeast Kluyveromyces lactis of Adh1 expressed

Put down in writing the clone of Kluyveromyces lactis KIACSI and KIACS2 gene among the embodiment 35.

Utilize gained plasmid pGV1429ACS1 and pGV1431ACS2 and empty carrier pGV1429 and pGV1431 to transform transformant and the contrast transformant of Kluyveromyces lactis (MATa, adh1 Δ, trp1, his3, leu2, ura3) by currently known methods to generate expressing K IACS1+ and KIACS2+.Select this two groups of transformant by TRP and URA prototroph.Use rough yeast protein extract and Western engram analysis that transformant assessment KIAcs1 and KIAcs2 are expressed as described in example 2 above.

As described in example 25 above those are proved expressing K lAcs1 and the proteic yeast conversion body assessment of KIAcs2 enhanced acetyl-CoA synthase activity.

Express how to cause higher butanols generation in order to assess crossing of KIACS1 and KIACS2, pGV1209KIACS1 and pGV1209KIACS2 are transformed into Kluyveromyces lactis (MatA, adh1, trp1, his3, leu2 and ura3) with pGV1208KI and pGV1227KI.Implementing butanols as described in example 4 above generates.The propyl carbinol productive rate is greater than 10%.

Embodiment 37:KIALD6 and KIACS1 or KIACS2 crossing in newborn Crewe Vickers yeast expressed

Clone KIALD6, KIACS1 and KIACS2 gene as mentioned described in the embodiment 33 and 35.

Utilize gained plasmid pGV1428ALD6 and pGV1429ACS1 or pGV1430 and carrier pGV1428 and pGV1429 or pGV1430 to transform transformant and the contrast transformant of Kluyveromyces lactis (MATa, trp1, his3, leu2, ura3) respectively by currently known methods to generate expressing K IALD6+KIACS1+, KIALD6+KIACS2+ and KIALD-KIACS-.Select this two groups of transformant by HIS, TRP and HIS, LEU prototroph respectively.

To transformant KIALD6+KIACS 1+ and KIALD6+KIACS2+ assessment enhanced acetyl-CoA synthase activity, compare with the contrast transformant (ALD-ACS-) that only contains carrier.For this reason, in SC-HIS, TRP and HIS, LEU substratum, cultivate KIALD6+KIACS1+, KIALD6+KIACS2+ and KIALD-KIACS-cell and assessment respectively to shake bottle pattern as described in example 25 above.

Express how to cause higher butanols generation in order to assess crossing of KIAld6 and KIAcs1 or KIAcs2, Kluyveromyces lactis (MATa, trp1, his3, leu2ura3) is used pGV1208KIALD6, pGV1209KIACS1 or pGV1209KIACS2, pGV1227KI, pGV1213KI conversion and selected HIS, LEU, TRP and URA prototroph.The transformant that use pGV1428, pGV1429, pGV1430 and pGV1431 conversion Kluyveromyces lactis (MATa, trp1, his3, leu2ura3) produce is conivium in contrast.Implementing butanols as described in example 4 above generates.The propyl carbinol productive rate is greater than 5%.

Embodiment 38:KIALD6, KIACS1 and KIACS2 crossing in the Kluyveromyces lactis that lacks KIAdh1 activity (Kladh1 Δ) expressed

Described in embodiment 33 and 35, clone KIALD6, KIACS1 and KIACS2 gene.

Utilize gained plasmid pGV1428ALD6 and pGV1429ACS1 or pGV1430ACS2 and carrier pGV1428 and pGV1429 or pGV1430 to transform transformant and the contrast transformant of Kluyveromyces lactis (MATa, Kladh1 Δ trp1, his3, leu2 ura3) respectively by currently known methods to generate expressing K IALD6+KIACS1+, KIALD6+KIACS2+ and KIALD-KIACS-.Select this two groups of transformant by HIS, TRP and HIS, LEU prototroph respectively.

As described in example 14 above to transformant KIALD6+KIACS1+ and KIALD6+KIACS2+ assessment cell acetyl-CoA level.

Whether cause higher butanols to generate in order to assess cross expressing of KIAld6 and KIAcs1 or KIAcs2, Kluyveromyces lactis (MATa, Kladh1 Δ trp1, his3, leu2 ura3) is transformed with pGV1208KIALD6, pGV1209KIACS1 or pGV1209KIACS2, pGV1227KI, pGV1213KI.Implementing butanols as described in example 4 above generates.The propyl carbinol productive rate is greater than 10%.

Sequence table

＜110〉Gevo Inc. (GEVO, INC.)

＜120〉production of butanol of being undertaken by the metabolic engineering yeast

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ctgaaggatg?ttccggcagt?cgacttaggg?gctacggcga?tcaaagaagc?cgtaaaaaag????120

gcaggaatta?aaccagagga?tgtgaatgaa?gttatcctgg?gcaacgtcct?gcaggctggt????180

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gcagagaaca?ttgcggaacg?ctggaatatc?tctcgggagg?aacaggatga?gttcgcttta????540

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ccggttgtga?ttaaagggcg?taaaggagaa?actgtcgttg?atacagacga?acacccgcgc????660

ttcggctcca?ccattgaggg?tctggctaag?ctgaaaccag?cctttaaaaa?ggatgggacg????720

gtaaccgcag?gcaacgcgtc?gggtttaaat?gattgtgccg?cagtgctggt?catcatgagc????780

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acgaaggtgg?aaattttaac?tcggatcagt?ggaacagttg?atctgaatat?ggccgctgac????240

tgcgatctgg?tcattgaagc?ggccgtagag?cgtatggata?tcaaaaaaca?aatttttgca????300

gacttagata?acatctgtaa?gccggaaacc?attctggctt?caaatacgtc?ctcgctgagc????360

atcactgagg?tggcgtctgc?cacaaaacgc?ccagacaaag?ttattggcat?gcatttcttt????420

aaccctgcac?cggtcatgaa?gttagtggaa?gtaatccgtg?ggattgctac?cagtcaggaa????480

acgttcgatg?cggttaaaga?gacctcaatc?gccattggaa?aagacccagt?ggaagtcgca????540

gaggcgcctg?gctttgttgt?aaatcgcatt?ctgatcccga?tgattaacga?agctgtggga????600

atcctggccg?aaggaattgc?atccgtcgag?gatatcgaca?aggcgatgaa?attaggcgct????660

aatcacccga?tgggtccact?ggaactgggc?gacttcattg?gtctggatat?ctgcttagcc????720

attatggacg?ttctgtattc?ggagactggg?gatagcaaat?accggcctca?tacactgtta????780

aagaaatatg?tgcgtgcagg?atggctgggc?cgcaaatctg?gtaagggttt?ctacgattat????840

tcaaaataa????????????????????????????????????????????????????????????849

<210>3

<211>786

<212>DNA

<213>Ca-crt-co

<400>3

atggaactaa?acaatgtcat?cctggaaaaa?gagggcaagg?tggcggttgt?caccattaat?????60

cgtccgaaag?ccttaaacgc?actgaatagc?gatacgctga?aagaaatgga?ctatgtaatc????120

ggtgagattg?aaaacgattc?tgaagtgtta?gctgttatcc?tgactggggc?gggagagaag????180

agttttgtcg?ccggcgcaga?catttcagaa?atgaaagaga?tgaatacaat?cgaaggtcgc????240

aaattcggga?ttctgggaaa?caaggtattt?cggcgtttag?aactgctgga?gaaaccagtg????300

atcgctgcgg?ttaatggctt?cgccttaggt?ggcggttgcg?aaattgcaat?gtcctgtgat????360

atccgcattg?cttcgagcaa?cgcgcgtttt?gggcagcctg?aggtcggact?gggcatcaca????420

ccgggtttcg?gcggtacgca?acgcctgtct?cggttagtgg?ggatgggaat?ggccaaacag????480

ctgattttta?ctgcacaaaa?tatcaaggct?gacgaagcgc?tgcgtattgg?cctggtaaac????540

aaagttgtgg?aaccaagtga?gttaatgaat?acagccaaag?aaatcgcaaa?caagattgtc????600

tcaaatgcgc?ctgttgctgt?aaaactgtcc?aaacaggcca?ttaaccgcgg?tatgcagtgc????660

gatatcgaca?ccgcactggc?gttcgagtcg?gaagcttttg?gggaatgttt?cagcacggag????720

gaccaaaagg?atgccatgac?cgcatttatt?gaaaaacgta?aaattgaagg?cttcaaaaat????780

agatag???????????????????????????????????????????????????????????????786

<210>4

<211>1140

<212>DNA

<213>Ca-bcd-co

<400>4

atggatttta?atttaacaag?agaacaggaa?ctggtccgtc?agatggtacg?tgaatttgca?????60

gaaaacgagg?ttaaaccgat?tgctgcagag?attgatgaga?ctgaacgctt?cccgatggaa????120

aacgtcaaaa?agatgggtca?gtatggcatg?atgggcattc?cgttctctaa?agagtacggc????180

ggtgcgggtg?gcgacgttct?gtcttatatc?atcgctgtag?aggaactgtc?caaagtatgt????240

ggcaccacgg?gcgtgatcct?gtccgcgcac?acctctctgt?gcgcaagcct?gatcaacgaa????300

cacggcaccg?aggaacagaa?gcaaaaatac?ctggtcccgc?tggccaaagg?tgaaaagatc????360

ggtgcatacg?gtctgacgga?accgaacgca?ggtacggaca?gcggcgcaca?acagacggtt????420

gcggtactgg?aaggcgacca?ctacgttatt?aacggtagca?aaatcttcat?cacgaacggt????480

ggcgtggctg?acacctttgt?tatcttcgcg?atgaccgacc?gcactaaagg?cactaaaggt????540

atctctgcgt?tcatcatcga?gaagggtttc?aagggttttt?ctatcggcaa?agtggaacag????600

aagctgggta?tccgtgcctc?ctctactacc?gagctggttt?tcgaagacat?gattgtgccg????660

gttgaaaata?tgatcggcaa?agaaggcaaa?ggcttcccga?tcgctatgaa?aaccctggat????720

ggcggccgta?tcggcattgc?agcacaggca?ctgggtatcg?cagaaggcgc?tttcaacgaa????780

gcacgtgcgt?acatgaaaga?acgtaaacag?tttggccgtt?ctctggataa?atttcaaggc????840

ctggcgtgga?tgatggcaga?catggacgta?gcgattgaat?ctgcgcgcta?cctggtctat????900

aaagcagctt?acctgaaaca?ggcaggtctg?ccttacaccg?ttgacgcagc?acgtgcgaaa????960

ctgcacgcgg?ccaacgttgc?catggatgtt?accaccaaag?ccgtgcaact?gtttggcggt???1020

tacggctata?ctaaggatta?tccggttgaa?cgtatgatgc?gtgacgcgaa?aatcaccgaa???1080

atctatgaag?gtacttccga?agtgcagaaa?ctggtcattt?caggaaaaat?ttttagttaa???1140

<210>5

<211>1011

<212>DNA

<213>Ca-eftA-co

<400>5

atgaataaag?cagattacaa?gggcgtttgg?gtctttgcgg?aacagcgtga?tggtgaactg?????60

cagaaagtgt?ccctggaact?gctgggcaaa?ggcaaggaga?tggcagaaaa?actgggtgtt????120

gaactgaccg?cagttctgct?gggtcacaac?actgaaaaga?tgtccaaaga?cctgctgtcc????180

catggcgcag?acaaggtgct?ggctgcggac?aacgaactgc?tggctcactt?tagcaccgac????240

ggttatgcaa?aagtaatctg?cgacctggtt?aacgagcgca?agccggaaat?cctgttcatc????300

ggcgccactt?ttattggtcg?cgacctgggc?cctcgtattg?ctgcgcgtct?gtccactggc????360

ctgactgcgg?attgcacctc?cctggacatt?gatgttgaaa?accgtgatct?gctggcaact????420

cgcccggcat?tcggcggcaa?cctgatcgcc?accatcgtat?gttccgacca?ccgtccgcaa????480

atggctactg?tacgtccggg?cgtatttgaa?aagctgccgg?tgaacgacgc?aaacgtttcc????540

gacgacaaaa?tcgaaaaagt?tgctatcaag?ctgaccgcta?gcgatatccg?taccaaagtt????600

tctaaagtag?tgaaactggc?gaaggacatc?gcagatattg?gtgaagcaaa?agttctggtg????660

gcaggcggtc?gtggcgtcgg?ttccaaagag?aacttcgaaa?aactggagga?actggcgtct????720

ctgctgggcg?gtactattgc?agcgtcccgt?gcagcaatcg?aaaaagaatg?ggtggacaag????780

gatctgcagg?tgggccagac?tggtaaaacc?gttcgtccga?ccctgtacat?cgcctgcggc????840

atctccggtg?ctattcagca?cctggccggc?atgcaggaca?gcgactacat?catcgccatc????900

aacaaagacg?ttgaagctcc?gatcatgaaa?gtggcggacc?tggcaatcgt?tggtgacgtg????960

aacaaagttg?ttccggaact?gatcgcgcag?gttaaagctg?ctaataatta?a????????????1011

<210>6

<211>780

<212>DNA

<213>Ca-eftB-co

<400>6

atgaatatag?ttgtttgttt?aaaacaggtc?ccggacaccg?cagaagttcg?tattgatcca?????60

gtaaagggta?cgctgattcg?cgagggcgtg?ccgtctatca?tcaacccaga?tgacaagaac????120

gccctggaag?aagcactggt?cctgaaagat?aattacggcg?ctcacgtaac?tgttatctct????180

atgggtccgc?cgcaagcgaa?aaatgcgctg?gttgaagctc?tggcgatggg?cgctgacgag????240

gctgttctgc?tgactgatcg?tgctttcggt?ggtgcggaca?ccctggccac?ttcccacact????300

atcgcggcag?gtatcaagaa?actgaaatat?gacattgtgt?ttgctggtcg?tcaggctatt????360

gacggtgaca?cggcacaggt?aggcccggaa?atcgccgaac?acctgggtat?tccgcaggtg????420

acctacgtag?aaaaagtaga?agtagacggt?gataccctga?aaatccgcaa?agcatgggaa????480

gatggctacg?aggtggttga?agtaaaaacc?ccggtactgc?tgaccgctat?caaagagctg????540

aatgtaccgc?gttacatgtc?tgttgagaaa?atcttcggcg?cgttcgacaa?ggaagtaaag????600

atgtggaccg?ctgatgatat?tgacgttgac?aaagcgaatc?tgggcctgaa?gggctcccca????660

actaaagtta?agaagtcctc?tactaaagaa?gtgaagggtc?agggtgaggt?gattgataaa????720

cctgttaaag?aagctgctgc?gtacgtggtt?tctaagctga?aagaagaaca?ctatatttaa????780

<210>7

<211>2577

<212>DNA

<213>Ca-adhE2-co

<400>7

atgaaagtta?caaatcaaaa?agaactgaaa?cagaagttaa?atgagctgcg?tgaggcgcaa?????60

aaaaaatttg?ccacctatac?gcaggaacaa?gtggataaga?ttttcaaaca?gtgcgcaatc????120

gctgcggcca?aagaacgcat?taacctggca?aagttagctg?ttgaagagac?tggcatcggt????180

ctggtcgagg?acaaaattat?caaaaatcat?tttgcggccg?agtacattta?taacaagtac????240

aaaaacgaga?aaacctgtgg?gatcattgac?cacgatgata?gcctgggaat?cacaaaggta?????300

gcagaaccga?ttggcatcgt?ggctgcgatt?gttccaacga?ctaatcctac?atctaccgcc?????360

atcttcaaaa?gtttaatttc?actgaaaacg?cggaatgcaa?tctttttctc?cccgcatcca?????420

cgtgctaaga?aatcgaccat?tgcggccgca?aaactgattt?tagacgcggc?tgtcaaggcc?????480

ggtgcaccta?aaaacatcat?tgggtggatc?gacgaaccga?gcattgaact?gtctcaggat?????540

ctgatgagtg?aggcggacat?cattttagct?actggaggcc?cgtcaatggt?aaaagccgca?????600

tattcctcgg?gtaagccagc?gatcggcgtg?ggtgctggga?atactcctgc?cattatcgac?????660

gaaagcgcag?acattgatat?ggcggtttct?agtatcattc?tgtcaaaaac?gtacgacaac?????720

ggagtcatct?gcgcctccga?acagtcgatt?ctggtgatga?atagcatcta?tgagaaagta?????780

aaggaagagt?ttgttaaacg?cggctcttac?attctgaacc?agaatgaaat?tgcaaaaatc?????840

aaggaaacca?tgttcaaaaa?cggtgcgatt?aatgctgata?tcgtgggcaa?aagtgcctat?????900

attatcgcga?agatggctgg?tattgaggtc?ccgcaaacta?caaaaatctt?aattggggaa?????960

gttcagtcag?tagaaaaatc?cgagctgttt?agccacgaaa?agctgtcgcc?ggtgttagca????1020

atgtataaag?tcaaagattt?cgacgaggcc?ctgaagaaag?cgcagcgtct?gatcgaatta????1080

ggaggctctg?gtcataccag?ttcactgtac?attgatagcc?aaaacaataa?agacaaggtt????1140

aaagaatttg?ggctggctat?gaaaacgtcc?cgcaccttta?tcaacatgcc?atcgtctcag????1200

ggcgcaagtg?gtgatttata?taatttcgcc?attgcgccta?gctttactct?gggatgtggc????1260

acatggggtg?ggaactcagt?gtcccaaaat?gtagagccga?agcatctgct?gaacatcaaa????1320

tcggtcgctg?aacggcgtga?gaatatgtta?tggttcaaag?ttccacagaa?gatttacttt????1380

aaatatggct?gcctgcgctt?cgcactgaaa?gaattaaagg?atatgaacaa?aaaacgtgcc????1440

tttatcgtga?cggacaagga?tctgttcaaa?ctgggttacg?taaataaaat?taccaaggtt????1500

ttagacgaaa?ttgatatcaa?atattctatt?tttactgaca?tcaaaagcga?tccgacaatt????1560

gatagtgtga?agaaaggagc?gaaagagatg?ctgaacttcg?aacctgacac?gatcatttca????1620

atcggcggtg?ggtccccgat?ggatgctgca?aaggtcatgc?atctgttata?cgagtatcca????1680

gaagccgaaa?ttgagaatct?ggcgatcaac?tttatggaca?ttcgcaaacg?gatctgtaat????1740

tttccgaaac?tgggaaccaa?ggctattagc?gttgcaatcc?ctactacggc?cggcaccggt????1800

tcggaagcga?caccgttcgc?tgtgattacc?aacgatgaga?ctgggatgaa?atatccactg????1860

acatcttacg?aattaacgcc?gaatatggca?atcattgata?ccgaactgat?gctgaacatg????1920

cctcgtaaat?taactgccgc?gacgggcatt?gacgcactgg?tacacgccat?cgaggcgtat????1980

gtcagtgtta?tggcaaccga?ttacacagac?gaactggcgt?tacgcgctat?taagatgatc????2040

tttaaatatc?tgccacgtgc?ctacaaaaat?ggtactaacg?atattgaagc?gcgcgagaag????2100

atggctcatg?catcaaatat?cgccggaatg?gcgttcgcta?acgcatttct?gggcgtgtgc????2160

cacagcatgg?cccataaatt?aggtgcgatg?caccatgtac?cgcatgggat?tgcttgtgca????2220

gtcctgatcg?aagaggttat?taaatataat?gccacggact?gccctaccaa?gcagacagcg????2280

ttcccgcaat?acaaatcccc?aaacgctaaa?cggaagtatg?cagaaatcgc?cgaatatctg????2340

aatctgaaag?gcacttcgga?tacggagaaa?gtgaccgcgt?taattgaagc?tatctctaag????2400

ctgaaaattg?atctgagtat?cccgcagaac?atttcagcag?ccggtattaa?taaaaaggac????2460

ttttacaaca?ccttagataa?aatgagcgag?ctggcgttcg?acgatcaatg?tacaactgct????2520

aatcctcgtt?atccgctgat?ctccgaatta?aaagatatct?atataaaatc?attttaa???????2577

<210>8

<211>1152

<212>DNA

<213>Me-bcd-co

<400>8

atggatttta?acttaacaga?tattcagcaa?gacttcctga?agctggcaca?cgactttggt?????60

gaaaagaaac?tggcccctac?tgttaccgaa?cgcgaccaca?aaggtatcta?cgataaagaa????120

ctgattgacg?aactgctgtc?tctgggtatc?accggcgcat?acttcgaaga?aaaatacggc????180

ggtagcggtg?acgacggtgg?cgatgtactg?tcttatatcc?tggccgtaga?agaactggcg????240

aaatacgacg?ctggtgttgc?tatcactctg?tctgccaccg?taagcctgtg?tgcgaatccg????300

atttggcagt?ttggtactga?ggctcagaaa?gaaaagtttc?tggttccact?ggtcgaaggt????360

actaaactgg?gtgcgtttgg?tctgaccgaa?ccgaacgcgg?gcactgatgc?gagcggccag????420

caaactattg?ctactaaaaa?cgatgacggc?acgtacaccc?tgaacggtag?caaaatcttc????480

atcaccaacg?gtggcgctgc?cgatatctac?atcgtatttg?cgatgaccga?caaaagcaag????540

ggtaaccatg?gcatcaccgc?gttcatcctg?gaagatggca?ctccgggttt?cacctacggc????600

aaaaaggaag?ataaaatggg?tatccacacc?tctcagacta?tggaactggt?tttccaggac????660

gttaaggtcc?cggccgagaa?catgctgggc?gaagaaggca?aaggcttcaa?gattgcaatg????720

atgaccctgg?acggcggtcg?cattggcgtt?gcggcccagg?cactgggcat?cgcagaggca????780

gcgctggccg?acgctgttga?atacagcaaa?cagcgtgttc?agtttggcaa?acctctgtgc????840

aaattccaat?ccattagctt?taagctggcc?gatatgaaaa?tgcagatcga?agccgcacgc????900

aacctggtat?ataaagctgc?atgcaagaaa?caagaaggta?aaccgttcac?cgtagacgct????960

gcgatcgcga?aacgtgtagc?cagcgatgtg?gcaatgcgcg?tgactaccga?agcagttcag???1020

attttcggtg?gctatggtta?ctctgaagaa?tacccggtgg?ctcgccacat?gcgcgacgca???1080

aaaatcactc?agatctacga?gggtacgaac?gaagtgcagc?tgatggtcac?cggcggtgct???1140

ctgttaagtt?aa???????????????????????????????????????????????????????1152

<210>9

<211>1017

<212>DNA

<213>Me-eftA-co

<400>9

atggatttag?cagaatacaa?aggcatctac?gtgatcgcag?agcagttcga?aggtaaactg?????60

cgtgacgttt?ctttcgaact?gctgggtcaa?gcgcgcatcc?tggcggacac?gatcggcgac????120

gaagtaggcg?caatcctgat?tggcaaagat?gtaaaaccac?tggcgcagga?actgatcgcg????180

catggtgctc?ataaagtgta?cgtctatgac?gacccgcagc?tggaacatta?caacacgact????240

gcctatgcca?aagtgatttg?cgacttcttt?catgaagaga?aaccaaacgt?tttcctggtt????300

ggtgcaacta?acatcggtcg?tgacctgggt?ccacgtgtag?cgaacagcct?gaaaaccggt????360

ctgactgcgg?attgtaccca?gctgggtgtt?gatgatgata?agaaaaccat?cgtttggacc????420

cgtccggcac?tgggcggcaa?catcatggcg?gaaattatct?gtccagataa?ccgcccgcag????480

atgggcactg?tgcgtcctca?tgtcttcaaa?aagccggaag?ccgacccgag?cgcaactggt????540

gaagtcattg?aaaagaaagc?gaacctgtct?gacgctgatt?tcatgactaa?gttcgtagaa????600

ctgatcaaac?tgggtggtga?aggcgttaaa?atcgaggatg?ccgatgttat?tgttgctggt????660

ggccgtggca?tgaatagcga?agagcctttt?aaaaccggta?tcctgaaaga?gtgcgcggac????720

gtactgggcg?gtgctgtcgg?tgccagccgt?gccgccgtgg?acgcgggctg?gatcgacgct????780

ctgcaccagg?tcggccagac?tggcaaaacc?gttggtccga?aaatctacat?tgcttgtgcg????840

attagcggtg?ctatccagcc?gctggcaggc?atgacgggct?ctgattgtat?tatcgcaatt????900

aacaaagatg?aagacgcgcc?tattttcaag?gtgtgcgact?atggcattgt?gggcgatgtg????960

ttcaaagtgc?tgccactgct?gactgaggcg?atcaagaaac?agaaaggcat?tgcataa??????1017

<210>10

<211>813

<212>DNA

<213>Me-eftB-co

<400>10

atggaaatat?tggtatgtgt?caaacaagtg?ccggatactg?cagaagtcaa?aattgatccg?????60

gttaaacaca?ccgtgattcg?tgcgggtgtg?ccgaatatct?tcaacccgtt?cgaccaaaac????120

gcgctggaag?cggcgctggc?gctgaaggac?gcggataaag?acgttaagat?tactctgctg????180

tctatgggcc?cggaccaggc?aaaagatgtt?ctgcgtgaag?gcctggccat?gggcgctgat????240

gacgcgtacc?tgctgtccga?tcgtaaactg?ggtggctccg?acactctggc?caccggttat????300

gctctggccc?aggctattaa?gaaactggct?gcggacaagg?gtattgagca?attcgacatc????360

atcctgtgtg?gtaagcaagc?gattgacggt?gataccgctc?aggtaggtcc?acagatcgct????420

tgtgagctgg?gcatcccgca?gatcacttat?gctcgtgaca?tcaaggttga?gggcgataag????480

gttactgtgc?agcaggaaaa?cgaagagggt?tacatcgtga?ccgaagcgca?gttcccggtt????540

ctgatcaccg?cggttaaaga?cctgaacgaa?cctcgtttcc?cgaccatccg?tggcaccatg????600

aaggcgaagc?gtcgtgaaat?cccgaacctg?gacgcagctg?cagttgccgc?ggacgacgcg????660

cagatcggcc?tgtccggttc?tccgaccaaa?gtacgcaaaa?ttttcacccc?accgcagcgt????720

tccggcggtc?tggtactgaa?agtggaagac?gacaacgaac?aggccattgt?cgaccaggtt????780

atggaaaaac?tggttgccca?gaaaatcatt??taa????????????????????????????????813

<210>11

<211>5024

<212>DNA

<213>pGV1090

<400>11

ctagtgcttg?gattctcacc?aataaaaaac?gcccggcggc?aaccgagcgt?tctgaacaaa??????60

tccagatgga?gttctgaggt?cattactgga?tctatcaaca?ggagtccaag?cgagctcgat?????120

atcaaattac?gccccgccct?gccactcatc?gcagtactgt?tgtaattcat?taagcattct?????180

gccgacatgg?aagccatcac?agacggcatg?atgaacctga?atcgccagcg?gcatcagcac?????240

cttgtcgcct?tgcgtataat?atttgcccat?ggtgaaaacg?ggggcgaaga?agttgtccat?????300

attggccacg?tttaaatcaa?aactggtgaa?actcacccag?ggattggctg?agacgaaaaa?????360

catattctca?ataaaccctt?tagggaaata?ggccaggttt?tcaccgtaac?acgccacatc?????420

ttgcgaatat?atgtgtagaa?actgccggaa?atcgtcgtgg?tattcactcc?agagcgatga?????480

aaacgtttca?gtttgctcat?ggaaaacggt?gtaacaaggg?tgaacactat?cccatatcac?????540

cagctcaccg?tctttcattg?ccatacgaaa?ctccggatga?gcattcatca?ggcgggcaag?????600

aatgtgaata?aaggccggat?aaaacttgtg?cttatttttc?tttacggtct?ttaaaaaggc?????660

cgtaatatcc?agctgaacgg?tctggttata?ggtacattga?gcaactgact?gaaatgcctc?????720

aaaatgttct?ttacgatgcc?attgggatat?atcaacggtg?gtatatccag?tgattttttt?????780

ctccatttta?gcttccttag?ctcctgaaaa?tctcgataac?tcaaaaaata?cgcccggtag?????840

tgatcttatt?tcattatggt?gaaagttgga?acctcttacg?tgccgatcaa?cgtctcattt?????900

tcgccagata?tcgacgtcta?agaaaccatt?attatcatga?cattaaccta?taaaaatagg?????960

cgtatcacga?ggccctttcg?tcttcacctc?gagaaatgtg?agcggataac?aattgacatt????1020

gtgagcggat?aacaagatac?tgagcacatc?agcaggacgc?actgaccgaa?ttcaaaattg????1080

aaggcttcaa?aaatagatag?gaggtaagtt?tatatggatt?ttaatttaac?aagagaacag????1140

gaactggtcc?gtcagatggt?acgtgaattt?gcagaaaacg?aggttaaacc?gattgctgca????1200

gagattgatg?agactgaacg?cttcccgatg?gaaaacgtca?aaaagatggg?tcagtatggc????1260

atgatgggca?ttccgttctc?taaagagtac?ggcggtgcgg?gtggcgacgt?tctgtcttat????1320

atcatcgctg?tagaggaact?gtccaaagta?tgtggcacca?cgggcgtgat?cctgtccgcg????1380

cacacctctc?tgtgcgcaag?cctgatcaac?gaacacggca?ccgaggaaca?gaagcaaaaa????1440

tacctggtcc?cgctggccaa?aggtgaaaag?atcggtgcat?acggtctgac?ggaaccgaac????1500

gcaggtacgg?acagcggcgc?acaacagacg?gttgcggtac?tggaaggcga?ccactacgtt????1560

attaacggta?gcaaaatctt?catcacgaac?ggtggcgtgg?ctgacacctt?tgttatcttc????1620

gcgatgaccg?accgcactaa?aggcactaaa?ggtatctctg?cgttcatcat?cgagaagggt????1680

ttcaagggtt?tttctatcgg?caaagtggaa?cagaagctgg?gtatccgtgc?ctcctctact????1740

accgagctgg?ttttcgaaga?catgattgtg?ccggttgaaa?atatgatcgg?caaagaaggc????1800

aaaggcttcc?cgatcgctat?gaaaaccctg?gatggcggcc?gtatcggcat?tgcagcacag????1860

gcactgggta?tcgcagaagg?cgctttcaac?gaagcacgtg?cgtacatgaa?agaacgtaaa????1920

cagtttggcc?gttctctgga?taaatttcaa?ggcctggcgt?ggatgatggc?agacatggac????1980

gtagcgattg?aatctgcgcg?ctacctggtc?tataaagcag?cttacctgaa?acaggcaggt????2040

ctgccttaca?ccgttgacgc?agcacgtgcg?aaactgcacg?cggccaacgt?tgccatggat????2100

gttaccacca?aagccgtgca?actgtttggc?ggttacggct?atactaagga?ttatccggtt????2160

gaacgtatga?tgcgtgacgc?gaaaatcacc?gaaatctatg?aaggtacttc?cgaagtgcag????2220

aaactggtca?tttcaggaaa?aatttttagt?taattaaagg?aggttaagag?gatgaatata????2280

gttgtttgtt?taaaacaggt?cccggacacc?gcagaagttc?gtattgatcc?agtaaagggt????2340

acgctgattc?gcgagggcgt?gccgtctatc?atcaacccag?atgacaagaa?cgccctggaa????2400

gaagcactgg?tcctgaaaga?taattacggc?gctcacgtaa?ctgttatctc?tatgggtccg????2460

ccgcaagcga?aaaatgcgct?ggttgaagct?ctggcgatgg?gcgctgacga?ggctgttctg????2520

ctgactgatc?gtgctttcgg?tggtgcggac?accctggcca?cttcccacac?tatcgcggca????2580

ggtatcaaga?aactgaaata?tgacattgtg?tttgctggtc?gtcaggctat?tgacggtgac????2640

acggcacagg?taggcccgga?aatcgccgaa?cacctgggta?ttccgcaggt?gacctacgta????2700

gaaaaagtag?aagtagacgg?tgataccctg?aaaatccgca?aagcatggga?agatggctac????2760

gaggtggttg?aagtaaaaac?cccggtactg?ctgaccgcta?tcaaagagct?gaatgtaccg????2820

cgttacatgt?ctgttgagaa?aatcttcggc?gcgttcgaca?aggaagtaaa?gatgtggacc????2880

gctgatgata?ttgacgttga?caaagcgaat?ctgggcctga?agggctcccc?aactaaagtt????2940

aagaagtcct?ctactaaaga?agtgaagggt?cagggtgagg?tgattgataa?acctgttaaa????3000

gaagctgctg?cgtacgtggt?ttctaagctg?aaagaagaac?actatattta?agttaggagg????3060

gatttttcaa?tgaataaagc?agattacaag?ggcgtttggg?tctttgcgga?acagcgtgat????3120

ggtgaactgc?agaaagtgtc?cctggaactg?ctgggcaaag?gcaaggagat?ggcagaaaaa????3180

ctgggtgttg?aactgaccgc?agttctgctg?ggtcacaaca?ctgaaaagat?gtccaaagac????3240

ctgctgtccc?atggcgcaga?caaggtgctg?gctgcggaca?acgaactgct?ggctcacttt????3300

agcaccgacg?gttatgcaaa?agtaatctgc?gacctggtta?acgagcgcaa?gccggaaatc????3360

ctgttcatcg?gcgccacttt?tattggtcgc?gacctgggcc?ctcgtattgc?tgcgcgtctg????3420

tccactggcc?tgactgcgga?ttgcacctcc?ctggacattg?atgttgaaaa?ccgtgatctg????3480

ctggcaactc?gcccggcatt?cggcggcaac?ctgatcgcca?ccatcgtatg?ttccgaccac????3540

cgtccgcaaa?tggctactgt?acgtccgggc?gtatttgaaa?agctgccggt?gaacgacgca????3600

aacgtttccg?acgacaaaat?cgaaaaagtt?gctatcaagc?tgaccgctag?cgatatccgt????3660

accaaagttt?ctaaagtagt?gaaactggcg?aaggacatcg?cagatattgg?tgaagcaaaa????3720

gttctggtgg?caggcggtcg?tggcgtcggt?tccaaagaga?acttcgaaaa?actggaggaa????3780

ctggcgtctc?tgctgggcgg?tactattgca?gcgtcccgtg?cagcaatcga?aaaagaatgg????3840

gtggacaagg?atctgcaggt?gggccagact?ggtaaaaccg?ttcgtccgac?cctgtacatc????3900

gcctgcggca?tctccggtgc?tattcagcac?ctggccggca?tgcaggacag?cgactacatc????3960

atcgccatca?acaaagacgt?tgaagctccg?atcatgaaag?tggcggacct?ggcaatcgtt????4020

ggtgacgtga?acaaagttgt?tccggaactg?atcgcgcagg?ttaaagctgc?taataattaa????4080

ggatcccatg?gtacgcgtgc?tagaggcatc?aaataaaacg?aaaggctcag?tcgaaagact????4140

gggcctttcg?ttttatctgt?tgtttgtcgg?tgaacgctct?cctgagtagg?acaaatccgc????4200

cgccctagac?ctaggcgttc?ggctgcggcg?agcggtatca?gctcactcaa?aggcggtaat????4260

acggttatcc?acagaatcag?gggataacgc?aggaaagaac?atgtgagcaa?aaggccagca????4320

aaaggccagg?aaccgtaaaa?aggccgcgtt?gctggcgttt?ttccataggc?tccgcccccc????4380

tgacgagcat?cacaaaaatc?gacgctcaag?tcagaggtgg?cgaaacccga?caggactata????4440

aagataccag?gcgtttcccc?ctggaagctc?cctcgtgcgc?tctcctgttc?cgaccctgcc????4500

gcttaccgga?tacctgtccg?cctttctccc?ttcgggaagc?gtggcgcttt?ctcaatgctc????4560

acgctgtagg?tatctcagtt?cggtgtaggt?cgttcgctcc?aagctgggct?gtgtgcacga????4620

accccccgtt?cagcccgacc?gctgcgcctt?atccggtaac?tatcgtcttg?agtccaaccc????4680

ggtaagacac?gacttatcgc?cactggcagc?agccactggt?aacaggatta?gcagagcgag????4740

gtatgtaggc?ggtgctacag?agttcttgaa?gtggtggcct?aactacggct?acactagaag????4800

gacagtattt?ggtatctgcg?ctctgctgaa?gccagttacc?ttcggaaaaa?gagttggtag????4860

ctcttgatcc?ggcaaacaaa?ccaccgctgg?tagcggtggt?ttttttgttt?gcaagcagca????4920

gattacgcgc?agaaaaaaag?gatctcaaga?agatcctttg?atcttttcta?cggggtctga????4980

cgctcagtgg?aacgaaaact?cacgttaagg?gattttggtc?atga?????????????????????5024

<210>12

<211>3206

<212>DNA

<213>pGV1095

<400>12

ctagtgcttg?gattctcacc?aataaaaaac?gcccggcggc?aaccgagcgt?tctgaacaaa?????60

tccagatgga?gttctgaggt?cattactgga?tctatcaaca?ggagtccaag?cgagctcgat????120

atcaaattac?gccccgccct?gccactcatc?gcagtactgt?tgtaattcat?taagcattct????180

gccgacatgg?aagccatcac?agacggcatg?atgaacctga?atcgccagcg?gcatcagcac????240

cttgtcgcct?tgcgtataat?atttgcccat?ggtgaaaacg?ggggcgaaga?agttgtccat????300

attggccacg?tttaaatcaa?aactggtgaa?actcacccag?ggattggctg?agacgaaaaa????360

catattctca?ataaaccctt?tagggaaata?ggccaggttt?tcaccgtaac?acgccacatc????420

ttgcgaatat?atgtgtagaa?actgccggaa?atcgtcgtgg?tattcactcc?agagcgatga????480

aaacgtttca?gtttgctcat?ggaaaacggt?gtaacaaggg?tgaacactat?cccatatcac????540

cagctcaccg?tctttcattg?ccatacgaaa?ctccggatga?gcattcatca?ggcgggcaag????600

aatgtgaata?aaggccggat?aaaacttgtg?cttatttttc?tttacggtct?ttaaaaaggc????660

cgtaatatcc?agctgaacgg?tctggttata?ggtacattga?gcaactgact?gaaatgcctc????720

aaaatgttct?ttacgatgcc?attgggatat?atcaacggtg?gtatatccag?tgattttttt?????780

ctccatttta?gcttccttag?ctcctgaaaa?tctcgataac?tcaaaaaata?cgcccggtag?????840

tgatcttatt?tcattatggt?gaaagttgga?acctcttacg?tgccgatcaa?cgtctcattt?????900

tcgccagata?tcgacgtcta?agaaaccatt?attatcatga?cattaaccta?taaaaatagg?????960

cgtatcacga?ggccctttcg?tcttcacctc?gagaaatgtg?agcggataac?aattgacatt????1020

gtgagcggat?aacaagatac?tgagcacatc?agcaggacgc?actgaccgaa?ttcaacagga????1080

ggggttaaag?tggttgattt?cgaatattca?ataccaacta?gaattttttt?cggtaaagat????1140

aagataaatg?tacttggaag?agagcttaaa?aaatatggtt?ctaaagtgct?tatagtttat????1200

ggtggaggaa?gtataaagag?aaatggaata?tatgataaag?ctgtaagtat?acttgaaaaa????1260

aacagtatta?aattttatga?acttgcagga?gtagagccaa?atccaagagt?aactacagtt????1320

gaaaaaggag?ttaaaatatg?tagagaaaat?ggagttgaag?tagtactagc?tataggtgga????1380

ggaagtgcaa?tagattgcgc?aaaggttata?gcagcagcat?gtgaatatga?tggaaatcca????1440

tgggatattg?tgttagatgg?ctcaaaaata?aaaagggtgc?ttcctatagc?tagtatatta????1500

accattgctg?caacaggatc?agaaatggat?acgtgggcag?taataaataa?tatggataca????1560

aacgaaaaac?taattgcggc?acatccagat?atggctccta?agttttctat?attagatcca????1620

acgtatacgt?ataccgtacc?taccaatcaa?acagcagcag?gaacagctga?tattatgagt????1680

catatatttg?aggtgtattt?tagtaataca?aaaacagcat?atttgcagga?tagaatggca????1740

gaagcgttat?taagaacttg?tattaaatat?ggaggaatag?ctcttgagaa?gccggatgat????1800

tatgaggcaa?gagccaatct?aatgtgggct?tcaagtcttg?cgataaatgg?acttttaaca????1860

tatggtaaag?acactaattg?gagtgtacac?ttaatggaac?atgaattaag?tgcttattac????1920

gacataacac?acggcgtagg?gcttgcaatt?ttaacaccta?attggatgga?gtatatttta????1980

aataatgata?cagtgtacaa?gtttgttgaa?tatggtgtaa?atgtttgggg?aatagacaaa????2040

gaaaaaaatc?actatgacat?agcacatcaa?gcaatacaaa?aaacaagaga?ttactttgta????2100

aatgtactag?gtttaccatc?tagactgaga?gatgttggaa?ttgaagaaga?aaaattggac????2160

ataatggcaa?aggaatcagt?aaagcttaca?ggaggaacca?taggaaacct?aagaccagta????2220

aacgcctccg?aagtcctaca?aatattcaaa?aaatctgtgt?aaggatccca?tggtacgcgt????2280

gctagaggca?tcaaataaaa?cgaaaggctc?agtcgaaaga?ctgggccttt?cgttttatct????2340

gttgtttgtc?ggtgaacgct?ctcctgagta?ggacaaatcc?gccgccctag?acctaggcgt????2400

tcggctgcgg?cgagcggtat?cagctcactc?aaaggcggta?atacggttat?ccacagaatc????2460

aggggataac?gcaggaaaga?acatgtgagc?aaaaggccag?caaaaggcca?ggaaccgtaa????2520

aaaggccgcg?ttgctggcgt?ttttccatag?gctccgcccc?cctgacgagc?atcacaaaaa????2580

tcgacgctca?agtcagaggt?ggcgaaaccc?gacaggacta?taaagatacc?aggcgtttcc????2640

ccctggaagc?tccctcgtgc?gctctcctgt?tccgaccctg?ccgcttaccg?gatacctgtc????2700

cgcctttctc?ccttcgggaa?gcgtggcgct?ttctcaatgc?tcacgctgta?ggtatctcag????2760

ttcggtgtag?gtcgttcgct?ccaagctggg?ctgtgtgcac?gaaccccccg?ttcagcccga????2820

ccgctgcgcc?ttatccggta?actatcgtct?tgagtccaac?ccggtaagac?acgacttatc????2880

gccactggca?gcagccactg?gtaacaggat?tagcagagcg?aggtatgtag?gcggtgctac????2940

agagttcttg?aagtggtggc?ctaactacgg?ctacactaga?aggacagtat?ttggtatctg????3000

cgctctgctg?aagccagtta?ccttcggaaa?aagagttggt?agctcttgat?ccggcaaaca????3060

aaccaccgct?ggtagcggtg?gtttttttgt?ttgcaagcag?cagattacgc?gcagaaaaaa????3120

aggatctcaa?gaagatcctt?tgatcttttc?tacggggtct?gacgctcagt?ggaacgaaaa????3180

ctcacgttaa?gggattttgg?tcatga?????????????????????????????????????????3206

<210>13

<211>2836

<212>DNA

<213>pGV1094

<400>13

ctagtgcttg?gattctcacc?aataaaaaac?gcccggcggc?aaccgagcgt?tctgaacaaa????60

tccagatgga?gttctgaggt?cattactgga?tctatcaaca?ggagtccaag?cgagctcgat????120

atcaaattac?gccccgccct?gccactcatc?gcagtactgt?tgtaattcat?taagcattct????180

gccgacatgg?aagccatcac?agacggcatg?atgaacctga?atcgccagcg?gcatcagcac????240

cttgtcgcct?tgcgtataat?atttgcccat?ggtgaaaacg?ggggcgaaga?agttgtccat????300

attggccacg?tttaaatcaa?aactggtgaa?actcacccag?ggattggctg?agacgaaaaa????360

catattctca?ataaaccctt?tagggaaata?ggccaggttt?tcaccgtaac?acgccacatc????420

ttgcgaatat?atgtgtagaa?actgccggaa?atcgtcgtgg?tattcactcc?agagcgatga????480

aaacgtttca?gtttgctcat?ggaaaacggt?gtaacaaggg?tgaacactat?cccatatcac????540

cagctcaccg?tctttcattg?ccatacgaaa?ctccggatga?gcattcatca?ggcgggcaag????600

aatgtgaata?aaggccggat?aaaacttgtg?cttatttttc?tttacggtct?ttaaaaaggc????660

cgtaatatcc?agctgaacgg?tctggttata?ggtacattga?gcaactgact?gaaatgcctc????720

aaaatgttct?ttacgatgcc?attgggatat?atcaacggtg?gtatatccag?tgattttttt????780

ctccatttta?gcttccttag?ctcctgaaaa?tctcgataac?tcaaaaaata?cgcccggtag????840

tgatcttatt?tcattatggt?gaaagttgga?acctcttacg?tgccgatcaa?cgtctcattt????900

tcgccagata?tcgacgtcta?agaaaccatt?attatcatga?cattaaccta?taaaaatagg????960

cgtatcacga?ggccctttcg?tcttcacctc?gagaaatgtg?agcggataac?aattgacatt???1020

gtgagcggat?aacaagatac?tgagcacatc?agcaggacgc?actgaccggg?aattcctatc???1080

tatttttgaa?gccttcaatt?tttcttttct?ctatgaaagc?tgtcattgca?tccttttgat???1140

cctctgttga?aaagcattct?ccaaatgctt?ctgattcaaa?tgctaaagca?gtatcaatat???1200

cacactgcat?tcctctatta?atagcctgtt?tgcttaactt?aacagctact?ggagcattgc???1260

tcacaatttt?gtttgcaatt?tcttttgctg?tattcattaa?ttcactaggt?tctactacct???1320

tatttacaag?tccgattctt?aatgcttcat?ctgcctttat?attttgtgca?gtaaatataa???1380

gctgctttgc?catgcccatt?ccaactaatc?ttgaaagtct?ttgtgtacca?ccaaaaccag????1440

gtgttattcc?gagacctact?tctggttgac?caaatcttgc?gttgcttgaa?gctattctta????1500

tatcacaaga?catagctatt?tcgcatccgc?ctcctaaagc?aaaaccatta?acagctgcta????1560

ttacaggctt?ttcaagaagt?tctaatcttc?taaacacttt?atttccaagt?atcccgaatt????1620

ttctaccttc?aatggtattc?atttccttca?tctcagaaat?atctgctcct?gctacaaatg????1680

atttttctcc?tgctccagtt?aaaattactg?caagtacttc?gctatcattt?tcaatttcac????1740

ctataacata?atccatttct?tttagtgtat?cactatttaa?cgcatttaat?gctttaggtc????1800

tgttaatggt?aactacagca?actttacctt?ccttttcaag?gatgacattg?tttagttcca????1860

tgactaatcc?tcctaaaata?ttggatccga?tccgatccca?tggtacgcgt?gctagaggca????1920

tcaaataaaa?cgaaaggctc?agtcgaaaga?ctgggccttt?cgttttatct?gttgtttgtc????1980

ggtgaacgct?ctcctgagta?ggacaaatcc?gccgccctag?acctaggcgt?tcggctgcgg????2040

cgagcggtat?cagctcactc?aaaggcggta?atacggttat?ccacagaatc?aggggataac????2100

gcaggaaaga?acatgtgagc?aaaaggccag?caaaaggcca?ggaaccgtaa?aaaggccgcg????2160

ttgctggcgt?ttttccatag?gctccgcccc?cctgacgagc?atcacaaaaa?tcgacgctca????2220

agtcagaggt?ggcgaaaccc?gacaggacta?taaagatacc?aggcgtttcc?ccctggaagc????2280

tccctcgtgc?gctctcctgt?tccgaccctg?ccgcttaccg?gatacctgtc?cgcctttctc????2340

ccttcgggaa?gcgtggcgct?ttctcaatgc?tcacgctgta?ggtatctcag?ttcggtgtag????2400

gtcgttcgct?ccaagctggg?ctgtgtgcac?gaaccccccg?ttcagcccga?ccgctgcgcc????2460

ttatccggta?actatcgtct?tgagtccaac?ccggtaagac?acgacttatc?gccactggca????2520

gcagccactg?gtaacaggat?tagcagagcg?aggtatgtag?gcggtgctac?agagttcttg????2580

aagtggtggc?ctaactacgg?ctacactaga?aggacagtat?ttggtatctg?cgctctgctg????2640

aagccagtta?ccttcggaaa?aagagttggt?agctcttgat?ccggcaaaca?aaccaccgct????2700

ggtagcggtg?gtttttttgt?ttgcaagcag?cagattacgc?gcagaaaaaa?aggatctcaa????2760

gaagatcctt?tgatcttttc?tacggggtct?gacgctcagt?ggaacgaaaa?ctcacgttaa????2820

gggattttgg?tcatga????????????????????????????????????????????????????2836

<210>14

<211>2908

<212>DNA

<213>pGV1037

<400>14

ctagtgcttg?gattctcacc?aataaaaaac?gcccggcggc?aaccgagcgt?tctgaacaaa?????60

tccagatgga?gttctgaggt?cattactgga?tctatcaaca?ggagtccaag?cgagctcgat????120

atcaaattac?gccccgccct?gccactcatc?gcagtactgt?tgtaattcat?taagcattct????180

gccgacatgg?aagccatcac?agacggcatg?atgaacctga?atcgccagcg?gcatcagcac????240

cttgtcgcct?tgcgtataat?atttgcccat?ggtgaaaacg?ggggcgaaga?agttgtccat????300

attggccacg?tttaaatcaa?aactggtgaa?actcacccag?ggattggctg?agacgaaaaa?????360

catattctca?ataaaccctt?tagggaaata?ggccaggttt?tcaccgtaac?acgccacatc?????420

ttgcgaatat?atgtgtagaa?actgccggaa?atcgtcgtgg?tattcactcc?agagcgatga?????480

aaacgtttca?gtttgctcat?ggaaaacggt?gtaacaaggg?tgaacactat?cccatatcac?????540

cagctcaccg?tctttcattg?ccatacgaaa?ctccggatga?gcattcatca?ggcgggcaag?????600

aatgtgaata?aaggccggat?aaaacttgtg?cttatttttc?tttacggtct?ttaaaaaggc?????660

cgtaatatcc?agctgaacgg?tctggttata?ggtacattga?gcaactgact?gaaatgcctc?????720

aaaatgttct?ttacgatgcc?attgggatat?atcaacggtg?gtatatccag?tgattttttt?????780

ctccatttta?gcttccttag?ctcctgaaaa?tctcgataac?tcaaaaaata?cgcccggtag?????840

tgatcttatt?tcattatggt?gaaagttgga?acctcttacg?tgccgatcaa?cgtctcattt?????900

tcgccagata?tcgacgtcta?agaaaccatt?attatcatga?cattaaccta?taaaaatagg?????960

cgtatcacga?ggccctttcg?tcttcacctc?gagaaatgtg?agcggataac?aattgacatt????1020

gtgagcggat?aacaagatac?tgagcacatc?agcaggacgc?actgaccgga?attcattgat????1080

agtttcttta?aatttaggga?ggtctgttta?atgaaaaagg?tatgtgttat?aggtgcaggt????1140

actatgggtt?caggaattgc?tcaggcattt?gcagctaaag?gatttgaagt?agtattaaga????1200

gatattaaag?atgaatttgt?tgatagagga?ttagatttta?tcaataaaaa?tctttctaaa????1260

ttagttaaaa?aaggaaagat?agaagaagct?actaaagttg?aaatcttaac?tagaatttcc????1320

ggaacagttg?accttaatat?ggcagctgat?tgcgatttag?ttatagaagc?agctgttgaa????1380

agaatggata?ttaaaaagca?gatttttgct?gacttagaca?atatatgcaa?gccagaaaca????1440

attcttgcat?caaatacatc?atcactttca?ataacagaag?tggcatcagc?aactaaaaga????1500

cctgataagg?ttataggtat?gcatttcttt?aatccagctc?ctgttatgaa?gcttgtagag????1560

gtaataagag?gaatagctac?atcacaagaa?acttttgatg?cagttaaaga?gacatctata????1620

gcaataggaa?aagatcctgt?agaagtagca?gaagcaccag?gatttgttgt?aaatagaata????1680

ttaataccaa?tgattaatga?agcagttggt?atattagcag?aaggaatagc?ttcagtagaa????1740

gacatagata?aagctatgaa?acttggagct?aatcacccaa?tgggaccatt?agaattaggt????1800

gattttatag?gtcttgatat?atgtcttgct?ataatggatg?ttttatactc?agaaactgga????1860

gattctaagt?atagaccaca?tacattactt?aagaagtatg?taagagcagg?atggcttgga????1920

agaaaatcag?gaaaaggttt?ctacgattat?tcaaaataag?gatccgatcc?catggtacgc????1980

gtgctagagg?catcaaataa?aacgaaaggc?tcagtcgaaa?gactgggcct?ttcgttttat????2040

ctgttgtttg?tcggtgaacg?ctctcctgag?taggacaaat?ccgccgccct?agacctaggc????2100

gttcggctgc?ggcgagcggt?atcagctcac?tcaaaggcgg?taatacggtt?atccacagaa????2160

tcaggggata?acgcaggaaa?gaacatgtga?gcaaaaggcc?agcaaaaggc?caggaaccgt????2220

aaaaaggccg?cgttgctggc?gtttttccat?aggctccgcc?cccctgacga?gcatcacaaa????2280

aatcgacgct?caagtcagag?gtggcgaaac?ccgacaggac?tataaagata?ccaggcgttt????2340

ccccctggaa?gctccctcgt?gcgctctcct?gttccgaccc?tgccgcttac?cggatacctg????2400

tccgcctttc?tcccttcggg?aagcgtggcg?ctttctcaat?gctcacgctg?taggtatctc????2460

agttcggtgt?aggtcgttcg?ctccaagctg?ggctgtgtgc?acgaaccccc?cgttcagccc????2520

gaccgctgcg?ccttatccgg?taactatcgt?cttgagtcca?acccggtaag?acacgactta????2580

tcgccactgg?cagcagccac?tggtaacagg?attagcagag?cgaggtatgt?aggcggtgct????2640

acagagttct?tgaagtggtg?gcctaactac?ggctacacta?gaaggacagt?atttggtatc????2700

tgcgctctgc?tgaagccagt?taccttcgga?aaaagagttg?gtagctcttg?atccggcaaa????2760

caaaccaccg?ctggtagcgg?tggttttttt?gtttgcaagc?agcagattac?gcgcagaaaa????2820

aaaggatctc?aagaagatcc?tttgatcttt?tctacggggt?ctgacgctca?gtggaacgaa????2880

aactcacgtt?aagggatttt?ggtcatga???????????????????????????????????????2908

<210>15

<211>6219

<212>DNA

<213>pGV1031

<400>15

tcgcgcgttt?cggtgatgac?ggtgaaaacc?tctgacacat?gcagctcccg?gagacggtca?????60

cagcttgtct?gtaagcggat?gccgggagca?gacaagcccg?tcagggcgcg?tcagcgggtg????120

ttggcgggtg?tcggggctgg?cttaactatg?cggcatcaga?gcagattgta?ctgagagtgc????180

accatatgcg?gtgtgaaata?ccgcacagat?gcgtaaggag?aaaataccgc?atcaggcgcc????240

attcgccatt?caggctgcgc?aactgttggg?aagggcgatc?ggtgcgggcc?tcttcgctat????300

tacgccagct?ggcgaaaggg?ggatgtgctg?caaggcgatt?aagttgggta?acgccagggt????360

tttcccagtc?acgacgttgt?aaaacgacgg?ccagtgaatt?cgagctcggt?accatatgca????420

taagtttaat?ttttttgtta?aaaaatatta?aactttgtgt?tttttttaac?aaaatatatt????480

gataaaaata?ataatagtgg?gtataattaa?gttgttagag?aaaacgtata?aattagggat????540

aaactatgga?acttatgaaa?tagattgaaa?tggtttatct?gttaccccgt?atcaaaattt????600

aggaggttag?ttagaatgaa?agaagttgta?atagctagtg?cagtaagaac?agcgattgga????660

tcttatggaa?agtctcttaa?ggatgtacca?gcagtagatt?taggagctac?agctataaag????720

gaagcagtta?aaaaagcagg?aataaaacca?gaggatgtta?atgaagtcat?tttaggaaat????780

gttcttcaag?caggtttagg?acagaatcca?gcaagacagg?catcttttaa?agcaggatta????840

ccagttgaaa?ttccagctat?gactattaat?aaggtttgtg?gttcaggact?tagaacagtt????900

agcttagcag?cacaaattat?aaaagcagga?gatgctgacg?taataatagc?aggtggtatg????960

gaaaatatgt?ctagagctcc?ttacttagcg?aataacgcta?gatggggata?tagaatggga???1020

aacgctaaat?ttgttgatga?aatgatcact?gacggattgt?gggatgcatt?taatgattac???1080

cacatgggaa?taacagcaga?aaacatagct?gagagatgga?acatttcaag?agaagaacaa???1140

gatgagtttg?ctcttgcatc?acaaaaaaaa?gctgaagaag?ctataaaatc?aggtcaattt???1200

aaagatgaaa?tagttcctgt?agtaattaaa?ggcagaaagg?gagaaactgt?agttgataca???1260

gatgagcacc?ctagatttgg?atcaactata?gaaggacttg?caaaattaaa?acctgccttc????1320

aaaaaagatg?gaacagttac?agctggtaat?gcatcaggat?taaatgactg?tgcagcagta????1380

cttgtaatca?tgagtgcaga?aaaagctaaa?gagcttggag?taaaaccact?tgctaagata????1440

gtttcttatg?gttcagcagg?agttgaccca?gcaataatgg?gatatggacc?tttctatgca????1500

acaaaagcag?ctattgaaaa?agcaggttgg?acagttgatg?aattagattt?aatagaatca????1560

aatgaagctt?ttgcagctca?aagtttagca?gtagcaaaag?atttaaaatt?tgatatgaat????1620

aaagtaaatg?taaatggagg?agctattgcc?cttggtcatc?caattggagc?atcaggtgca????1680

agaatactcg?ttactcttgt?acacgcaatg?caaaaaagag?atgcaaaaaa?aggcttagca????1740

actttatgta?taggtggcgg?acaaggaaca?gcaatattgc?tagaaaagtg?ctagaaagga????1800

tccagaattt?aaaaggaggg?attaaaatga?actctaaaat?aattagattt?gaaaatttaa????1860

ggtcattctt?taaagatggg?atgacaatta?tgattggagg?ttttttaaac?tgtggcactc????1920

caaccaaatt?aattgatttt?ttagttaatt?taaatataaa?gaatttaacg?attataagta????1980

atgatacatg?ttatcctaat?acaggtattg?gtaagttaat?atcaaataat?caagtaaaaa????2040

agcttattgc?ttcatatata?ggcagcaacc?cagatactgg?caaaaaactt?tttaataatg????2100

aacttgaagt?agagctctct?ccccaaggaa?ctctagtgga?aagaatacgt?gcaggcggat????2160

ctggcttagg?tggtgtacta?actaaaacag?gtttaggaac?tttgattgaa?aaaggaaaga????2220

aaaaaatatc?tataaatgga?acggaatatt?tgttagagct?acctcttaca?gccgatgtag????2280

cattaattaa?aggtagtatt?gtagatgagg?ccggaaacac?cttctataaa?ggtactacta????2340

aaaactttaa?tccctatatg?gcaatggcag?ctaaaaccgt?aatagttgaa?gctgaaaatt????2400

tagttagctg?tgaaaaacta?gaaaaggaaa?aagcaatgac?ccccggagtt?cttataaatt????2460

atatagtaaa?ggagcctgca?taaaatgatt?aatgataaaa?acctagcgaa?agaaataata????2520

gccaaaagag?ttgcaagaga?attaaaaaat?ggtcaacttg?taaacttagg?tgtaggtctt????2580

cctaccatgg?ttgcagatta?tataccaaaa?aatttcaaaa?ttactttcca?atcagaaaac????2640

ggaatagttg?gaatgggcgc?tagtcctaaa?ataaatgagg?cagataaaga?tgtagtaaat????2700

gcaggaggag?actatacaac?agtacttcct?gacggcacat?ttttcgatag?ctcagtttcg????2760

ttttcactaa?tccgtggtgg?tcacgtagat?gttactgttt?taggggctct?ccaggtagat????2820

gaaaagggta?atatagccaa?ttggattgtt?cctggaaaaa?tgctctctgg?tatgggtgga????2880

gctatggatt?tagtaaatgg?agctaagaaa?gtaataattg?caatgagaca?tacaaataaa????2940

ggtcaaccta?aaattttaaa?aaaatgtaca?cttcccctca?cggcaaagtc?tcaagcaaat????3000

ctaattgtaa?cagaacttgg?agtaattgag?gttattaatg?atggtttact?tctcactgaa????3060

attaataaaa?acacaaccat?tgatgaaata?aggtctttaa?ctgctgcaga?tttactcata????3120

tccaatgaac?ttagacccat?ggctgtttag?aaagaattct?tgatatcagg?aaggtgactt????3180

ttatgttaaa?ggatgaagta?attaaacaaa?ttagcacgcc?attaacttcg?cctgcatttc????3240

ctagaggacc?ctataaattt?cataatcgtg?agtattttaa?cattgtatat?cgtacagata????3300

tggatgctct?tcgtaaagtt?gtgccagagc?ctttagaaat?tgatgagccc?ttagtcaggt????3360

ttgaaattat?ggcaatgcat?gatacgagtg?gacttggttg?ttatacagaa?agcggacagg????3420

ctattcccgt?aagctgtaat?ggagttaagg?gagattatct?tcatatgatg?tatttagata????3480

atgagcctgc?aattgcagta?ggaagggaat?taagtgcata?tcctaaaaag?ctcgggtatc????3540

caaagctttt?tgtggattca?gatactttag?taggaacttt?agactatgga?aaacttagag????3600

ttgcgacagc?tacaatgggg?tacaaacata?aagccttaga?tgctaatgaa?gcaaaggatc????3660

aaatttgtcg?ccctaattat?atgttgaaaa?taatacccaa?ttatgatgga?agccctagga????3720

tatgtgagct?tataaatgcg?aaaatcacag?atgttaccgt?acatgaagct?tggacaggac????3780

caactcgact?gcagttattt?gatcacgcta?tggcgccact?taatgatttg?ccagtaaaag????3840

agattgtttc?tagctctcac?attcttgcag?atataatatt?gcctagagct?gaagttatat????3900

atgattatct?taagtaataa?aaataagagt?taccttaaat?ggtaactctt?atttttttaa????3960

tgtcgacctg?caggcatgca?agcttggcgt?aatcatggtc?atagctgttt?cctgtgtgaa????4020

attgttatcc?gctcacaatt?ccacacaaca?tacgagccgg?aagcataaag?tgtaaagcct????4080

ggggtgccta?atgagtgagc?taactcacat?taattgcgtt?gcgctcactg?cccgctttcc????4140

agtcgggaaa?cctgtcgtgc?cagctgcatt?aatgaatcgg?ccaacgcgcg?gggagaggcg????4200

gtttgcgtat?tgggcgctct?tccgcttcct?cgctcactga?ctcgctgcgc?tcggtcgttc????4260

ggctgcggcg?agcggtatca?gctcactcaa?aggcggtaat?acggttatcc?acagaatcag????4320

gggataacgc?aggaaagaac?atgtgagcaa?aaggccagca?aaaggccagg?aaccgtaaaa????4380

aggccgcgtt?gctggcgttt?ttccataggc?tccgcccccc?tgacgagcat?cacaaaaatc????4440

gacgctcaag?tcagaggtgg?cgaaacccga?caggactata?aagataccag?gcgtttcccc????4500

ctggaagctc?cctcgtgcgc?tctcctgttc?cgaccctgcc?gcttaccgga?tacctgtccg????4560

cctttctccc?ttcgggaagc?gtggcgcttt?ctcatagctc?acgctgtagg?tatctcagtt????4620

cggtgtaggt?cgttcgctcc?aagctgggct?gtgtgcacga?accccccgtt?cagcccgacc????4680

gctgcgcctt?atccggtaac?tatcgtcttg?agtccaaccc?ggtaagacac?gacttatcgc????4740

cactggcagc?agccactggt?aacaggatta?gcagagcgag?gtatgtaggc?ggtgctacag????4800

agttcttgaa?gtggtggcct?aactacggct?acactagaag?gacagtattt?ggtatctgcg????4860

ctctgctgaa?gccagttacc?ttcggaaaaa?gagttggtag?ctcttgatcc?ggcaaacaaa????4920

ccaccgctgg?tagcggtggt?ttttttgttt?gcaagcagca?gattacgcgc?agaaaaaaag????4980

gatctcaaga?agatcctttg?atcttttcta?cggggtctga?cgctcagtgg?aacgaaaact????5040

cacgttaagg?gattttggtc?atgagattat?caaaaaggat?cttcacctag?atccttttaa????5100

attaaaaatg?aagttttaaa?tcaatctaaa?gtatatatga?gtaaacttgg?tctgacagtt????5160

accaatgctt?aatcagtgag?gcacctatct?cagcgatctg?tctatttcgt?tcatccatag????5220

ttgcctgact?ccccgtcgtg?tagataacta?cgatacggga?gggcttacca?tctggcccca????5280

gtgctgcaat?gataccgcga?gacccacgct?caccggctcc?agatttatca?gcaataaacc????5340

agccagccgg?aagggccgag?cgcagaagtg?gtcctgcaac?tttatccgcc?tccatccagt????5400

ctattaattg?ttgccgggaa?gctagagtaa?gtagttcgcc?agttaatagt?ttgcgcaacg????5460

ttgttgccat?tgctacaggc?atcgtggtgt?cacgctcgtc?gtttggtatg?gcttcattca????5520

gctccggttc?ccaacgatca?aggcgagtta?catgatcccc?catgttgtgc?aaaaaagcgg????5580

ttagctcctt?cggtcctccg?atcgttgtca?gaagtaagtt?ggccgcagtg?ttatcactca????5640

tggttatggc?agcactgcat?aattctctta?ctgtcatgcc?atccgtaaga?tgcttttctg????5700

tgactggtga?gtactcaacc?aagtcattct?gagaatagtg?tatgcggcga?ccgagttgct????5760

cttgcccggc?gtcaatacgg?gataataccg?cgccacatag?cagaacttta?aaagtgctca????5820

tcattggaaa?acgttcttcg?gggcgaaaac?tctcaaggat?cttaccgctg?ttgagatcca????5880

gttcgatgta?acccactcgt?gcacccaact?gatcttcagc?atcttttact?ttcaccagcg????5940

tttctgggtg?agcaaaaaca?ggaaggcaaa?atgccgcaaa?aaagggaata?agggcgacac????6000

ggaaatgttg?aatactcata?ctcttccttt?ttcaatatta?ttgaagcatt?tatcagggtt????6060

attgtctcat?gagcggatac?atatttgaat?gtatttagaa?aaataaacaa?ataggggttc????6120

cgcgcacatt?tccccgaaaa?gtgccacctg?acgtctaaga?aaccattatt?atcatgacat????6180

taacctataa?aaataggcgt?atcacgaggc?cctttcgtc???????????????????????????6219

<210>16

<211>2855

<212>DNA

<213>pGV1049

<400>16

ctagtgcttg?gattctcacc?aataaaaaac?gcccggcggc?aaccgagcgt?tctgaacaaa?????60

tccagatgga?gttctgaggt?cattactgga?tctatcaaca?ggagtccaag?cgagctcgat????120

atcaaattac?gccccgccct?gccactcatc?gcagtactgt?tgtaattcat?taagcattct????180

gccgacatgg?aagccatcac?agacggcatg?atgaacctga?atcgccagcg?gcatcagcac????240

cttgtcgcct?tgcgtataat?atttgcccat?ggtgaaaacg?ggggcgaaga?agttgtccat????300

attggccacg?tttaaatcaa?aactggtgaa?actcacccag?ggattggctg?agacgaaaaa????360

catattctca?ataaaccctt?tagggaaata?ggccaggttt?tcaccgtaac?acgccacatc????420

ttgcgaatat?atgtgtagaa?actgccggaa?atcgtcgtgg?tattcactcc?agagcgatga????480

aaacgtttca?gtttgctcat?ggaaaacggt?gtaacaaggg?tgaacactat?cccatatcac????540

cagctcaccg?tctttcattg?ccatacgaaa?ctccggatga?gcattcatca?ggcgggcaag????600

aatgtgaata?aaggccggat?aaaacttgtg?cttatttttc?tttacggtct?ttaaaaaggc????660

cgtaatatcc?agctgaacgg?tctggttata?ggtacattga?gcaactgact?gaaatgcctc????720

aaaatgttct?ttacgatgcc?attgggatat?atcaacggtg?gtatatccag?tgattttttt????780

ctccatttta?gcttccttag?ctcctgaaaa?tctcgataac?tcaaaaaata?cgcccggtag????840

tgatcttatt?tcattatggt?gaaagttgga?acctcttacg?tgccgatcaa?cgtctcattt????900

tcgccagata?tcgacgtcta?agaaaccatt?attatcatga?cattaaccta?taaaaatagg?????960

cgtatcacga?ggccctttcg?tcttcacctc?gagaaatgtg?agcggataac?aattgacatt????1020

gtgagcggat?aacaagatac?tgagcacatc?agcaggacgc?actgaccgaa?ttcattaaag????1080

aggagaaagg?taccaaaata?agcaagtttg?aaggaggtcc?ttagaatgga?attaaaaaat????1140

gttattcttg?aaaaagaagg?gcatttagct?attgttacaa?tcaatagacc?aaaggcatta????1200

aatgcattga?attcagaaac?actaaaagat?ttaaatgttg?ttttagatga?tttagaagca????1260

gacaacaatg?tgtatgcagt?tatagttaca?ggtgctggtg?agaaatcttt?tgttgctgga????1320

gcagatattt?cagaaatgaa?agatcttaat?gaagaacaag?gtaaagaatt?tggtatttta????1380

ggaaacaatg?tcttcagaag?attagaaaaa?ttggataagc?cagttatcgc?agctatatca????1440

ggatttgctc?ttggtggtgg?atgtgaactt?gctatgtcat?gtgacataag?aatagcttca????1500

gttaaagcta?aatttggtca?accagaagca?ggacttggaa?taactccagg?atttggtgga????1560

actcaaagat?tagctagaat?tgtagggcca?ggaaaagcta?aagaattaat?ttatacttgt????1620

gaccttataa?atgcagaaga?agcttataga?ataggtttag?ttaataaagt?agttgaatta????1680

gaaaaattga?tggaagaagc?aaaagcaatg?gctaacaaga?ttgcagctaa?tgctccaaaa????1740

gcagttgcat?attgtaaaga?tgctatagac?agaggaatgc?aagttgatat?agatgcagct????1800

atattaatag?aagcagaaga?ctttggaaag?tgctttgcaa?cagaagatca?aacagaagga????1860

atgactgcgt?tcttagaaag?aagagcagaa?aagaattttc?aaaataaata?aggatcccat????1920

ggtacgcgtg?ctagaggcat?caaataaaac?gaaaggctca?gtcgaaagac?tgggcctttc????1980

gttttatctg?ttgtttgtcg?gtgaacgctc?tcctgagtag?gacaaatccg?ccgccctaga????2040

cctaggcgtt?cggctgcggc?gagcggtatc?agctcactca?aaggcggtaa?tacggttatc????2100

cacagaatca?ggggataacg?caggaaagaa?catgtgagca?aaaggccagc?aaaaggccag????2160

gaaccgtaaa?aaggccgcgt?tgctggcgtt?tttccatagg?ctccgccccc?ctgacgagca????2220

tcacaaaaat?cgacgctcaa?gtcagaggtg?gcgaaacccg?acaggactat?aaagatacca????2280

ggcgtttccc?cctggaagct?ccctcgtgcg?ctctcctgtt?ccgaccctgc?cgcttaccgg????2340

atacctgtcc?gcctttctcc?cttcgggaag?cgtggcgctt?tctcaatgct?cacgctgtag????2400

gtatctcagt?tcggtgtagg?tcgttcgctc?caagctgggc?tgtgtgcacg?aaccccccgt????2460

tcagcccgac?cgctgcgcct?tatccggtaa?ctatcgtctt?gagtccaacc?cggtaagaca????2520

cgacttatcg?ccactggcag?cagccactgg?taacaggatt?agcagagcga?ggtatgtagg????2580

cggtgctaca?gagttcttga?agtggtggcc?taactacggc?tacactagaa?ggacagtatt????2640

tggtatctgc?gctctgctga?agccagttac?cttcggaaaa?agagttggta?gctcttgatc????2700

cggcaaacaa?accaccgctg?gtagcggtgg?tttttttgtt?tgcaagcagc?agattacgcg????2760

cagaaaaaaa?ggatctcaag?aagatccttt?gatcttttct?acggggtctg?acgctcagtg????2820

gaacgaaaac?tcacgttaag?ggattttggt?catga???????????????????????????????2855

<210>17

<211>2891

<212>DNA

<213>pGV1050

<400>17

ctagtgcttg?gattctcacc?aataaaaaac?gcccggcggc?aaccgagcgt?tctgaacaaa??????60

tccagatgga?gttctgaggt?cattactgga?tctatcaaca?ggagtccaag?cgagctcgat?????120

atcaaattac?gccccgccct?gccactcatc?gcagtactgt?tgtaattcat?taagcattct?????180

gccgacatgg?aagccatcac?agacggcatg?atgaacctga?atcgccagcg?gcatcagcac?????240

cttgtcgcct?tgcgtataat?atttgcccat?ggtgaaaacg?ggggcgaaga?agttgtccat?????300

attggccacg?tttaaatcaa?aactggtgaa?actcacccag?ggattggctg?agacgaaaaa?????360

catattctca?ataaaccctt?tagggaaata?ggccaggttt?tcaccgtaac?acgccacatc?????420

ttgcgaatat?atgtgtagaa?actgccggaa?atcgtcgtgg?tattcactcc?agagcgatga?????480

aaacgtttca?gtttgctcat?ggaaaacggt?gtaacaaggg?tgaacactat?cccatatcac?????540

cagctcaccg?tctttcattg?ccatacgaaa?ctccggatga?gcattcatca?ggcgggcaag?????600

aatgtgaata?aaggccggat?aaaacttgtg?cttatttttc?tttacggtct?ttaaaaaggc?????660

cgtaatatcc?agctgaacgg?tctggttata?ggtacattga?gcaactgact?gaaatgcctc?????720

aaaatgttct?ttacgatgcc?attgggatat?atcaacggtg?gtatatccag?tgattttttt?????780

ctccatttta?gcttccttag?ctcctgaaaa?tctcgataac?tcaaaaaata?cgcccggtag?????840

tgatcttatt?tcattatggt?gaaagttgga?acctcttacg?tgccgatcaa?cgtctcattt?????900

tcgccagata?tcgacgtcta?agaaaccatt?attatcatga?cattaaccta?taaaaatagg?????960

cgtatcacga?ggccctttcg?tcttcacctc?gagaaatgtg?agcggataac?aattgacatt????1020

gtgagcggat?aacaagatac?tgagcacatc?agcaggacgc?actgaccgaa?ttcaaaagat????1080

ttagaggagg?aataattcat?gaaaaagatt?tttgtacttg?gagcaggaac?aatgggtgct????1140

ggtatcgttc?aagcattcgc?tcaaaaaggt?tgtgaagtaa?ttgtaagaga?cataaaggaa????1200

gaatttgttg?acagaggaat?agctggaatc?actaaaggat?tagaaaagca?agttgctaaa????1260

ggaaaaatgt?ctgaagaaga?taaagaagct?atactttcaa?gaatttcagg?aacaactgat????1320

atgaaattag?ctgctgactg?tgatttagta?gttgaagctg?caatcgaaaa?catgaaaatt????1380

aagaaggaaa?tcttcgctga?attagatgga?atttgtaagc?cagaagcgat?tttagcttca????1440

aacacttcat?ctttatcaat?tactgaagtt?gcttcagcta?caaagagacc?tgataaagtt????1500

atcggaatgc?atttctttaa?tccagctcca?gtaatgaagc?ttgttgaaat?tattaaagga????1560

atagctactt?ctcaagaaac?ttttgatgct?gttaaggaat?tatcagttgc?tattggaaaa????1620

gaaccagtag?aagttgcaga?agctccagga?ttcgttgtaa?acagaatatt?aatcccaatg????1680

attaacgaag?cttcatttat?cctacaagaa?ggaatagctt?cagttgaaga?tattgataca????1740

gctatgaaat?atggtgctaa?ccatccaatg?ggacctttag?ctttaggaga?tcttattgga????1800

ttagacgttt?gcttagctat?catggatgtt?ttattcactg?aaacaggtga?taacaagtac????1860

agagctagca?gcatattaag?aaaatatgtt?agagctggat?ggcttggaag?aaaatcagga????1920

aaaggattct?atgattattc?taaataagga?tcccatggta?cgcgtgctag?aggcatcaaa????1980

taaaacgaaa?ggctcagtcg?aaagactggg?cctttcgttt?tatctgttgt?ttgtcggtga????2040

acgctctcct?gagtaggaca?aatccgccgc?cctagaccta?ggcgttcggc?tgcggcgagc????2100

ggtatcagct?cactcaaagg?cggtaatacg?gttatccaca?gaatcagggg?ataacgcagg????2160

aaagaacatg?tgagcaaaag?gccagcaaaa?ggccaggaac?cgtaaaaagg?ccgcgttgct????2220

ggcgtttttc?cataggctcc?gcccccctga?cgagcatcac?aaaaatcgac?gctcaagtca????2280

gaggtggcga?aacccgacag?gactataaag?ataccaggcg?tttccccctg?gaagctccct????2340

cgtgcgctct?cctgttccga?ccctgccgct?taccggatac?ctgtccgcct?ttctcccttc????2400

gggaagcgtg?gcgctttctc?aatgctcacg?ctgtaggtat?ctcagttcgg?tgtaggtcgt????2460

tcgctccaag?ctgggctgtg?tgcacgaacc?ccccgttcag?cccgaccgct?gcgccttatc????2520

cggtaactat?cgtcttgagt?ccaacccggt?aagacacgac?ttatcgccac?tggcagcagc????2580

cactggtaac?aggattagca?gagcgaggta?tgtaggcggt?gctacagagt?tcttgaagtg????2640

gtggcctaac?tacggctaca?ctagaaggac?agtatttggt?atctgcgctc?tgctgaagcc????2700

agttaccttc?ggaaaaagag?ttggtagctc?ttgatccggc?aaacaaacca?ccgctggtag????2760

cggtggtttt?tttgtttgca?agcagcagat?tacgcgcaga?aaaaaaggat?ctcaagaaga????2820

tcctttgatc?ttttctacgg?ggtctgacgc?tcagtggaac?gaaaactcac?gttaagggat????2880

tttggtcatg?a?????????????????????????????????????????????????????????2891

<210>18

<211>3205

<212>DNA

<213>pGV1091

<400>18

ctagtgcttg?gattctcacc?aataaaaaac?gcccggcggc?aaccgagcgt?tctgaacaaa?????60

tccagatgga?gttctgaggt?cattactgga?tctatcaaca?ggagtccaag?cgagctcgat????120

atcaaattac?gccccgccct?gccactcatc?gcagtactgt?tgtaattcat?taagcattct????180

gccgacatgg?aagccatcac?agacggcatg?atgaacctga?atcgccagcg?gcatcagcac????240

cttgtcgcct?tgcgtataat?atttgcccat?ggtgaaaacg?ggggcgaaga?agttgtccat????300

attggccacg?tttaaatcaa?aactggtgaa?actcacccag?ggattggctg?agacgaaaaa????360

catattctca?ataaaccctt?tagggaaata?ggccaggttt?tcaccgtaac?acgccacatc????420

ttgcgaatat?atgtgtagaa?actgccggaa?atcgtcgtgg?tattcactcc?agagcgatga????480

aaacgtttca?gtttgctcat?ggaaaacggt?gtaacaaggg?tgaacactat?cccatatcac????540

cagctcaccg?tctttcattg?ccatacgaaa?ctccggatga?gcattcatca?ggcgggcaag????600

aatgtgaata?aaggccggat?aaaacttgtg?cttatttttc?tttacggtct?ttaaaaaggc????660

cgtaatatcc?agctgaacgg?tctggttata?ggtacattga?gcaactgact?gaaatgcctc????720

aaaatgttct?ttacgatgcc?attgggatat?atcaacggtg?gtatatccag?tgattttttt????780

ctccatttta?gcttccttag?ctcctgaaaa?tctcgataac?tcaaaaaata?cgcccggtag?????840

tgatcttatt?tcattatggt?gaaagttgga?acctcttacg?tgccgatcaa?cgtctcattt?????900

tcgccagata?tcgacgtcta?agaaaccatt?attatcatga?cattaaccta?taaaaatagg?????960

cgtatcacga?ggccctttcg?tcttcacctc?gagaaatgtg?agcggataac?aattgacatt????1020

gtgagcggat?aacaagatac?tgagcacatc?agcaggacgc?actgaccgaa?ttcattaaag????1080

aggagaaagg?taccatggca?cgttttactt?taccaagaga?catttatcat?ggagaaggag????1140

cacttgaggc?acttaaaact?ttaaaaggta?agaaagcttt?cttagtagtt?ggtggcggat????1200

caatgaaaag?atttggattt?cttaaacaag?ttgaagatta?tttaaaagaa?gcaggaatgg????1260

aagtagaatt?atttgaaggt?gttgaaccag?atccatcagt?ggaaacagta?atgaaaggcg????1320

cagaagctat?gagaaacttt?gagcctgatt?ggatagttgc?aatgggtgga?ggatcaccaa????1380

ttgatgctgc?aaaggctatg?tggatattct?acgaataccc?agattttact?tttgaacaag????1440

cagttgttcc?atttggatta?ccagacctta?gacaaaaagc?taagtttgta?gctattccat????1500

caacaagcgg?tacagctaca?gaagttacag?cattctcagt?tatcacaaat?tattcagaaa????1560

aaattaaata?tcctttagct?gattttaaca?taactccaga?tatagcaata?gttgatccag????1620

cacttgctca?aactatgcca?aaaactttaa?cagctcatac?tggaatggat?gcattaactc????1680

acgctataga?agcatacact?gcatcacttc?aatcaaattt?ctcagatcca?ttagcaatta????1740

aagctgtaga?aatggttcaa?gaaaatttaa?tcaaatcatt?tgaaggagat?aaagaagcta????1800

gaaatctaat?gcatgaagct?caatgtttag?ctggaatggc?attttctaat?gcattacttg????1860

gaatagttca?ctcaatggct?cataaggttg?gtgctgtatt?ccatattcct?catggatgtg????1920

caaatgctat?atttttacca?tatgtaattg?agtataacag?aacaaaatgc?gaaaatagat????1980

atggagatat?tgcgagagcc?ttaaaattaa?aaggaaacaa?tgatgccgag?ttaactgatt????2040

cattaattga?attaattaat?ggattaaatg?ataagttaga?gattcctcac?tcaatgaaag????2100

agtatggagt?tactgaagaa?gattttaaag?ctaatctttc?atttatcgct?cataacgcag????2160

tattagatgc?atgcacagga?tcaaatccta?gagaaataga?tgatgctaca?atggaaaaat????2220

tatttgaatg?cacatactat?ggaactaaag?ttaatttgta?aggatcccat?ggtacgcgtg????2280

ctagaggcat?caaataaaac?gaaaggctca?gtcgaaagac?tgggcctttc?gttttatctg????2340

ttgtttgtcg?gtgaacgctc?tcctgagtag?gacaaatccg?ccgccctaga?cctaggcgtt????2400

cggctgcggc?gagcggtatc?agctcactca?aaggcggtaa?tacggttatc?cacagaatca????2460

ggggataacg?caggaaagaa?catgtgagca?aaaggccagc?aaaaggccag?gaaccgtaaa????2520

aaggccgcgt?tgctggcgtt?tttccatagg?ctccgccccc?ctgacgagca?tcacaaaaat????2580

cgacgctcaa?gtcagaggtg?gcgaaacccg?acaggactat?aaagatacca?ggcgtttccc????2640

cctggaagct?ccctcgtgcg?ctctcctgtt?ccgaccctgc?cgcttaccgg?atacctgtcc????2700

gcctttctcc?cttcgggaag?cgtggcgctt?tctcaatgct?cacgctgtag?gtatctcagt????2760

tcggtgtagg?tcgttcgctc?caagctgggc?tgtgtgcacg?aaccccccgt?tcagcccgac????2820

cgctgcgcct?tatccggtaa?ctatcgtctt?gagtccaacc?cggtaagaca?cgacttatcg????2880

ccactggcag?cagccactgg?taacaggatt?agcagagcga?ggtatgtagg?cggtgctaca????2940

gagttcttga?agtggtggcc?taactacggc?tacactagaa?ggacagtatt?tggtatctgc????3000

gctctgctga?agccagttac?cttcggaaaa?agagttggta?gctcttgatc?cggcaaacaa????3060

accaccgctg?gtagcggtgg?tttttttgtt?tgcaagcagc?agattacgcg?cagaaaaaaa????3120

ggatctcaag?aagatccttt?gatcttttct?acggggtctg?acgctcagtg?gaacgaaaac????3180

tcacgttaag?ggattttggt?catga??????????????????????????????????????????3205

<210>19

<211>3449

<212>DNA

<213>pGV1096

<400>19

ctagtgcttg?gattctcacc?aataaaaaac?gcccggcggc?aaccgagcgt?tctgaacaaa?????60

tccagatgga?gttctgaggt?cattactgga?tctatcaaca?ggagtccaag?cgagctcgat????120

atcaaattac?gccccgccct?gccactcatc?gcagtactgt?tgtaattcat?taagcattct????180

gccgacatgg?aagccatcac?agacggcatg?atgaacctga?atcgccagcg?gcatcagcac????240

cttgtcgcct?tgcgtataat?atttgcccat?ggtgaaaacg?ggggcgaaga?agttgtccat????300

attggccacg?tttaaatcaa?aactggtgaa?actcacccag?ggattggctg?agacgaaaaa????360

catattctca?ataaaccctt?tagggaaata?ggccaggttt?tcaccgtaac?acgccacatc????420

ttgcgaatat?atgtgtagaa?actgccggaa?atcgtcgtgg?tattcactcc?agagcgatga????480

aaacgtttca?gtttgctcat?ggaaaacggt?gtaacaaggg?tgaacactat?cccatatcac????540

cagctcaccg?tctttcattg?ccatacgaaa?ctccggatga?gcattcatca?ggcgggcaag????600

aatgtgaata?aaggccggat?aaaacttgtg?cttatttttc?tttacggtct?ttaaaaaggc????660

cgtaatatcc?agctgaacgg?tctggttata?ggtacattga?gcaactgact?gaaatgcctc????720

aaaatgttct?ttacgatgcc?attgggatat?atcaacggtg?gtatatccag?tgattttttt????780

ctccatttta?gcttccttag?ctcctgaaaa?tctcgataac?tcaaaaaata?cgcccggtag????840

tgatcttatt?tcattatggt?gaaagttgga?acctcttacg?tgccgatcaa?cgtctcattt????900

tcgccagata?tcgacgtcta?agaaaccatt?attatcatga?cattaaccta?taaaaatagg????960

cgtatcacga?ggccctttcg?tcttcacctc?gagaaatgtg?agcggataac?aattgacatt???1020

gtgagcggat?aacaagatac?tgagcacatc?agcaggacgc?actgaccggg?aattcggagg???1080

aatagttcat?gaataaagac?acactaatac?ctacaactaa?agatttaaaa?gtaaaaacaa???1140

atggtgaaaa?cattaattta?aagaactaca?aggataattc?ttcatgtttc?ggagtattcg???1200

aaaatgttga?aaatgctata?agcagcgctg?tacacgcaca?aaagatatta?tcccttcatt???1260

atacaaaaga?gcaaagagaa?aaaatcataa?ctgagataag?aaaggccgca?ttacaaaata???1320

aagaggtctt?ggctacaatg?attctagaag?aaacacatat?gggaagatat?gaggataaaa???1380

tattaaaaca?tgaattggta?gctaaatata?ctcctggtac?agaagattta?actactactg???1440

cttggtcagg?tgataatggt?cttacagttg?tagaaatgtc?tccatatggt?gttataggtg????1500

caataactcc?ttctacgaat?ccaactgaaa?ctgtaatatg?taatagcata?ggcatgatag????1560

ctgctggaaa?tgctgtagta?tttaacggac?acccatgcgc?taaaaaatgt?gttgcctttg????1620

ctgttgaaat?gataaataag?gcaattattt?catgtggcgg?tcctgaaaat?ctagtaacaa????1680

ctataaaaaa?tccaactatg?gagtctctag?atgcaattat?taagcatcct?tcaataaaac????1740

ttctttgcgg?aactgggggt?ccaggaatgg?taaaaaccct?cttaaattct?ggtaagaaag????1800

ctataggtgc?tggtgctgga?aatccaccag?ttattgtaga?tgatactgct?gatatagaaa????1860

aggctggtag?gagcatcatt?gaaggctgtt?cttttgataa?taatttacct?tgtattgcag????1920

aaaaagaagt?atttgttttt?gagaatgttg?cagatgattt?aatatctaac?atgctaaaaa????1980

ataatgctgt?aattataaat?gaagatcaag?tatcaaaatt?aatagattta?gtattacaaa????2040

aaaataatga?aactcaagaa?tactttataa?acaaaaaatg?ggtaggaaaa?gatgcaaaat????2100

tattcttaga?tgaaatagat?gttgagtctc?cttcaaatgt?taaatgcata?atctgcgaag????2160

taaatgcaaa?tcatccattt?gttatgacag?aactcatgat?gccaatattg?ccaattgtaa????2220

gagttaaaga?tatagatgaa?gctattaaat?atgcaaagat?agcagaacaa?aatagaaaac????2280

atagtgccta?tatttattct?aaaaatatag?acaacctaaa?tagatttgaa?agagaaatag????2340

atactactat?ttttgtaaag?aatgctaaat?cttttgctgg?tgttggttat?gaagcagaag????2400

gatttacaac?tttcactatt?gctggatcta?ctggtgaggg?aataacctct?gcaaggaatt????2460

ttacaagaca?aagaagatgt?gtacttgccg?gctaaggatc?cgatccgatc?ccatggtacg????2520

cgtgctagag?gcatcaaata?aaacgaaagg?ctcagtcgaa?agactgggcc?tttcgtttta????2580

tctgttgttt?gtcggtgaac?gctctcctga?gtaggacaaa?tccgccgccc?tagacctagg????2640

cgttcggctg?cggcgagcgg?tatcagctca?ctcaaaggcg?gtaatacggt?tatccacaga????2700

atcaggggat?aacgcaggaa?agaacatgtg?agcaaaaggc?cagcaaaagg?ccaggaaccg????2760

taaaaaggcc?gcgttgctgg?cgtttttcca?taggctccgc?ccccctgacg?agcatcacaa????2820

aaatcgacgc?tcaagtcaga?ggtggcgaaa?cccgacagga?ctataaagat?accaggcgtt????2880

tccccctgga?agctccctcg?tgcgctctcc?tgttccgacc?ctgccgctta?ccggatacct????2940

gtccgccttt?ctcccttcgg?gaagcgtggc?gctttctcaa?tgctcacgct?gtaggtatct????3000

cagttcggtg?taggtcgttc?gctccaagct?gggctgtgtg?cacgaacccc?ccgttcagcc????3060

cgaccgctgc?gccttatccg?gtaactatcg?tcttgagtcc?aacccggtaa?gacacgactt????3120

atcgccactg?gcagcagcca?ctggtaacag?gattagcaga?gcgaggtatg?taggcggtgc????3180

tacagagttc?ttgaagtggt?ggcctaacta?cggctacact?agaaggacag?tatttggtat????3240

ctgcgctctg?ctgaagccag?ttaccttcgg?aaaaagagtt?ggtagctctt?gatccggcaa????3300

acaaaccacc?gctggtagcg?gtggtttttt?tgtttgcaag?cagcagatta?cgcgcagaaa????3360

aaaaggatct?caagaagatc?ctttgatctt?ttctacgggg?tctgacgctc?agtggaacga????3420

aaactcacgt?taagggattt?tggtcatga??????????????????????????????????????3449

<210>20

<211>1425

<212>DNA

<213>1pdA

<400>20

atgagtactg?aaatcaaaac?tcaggtcgtg?gtacttgggg?caggccccgc?aggttactcc??????60

gctgccttcc?gttgcgctga?tttaggtctg?gaaaccgtaa?tcgtagaacg?ttacaacacc?????120

cttggcggtg?tttgcctgaa?cgtcggctgt?atcccttcta?aagcactgct?gcacgtagca?????180

aaagttatcg?aagaagccaa?agcgctggct?gaacacggta?tcgtcttcgg?cgaaccgaaa?????240

accgatatcg?acaagattcg?tacctggaaa?gagaaagtga?tcaatcagct?gaccggtggt?????300

ctggctggta?tggcgaaagg?ccgcaaagtc?aaagtggtca?acggtctggg?taaattcacc?????360

ggggctaaca?ccctggaagt?tgaaggtgag?aacggcaaaa?ccgtgatcaa?cttcgacaac?????420

gcgatcattg?cagcgggttc?tcgcccgatc?caactgccgt?ttattccgca?tgaagatccg?????480

cgtatctggg?actccactga?cgcgctggaa?ctgaaagaag?taccagaacg?cctgctggta?????540

atgggtggcg?gtatcatcgg?tctggaaatg?ggcaccgttt?accacgcgct?gggttcacag?????600

attgacgtgg?ttgaaatgtt?cgaccaggtt?atcccggcag?ctgacaaaga?catcgttaaa?????660

gtcttcacca?agcgtatcag?caagaaattc?aacctgatgc?tggaaaccaa?agttaccgcc?????720

gttgaagcga?aagaagacgg?catttatgtg?acgatggaag?gcaaaaaagc?acccgctgaa?????780

ccgcagcgtt?acgacgccgt?gctggtagcg?attggtcgtg?tgccgaacgg?taaaaacctc?????840

gacgcaggca?aagcaggcgt?ggaagttgac?gaccgtggtt?tcatccgcgt?tgacaaacag?????900

ctgcgtacca?acgtaccgca?catctttgct?atcggcgata?tcgtcggtca?accgatgctg?????960

gcacacaaag?gtgttcacga?aggtcacgtt?gccgctgaag?ttatcgccgg?taagaaacac????1020

tacttcgatc?cgaaagttat?cccgtccatc?gcctataccg?aaccagaagt?tgcatgggtg????1080

ggtctgactg?agaaagaagc?gaaagagaaa?ggcatcagct?atgaaaccgc?caccttcccg????1140

tgggctgctt?ctggtcgtgc?tatcgcttcc?gactgcgcag?acggtatgac?caagctgatt????1200

ttcgacaaag?aatctcaccg?tgtgatcggt?ggtgcgattg?tcggtactaa?cggcggcgag????1260

ctgctgggtg?aaatcggcct?ggcaatcgaa?atgggttgtg?atgctgaaga?catcgcactg????1320

accatccacg?cgcacccgac?tctgcacgag?tctgtgggcc?tggcggcaga?agtgttcgaa????1380

ggtagcatta?ccgacctgcc?gaacccgaaa?gcgaagaaga?agtaa????????????????????1425

<210>21

<211>2664

<212>DNA

<213>aceE

<400>21

atgtcagaac?gtttcccaaa?tgacgtggat?ccgatcgaaa?ctcgcgactg?gctccaggcg?????60

atcgaatcgg?tcatccgtga?agaaggtgtt?gagcgtgctc?agtatctgat?cgaccaactg????120

cttgctgaag?cccgcaaagg?cggtgtaaac?gtagccgcag?gcacaggtat?cagcaactac????180

atcaacacca?tccccgttga?agaacaaccg?gagtatccgg?gtaatctgga?actggaacgc?????240

cgtattcgtt?cagctatccg?ctggaacgcc?atcatgacgg?tgctgcgtgc?gtcgaaaaaa?????300

gacctcgaac?tgggcggcca?tatggcgtcc?ttccagtctt?ccgcaaccat?ttatgatgtg?????360

tgctttaacc?acttcttccg?tgcacgcaac?gagcaggatg?gcggcgacct?ggtttacttc?????420

cagggccaca?tctccccggg?cgtgtacgct?cgtgctttcc?tggaaggtcg?tctgactcag?????480

gagcagctgg?ataacttccg?tcaggaagtt?cacggcaatg?gcctctcttc?ctatccgcac?????540

ccgaaactga?tgccggaatt?ctggcagttc?ccgaccgtat?ctatgggtct?gggtccgatt?????600

ggtgctattt?accaggctaa?attcctgaaa?tatctggaac?accgtggcct?gaaagatacc?????660

tctaaacaaa?ccgtttacgc?gttcctcggt?gacggtgaaa?tggacgaacc?ggaatccaaa?????720

ggtgcgatca?ccatcgctac?ccgtgaaaaa?ctggataacc?tggtcttcgt?tatcaactgt?????780

aacctgcagc?gtcttgacgg?cccggtcacc?ggtaacggca?agatcatcaa?cgaactggaa?????840

ggcatcttcg?aaggtgctgg?ctggaacgtg?atcaaagtga?tgtggggtag?ccgttgggat?????900

gaactgctgc?gtaaggatac?cagcggtaaa?ctgatccagc?tgatgaacga?aaccgttgac?????960

ggcgactacc?agaccttcaa?atcgaaagat?ggtgcgtacg?ttcgtgaaca?cttcttcggt????1020

aaatatcctg?aaaccgcagc?actggttgca?gactggactg?acgagcagat?ctgggcactg????1080

aaccgtggtg?gtcacgatcc?gaagaaaatc?tacgctgcat?tcaagaaagc?gcaggaaacc????1140

aaaggcaaag?cgacagtaat?ccttgctcat?accattaaag?gttacggcat?gggcgacgcg????1200

gctgaaggta?aaaacatcgc?gcaccaggtt?aagaaaatga?acatggacgg?tgtgcgtcat????1260

atccgcgacc?gtttcaatgt?gccggtgtct?gatgcagata?tcgaaaaact?gccgtacatc????1320

accttcccgg?aaggttctga?agagcatacc?tatctgcacg?ctcagcgtca?gaaactgcac????1380

ggttatctgc?caagccgtca?gccgaacttc?accgagaagc?ttgagctgcc?gagcctgcaa????1440

gacttcggcg?cgctgttgga?agagcagagc?aaagagatct?ctaccactat?cgctttcgtt????1500

cgtgctctga?acgtgatgct?gaagaacaag?tcgatcaaag?atcgtctggt?accgatcatc????1560

gccgacgaag?cgcgtacttt?cggtatggaa?ggtctgttcc?gtcagattgg?tatttacagc????1620

ccgaacggtc?agcagtacac?cccgcaggac?cgcgagcagg?ttgcttacta?taaagaagac????1680

gagaaaggtc?agattctgca?ggaagggatc?aacgagctgg?gcgcaggttg?ttcctggctg????1740

gcagcggcga?cctcttacag?caccaacaat?ctgccgatga?tcccgttcta?catctattac????1800

tcgatgttcg?gcttccagcg?tattggcgat?ctgtgctggg?cggctggcga?ccagcaagcg????1860

cgtggcttcc?tgatcggcgg?tacttccggt?cgtaccaccc?tgaacggcga?aggtctgcag????1920

cacgaagatg?gtcacagcca?cattcagtcg?ctgactatcc?cgaactgtat?ctcttacgac????1980

ccggcttacg?cttacgaagt?tgctgtcatc?atgcatgacg?gtctggagcg?tatgtacggt????2040

gaaaaacaag?agaacgttta?ctactacatc?actacgctga?acgaaaacta?ccacatgccg????2100

gcaatgccgg?aaggtgctga?ggaaggtatc?cgtaaaggta?tctacaaact?cgaaactatt????2160

gaaggtagca?aaggtaaagt?tcagctgctc?ggctccggtt?ctatcctgcg?tcacgtccgt????2220

gaagcagctg?agatcctggc?gaaagattac?ggcgtaggtt?ctgacgttta?tagcgtgacc????2280

tccttcaccg?agctggcgcg?tgatggtcag?gattgtgaac?gctggaacat?gctgcacccg????2340

ctggaaactc?cgcgcgttcc?gtatatcgct?caggtgatga?acgacgctcc?ggcagtggca????2400

tctaccgact?atatgaaact?gttcgctgag?caggtccgta?cttacgtacc?ggctgacgac????2460

taccgcgtac?tgggtactga?tggcttcggt?cgttccgaca?gccgtgagaa?cctgcgtcac????2520

cacttcgaag?ttgatgcttc?ttatgtcgtg?gttgcggcgc?tgggcgaact?ggctaaacgt????2580

ggcgaaatcg?ataagaaagt?ggttgctgac?gcaatcgcca?aattcaacat?cgatgcagat????2640

aaagttaacc?cgcgtctggc?gtaa???????????????????????????????????????????2664

<210>22

<211>1893

<212>DNA

<213>aceF

<400>22

atggctatcg?aaatcaaagt?accggacatc?ggggctgatg?aagttgaaat?caccgagatc?????60

ctggtcaaag?tgggcgacaa?agttgaagcc?gaacagtcgc?tgatcaccgt?agaaggcgac????120

aaagcctcta?tggaagttcc?gtctccgcag?gcgggtatcg?ttaaagagat?caaagtctct????180

gttggcgata?aaacccagac?cggcgcactg?attatgattt?tcgattccgc?cgacggtgca????240

gcagacgctg?cacctgctca?ggcagaagag?aagaaagaag?cagctccggc?agcagcacca????300

gcggctgcgg?cggcaaaaga?cgttaacgtt?ccggatatcg?gcagcgacga?agttgaagtg????360

accgaaatcc?tggtgaaagt?tggcgataaa?gttgaagctg?aacagtcgct?gatcaccgta????420

gaaggcgaca?aggcttctat?ggaagttccg?gctccgtttg?ctggcaccgt?gaaagagatc????480

aaagtgaacg?tgggtgacaa?agtgtctacc?ggctcgctga?ttatggtctt?cgaagtcgcg????540

ggtgaagcag?gcgcggcagc?tccggccgct?aaacaggaag?cagctccggc?agcggcccct????600

gcaccagcgg?ctggcgtgaa?agaagttaac?gttccggata?tcggcggtga?cgaagttgaa????660

gtgactgaag?tgatggtgaa?agtgggcgac?aaagttgccg?ctgaacagtc?actgatcacc????720

gtagaaggcg?acaaagcttc?tatggaagtt?ccggcgccgt?ttgcaggcgt?cgtgaaggaa????780

ctgaaagtca?acgttggcga?taaagtgaaa?actggctcgc?tgattatgat?cttcgaagtt????840

gaaggcgcag?cgcctgcggc?agctcctgcg?aaacaggaag?cggcagcgcc?ggcaccggca????900

gcaaaagctg?aagccccggc?agcagcacca?gctgcgaaag?cggaaggcaa?atctgaattt????960

gctgaaaacg?acgcttatgt?tcacgcgact?ccgctgatcc?gccgtctggc?acgcgagttt???1020

ggtgttaacc?ttgcgaaagt?gaagggcact?ggccgtaaag?gtcgtatcct?gcgcgaagac???1080

gttcaggctt?acgtgaaaga?agctatcaaa?cgtgcagaag?cagctccggc?agcgactggc???1140

ggtggtatcc?ctggcatgct?gccgtggccg?aaggtggact?tcagcaagtt?tggtgaaatc???1200

gaagaagtgg?aactgggccg?catccagaaa?atctctggtg?cgaacctgag?ccgtaactgg???1260

gtaatgatcc?cgcatgttac?tcacttcgac?aaaaccgata?tcaccgagtt?ggaagcgttc???1320

cgtaaacagc?agaacgaaga?agcggcgaaa?cgtaagctgg?atgtgaagat?caccccggtt????1380

gtcttcatca?tgaaagccgt?tgctgcagct?cttgagcaga?tgcctcgctt?caatagttcg????1440

ctgtcggaag?acggtcagcg?tctgaccctg?aagaaataca?tcaacatcgg?tgtggcggtg????1500

gataccccga?acggtctggt?tgttccggta?ttcaaagacg?tcaacaagaa?aggcatcatc????1560

gagctgtctc?gcgagctgat?gactatttct?aagaaagcgc?gtgacggtaa?gctgactgcg????1620

ggcgaaatgc?agggcggttg?cttcaccatc?tccagcatcg?gcggcctggg?tactacccac????1680

ttcgcgccga?ttgtgaacgc?gccggaagtg?gctatcctcg?gcgtttccaa?gtccgcgatg????1740

gagccggtgt?ggaatggtaa?agagttcgtg?ccgcgtctga?tgctgccgat?ttctctctcc????1800

ttcgaccacc?gcgtgatcga?cggtgctgat?ggtgcccgtt?tcattaccat?cattaacaac????1860

acgctgtctg?acattcgccg?tctggtgatg?taa?????????????????????????????????1893

<210>23

<211>1263

<212>DNA

<213>PDA1

<400>23

atgcttgctg?cttcattcaa?acgccaacca?tcacaattgg?tccgcgggtt?aggagctgtt?????60

cttcgcactc?ccaccaggat?aggtcatgtt?cgtaccatgg?caactttaaa?aacaactgat????120

aagaaggccc?ctgaggacat?cgagggctcg?gacacagtgc?aaattgagtt?gcctgaatct????180

tccttcgagt?cgtatatgct?agagcctcca?gacttgtctt?atgagacttc?gaaagccacc????240

ttgttacaga?tgtataaaga?tatggtcatc?atcagaagaa?tggagatggc?ttgtgacgcc????300

ttgtacaagg?ccaagaaaat?cagaggtttt?tgccatctat?ctgttggtca?ggaggccatt????360

gctgtcggta?tcgagaatgc?catcacaaaa?ttggattcca?tcatcacatc?ttacagatgt????420

cacggtttca?cttttatgag?aggtgcctca?gtgaaagccg?ttctggctga?attgatgggt????480

agaagagccg?gtgtctctta?tggtaagggt?ggttccatgc?acctttacgc?tccaggcttc????540

tatggtggta?atggtatcgt?gggtgcccag?gttcctttag?gtgcaggttt?agcttttgct????600

caccaataca?agaacgagga?cgcctgctct?ttcactttgt?atggtgatgg?tgcctctaat????660

caaggtcaag?tttttgaatc?tttcaacatg?gccaaattat?ggaatttgcc?cgtcgtgttt????720

tgctgtgaga?acaacaagta?cggtatgggt?accgccgctt?caagatcctc?cgcgatgact????780

gaatatttca?agcgtggtca?atatattcca?ggtttaaaag?ttaacggtat?ggatattcta????840

gctgtctacc?aagcatccaa?gtttgctaag?gactggtgtc?tatccggcaa?aggtcctctc????900

gttctagaat?atgaaaccta?taggtacggt?ggccattcta?tgtctgatcc?cggtactacc????960

tacagaacta?gagacgagat?tcagcatatg?agatccaaga?acgatccaat?tgctggtctt???1020

aagatgcatt?tgattgatct?aggtattgcc?actgaagctg?aagtcaaagc?ttacgacaag???1080

tccgctagaa?aatacgttga?cgaacaagtt?gaattagctg?atgctgctcc?tcctccagaa???1140

gccaaattat?ccatcttgtt?tgaagacgtc?tacgtgaaag?gtacagaaac?tccaacccta???1200

agaggtagga?tccctgaaga?tacttgggac?ttcaaaaagc?aaggttttgc?ctctagggat???1260

taa?????????????????????????????????????????????????????????????????1163

<210>24

<211>1101

<212>DNA

<213>PDB1

<400>24

atgttttcca?gactgccaac?atcattggcc?agaaatgttg?cacgtcgtgc?cccaacttct?????60

tttgtaagac?cctctgcagc?agcagcagca?ttgagattct?catcaacaaa?gacgatgacc????120

gtcagagagg?ccttgaatag?tgccatggcg?gaagaattgg?accgtgatga?tgatgtcttc????180

cttattggtg?aagaagttgc?acaatataac?ggggcttata?aggtgtcaaa?gggtttattg????240

gacaggttcg?gtgaacgtcg?tgtggttgac?acacctatta?ccgaatacgg?gttcacaggt????300

ttggccgttg?gtgccgcttt?gaagggtttg?aagccaattg?tagagtttat?gtcgttcaat????360

ttctctatgc?aagctatcga?tcatgttgtc?aattccgctg?caaagactca?ctacatgtct????420

ggtggtactc?aaaaatgtca?aatggtcttc?agaggtccta?atggtgctgc?agtgggtgtt????480

ggtgctcaac?attcacagga?cttttctcct?tggtacggtt?ccattccagg?gttaaaggtc????540

cttgtccctt?attctgctga?agatgctagg?ggtttgttaa?aggccgccat?cagagatcca????600

aaccctgttg?tatttttaga?gaacgaattg?ttgtacggtg?aatcttttga?aatctcagaa????660

gaagctttat?cccctgagtt?caccttgcca?tacaaggcta?agatcgaaag?agaaggtacc????720

gatatttcca?ttgttacgta?cacaagaaac?gttcagtttt?ctttggaagc?cgctgaaatt????780

ctacaaaaga?aatatggtgt?ctctgcagaa?gttatcaact?tgcgttctat?tagaccttta????840

gatactgaag?ctatcatcaa?aactgtcaag?aagacaaacc?acttgattac?tgttgaatcc????900

actttcccat?catttggtgt?tggtgctgaa?attgtcgccc?aagttatgga?gtctgaagcc????960

tttgattact?tggatgctcc?aatccaaaga?gttactggtg?ccgatgttcc?aacaccttac???1020

gctaaagaat?tagaagattt?cgctttccct?gatactccaa?ccatcgttaa?agctgtcaaa???1080

gaagtcttgt?caattgaata?a?????????????????????????????????????????????1101

<210>25

<211>1233

<212>DNA

<213>PDX1

<400>25

atgctaagtg?caatttccaa?agtctccact?ttaaaatcat?gtacaagata?tttaaccaaa?????60

tgcaactatc?atgcatcagc?taaattactt?gctgtaaaga?cattttcaat?gcctgcaatg????120

tctcctacta?tggagaaagg?ggggattgtg?tcttggaaat?ataaagttgg?cgaaccattc????180

agcgcgggcg?atgtgatatt?agaagtggaa?acagataaat?ctcaaattga?tgtggaagca????240

ctggacgatg?gtaaactagc?taagatcctg?aaagatgaag?gctctaaaga?tgttgatgtt????300

ggtgaaccta?ttgcttatat?tgctgatgtt?gatgatgatt?tagctactat?aaagttaccc????360

caagaggcca?acaccgcaaa?tgcgaaatct?attgaaatta?agaagccatc?cgcagatagt????420

actgaagcaa?cacaacaaca?tttaaaaaaa?gccacagtta?caccaataaa?aaccgttgac????480

ggcagccaag?ccaatcttga?acagacgcta?ttaccatccg?tgtcattact?actggctgag????540

aacaatatat?ccaaacaaaa?ggctttgaag?gaaattgcgc?catctggttc?caacggtaga????600

ctattaaagg?gtgatgtgct?agcataccta?gggaaaatac?cacaagattc?ggttaacaag????660

gtaacagaat?ttatcaagaa?gaacgaacgt?ctcgatttat?cgaacattaa?acctatacag????720

ctcaaaccaa?aaatagccga?gcaagctcaa?acaaaagctg?ccgacaagcc?aaagattact????780

cctgtagaat?ttgaagagca?attagtgttc?catgctcccg?cctctattcc?gtttgacaaa????840

ctgagtgaat?cattgaactc?tttcatgaaa?gaagcttacc?agttctcaca?cggaacacca????900

ctaatggaca?caaattcgaa?atactttgac?cctattttcg?aggaccttgt?caccttgagc????960

ccaagagagc?caagatttaa?attttcctat?gacttgatgc?aaattcccaa?agctaataac???1020

atgcaagaca?cgtacggtca?agaagacata?tttgacctct?taacaggttc?agacgcgact???1080

gcctcatcag?taagacccgt?tgaaaagaac?ttacctgaaa?aaaacgaata?tatactagcg???1140

ttgaatgtta?gcgtcaacaa?caagaagttt?aatgacgcgg?aggccaaggc?aaaaagattc???1200

cttgattacg?taagggagtt?agaatcattt??tga???????????????????????????????1233

<210>26

<211>1449

<212>DNA

<213>LAT1

<400>26

atgtctgcct?ttgtcagggt?ggttccaaga?atatccagaa?gttcagtact?caccagatca?????60

ttgagactgc?aattgagatg?ctacgcatcg?tacccagagc?acaccattat?tggtatgccg????120

gcactgtctc?ctacgatgac?gcaaggtaat?cttgctgctt?ggactaagaa?ggaaggtgac????180

caattgtctc?ccggtgaagt?tattgccgaa?atagaaacag?acaaggctca?aatggacttt????240

gagttccaag?aagatggtta?cttagccaag?attctagttc?ctgaaggtac?aaaggacatt????300

cctgtcaaca?agcctattgc?cgtctatgtg?gaggacaaag?ctgatgtgcc?agcttttaag????360

gactttaagc?tggaggattc?aggttctgat?tcaaagacca?gtacgaaggc?tcagcctgcc????420

gaaccacagg?cagaaaagaa?acaagaagcg?ccagctgaag?agaccaagac?ttctgcacct????480

gaagctaaga?aatctgacgt?tgctgctcct?caaggtagga?tttttgcctc?tccacttgcc????540

aagactatcg?ccttggaaaa?gggtatttct?ttgaaggatg?ttcacggcac?tggaccccgc????600

ggtagaatta?ccaaggctga?cattgagtca?tatctagaaa?agtcgtctaa?gcagtcttct????660

caaaccagtg?gtgctgccgc?cgccactcct?gccgccgcta?cctcaagcac?tactgctggc????720

tctgctccat?cgccttcttc?tacagcatca?tatgaggatg?ttccaatttc?aaccatgaga????780

agcatcattg?gagaacgttt?attgcaatct?actcaaggca?ttccatcata?catcgtttcc????840

tccaagatat?ccatctccaa?acttttgaaa?ttgagacagt?ccttgaacgc?tacagcaaac????900

gacaagtaca?aactgtccat?taatgaccta?ttagtaaaag?ccatcactgt?tgcggctaag????960

agggtgccag?atgccaatgc?ctactggtta?cctaatgaga?acgttatccg?taaattcaag????1020

aatgtcgatg?tctcagtcgc?tgttgccaca?ccaacaggat?tattgacacc?aattgtcaag????1080

aattgtgagg?ccaagggctt?gtcgcaaatc?tctaacgaaa?tcaaggaact?agtcaagcgt????1140

gccagaataa?acaaattggc?accagaggaa?ttccaaggtg?ggaccatttg?catatccaat????1200

atgggcatga?ataatgctgt?taacatgttt?acttcgatta?tcaacccacc?acagtctaca????1260

atcttggcca?tcgctactgt?tgaaagggtc?gctgtggaag?acgccgctgc?tgagaacgga????1320

ttctcctttg?ataaccaggt?taccataaca?gggacctttg?atcatagaac?cattgatggc????1380

gccaaaggtg?cagaattcat?gaaggaattg?aaaactgtta?ttgaaaatcc?tttggaaatg????1440

ctattgtga????????????????????????????????????????????????????????????1449

<210>27

<211>1500

<212>DNA

<213>LPD1

<400>27

atgttaagaa?tcagatcact?cctaaataat?aagcgtgcct?tttcgtccac?agtcaggaca?????60

ttgaccatta?acaagtcaca?tgatgtagtc?atcatcggtg?gtggccctgc?tggttacgtg????120

gctgctatca?aagctgctca?attgggattt?aacactgcat?gtgtagaaaa?aagaggcaaa????180

ttaggcggta?cctgtcttaa?cgttggatgt?atcccctcca?aagcacttct?aaataattct????240

catttattcc?accaaatgca?tacggaagcg?caaaagagag?gtattgacgt?caacggtgat????300

atcaaaatta?acgtagcaaa?cttccaaaag?gctaaggatg?acgctgttaa?gcaattaact????360

ggaggtattg?agcttctgtt?caagaaaaat?aaggtcacct?attataaagg?taatggttca????420

ttcgaagacg?aaacgaagat?cagagtaact?cccgttgatg?ggttggaagg?cactgtcaag????480

gaagaccaca?tactagatgt?taagaacatc?atagtcgcca?cgggctctga?agttacaccc????540

ttccccggta?ttgaaataga?tgaggaaaaa?attgtctctt?caacaggtgc?tctttcgtta????600

aaggaaattc?ccaaaagatt?aaccatcatt?ggtggaggaa?tcatcggatt?ggaaatgggt????660

tcagtttact?ctagattagg?ctccaaggtt?actgtagtag?aatttcaacc?tcaaattggt????720

gcatctatgg?acggcgaggt?tgccaaagcc?acccaaaagt?tcttgaaaaa?gcaaggtttg????780

gacttcaaat?taagcaccaa?agttatttct?gcaaagagaa?acgacgacaa?gaacgtcgtc????840

gaaattgttg?tagaagatac?taaaacgaat?aagcaagaaa?atttggaagc?tgaagttttg????900

ctggttgctg?ttggtagaag?accttacatt?gctggcttag?gggctgaaaa?gattggatta????960

gaagtagaca?aaaggggacg?cctagtcatt?gatgaccaat?ttaattccaa?gttcccacac???1020

attaaagtgg?taggagatgt?tacatttggt?ccaatgctgg?ctcacaaagc?cgaagaggaa???1080

ggtattgcag?ctgtcgaaat?gttgaaaact?ggtcacggtc?atgtcaacta?taacaacatt???1140

ccttcggtca?tgtattctca?cccagaagta?gcatgggttg?gtaaaaccga?agagcaattg???1200

aaagaagccg?gcattgacta?taaaattggt?aagttcccct?ttgcggccaa?ttcaagagcc???1260

aagaccaacc?aagacactga?aggtttcgtg?aagattttga?tcgattccaa?gaccgagcgt???1320

attttggggg?ctcacattat?cggtccaaat?gccggtgaaa?tgattgctga?agctggctta????1380

gccttagaat?atggcgcttc?cgcagaagat?gttgctaggg?tctgccatgc?tcatcctact????1440

ttgtccgaag?catttaagga?agctaacatg?gctgcctatg?ataaagctat?tcattgttga????1500

<210>28

<211>1692

<212>DNA

<213>PDC1

<400>28

atgtctgaaa?ttactttggg?taaatatttg?ttcgaaagat?taaagcaagt?caacgttaac?????60

accgttttcg?gtttgccagg?tgacttcaac?ttgtccttgt?tggacaagat?ctacgaagtt????120

gaaggtatga?gatgggctgg?taacgccaac?gaattgaacg?ctgcttacgc?cgctgatggt????180

tacgctcgta?tcaagggtat?gtcttgtatc?atcaccacct?tcggtgtcgg?tgaattgtct????240

gctttgaacg?gtattgccgg?ttcttacgct?gaacacgtcg?gtgttttgca?cgttgttggt????300

gtcccatcca?tctctgctca?agctaagcaa?ttgttgttgc?accacacctt?gggtaacggt????360

gacttcactg?ttttccacag?aatgtctgcc?aacatttctg?aaaccactgc?tatgatcact????420

gacattgcta?ccgccccagc?tgaaattgac?agatgtatca?gaaccactta?cgtcacccaa????480

agaccagtct?acttaggttt?gccagctaac?ttggtcgact?tgaacgtccc?agctaagttg????540

ttgcaaactc?caattgacat?gtctttgaag?ccaaacgatg?ctgaatccga?aaaggaagtc????600

attgacacca?tcttggcttt?ggtcaaggat?gctaagaacc?cagttatctt?ggctgatgct????660

tgttgttcca?gacacgacgt?caaggctgaa?actaagaagt?tgattgactt?gactcaattc????720

ccagctttcg?tcaccccaat?gggtaagggt?tccattgacg?aacaacaccc?aagatacggt????780

ggtgtttacg?tcggtacctt?gtccaagcca?gaagttaagg?aagccgttga?atctgctgac????840

ttgattttgt?ctgtcggtgc?tttgttgtct?gatttcaaca?ccggttcttt?ctcttactct????900

tacaagacca?agaacattgt?cgaattccac?tccgaccaca?tgaagatcag?aaacgccact????960

ttcccaggtg?tccaaatgaa?attcgttttg?caaaagttgt?tgaccactat?tgctgacgcc???1020

gctaagggtt?acaagccagt?tgctgtccca?gctagaactc?cagctaacgc?tgctgtccca???1080

gcttctaccc?cattgaagca?agaatggatg?tggaaccaat?tgggtaactt?cttgcaagaa???1140

ggtgatgttg?tcattgctga?aaccggtacc?tccgctttcg?gtatcaacca?aaccactttc???1200

ccaaacaaca?cctacggtat?ctctcaagtc?ttatggggtt?ccattggttt?caccactggt???1260

gctaccttgg?gtgctgcttt?cgctgctgaa?gaaattgatc?caaagaagag?agttatctta???1320

ttcattggtg?acggttcttt?gcaattgact?gttcaagaaa?tctccaccat?gatcagatgg???1380

ggcttgaagc?catacttgtt?cgtcttgaac?aacgatggtt?acaccattga?aaagttgatt???1440

cacggtccaa?aggctcaata?caacgaaatt?caaggttggg?accacctatc?cttgttgcca???1500

actttcggtg?ctaaggacta?tgaaacccac?agagtcgcta?ccaccggtga?atgggacaag???1560

ttgacccaag?acaagtcttt?caacgacaac?tctaagatca?gaatgattga?aatcatgttg???1620

ccagtcttcg?atgctccaca?aaacttggtt?gaacaagcta?agttgactgc?tgctaccaac???1680

gctaagcaat??aa??????????????????????????????????????????????????????1692

<210>29

<211>1503

<212>DNA

<213>ALD6

<400>29

atgactaagc?tacactttga?cactgctgaa?ccagtcaaga?tcacacttcc?aaatggtttg?????60

acatacgagc?aaccaaccgg?tctattcatt?aacaacaagt?ttatgaaagc?tcaagacggt????120

aagacctatc?ccgtcgaaga?tccttccact?gaaaacaccg?tttgtgaggt?ctcttctgcc????180

accactgaag?atgttgaata?tgctatcgaa?tgtgccgacc?gtgctttcca?cgacactgaa????240

tgggctaccc?aagacccaag?agaaagaggc?cgtctactaa?gtaagttggc?tgacgaattg????300

gaaagccaaa?ttgacttggt?ttcttccatt?gaagctttgg?acaatggtaa?aactttggcc????360

ttagcccgtg?gggatgttac?cattgcaatc?aactgtctaa?gagatgctgc?tgcctatgcc????420

gacaaagtca?acggtagaac?aatcaacacc?ggtgacggct?acatgaactt?caccacctta????480

gagccaatcg?gtgtctgtgg?tcaaattatt?ccatggaact?ttccaataat?gatgttggct????540

tggaagatcg?ccccagcatt?ggccatgggt?aacgtctgta?tcttgaaacc?cgctgctgtc????600

acacctttaa?atgccctata?ctttgcttct?ttatgtaaga?aggttggtat?tccagctggt????660

gtcgtcaaca?tcgttccagg?tcctggtaga?actgttggtg?ctgctttgac?caacgaccca????720

agaatcagaa?agctggcttt?taccggttct?acagaagtcg?gtaagagtgt?tgctgtcgac????780

tcttctgaat?ctaacttgaa?gaaaatcact?ttggaactag?gtggtaagtc?cgcccatttg????840

gtctttgacg?atgctaacat?taagaagact?ttaccaaatc?tagtaaacgg?tattttcaag????900

aacgctggtc?aaatttgttc?ctctggttct?agaatttacg?ttcaagaagg?tatttacgac????960

gaactattgg?ctgctttcaa?ggcttacttg?gaaaccgaaa?tcaaagttgg?taatccattt???1020

gacaaggcta?acttccaagg?tgctatcact?aaccgtcaac?aattcgacac?aattatgaac???1080

tacatcgata?tcggtaagaa?agaaggcgcc?aagatcttaa?ctggtggcga?aaaagttggt???1140

gacaagggtt?acttcatcag?accaaccgtt?ttctacgatg?ttaatgaaga?catgagaatt???1200

gttaaggaag?aaatttttgg?accagttgtc?actgtcgcaa?agttcaagac?tttagaagaa???1260

ggtgtcgaaa?tggctaacag?ctctgaattc?ggtctaggtt?ctggtatcga?aacagaatct???1320

ttgagcacag?gtttgaaggt?ggccaagatg?ttgaaggccg?gtaccgtctg?gatcaacaca???1380

tacaacgatt?ttgactccag?agttccattc?ggtggtgtta?agcaatctgg?ttacggtaga???1440

gaaatgggtg?aagaagtcta?ccatgcatac?actgaagtaa?aagctgtcag?aattaagttg???1500

taa?????????????????????????????????????????????????????????????????1503

<210>30

<211>2142

<212>DNA

<213>ACS1

atgtcgccct?ctgccgtaca?atcatcaaaa?ctagaagaac?agtcaagtga?aattgacaag??????60

ttgaaagcaa?aaatgtccca?gtctgccgcc?actgcgcagc?agaagaagga?acatgagtat?????120

gaacatttga?cttcggtcaa?gatcgtgcca?caacggccca?tctcagatag?actgcagccc?????180

gcaattgcta?cccactattc?tccacacttg?gacgggttgc?aggactatca?gcgcttgcac?????240

aaggagtcta?ttgaagaccc?tgctaagttc?ttcggttcta?aagctaccca?atttttaaac?????300

tggtctaagc?cattcgataa?ggtgttcatc?ccagacccta?aaacgggcag?gccctccttc?????360

cagaacaatg?catggttcct?caacggccaa?ttaaacgcct?gttacaactg?tgttgacaga?????420

catgccttga?agactcctaa?caagaaagcc?attattttcg?aaggtgacga?gcctggccaa?????480

ggctattcca?ttacctacaa?ggaactactt?gaagaagttt?gtcaagtggc?acaagtgctg?????540

acttactcta?tgggcgttcg?caagggcgat?actgttgccg?tgtacatgcc?tatggtccca?????600

gaagcaatca?taaccttgtt?ggccatttcc?cgtatcggtg?ccattcactc?cgtagtcttt?????660

gccgggtttt?cttccaactc?cttgagagat?cgtatcaacg?atggggactc?taaagttgtc?????720

atcactacag?atgaatccaa?cagaggtggt?aaagtcattg?agactaaaag?aattgttgat?????780

gacgcgctaa?gagagacccc?aggcgtgaga?cacgtcttgg?tttatagaaa?gaccaacaat?????840

ccatctgttg?ctttccatgc?ccccagagat?ttggattggg?caacagaaaa?gaagaaatac?????900

aagacctact?atccatgcac?acccgttgat?tctgaggatc?cattattctt?gttgtatacg?????960

tctggttcta?ctggtgcccc?caagggtgtt?caacattcta?ccgcaggtta?cttgctggga????1020

gctttgttga?ccatgcgcta?cacttttgac?actcaccaag?aagacgtttt?cttcacagct????1080

ggagacattg?gctggattac?aggccacact?tatgtggttt?atggtccctt?actatatggt????1140

tgtgccactt?tggtctttga?agggactcct?gcgtacccaa?attactcccg?ttattgggat????1200

attattgatg?aacacaaagt?cacccaattt?tatgttgcgc?caactgcttt?gcgtttgttg????1260

aaaagagctg?gtgattccta?catcgaaaat?cattccttaa?aatctttgcg?ttgcttgggt????1320

tcggtcggtg?agccaattgc?tgctgaagtt?tgggagtggt?actctgaaaa?aataggtaaa????1380

aatgaaatcc?ccattgtaga?cacctactgg?caaacagaat?ctggttcgca?tctggtcacc????1440

ccgctggctg?gtggtgttac?accaatgaaa?ccgggttctg?cctcattccc?cttcttcggt????1500

attgatgcag?ttgttcttga?ccctaacact?ggtgaagaac?ttaacaccag?ccacgcagag????1560

ggtgtccttg?ccgtcaaagc?tgcatggcca?tcatttgcaa?gaactatttg?gaaaaatcat????1620

gataggtatc?tagacactta?tttgaaccct?taccctggct?actatttcac?tggtgatggt????1680

gctgcaaagg?ataaggatgg?ttatatctgg?attttgggtc?gtgtagacga?tgtggtgaac????1740

gtctctggtc?accgtctgtc?taccgctgaa?attgaggctg?ctattatcga?agatccaatt????1800

gtggccgagt?gtgctgttgt?cggattcaac?gatgacttga?ctggtcaagc?agttgctgca????1860

tttgtggtgt?tgaaaaacaa?atctagttgg?tccaccgcaa?cagatgatga?attacaagat????1920

atcaagaagc?atttggtctt?tactgttaga?aaagacatcg?ggccatttgc?cgcaccaaaa????1980

ttgatcattt?tagtggatga?cttgcccaag?acaagatccg?gcaaaattat?gagacgtatt????2040

ttaagaaaaa?tcctagcagg?agaaagtgac?caactaggcg?acgtttctac?attgtcaaac????2100

cctggcattg?ttagacatct?aattgattcg?gtcaagttgt?aa???????????????????????2142

<210>31

<211>2052

<212>DNA

<213>ACS2

<400>31

atgacaatca?aggaacataa?agtagtttat?gaagctcaca?acgtaaaggc?tcttaaggct?????60

cctcaacatt?tttacaacag?ccaacccggc?aagggttacg?ttactgatat?gcaacattat????120

caagaaatgt?atcaacaatc?tatcaatgag?ccagaaaaat?tctttgataa?gatggctaag????180

gaatacttgc?attgggatgc?tccatacacc?aaagttcaat?ctggttcatt?gaacaatggt????240

gatgttgcat?ggtttttgaa?cggtaaattg?aatgcatcat?acaattgtgt?tgacagacat????300

gcctttgcta?atcccgacaa?gccagctttg?atctatgaag?ctgatgacga?atccgacaac????360

aaaatcatca?catttggtga?attactcaga?aaagtttccc?aaatcgctgg?tgtcttaaaa????420

agctggggcg?ttaagaaagg?tgacacagtg?gctatctatt?tgccaatgat?tccagaagcg????480

gtcattgcta?tgttggctgt?ggctcgtatt?ggtgctattc?actctgttgt?ctttgctggg????540

ttctccgctg?gttcgttgaa?agatcgtgtc?gttgacgcta?attctaaagt?ggtcatcact????600

tgtgatgaag?gtaaaagagg?tggtaagacc?atcaacacta?aaaaaattgt?tgacgaaggt????660

ttgaacggag?tcgatttggt?ttcccgtatc?ttggttttcc?aaagaactgg?tactgaaggt????720

attccaatga?aggccggtag?agattactgg?tggcatgagg?aggccgctaa?gcagagaact????780

tacctacctc?ctgtttcatg?tgacgctgaa?gatcctctat?ttttattata?cacttccggt????840

tccactggtt?ctccaaaggg?tgtcgttcac?actacaggtg?gttatttatt?aggtgccgct????900

ttaacaacta?gatacgtttt?tgatattcac?ccagaagatg?ttctcttcac?tgccggtgac????960

gtcggctgga?tcacgggtca?cacctatgct?ctatatggtc?cattaacctt?gggtaccgcc???1020

tcaataattt?tcgaatccac?tcctgcctac?ccagattatg?gtagatattg?gagaattatc???1080

caacgtcaca?aggctaccca?tttctatgtg?gctccaactg?ctttaagatt?aatcaaacgt???1140

gtaggtgaag?ccgaaattgc?caaatatgac?acttcctcat?tacgtgtctt?gggttccgtc???1200

ggtgaaccaa?tctctccaga?cttatgggaa?tggtatcatg?aaaaagtggg?taacaaaaac???1260

tgtgtcattt?gtgacactat?gtggcaaaca?gagtctggtt?ctcatttaat?tgctcctttg???1320

gcaggtgctg?tcccaacaaa?acctggttct?gctaccgtgc?cattctttgg?tattaacgct???1380

tgtatcattg?accctgttac?aggtgtggaa?ttagaaggta?atgatgtcga?aggtgtcctt???1440

gccgttaaat?caccatggcc?atcaatggct?agatctgttt?ggaaccacca?cgaccgttac???1500

atggatactt?acttgaaacc?ttatcctggt?cactatttca?caggtgatgg?tgctggtaga???1560

gatcatgatg?gttactactg?gatcaggggt?agagttgacg?acgttgtaaa?tgtttccggt???1620

catagattat?ccacatcaga?aattgaagca?tctatctcaa?atcacgaaaa?cgtctcggaa???1680

gctgctgttg?tcggtattcc?agatgaattg?accggtcaaa?ccgtcgttgc?atatgtttcc????1740

ctaaaagatg?gttatctaca?aaacaacgct?actgaaggtg?atgcagaaca?catcacacca????1800

gataatttac?gtagagaatt?gatcttacaa?gttaggggtg?agattggtcc?tttcgcctca????1860

ccaaaaacca?ttattctagt?tagagatcta?ccaagaacaa?ggtcaggaaa?gattatgaga????1920

agagttctaa?gaaaggttgc?ttctaacgaa?gccgaacagc?taggtgacct?aactactttg????1980

gccaacccag?aagttgtacc?tgccatcatt?tctgctgtag?agaaccaatt?tttctctcaa????2040

aaaaagaaat??aa???????????????????????????????????????????????????????2052

<210>32

<211>5206

<212>DNA

<213>pGV1428

<400>32

ccataacaca?gtcctttccc?gcaattttct?ttttctatta?ctcttggcct?cctctagtac?????60

actctatatt?tttttatgcc?tcggtaatga?ttttcatttt?tttttttccc?ctagcggatg????120

actctttttt?tttcttagcg?attggcatta?tcacataatg?aattatacat?tatataaagt????180

aatgtgattt?cttcgaagaa?tatactaaaa?aatgagcagg?caagataaac?gaaggcaaag????240

atgacagagc?agaaagccct?agtaaagcgt?attacaaatg?aaaccaagat?tcagattgcg????300

atctctttaa?agggtggtcc?cctagcgata?gagcactcga?tcttcccaga?aaaagaggca????360

gaagcagtag?cagaacaggc?cacacaatcg?caagtgatta?acgtccacac?aggtataggg????420

tttctggacc?atatgataca?tgctctggcc?aagcattccg?gctggtcgct?aatcgttgag????480

tgcattggtg?acttacacat?agacgaccat?cacaccactg?aagactgcgg?gattgctctc????540

ggtcaagctt?ttaaagaggc?cctaggggcc?gtgcgtggag?taaaaaggtt?tggatcagga????600

tttgcgcctt?tggatgaggc?actttccaga?gcggtggtag?atctttcgaa?caggccgtac????660

gcagttgtcg?aacttggttt?gcaaagggag?aaagtaggag?atctctcttg?cgagatgatc????720

ccgcattttc?ttgaaagctt?tgcagaggct?agcagaatta?ccctccacgt?tgattgtctg????780

cgaggcaaga?atgatcatca?ccgtagtgag?agtgcgttca?aggctcttgc?ggttgccata????840

agagaagcca?cctcgcccaa?tggtaccaac?gatgttccct?ccaccaaagg?tgttcttatg????900

tagtgacacc?gattatttaa?agctgcagca?tacgatatat?atacatgtgt?atatatgtat????960

acctatgaat?gtcagtaagt?atgtatacga?acagtatgat?actgaagatg?acaaggtaat???1020

gcatcattct?atacgtgtca?ttctgaacga?ggcgacgtcg?ccggcgatca?cagcggacgg???1080

tggtggcatg?atggggcttg?cgatgctatg?tttgtttgtt?ttgtgatgat?gtatattatt???1140

attgaaaaac?gatatcagac?atttgtctga?taatgcttca?ttatcagaca?aatgtctgat???1200

atcgtttgga?gaaaaagaaa?aggaaaacaa?actaaatatc?tactatatac?cactgtattt???1260

tatactaatg?actttctacg?cctagtgtca?ccctctcgtg?tacccattga?ccctgtatcg???1320

gcgcgttgcc?tcgcgttcct?gtaccatata?tttttgttta?tttaggtatt?aaaatttact???1380

ttcctcatac?aaatattaaa?ttcaccaaac?ttctcaaaaa?ctaattattc?gtagttacaa????1440

actctatttt?acaatcacgt?ttattcaacc?attctacatc?caataaccaa?aatgcccatg????1500

tacctctcag?cgaagtccaa?cggtactgtc?caatattctc?attaaatagt?ctttcatcta????1560

tatatcagaa?ggtaattata?attagagatt?tcgaatcatt?accgtgccga?ttcgcacgct????1620

gcaacggcat?gcatcactaa?tgaaaagcat?acgacgcctg?cgtctgacat?gcactcattc????1680

tgaagaagat?tctgggcgcg?tttcgttctc?gttttcctct?gtatattgta?ctctggtgga????1740

caatttgaac?ataacgtctt?tcacctcgcc?attctcaata?atgggttcca?attctatcca????1800

ggtagcggtt?aattgacggt?gcttaagccg?tatgctcact?ctaacgctac?cgttgtccaa????1860

acaacggacc?cctttgtgac?gggtgtaaga?cccatcatga?agtaaaacat?ctctaacggt????1920

atggaaaaga?gtggtacggt?caagtttcct?ggcacgagtc?aattttccct?cttcgtgtag????1980

atcggtaccg?gccgcaaatt?aaagccttcg?agcgtcccaa?aaccttctca?agcaaggttt????2040

tcagtataat?gttacatgcg?tacacgcgtc?tgtacagaaa?aaaaagaaaa?atttgaaata????2100

taaataacgt?tcttaatact?aacataacta?taaaaaaata?aatagggacc?tagacttcag????2160

gttgtctaac?tccttccttt?tcggttagag?cggatgtggg?gggagggcgt?gaatgtaagc????2220

gtgacataac?taattacatg?actcgagcgg?ccgcggatcc?cgggaattcg?tcgacaccat????2280

cttcttctga?gatgagtttt?tgttccatgc?tagttctaga?atccgtcgaa?actaagttct????2340

ggtgttttaa?aactaaaaaa?aagactaact?ataaaagtag?aatttaagaa?gtttaagaaa????2400

tagatttaca?gaattacaat?caatacctac?cgtctttata?tacttattag?tcaagtaggg????2460

gaataatttc?agggaactgg?tttcaacctt?ttttttcagc?tttttccaaa?tcagagagag????2520

cagaaggtaa?tagaaggtgt?aagaaaatga?gatagataca?tgcgtgggtc?aattgccttg????2580

tgtcatcatt?tactccaggc?aggttgcatc?actccattga?ggttgtgccc?gttttttgcc????2640

tgtttgtgcc?cctgttctct?gtagttgcgc?taagagaatg?gacctatgaa?ctgatggttg????2700

gtgaagaaaa?caatattttg?gtgctgggat?tctttttttt?tctggatgcc?agcttaaaaa????2760

gcgggctcca?ttatatttag?tggatgccag?gaataaactg?ttcacccaga?cacctacgat????2820

gttatatatt?ctgtgtaacc?cgccccctat?tttgggcatg?tacgggttac?agcagaatta????2880

aaaggctaat?tttttgacta?aataaagtta?ggaaaatcac?tactattaat?tatttacgta????2940

ttctttgaaa?tggcgagtat?tgataatgat?aaactgagct?agatctgggc?ccgagctcca????3000

gcttttgttc?cctttagtga?gggttaattg?cgcgcttggc?gtaatcatgg?tcatagctgt????3060

ttcctgtgtg?aaattgttat?ccgctcacaa?ttccacacaa?cataggagcc?ggaagcataa????3120

agtgtaaagc?ctggggtgcc?taatgagtga?ggtaactcac?attaattgcg?ttgcgctcac????3180

tgcccgcttt?ccagtcggga?aacctgtcgt?gccagctgca?ttaatgaatc?ggccaacgcg????3240

cggggagagg?cggtttgcgt?attgggcgct?cttccgcttc?ctcgctcact?gactcgctgc????3300

gctcggtcgt?tcggctgcgg?cgagcggtat?cagctcactc?aaaggcggta?atacggttat????3360

ccacagaatc?aggggataac?gcaggaaaga?acatgtgagc?aaaaggccag?caaaaggcca????3420

ggaaccgtaa?aaaggccgcg?ttgctggcgt?ttttccatag?gctccgcccc?cctgacgagc????3480

atcacaaaaa?tcgacgctca?agtcagaggt?ggcgaaaccc?gacaggacta?taaagatacc????3540

aggcgtttcc?ccctggaagc?tccctcgtgc?gctctcctgt?tccgaccctg?ccgcttaccg????3600

gatacctgtc?cgcctttctc?ccttcgggaa?gcgtggcgct?ttctcatagc?tcacgctgta????3660

ggtatctcag?ttcggtgtag?gtcgttcgct?ccaagctggg?ctgtgtgcac?gaaccccccg????3720

ttcagcccga?ccgctgcgcc?ttatccggta?actatcgtct?tgagtccaac?ccggtaagac????3780

acgacttatc?gccactggca?gcagccactg?gtaacaggat?tagcagagcg?aggtatgtag????3840

gcggtgctac?agagttcttg?aagtggtggc?ctaactacgg?ctacactaga?aggacagtat????3900

ttggtatctg?cgctctgctg?aagccagtta?ccttcggaaa?aagagttggt?agctcttgat????3960

ccggcaaaca?aaccaccgct?ggtagcggtg?gtttttttgt?ttgcaagcag?cagattacgc????4020

gcagaaaaaa?aggatctcaa?gaagatcctt?tgatcttttc?tacggggtct?gacgctcagt????4080

ggaacgaaaa?ctcacgttaa?gggattttgg?tcatgagatt?atcaaaaagg?atcttcacct????4140

agatcctttt?aaattaaaaa?tgaagtttta?aatcaatcta?aagtatatat?gagtaaactt????4200

ggtctgacag?ttaccaatgc?ttaatcagtg?aggcacctat?ctcagcgatc?tgtctatttc????4260

gttcatccat?agttgcctga?ctccccgtcg?tgtagataac?tacgatacgg?gagggcttac????4320

catctggccc?cagtgctgca?atgataccgc?gagacccacg?ctcaccggct?ccagatttat????4380

cagcaataaa?ccagccagcc?ggaagggccg?agcgcagaag?tggtcctgca?actttatccg????4440

cctccatcca?gtctattaat?tgttgccggg?aagctagagt?aagtagttcg?ccagttaata????4500

gtttgcgcaa?cgttgttgcc?attgctacag?gcatcgtggt?gtcacgctcg?tcgtttggta????4560

tggcttcatt?cagctccggt?tcccaacgat?caaggcgagt?tacatgatcc?cccatgttgt????4620

gcaaaaaagc?ggttagctcc?ttcggtcctc?cgatcgttgt?cagaagtaag?ttggccgcag????4680

tgttatcact?catggttatg?gcagcactgc?ataattctct?tactgtcatg?ccatccgtaa????4740

gatgcttttc?tgtgactggt?gagtactcaa?ccaagtcatt?ctgagaatag?tgtatgcggc????4800

gaccgagttg?ctcttgcccg?gcgtcaatac?gggataatac?cgcgccacat?agcagaactt????4860

taaaagtgct?catcattgga?aaacgttctt?cggggcgaaa?actctcaagg?atcttaccgc????4920

tgttgagatc?cagttcgatg?taacccactc?gtgcacccaa?ctgatcttca?gcatctttta????4980

ctttcaccag?cgtttctggg?tgagcaaaaa?caggaaggca?aaatgccgca?aaaaagggaa????5040

taagggcgac?acggaaatgt?tgaatactca?tactcttcct?ttttcaatat?tattgaagca????5100

tttatcaggg?ttattgtctc?atgagcggat?acatatttga?atgtatttag?aaaaataaac????5160

aaataggggt?tccgcgcaca?tttccccgaa?aagtgccacc?tgacgt???????????????????5206

<210>33

<211>5157

<212>DNA

<213>pGV1429

<400>33

caggcaagtg?cacaaacaat?acttaaataa?atactactca?gtaataacct?atttcttagc??????60

atttttgacg?aaatttgcta?ttttgttaga?gtcttttaca?ccatttgtct?ccacacctcc????120

gcttacatca?acaccaataa?cgccatttaa?tctaagcgca?tcaccaacat?tttctggcgt????180

cagtccacca?gctaacataa?aatgtaagct?ttcggggctc?tcttgccttc?caacccagtc????240

agaaatcgag?ttccaatcca?aaagttcacc?tgtcccacct?gcttctgaat?caaacaaggg????300

aataaacgaa?tgaggtttct?gtgaagctgc?actgagtagt?atgttgcagt?cttttggaaa????360

tacgagtctt?ttaataactg?gcaaaccgag?gaactcttgg?tattcttgcc?acgactcatc????420

tccatgcagt?tggacgatat?caatgccgta?atcattgacc?agagccaaaa?catcctcctt????480

aggttgatta?cgaaacacgc?caaccaagta?tttcggagtg?cctgaactat?ttttatatgc????540

ttttacaaga?cttgaaattt?tccttgcaat?aaccgggtca?attgttctct?ttctattggg????600

cacacatata?atacccagca?agtcagcatc?ggaatctaga?gcacattctg?cggcctctgt????660

gctctgcaag?ccgcaaactt?tcaccaatgg?accagaacta?cctgtgaaat?taataacaga????720

catactccaa?gctgcctttg?tgtgcttaat?cacgtatact?cacgtgctca?atagtcacca????780

atgccctccc?tcttggccct?ctccttttct?tttttcgacc?gaattaattc?ttaatcggca????840

aaaaaagaaa?agctccggat?caagattgta?cgtaaggtga?caagctattt?ttcaataaag????900

aatatcttcc?actactgcca?tctggcgtca?taactgcaaa?gtacacatat?attacgatgc????960

tgtctattaa?atgcttccta?tattatatat?atagtaatgt?cgttgacgtc?gccggcgatc???1020

acagcggacg?gtggtggcat?gatggggctt?gcgatgctat?gtttgtttgt?tttgtgatga???1080

tgtatattat?tattgaaaaa?cgatatcaga?catttgtctg?ataatgcttc?attatcagac???1140

aaatgtctga?tatcgtttgg?agaaaaagaa?aaggaaaaca?aactaaatat?ctactatata???1200

ccactgtatt?ttatactaat?gactttctac?gcctagtgtc?accctctcgt?gtacccattg???1260

accctgtatc?ggcgcgttgc?ctcgcgttcc?tgtaccatat?atttttgttt?atttaggtat???1320

taaaatttac?tttcctcata?caaatattaa?attcaccaaa?cttctcaaaa?actaattatt???1380

cgtagttaca?aactctattt?tacaatcacg?tttattcaac?cattctacat?ccaataacca???1440

aaatgcccat?gtacctctca?gcgaagtcca?acggtactgt?ccaatattct?cattaaatag???1500

tctttcatct?atatatcaga?aggtaattat?aattagagat?ttcgaatcat?taccgtgccg???1560

attcgcacgc?tgcaacggca?tgcatcacta?atgaaaagca?tacgacgcct?gcgtctgaca???1620

tgcactcatt?ctgaagaaga?ttctgggcgc?gtttcgttct?cgttttcctc?tgtatattgt???1680

actctggtgg?acaatttgaa?cataacgtct?ttcacctcgc?cattctcaat?aatgggttcc???1740

aattctatcc?aggtagcggt?taattgacgg?tgcttaagcc?gtatgctcac?tctaacgcta???1800

ccgttgtcca?aacaacggac?ccctttgtga?cgggtgtaag?acccatcatg?aagtaaaaca???1860

tctctaacgg?tatggaaaag?agtggtacgg?tcaagtttcc?tggcacgagt?caattttccc???1920

tcttcgtgta?gatcggtacc?ggccgcaaat?taaagccttc?gagcgtccca?aaaccttctc???1980

aagcaaggtt?ttcagtataa?tgttacatgc?gtacacgcgt?ctgtacagaa?aaaaaagaaa???2040

aatttgaaat?ataaataacg?ttcttaatac?taacataact?ataaaaaaat?aaatagggac???2100

ctagacttca?ggttgtctaa?ctccttcctt?ttcggttaga?gcggatgtgg?ggggagggcg????2160

tgaatgtaag?cgtgacataa?ctaattacat?gactcgagcg?gccgcggatc?ccgggaattc????2220

gtcgacacca?tcttcttctg?agatgagttt?ttgttccatg?ctagttctag?aatccgtcga????2280

aactaagttc?tggtgtttta?aaactaaaaa?aaagactaac?tataaaagta?gaatttaaga????2340

agtttaagaa?atagatttac?agaattacaa?tcaataccta?ccgtctttat?atacttatta????2400

gtcaagtagg?ggaataattt?cagggaactg?gtttcaacct?tttttttcag?ctttttccaa????2460

atcagagaga?gcagaaggta?atagaaggtg?taagaaaatg?agatagatac?atgcgtgggt????2520

caattgcctt?gtgtcatcat?ttactccagg?caggttgcat?cactccattg?aggttgtgcc????2580

cgttttttgc?ctgtttgtgc?ccctgttctc?tgtagttgcg?ctaagagaat?ggacctatga????2640

actgatggtt?ggtgaagaaa?acaatatttt?ggtgctggga?ttcttttttt?ttctggatgc????2700

cagcttaaaa?agcgggctcc?attatattta?gtggatgcca?ggaataaact?gttcacccag????2760

acacctacga?tgttatatat?tctgtgtaac?ccgcccccta?ttttgggcat?gtacgggtta????2820

cagcagaatt?aaaaggctaa?ttttttgact?aaataaagtt?aggaaaatca?ctactattaa????2880

ttatttacgt?attctttgaa?atggcgagta?ttgataatga?taaactgagc?tagatctggg????2940

cccgagctcc?agcttttgtt?ccctttagtg?agggttaatt?gcgcgcttgg?cgtaatcatg????3000

gtcatagctg?tttcctgtgt?gaaattgtta?tccgctcaca?attccacaca?acataggagc????3060

cggaagcata?aagtgtaaag?cctggggtgc?ctaatgagtg?aggtaactca?cattaattgc????3120

gttgcgctca?ctgcccgctt?tccagtcggg?aaacctgtcg?tgccagctgc?attaatgaat????3180

cggccaacgc?gcggggagag?gcggtttgcg?tattgggcgc?tcttccgctt?cctcgctcac????3240

tgactcgctg?cgctcggtcg?ttcggctgcg?gcgagcggta?tcagctcact?caaaggcggt????3300

aatacggtta?tccacagaat?caggggataa?cgcaggaaag?aacatgtgag?caaaaggcca????3360

gcaaaaggcc?aggaaccgta?aaaaggccgc?gttgctggcg?tttttccata?ggctccgccc????3420

ccctgacgag?catcacaaaa?atcgacgctc?aagtcagagg?tggcgaaacc?cgacaggact????3480

ataaagatac?caggcgtttc?cccctggaag?ctccctcgtg?cgctctcctg?ttccgaccct????3540

gccgcttacc?ggatacctgt?ccgcctttct?cccttcggga?agcgtggcgc?tttctcatag????3600

ctcacgctgt?aggtatctca?gttcggtgta?ggtcgttcgc?tccaagctgg?gctgtgtgca????3660

cgaacccccc?gttcagcccg?accgctgcgc?cttatccggt?aactatcgtc?ttgagtccaa????3720

cccggtaaga?cacgacttat?cgccactggc?agcagccact?ggtaacagga?ttagcagagc????3780

gaggtatgta?ggcggtgcta?cagagttctt?gaagtggtgg?cctaactacg?gctacactag????3840

aaggacagta?tttggtatct?gcgctctgct?gaagccagtt?accttcggaa?aaagagttgg????3900

tagctcttga?tccggcaaac?aaaccaccgc?tggtagcggt?ggtttttttg?tttgcaagca????3960

gcagattacg?cgcagaaaaa?aaggatctca?agaagatcct?ttgatctttt?ctacggggtc????4020

tgacgctcag?tggaacgaaa?actcacgtta?agggattttg?gtcatgagat?tatcaaaaag????4080

gatcttcacc?tagatccttt?taaattaaaa?atgaagtttt?aaatcaatct?aaagtatata????4140

tgagtaaact?tggtctgaca?gttaccaatg?cttaatcagt?gaggcaccta?tctcagcgat????4200

ctgtctattt?cgttcatcca?tagttgcctg?actccccgtc?gtgtagataa?ctacgatacg????4260

ggagggctta?ccatctggcc?ccagtgctgc?aatgataccg?cgagacccac?gctcaccggc????4320

tccagattta?tcagcaataa?accagccagc?cggaagggcc?gagcgcagaa?gtggtcctgc????4380

aactttatcc?gcctccatcc?agtctattaa?ttgttgccgg?gaagctagag?taagtagttc????4440

gccagttaat?agtttgcgca?acgttgttgc?cattgctaca?ggcatcgtgg?tgtcacgctc????4500

gtcgtttggt?atggcttcat?tcagctccgg?ttcccaacga?tcaaggcgag?ttacatgatc????4560

ccccatgttg?tgcaaaaaag?cggttagctc?cttcggtcct?ccgatcgttg?tcagaagtaa????4620

gttggccgca?gtgttatcac?tcatggttat?ggcagcactg?cataattctc?ttactgtcat????4680

gccatccgta?agatgctttt?ctgtgactgg?tgagtactca?accaagtcat?tctgagaata????4740

gtgtatgcgg?cgaccgagtt?gctcttgccc?ggcgtcaata?cgggataata?ccgcgccaca????4800

tagcagaact?ttaaaagtgc?tcatcattgg?aaaacgttct?tcggggcgaa?aactctcaag????4860

gatcttaccg?ctgttgagat?ccagttcgat?gtaacccact?cgtgcaccca?actgatcttc????4920

agcatctttt?actttcacca?gcgtttctgg?gtgagcaaaa?acaggaaggc?aaaatgccgc????4980

aaaaaaggga?ataagggcga?cacggaaatg?ttgaatactc?atactcttcc?tttttcaata????5040

ttattgaagc?atttatcagg?gttattgtct?catgagcgga?tacatatttg?aatgtattta????5100

gaaaaataaa?caaatagggg?ttccgcgcac?atttccccga?aaagtgccac?ctgacgt???????5157

<210>34

<211>6041

<212>DNA

<213>pGV1430

<400>34

ccagttaact?gtgggaatac?tcaggtatcg?taagatgcaa?gagttcgaat?ctcttagcaa?????60

ccattatttt?tttcctcaac?ataacgagaa?cacacagggg?cgctatcgca?cagaatcaaa????120

ttcgatgact?ggaaattttt?tgttaatttc?agaggtcgcc?tgacgcatat?acctttttca????180

actgaaaaat?tgggagaaaa?aggaaaggtg?agagcgccgg?aaccggcttt?tcatatagaa????240

tagagaagcg?ttcatgacta?aatgcttgca?tcacaatact?tgaagttgac?aatattattt????300

aaggacctat?tgttttttcc?aataggtggt?tagcaatcgt?cttactttct?aacttttctt????360

accttttaca?tttcagcaat?atatatatat?atatttcaag?gatataccat?tctaatgtct????420

gcccctaaga?agatcgtcgt?tttgccaggt?gaccacgttg?gtcaagaaat?cacagccgaa????480

gccattaagg?ttcttaaagc?tatttctgat?gttcgttcca?atgtcaagtt?cgatttcgaa????540

aatcatttaa?ttggtggtgc?tgctatcgat?gctacaggtg?ttccacttcc?agatgaggcg????600

ctggaagcct?ccaagaaggc?tgatgccgtt?ttgttaggtg?ctgtgggtgg?tcctaaatgg????660

ggtaccggta?gtgttagacc?tgaacaaggt?ttactaaaaa?tccgtaaaga?acttcaattg????720

tacgccaact?taagaccatg?taactttgca?tccgactctc?ttttagactt?atctccaatc????780

aagccacaat?ttgctaaagg?tactgacttc?gttgttgtca?gagaattagt?gggaggtatt?????840

tactttggta?agagaaagga?agacgatggt?gatggtgtcg?cttgggatag?tgaacaatac?????900

accgttccag?aagtgcaaag?aatcacaaga?atggccgctt?tcatggccct?acaacatgag?????960

ccaccattgc?ctatttggtc?cttggataaa?gctaatgttt?tggcctcttc?aagattatgg????1020

agaaaaactg?tggaggaaac?catcaagaac?gaattcccta?cattgaaggt?tcaacatcaa????1080

ttgattgatt?ctgccgccat?gatcctagtt?aagaacccaa?cccacctaaa?tggtattata????1140

atcaccagca?acatgtttgg?tgatatcatc?tccgatgaag?cctccgttat?cccaggttcc????1200

ttgggtttgt?tgccatctgc?gtccttggcc?tctttgccag?acaagaacac?cgcatttggt????1260

ttgtacgaac?catgccacgg?ttctgctcca?gatttgccaa?agaataaggt?caaccctatc????1320

gccactatct?tgtctgctgc?aatgatgttg?aaattgtcat?tgaacttgcc?tgaagaaggt????1380

aaggccattg?aagatgcagt?taaaaaggtt?ttggatgcag?gtatcagaac?tggtgattta????1440

ggtggttcca?acagtaccac?cgaagtcggt?gatgctgtcg?ccgaagaagt?taagaaaatc????1500

cttgcttaaa?aagattctct?ttttttatga?tatttgtaca?taaactttat?aaatgaaatt????1560

cataatagaa?acgacacgaa?attacaaaat?ggaatatgtt?catagggtag?acgaaactat????1620

atacgcaatc?tacatacatt?tatcaagaag?gagaaaaagg?aggatgtaaa?ggaatacagg????1680

taagcaaatt?gatactaatg?gctcaacgtg?ataaggaaaa?agaattgcac?tttaacatta????1740

atattgacaa?ggaggagggc?accacacaaa?aagttaggtg?taacagaaaa?tcatgaaact????1800

atgattccta?atttatatat?tggaggattt?tctctaaaaa?aaaaaaaata?caacaaataa????1860

aaaacactca?atgacctgac?catttgatgg?agttgccggc?gatcacagcg?gacggtggtg????1920

gcatgatggg?gcttgcgatg?ctatgtttgt?ttgttttgtg?atgatgtata?ttattattga????1980

aaaacgatat?cagacatttg?tctgataatg?cttcattatc?agacaaatgt?ctgatatcgt????2040

ttggagaaaa?agaaaaggaa?aacaaactaa?atatctacta?tataccactg?tattttatac????2100

taatgacttt?ctacgcctag?tgtcaccctc?tcgtgtaccc?attgaccctg?tatcggcgcg????2160

ttgcctcgcg?ttcctgtacc?atatattttt?gtttatttag?gtattaaaat?ttactttcct????2220

catacaaata?ttaaattcac?caaacttctc?aaaaactaat?tattcgtagt?tacaaactct????2280

attttacaat?cacgtttatt?caaccattct?acatccaata?accaaaatgc?ccatgtacct????2340

ctcagcgaag?tccaacggta?ctgtccaata?ttctcattaa?atagtctttc?atctatatat????2400

cagaaggtaa?ttataattag?agatttcgaa?tcattaccgt?gccgattcgc?acgctgcaac????2460

ggcatgcatc?actaatgaaa?agcatacgac?gcctgcgtct?gacatgcact?cattctgaag????2520

aagattctgg?gcgcgtttcg?ttctcgtttt?cctctgtata?ttgtactctg?gtggacaatt????2580

tgaacataac?gtctttcacc?tcgccattct?caataatggg?ttccaattct?atccaggtag????2640

cggttaattg?acggtgctta?agccgtatgc?tcactctaac?gctaccgttg?tccaaacaac????2700

ggaccccttt?gtgacgggtg?taagacccat?catgaagtaa?aacatctcta?acggtatgga????2760

aaagagtggt?acggtcaagt?ttcctggcac?gagtcaattt?tccctcttcg?tgtagatcgg????2820

taccggccgc?aaattaaagc?cttcgagcgt?cccaaaacct?tctcaagcaa?ggttttcagt????2880

ataatgttac?atgcgtacac?gcgtctgtac?agaaaaaaaa?gaaaaatttg?aaatataaat????2940

aacgttctta?atactaacat?aactataaaa?aaataaatag?ggacctagac?ttcaggttgt????3000

ctaactcctt?ccttttcggt?tagagcggat?gtggggggag?ggcgtgaatg?taagcgtgac????3060

ataactaatt?acatgactcg?agcggccgcg?gatcccggga?attcgtcgac?accatcttct????3120

tctgagatga?gtttttgttc?catgctagtt?ctagaatccg?tcgaaactaa?gttctggtgt????3180

tttaaaacta?aaaaaaagac?taactataaa?agtagaattt?aagaagttta?agaaatagat????3240

ttacagaatt?acaatcaata?cctaccgtct?ttatatactt?attagtcaag?taggggaata????3300

atttcaggga?actggtttca?accttttttt?tcagcttttt?ccaaatcaga?gagagcagaa????3360

ggtaatagaa?ggtgtaagaa?aatgagatag?atacatgcgt?gggtcaattg?ccttgtgtca????3420

tcatttactc?caggcaggtt?gcatcactcc?attgaggttg?tgcccgtttt?ttgcctgttt????3480

gtgcccctgt?tctctgtagt?tgcgctaaga?gaatggacct?atgaactgat?ggttggtgaa????3540

gaaaacaata?ttttggtgct?gggattcttt?ttttttctgg?atgccagctt?aaaaagcggg????3600

ctccattata?tttagtggat?gccaggaata?aactgttcac?ccagacacct?acgatgttat????3660

atattctgtg?taacccgccc?cctattttgg?gcatgtacgg?gttacagcag?aattaaaagg????3720

ctaatttttt?gactaaataa?agttaggaaa?atcactacta?ttaattattt?acgtattctt????3780

tgaaatggcg?agtattgata?atgataaact?gagctagatc?tgggcccgag?ctccagcttt????3840

tgttcccttt?agtgagggtt?aattgcgcgc?ttggcgtaat?catggtcata?gctgtttcct????3900

gtgtgaaatt?gttatccgct?cacaattcca?cacaacatag?gagccggaag?cataaagtgt????3960

aaagcctggg?gtgcctaatg?agtgaggtaa?ctcacattaa?ttgcgttgcg?ctcactgccc????4020

gctttccagt?cgggaaacct?gtcgtgccag?ctgcattaat?gaatcggcca?acgcgcgggg????4080

agaggcggtt?tgcgtattgg?gcgctcttcc?gcttcctcgc?tcactgactc?gctgcgctcg????4140

gtcgttcggc?tgcggcgagc?ggtatcagct?cactcaaagg?cggtaatacg?gttatccaca????4200

gaatcagggg?ataacgcagg?aaagaacatg?tgagcaaaag?gccagcaaaa?ggccaggaac????4260

cgtaaaaagg?ccgcgttgct?ggcgtttttc?cataggctcc?gcccccctga?cgagcatcac????4320

aaaaatcgac?gctcaagtca?gaggtggcga?aacccgacag?gactataaag?ataccaggcg????4380

tttccccctg?gaagctccct?cgtgcgctct?cctgttccga?ccctgccgct?taccggatac????4440

ctgtccgcct?ttctcccttc?gggaagcgtg?gcgctttctc?atagctcacg?ctgtaggtat????4500

ctcagttcgg?tgtaggtcgt?tcgctccaag?ctgggctgtg?tgcacgaacc?ccccgttcag????4560

cccgaccgct?gcgccttatc?cggtaactat?cgtcttgagt?ccaacccggt?aagacacgac????4620

ttatcgccac?tggcagcagc?cactggtaac?aggattagca?gagcgaggta?tgtaggcggt????4680

gctacagagt?tcttgaagtg?gtggcctaac?tacggctaca?ctagaaggac?agtatttggt????4740

atctgcgctc?tgctgaagcc?agttaccttc?ggaaaaagag?ttggtagctc?ttgatccggc????4800

aaacaaacca?ccgctggtag?cggtggtttt?tttgtttgca?agcagcagat?tacgcgcaga????4860

aaaaaaggat?ctcaagaaga?tcctttgatc?ttttctacgg?ggtctgacgc?tcagtggaac????4920

gaaaactcac?gttaagggat?tttggtcatg?agattatcaa?aaaggatctt?cacctagatc????4980

cttttaaatt?aaaaatgaag?ttttaaatca?atctaaagta?tatatgagta?aacttggtct????5040

gacagttacc?aatgcttaat?cagtgaggca?cctatctcag?cgatctgtct?atttcgttca????5100

tccatagttg?cctgactccc?cgtcgtgtag?ataactacga?tacgggaggg?cttaccatct????5160

ggccccagtg?ctgcaatgat?accgcgagac?ccacgctcac?cggctccaga?tttatcagca????5220

ataaaccagc?cagccggaag?ggccgagcgc?agaagtggtc?ctgcaacttt?atccgcctcc????5280

atccagtcta?ttaattgttg?ccgggaagct?agagtaagta?gttcgccagt?taatagtttg????5340

cgcaacgttg?ttgccattgc?tacaggcatc?gtggtgtcac?gctcgtcgtt?tggtatggct????5400

tcattcagct?ccggttccca?acgatcaagg?cgagttacat?gatcccccat?gttgtgcaaa????5460

aaagcggtta?gctccttcgg?tcctccgatc?gttgtcagaa?gtaagttggc?cgcagtgtta????5520

tcactcatgg?ttatggcagc?actgcataat?tctcttactg?tcatgccatc?cgtaagatgc????5580

ttttctgtga?ctggtgagta?ctcaaccaag?tcattctgag?aatagtgtat?gcggcgaccg????5640

agttgctctt?gcccggcgtc?aatacgggat?aataccgcgc?cacatagcag?aactttaaaa????5700

gtgctcatca?ttggaaaacg?ttcttcgggg?cgaaaactct?caaggatctt?accgctgttg????5760

agatccagtt?cgatgtaacc?cactcgtgca?cccaactgat?cttcagcatc?ttttactttc????5820

accagcgttt?ctgggtgagc?aaaaacagga?aggcaaaatg?ccgcaaaaaa?gggaataagg????5880

gcgacacgga?aatgttgaat?actcatactc?ttcctttttc?aatattattg?aagcatttat????5940

cagggttatt?gtctcatgag?cggatacata?tttgaatgta?tttagaaaaa?taaacaaata????6000

ggggttccgc?gcacatttcc?ccgaaaagtg?ccacctgacg?t????????????????????????6041

<210>35

<211>5639

<212>DNA

<213>pGV1431

<400>35

ctgattggaa?agaccattct?gctttacttt?tagagcatct?tggtcttctg?agctcattat?????60

acctcaatca?aaactgaaat?taggtgcctg?tcacggctct?ttttttactg?tacctgtgac????120

ttcctttctt?atttccaagg?atgctcatca?caatacgctt?ctagatctat?tatgcattat????180

aattaatagt?tgtagctaca?aaaggtaaaa?gaaagtccgg?ggcaggcaac?aatagaaatc????240

ggcaaaaaaa?actacagaaa?tactaagagc?ttcttcccca?ttcagtcatc?gcatttcgaa????300

acaagagggg?aatggctctg?gctagggaac?taaccaccat?cgcctgactc?tatgcactaa????360

ccacgtgact?acatatatgt?gatcgttttt?aacatttttc?aaaggctgtg?tgtctggctg????420

tttccattaa?ttttcactga?ttaagcagtc?atattgaatc?tgagctcatc?accaacaaga????480

aatactaccg?taaaagtgta?aaagttcgtt?taaatcattt?gtaaactgga?acagcaagag????540

gaagtatcat?cagctagccc?cataaactaa?tcaaaggagg?atgtctacta?agagttactc????600

ggaaagagca?gctgctcata?gaagtccagt?tgctgccaag?cttttaaact?tgatggaaga????660

gaagaagtca?aacttatgtg?cttctcttga?tgttcgtaaa?acagcagagt?tgttaagatt?????720

agttgaggtt?ttgggtccat?atatctgtct?attgaagaca?catgtagata?tcttggagga?????780

tttcagcttt?gagaatacca?ttgtgccgtt?gaagcaatta?gcagagaaac?acaagttttt?????840

gatatttgaa?gacaggaagt?ttgccgacat?tgggaacact?gttaaattac?aatacacgtc?????900

tggtgtatac?cgtatcgccg?aatggtctga?tatcaccaat?gcacacggtg?tgactggtgc?????960

gggcattgtt?gctggtttga?agcaaggtgc?cgaggaagtt?acgaaagaac?ctagagggtt????1020

gttaatgctt?gccgagttat?cgtccaaggg?gtctctagcg?cacggtgaat?acactcgtgg????1080

gaccgtggaa?attgccaaga?gtgataagga?ctttgttatt?ggatttattg?ctcaaaacga????1140

tatgggtgga?agagaagagg?gctacgattg?gttgatcatg?acgccaggtg?ttggtcttga????1200

tgacaaaggt?gatgctttgg?gacaacaata?cagaactgtg?gatgaagttg?ttgccggtgg????1260

atcagacatc?attattgttg?gtagaggtct?tttcgcaaag?ggaagagatc?ctgtagtgga????1320

aggtgagaga?tacagaaagg?cgggatggga?cgcttacttg?aagagagtag?gcagatccgc????1380

ttaagagttc?tccgagaaca?agcagaggtt?cgagtgtact?cggatcagaa?gttacaagtt????1440

gatcgtttat?atataaacta?tacagagatg?ttagagtgta?atggcattgc?gtgccggcga????1500

tcacagcgga?cggtggtggc?atgatggggc?ttgcgatgct?atgtttgttt?gttttgtgat????1560

gatgtatatt?attattgaaa?aacgatatca?gacatttgtc?tgataatgct?tcattatcag????1620

acaaatgtct?gatatcgttt?ggagaaaaag?aaaaggaaaa?caaactaaat?atctactata????1680

taccactgta?ttttatacta?atgactttct?acgcctagtg?tcaccctctc?gtgtacccat????1740

tgaccctgta?tcggcgcgtt?gcctcgcgtt?cctgtaccat?atatttttgt?ttatttaggt????1800

attaaaattt?actttcctca?tacaaatatt?aaattcacca?aacttctcaa?aaactaatta????1860

ttcgtagtta?caaactctat?tttacaatca?cgtttattca?accattctac?atccaataac????1920

caaaatgccc?atgtacctct?cagcgaagtc?caacggtact?gtccaatatt?ctcattaaat????1980

agtctttcat?ctatatatca?gaaggtaatt?ataattagag?atttcgaatc?attaccgtgc????2040

cgattcgcac?gctgcaacgg?catgcatcac?taatgaaaag?catacgacgc?ctgcgtctga????2100

catgcactca?ttctgaagaa?gattctgggc?gcgtttcgtt?ctcgttttcc?tctgtatatt????2160

gtactctggt?ggacaatttg?aacataacgt?ctttcacctc?gccattctca?ataatgggtt????2220

ccaattctat?ccaggtagcg?gttaattgac?ggtgcttaag?ccgtatgctc?actctaacgc????2280

taccgttgtc?caaacaacgg?acccctttgt?gacgggtgta?agacccatca?tgaagtaaaa????2340

catctctaac?ggtatggaaa?agagtggtac?ggtcaagttt?cctggcacga?gtcaattttc????2400

cctcttcgtg?tagatcggta?ccggccgcaa?attaaagcct?tcgagcgtcc?caaaaccttc????2460

tcaagcaagg?ttttcagtat?aatgttacat?gcgtacacgc?gtctgtacag?aaaaaaaaga????2520

aaaatttgaa?atataaataa?cgttcttaat?actaacataa?ctataaaaaa?ataaataggg????2580

acctagactt?caggttgtct?aactccttcc?ttttcggtta?gagcggatgt?ggggggaggg????2640

cgtgaatgta?agcgtgacat?aactaattac?atgactcgag?cggccgcgga?tcccgggaat????2700

tcgtcgacac?catcttcttc?tgagatgagt?ttttgttcca?tgctagttct?agaatccgtc????2760

gaaactaagt?tctggtgttt?taaaactaaa?aaaaagacta?actataaaag?tagaatttaa????2820

gaagtttaag?aaatagattt?acagaattac?aatcaatacc?taccgtcttt?atatacttat????2880

tagtcaagta?ggggaataat?ttcagggaac?tggtttcaac?cttttttttc?agctttttcc????2940

aaatcagaga?gagcagaagg?taatagaagg?tgtaagaaaa?tgagatagat?acatgcgtgg????3000

gtcaattgcc?ttgtgtcatc?atttactcca?ggcaggttgc?atcactccat?tgaggttgtg????3060

cccgtttttt?gcctgtttgt?gcccctgttc?tctgtagttg?cgctaagaga?atggacctat????3120

gaactgatgg?ttggtgaaga?aaacaatatt?ttggtgctgg?gattcttttt?ttttctggat????3180

gccagcttaa?aaagcgggct?ccattatatt?tagtggatgc?caggaataaa?ctgttcaccc????3240

agacacctac?gatgttatat?attctgtgta?acccgccccc?tattttgggc?atgtacgggt????3300

tacagcagaa?ttaaaaggct?aattttttga?ctaaataaag?ttaggaaaat?cactactatt????3360

aattatttac?gtattctttg?aaatggcgag?tattgataat?gataaactga?gctagatctg????3420

ggcccgagct?ccagcttttg?ttccctttag?tgagggttaa?ttgcgcgctt?ggcgtaatca????3480

tggtcatagc?tgtttcctgt?gtgaaattgt?tatccgctca?caattccaca?caacatagga????3540

gccggaagca?taaagtgtaa?agcctggggt?gcctaatgag?tgaggtaact?cacattaatt????3600

gcgttgcgct?cactgcccgc?tttccagtcg?ggaaacctgt?cgtgccagct?gcattaatga????3660

atcggccaac?gcgcggggag?aggcggtttg?cgtattgggc?gctcttccgc?ttcctcgctc????3720

actgactcgc?tgcgctcggt?cgttcggctg?cggcgagcgg?tatcagctca?ctcaaaggcg????3780

gtaatacggt?tatccacaga?atcaggggat?aacgcaggaa?agaacatgtg?agcaaaaggc????3840

cagcaaaagg?ccaggaaccg?taaaaaggcc?gcgttgctgg?cgtttttcca?taggctccgc????3900

ccccctgacg?agcatcacaa?aaatcgacgc?tcaagtcaga?ggtggcgaaa?cccgacagga????3960

ctataaagat?accaggcgtt?tccccctgga?agctccctcg?tgcgctctcc?tgttccgacc????4020

ctgccgctta?ccggatacct?gtccgccttt?ctcccttcgg?gaagcgtggc?gctttctcat????4080

agctcacgct?gtaggtatct?cagttcggtg?taggtcgttc?gctccaagct?gggctgtgtg????4140

cacgaacccc?ccgttcagcc?cgaccgctgc?gccttatccg?gtaactatcg?tcttgagtcc????4200

aacccggtaa?gacacgactt?atcgccactg?gcagcagcca?ctggtaacag?gattagcaga????4260

gcgaggtatg?taggcggtgc?tacagagttc?ttgaagtggt?ggcctaacta?cggctacact????4320

agaaggacag?tatttggtat?ctgcgctctg?ctgaagccag?ttaccttcgg?aaaaagagtt????4380

ggtagctctt?gatccggcaa?acaaaccacc?gctggtagcg?gtggtttttt?tgtttgcaag????4440

cagcagatta?cgcgcagaaa?aaaaggatct?caagaagatc?ctttgatctt?ttctacgggg????4500

tctgacgctc?agtggaacga?aaactcacgt?taagggattt?tggtcatgag?attatcaaaa????4560

aggatcttca?cctagatcct?tttaaattaa?aaatgaagtt?ttaaatcaat?ctaaagtata????4620

tatgagtaaa?cttggtctga?cagttaccaa?tgcttaatca?gtgaggcacc?tatctcagcg????4680

atctgtctat?ttcgttcatc?catagttgcc?tgactccccg?tcgtgtagat?aactacgata????4740

cgggagggct?taccatctgg?ccccagtgct?gcaatgatac?cgcgagaccc?acgctcaccg????4800

gctccagatt?tatcagcaat?aaaccagcca?gccggaaggg?ccgagcgcag?aagtggtcct????4860

gcaactttat?ccgcctccat?ccagtctatt?aattgttgcc?gggaagctag?agtaagtagt????4920

tcgccagtta?atagtttgcg?caacgttgtt?gccattgcta?caggcatcgt?ggtgtcacgc????4980

tcgtcgtttg?gtatggcttc?attcagctcc?ggttcccaac?gatcaaggcg?agttacatga????5040

tcccccatgt?tgtgcaaaaa?agcggttagc?tccttcggtc?ctccgatcgt?tgtcagaagt????5100

aagttggccg?cagtgttatc?actcatggtt?atggcagcac?tgcataattc?tcttactgtc????5160

atgccatccg?taagatgctt?ttctgtgact?ggtgagtact?caaccaagtc?attctgagaa????5220

tagtgtatgc?ggcgaccgag?ttgctcttgc?ccggcgtcaa?tacgggataa?taccgcgcca????5280

catagcagaa?ctttaaaagt?gctcatcatt?ggaaaacgtt?cttcggggcg?aaaactctca????5340

aggatcttac?cgctgttgag?atccagttcg?atgtaaccca?ctcgtgcacc?caactgatct????5400

tcagcatctt?ttactttcac?cagcgtttct?gggtgagcaa?aaacaggaag?gcaaaatgcc????5460

gcaaaaaagg?gaataagggc?gacacggaaa?tgttgaatac?tcatactctt?cctttttcaa????5520

tattattgaa?gcatttatca?gggttattgt?ctcatgagcg?gatacatatt?tgaatgtatt????5580

tagaaaaata?aacaaatagg?ggttccgcgc?acatttcccc?gaaaagtgcc?acctgacgt?????5639

<210>36

<211>741

<212>DNA

<213>pf1A

<400>36

atgtcagtta?ttggtcgcat?tcactccttt?gaatcctgtg?gaaccgtaga?cggcccaggt?????60

attcgcttta?tcaccttttt?ccagggctgc?ctgatgcgct?gcctgtattg?tcataaccgc????120

gacacctggg?acacgcatgg?cggtaaagaa?gttaccgttg?aagatttgat?gaaggaagtg????180

gtgacctatc?gccactttat?gaacgcttcc?ggcggcggcg?ttaccgcatc?cggcggtgaa????240

gcaatcctgc?aagctgagtt?tgttcgtgac?tggttccgcg?cctgcaaaaa?agaaggcatt????300

catacctgtc?tggacaccaa?cggttttgtt?cgtcgttacg?atccggtgat?tgatgaactg????360

ctggaagtaa?ccgacctggt?aatgctcgat?ctcaaacaga?tgaacgacga?gatccaccaa????420

aatctggttg?gagtttccaa?ccaccgcacg?ctggagttcg?ctaaatatct?ggcgaacaaa????480

aatgtgaagg?tgtggatccg?ctacgttgtt?gtcccaggct?ggtctgacga?tgacgattca????540

gcgcatcgcc?tcggtgaatt?tacccgtgat?atgggcaacg?ttgagaaaat?cgagcttctc????600

ccctaccacg?agctgggcaa?acacaaatgg?gtggcaatgg?gtgaagagta?caaactcgac????660

ggtgttaaac?caccgaagaa?agagaccatg?gaacgcgtga?aaggcattct?tgagcagtac????720

ggtcataagg?taatgttcta?a??????????????????????????????????????????????741

<210>37

<211>2283

<212>DNA

<213>pflB

<400>37

atgtccgagc?ttaatgaaaa?gttagccaca?gcctgggaag?gttttaccaa?aggtgactgg??????60

cagaatgaag?taaacgtccg?tgacttcatt?cagaaaaact?acactccgta?cgagggtgac?????120

gagtccttcc?tggctggcgc?tactgaagcg?accaccaccc?tgtgggacaa?agtaatggaa?????180

ggcgttaaac?tggaaaaccg?cactcacgcg?ccagttgact?ttgacaccgc?tgttgcttcc?????240

accatcacct?ctcacgacgc?tggctacatc?aacaagcagc?ttgagaaaat?cgttggtctg?????300

cagactgaag?ctccgctgaa?acgtgctctt?atcccgttcg?gtggtatcaa?aatgatcgaa?????360

ggttcctgca?aagcgtacaa?ccgcgaactg?gatccgatga?tcaaaaaaat?cttcactgaa?????420

taccgtaaaa?ctcacaacca?gggcgtgttc?gacgtttaca?ctccggacat?cctgcgttgc?????480

cgtaaatctg?gtgttctgac?cggtctgcca?gatgcatatg?gccgtggccg?tatcatcggt?????540

gactaccgtc?gcgttgcgct?gtacggtatc?gactacctga?tgaaagacaa?actggcacag?????600

ttcacttctc?tgcaggctga?tctggaaaac?ggcgtaaacc?tggaacagac?tatccgtctg?????660

cgcgaagaaa?tcgctgaaca?gcaccgcgct?ctgggtcaga?tgaaagaaat?ggctgcgaaa?????720

tacggctacg?acatctctgg?tccggctacc?aacgctcagg?aagctatcca?gtggacttac?????780

ttcggctacc?tggctgctgt?taagtctcag?aacggtgctg?caatgtcctt?cggtcgtacc?????840

tccaccttcc?tggatgtgta?catcgaacgt?gacctgaaag?ctggcaagat?caccgaacaa?????900

gaagcgcagg?aaatggttga?ccacctggtc?atgaaactgc?gtatggttcg?cttcctgcgt?????960

actccggaat?acgatgaact?gttctctggc?gacccgatct?gggcaaccga?atctatcggt????1020

ggtatgggcc?tcgacggtcg?taccctggtt?accaaaaaca?gcttccgttt?cctgaacacc????1080

ctgtacacca?tgggtccgtc?tccggaaccg?aacatgacca?ttctgtggtc?tgaaaaactg????1140

ccgctgaact?tcaagaaatt?cgccgctaaa?gtgtccatcg?acacctcttc?tctgcagtat????1200

gagaacgatg?acctgatgcg?tccggacttc?aacaacgatg?actacgctat?tgcttgctgc????1260

gtaagcccga?tgatcgttgg?taaacaaatg?cagttcttcg?gtgcgcgtgc?aaacctggcg????1320

aaaaccatgc?tgtacgcaat?caacggcggc?gttgacgaaa?aactgaaaat?gcaggttggt????1380

ccgaagtctg?aaccgatcaa?aggcgatgtc?ctgaactatg?atgaagtgat?ggagcgcatg????1440

gatcacttca?tggactggct?ggctaaacag?tacatcactg?cactgaacat?catccactac????1500

atgcacgaca?agtacagcta?cgaagcctct?ctgatggcgc?tgcacgaccg?tgacgttatc????1560

cgcaccatgg?cgtgtggtat?cgctggtctg?tccgttgctg?ctgactccct?gtctgcaatc????1620

aaatatgcga?aagttaaacc?gattcgtgac?gaagacggtc?tggctatcga?cttcgaaatc????1680

gaaggcgaat?acccgcagtt?tggtaacaat?gatccgcgtg?tagatgacct?ggctgttgac????1740

ctggtagaac?gtttcatgaa?gaaaattcag?aaactgcaca?cctaccgtga?cgctatcccg????1800

actcagtctg?ttctgaccat?cacttctaac?gttgtgtatg?gtaagaaaac?gggtaacacc????1860

ccagacggtc?gtcgtgctgg?cgcgccgttc?ggaccgggtg?ctaacccgat?gcacggtcgt????1920

gaccagaaag?gtgcagtagc?ctctctgact?tccgttgcta?aactgccgtt?tgcttacgct????1980

aaagatggta?tctcctacac?cttctctatc?gttccgaacg?cactgggtaa?agacgacgaa????2040

gttcgtaaga?ccaacctggc?tggtctgatg?gatggttact?tccaccacga?agcatccatc????2100

gaaggtggtc?agcacctgaa?cgttaacgtg?atgaaccgtg?aaatgctgct?cgacgcgatg????2160

gaaaacccgg?aaaaatatcc?gcagctgacc?atccgtgtat?ctggctacgc?agtacgtttc????2220

aactcgctga?ctaaagaaca?gcagcaggac?gttattactc?gtaccttcac?tcaatctatg????2280

taa??????????????????????????????????????????????????????????????????2283

<210>38

<211>1095

<212>DNA

<213>Cb-FDH1

<400>38

atgaagatcg?ttttagtctt?atatggtgct?ggtaaacacg?ctgccgatga?agaaaaatta?????60

tacggttgta?ctgaaaacaa?attaggtatt?gccaattggt?tgaaagatca?aggacatgaa????120

ctaatcacca?cgtctgataa?agaaggcgga?aacagtgtgt?tggatcaaca?tataccagat????180

gccgatatta?tcattacaac?tcctttccat?cctgcttata?tcactaagga?aagaatcgac????240

aaggctaaaa?aattgaaatt?agttgttgtc?gctggtgtcg?gttctgatca?tattgatttg????300

gattatatca?accaaacagg?taggaaaatc?tccgtcttgg?aagttaccgg?ttctaatgtt????360

gtctctgttg?cagaacacgt?tgtcatgacc?atgcttgtct?tggttagaaa?ttttgttcca????420

gctcacgaac?aaaacattaa?ccacgattgg?gaggttgctg?ctatcgctaa?ggatgcttac????480

gatatcgaag?gtaaaactat?cgccaccatt?ggtgccggta?gaattggtta?cagagtcttg????540

gaaagattag?tcccattcaa?tcctaaagaa?ttattatact?acgattatca?agctttacca????600

aaagatgctg?aagaaaaagt?tggtgctaga?agggttgaaa?atattgaaga?attggttgcc????660

caagctgata?tagttacagt?taatgctcca?ttacacgctg?gtacaaaagg?tttaattaac????720

aaggaattat?tgtctaaatt?caagaaaggt?gcttggttag?tcaatactgc?aagaggtgcc????780

atttgtgttg?ccgaagatgt?tgctgcagct?ttagaatctg?gtcaattaag?aggttatggt????840

ggtgatgttt?ggttcccaca?accagctcca?aaagatcacc?catggagaga?tatgagaaac????900

aaatatggtg?ctggtaacgc?cacgactcct?cattactctg?gtactacttt?agatgctcaa????960

actagatacg?ctcaaggtac?taaaaatatc?ttggagtcat?tctttactgg?taagtttgat???1020

tacagaccac?aagatatcat?cttattaaac?ggtgaatacg?ttaccaaagc?ttacggtaaa???1080

cacgataaga?aataa????????????????????????????????????????????????????1095

<210>39

<211>1524

<212>DNA

<213>K1ALD6

<400>39

atgtcctcta?caattgctga?gaaattgaac?ctcaagatcg?tcgaacaaga?cgctgttagc?????60

atcactttgc?caaacggttt?gacttaccaa?caaccaactg?gtttgttcat?caacaatcag????120

ttcatcaagt?ctcaagacgg?taagactttg?aaggttgaaa?acccatctac?tgaggaaatc????180

attgtcgaag?tccaatctgc?tacttctcaa?gacgtcgagt?acgccgttga?agctgccgat????240

gctgctttca?actccgaatg?gtctactatg?gacccaaaaa?agcgtggttc?tttgttgttt????300

aagttggctg?acttgattga?agctcaaaag?gaattgattg?cttctatcga?atctgctgac????360

aacggtaaga?ctttggccct?agccagaggt?gatgttggtt?tggtcattga?ctacatcaga????420

tctgctgctg?gttatgctga?caagttgggt?ggtagaacta?tcaacactgg?tgatggttac????480

gctaacttca?cttacaagga?acctctaggt?gtctgtggtc?aaatcatccc?atggaacttc????540

ccattgatga?tgctttcttg?gaagatcgcc?cctgctttgg?ttgctggtaa?caccgttatc????600

ttgaagccag?cttccccaac?cccattgaac?gctttgttct?ttgcttcttt?gtgtaaggaa????660

gcaggtatcc?cagctggtgt?cgttaacatc?gttccaggtc?caggtagatc?cgttggtgac????720

accatcacca?accatccaaa?aattagaaag?attgccttca?ctggttccac?tgacattggt????780

agagacgttg?ctatcaaggc?tgcccaatct?aacttgaaga?aggtcacctt?ggaattgggt????840

ggtaaatccg?ctcatttggt?ctttgaagat?gccaacatta?agaagactat?tccaaacttg????900

gtcaacggta?ttttcaagaa?tgctggtcaa?atttgttcct?ctggttccag?aatctatgtc????960

caagacacca?tctacgatca?actattgtct?gaattcaaga?cttacctgga?aactgaaatt???1020

aaggtcggtt?ccccattcga?tgaatctaac?ttccaagctg?ctatcaacaa?caaggctcaa???1080

ttcgaaacta?tcttgaacta?catcgacatc?ggtaagaagg?aaggtgcttc?tatcttgact???1140

ggtggtgaaa?gagtaggcaa?caagggttac?ttcattaaac?caactgtatt?ctacaacgtt???1200

aaggaagata?tgagaatcgt?caaggaagaa?atctttggtc?ctgtcgtcac?catctccaag???1260

ttctctactg?tcgacgaagc?tgtcgctttg?gctaacgact?ccgaattcgg?tttgggtgct???1320

ggtatcgaaa?ctgaaaacat?ctccgttgcc?ttgaaggtcg?caaagagact?aaaagctggt???1380

accgtctgga?tcaacactta?caacgatttc?gacgctgccg?ttccattcgg?tggttacaag???1440

caatctggtt?acggtagaga?aatgggtgaa?gaagctttcg?aatcttacac?tcaaatcaag???1500

gccgtcagga?tcaagttgga?ttaa??????????????????????????????????????????1524

<210>40

<211>2124

<212>DNA

<213>K1ACS1

<400>40

atgtctcctg?ctgttgatac?cgcttccacc?gccaaagatc?caatctcagt?catgaaatct?????60

aacgcttcag?ctgccgctgc?agaccaaatt?aagacccatg?aatacgaaca?tttaacttct????120

gtgcctatag?tgcagcctct?accaattact?gataggttga?gcagcgaagc?agctcaaaaa????180

tataaaccta?atttgccagg?tgggttcgaa?gagtacaagt?ctttgcacaa?ggaatcactt????240

gaaaatccag?ccaagtttta?ccatgaacgt?gctcagctgt?tgaattggtt?caaaccatac????300

gatcaagttt?tcatcccaga?taccgaaggt?aaaccaactt?ttgagaacaa?cgcttggttt????360

accaacggtc?aattgaacgc?ttgttacaat?ttggtagaca?gacatgcctt?cactcaacca?????420

aacaaggttg?ccattcttta?tgaagctgat?gaaccaggtc?aaggttatag?tctcacttat?????480

gcggaattgt?tagaacaagt?ctgtaaagtt?gctcaaatct?tgcaatactc?gatgaacgtc?????540

aagaaaggtg?acacggtcgc?agtttatatg?ccaatgatcc?cacaggcttt?gattaccttg?????600

ttggcaatta?ctcgtatcgg?tgccattcat?tccgttgttt?ttgctgggtt?ctcttcgaat?????660

tcattgcgtg?atcgtattaa?cgatgcttac?tcaaagacag?tcatcaccac?cgatgaatct?????720

aagagaggtg?gtaagaccat?cgaaaccaag?cgtatcgtcg?atgaagcctt?gaaggatacc?????780

cctcaagtaa?caaacgtttt?ggtcttcaaa?cgtactcata?acgaaaatat?caagtacatt?????840

ccaggtaggg?atttggactg?ggatgaggaa?gtcaagaagt?acaaatctta?caccccatgc?????900

gaacctgttg?actctgaaca?tcctttgttc?ttattgtata?cttcgggttc?caccggtgct?????960

ccaaagggtg?ttcaacattc?tacagcaggt?tacttgctcc?aagcattatt?aagtatgaaa????1020

tacacctttg?acatccaaaa?cgatgacatc?ttcttcaccg?caggtgacat?tggttggatc????1080

actggtcaca?catactgtgt?ttacggtcca?ttgttacaag?gttgtactac?tttggtgttc????1140

gaaggtacac?ctgcctatcc?aaacttttct?cgttattggg?aaattgttga?caagtaccaa????1200

gtgactcaat?tctatgtagc?cccaactgca?ctacgtctat?tgaagagagc?tggtgattcc????1260

tttactgaag?gattctctct?caagtcattg?cgctccttgg?gttccgttgg?tgaacctatc????1320

gctgctgaag?tttgggaatg?gtactctgaa?aagattggta?agaatgagct?accaatcgta????1380

gacacatact?ggcaaactga?atctggctcc?cacttggtca?ctccattggc?tggtggtgct????1440

actccaatga?aaccaggtgc?agcggcattc?ccattctttg?gtattgattt?ggcagtgttg????1500

gatccaacca?caggtatcga?gcaaactggt?gaacatgcag?aaggtgttct?tgccattaaa????1560

agaccttggc?catctttcgc?aagaaccatt?tggaagaata?acgataggtt?cttagacacg????1620

tacttgaaac?catacccggg?ctattacttc?actggtgatg?gtgttgcccg?tgataaagat????1680

ggattcttct?ggatcttggg?tcgtgttgat?gatgttgtta?acgtctcagg?tcacaggttg????1740

tctactgctg?aaattgaagc?tgctatcatt?gaagatgata?tggttgccga?atgtgcagtt????1800

gttgggttta?acgacgaatt?gactggtcaa?gccgttgctg?cctttgtagt?attgaagaac????1860

aagtctagtt?taactgctgc?aagcgagtcc?gagttacaag?acatcaaaaa?gcatttgatc????1920

atcaccgtta?gaaaggatat?tggtccattc?gctgctccta?agttgatcgt?cctagttgat????1980

gatctaccaa?agactagatc?tggcaagatt?atgagacgta?ttttgagaaa?gatcctagcc????2040

ggtgaatctg?atcaattggg?cgacgtctcc?acattatcca?accctggtat?cgttaagcac????2100

ttgatcgatt?ccgtgaaatt?ataa???????????????????????????????????????????2124

<210>41

<211>2055

<212>DNA

<213>K1ACS2

<400>41

atgtcgtcgg?ataaattgca?taaggttgtg?catgaagctc?acgatgttga?agctcgtcat??????60

gctccagaac?atttctacaa?ttctcaaccg?ggtaaatcgt?actgtactga?tgaagaacat????120

taccgtgaga?tgtacactca?gtccattgag?gacccagcag?ggtttttcgg?tccattggcc????180

aaggaatatc?tagattggga?tcgtccattc?acccaagtcc?aaagcggttc?tttggaacac????240

ggtgacattg?cctggttctt?aaatggtgaa?ctgaatgctt?cttataactg?tgttgacaga????300

cacgcttttg?ccaacccaga?caagccagct?ttaatctacg?aagccgacga?tgaatctgaa????360

aacaaggtga?tcacttttgg?cgaattgttg?agacaggtct?ctgaagtggc?tggtgtcttg????420

caatcttggg?gtgtcaagaa?aggagacacc?gtcgccgttt?acttgccaat?gattcctgct????480

gcagttgttg?ctatgttggc?cgttgcaaga?ttaggtgcca?ttcattcggt?tatctttgcc????540

ggtttctctg?ccggttcctt?gaaggaaaga?gttgtcgatg?caggctgtaa?agtggtcatc????600

acttgcgatg?aaggtaagag?aggtggtaag?accgttcata?ccaagaagat?tgtcgacgaa????660

ggtttggccg?gtgtcgattc?cgtttccaag?atcttggttt?tccaaagaac?tggtactcaa????720

ggtatcccaa?tgaagccagc?tagagatttc?tggtggcacg?aagagtgtgt?caagcaaaga????780

ggttacttgc?cacctgtccc?agtcaactcc?gaagatccat?tgttcttgtt?gtacacctct????840

ggttccaccg?gttctccaaa?aggtgtcgtg?cactctactg?ctggttactt?gttaggttct????900

gctttgacca?ccagattcgt?tttcgatatt?catccagaag?atgttttgtt?cactgctggt????960

gacgtcggtt?ggattaccgg?ccacacttac?gccttgtacg?gtccattgac?cttaggtacc???1020

gctaccatta?ttttcgaatc?tactccagct?tacccagatt?atggtagata?ctggagaatc???1080

attgaacgtc?atagagctac?ccacttctac?gtcgccccaa?ctgccctaag?attgatcaaa???1140

cgtgtcggtg?aagaagaaat?tgccaagtat?gatacctcct?ccttaagagt?cttgggttct???1200

gtcggtgaac?caatctctcc?agatctatgg?gaatggtacc?acgaaaaggt?tggtaagaat???1260

aactgtgtta?tctgtgacac?catgtggcaa?accgaatccg?gttcacactt?gattgcccca???1320

ttggctggtg?ctgtcccaac?caaaccaggt?tccgctaccg?tcccattctt?cggtattaac???1380

gcctgtatca?tcgacccagt?ttctggtgaa?gaattgaagg?gtaacgatgt?tgaaggtgtc???1440

ttggcagtga?agtccccatg?gccttctatg?gccagatctg?tctggaacaa?ccatgctcgt???1500

tacttcgaaa?cttatttgaa?gccataccca?ggatactact?tcacaggtga?tggtgctggt???1560

agagatcacg?acggttacta?ctggatcaga?ggtagagttg?acgatgtcgt?taacgtttcc???1620

ggtcacagac?tttctactgc?tgaaatcgaa?gctgctctag?ctgaacacga?aggtgtttct???1680

gaagctgccg?ttgttggtat?cactgatgaa?ctaacaggtc?aagctgtcat?tgcattcgtt???1740

tccttgaagg?acggctatct?gtctgaaaat?gcggtagagg?gtgacagtac?ccacatctct???1800

ccagacaact?tacgtcgtga?gttgattcta?caagtcagag?gtgaaattgg?tccattcgct???1860

gcaccaaaga?ccgttgttgt?tgtcaacgat?ttgccaaaaa?ctagatccgg?taaaattatg???1920

agaagagtct?tgagaaaggt?tgcatccaag?gaagctgatc?aattgggtga?tctaagtacc???1980

ttagccaatg?cagacgttgt?accatctatc?atttctgcag?tagaaaatca?atttttcagt???2040

cagcagaaga?aataa????????????????????????????????????????????????????2055

<210>42

<211>37

<212>DNA

<213>Gevo-311

<400>42

gaggt?tgtcg?acatgaaaaa?gatttttgta?cttggag?????????????????????37

<210>43

<211>35

<212>DNA

<213>Gevo-175

<400>43

aattggatcc?ttatttagaa?taatcataga?atcct????????????????????????35

<210>44

<211>37

<212>DNA

<213>Gevo-312

<400>44

gttcttgtcg?acatggaatt?aaaaaatgtt?attcttg??????????????????????37

<210>45

<211>37

<212>DNA

<213>Gevo-171

<400>45

aattggatcc?ttatttattt?tgaaaattct?tttctgc??????????????????????37

<210>46

<211>37

<212>DNA

<213>Gevo-313

<400>46

caagaggtcg?acatgaattt?ccaattaact?agagaac??????????????????????37

<210>47

<211>34

<212>DNA

<213>Gevo-314

<400>47

gcgtccggat?ccctatctta?aaatgcttcc?tgcg?????????????????????????34

<210>48

<211>36

<212>DNA

<213>Gevo-315

<400>48

cggaaagtcg?acatgaatat?agcagattac?aaaggc???????????????????????36

<210>49

<211>35

<212>DNA

<213>Gevo-173

<400>49

aattggatcc?ttattcagcg?ctctttattt?cttta??????????????????????????????35

<210>50

<211>37

<212>DNA

<213>Gevo-316

<400>50

caaaatgtcg?acatgaatat?agtagtttgt?gtaaaac????????????????????????????37

<210>51

<211>37

<212>DNA

<213>Gevo-317

<400>51

taatttggat?ccttagatgt?agtgtttttc?ttttaat????????????????????????????37

<210>52

<211>35

<212>DNA

<213>Gevo-319

<400>52

gaaccagtcg?acatggcacg?ttttacttta?ccaag??????????????????????????????35

<210>53

<211>35

<212>DNA

<213>Gevo-177

<400>53

aattggatcc?ttacaaatta?actttagttc?catag??????????????????????????????35

<210>54

<211>36

<212>DNA

<213>Gevo-318

<400>54

tccatagtcg?acatgaataa?agacacacta?atacct?????????????????????????????36

<210>55

<211>40

<212>DNA

<213>Gevo-249

<400>55

aattggatcc?ttagccggca?agtacacatc?ttctttgtct?????????????????????????40

<210>56

<211>35

<212>DNA

<213>Gevo-308

<400>56

gatcgagtcg?acatgaaaga?agttgtaata?gctag??????????????????????????????35

<210>57

<211>35

<212>DNA

<213>Gevo-309

<400>57

gttataggat?ccctagcact?tttctagcaa?tattg???????????????????????????????35

<210>58

<211>37

<212>DNA

<213>Gevo-281

<400>58

gtggatgtcg?acatgaaaaa?ggtatgtgtt?ataggtg?????????????????????????????37

<210>59

<211>35

<212>DNA

<213>Gevo-161

<400>59

aattggatcc?ttattttgaa?taatcgtaga?aacct???????????????????????????????35

<210>60

<211>35

<212>DNA

<213>Gevo-282

<400>60

tcctacgtcg?acatggaact?aaacaatgtc?atcct???????????????????????????????35

<210>61

<211>36

<212>DNA

<213>Gevo-283

<400>61

taacttggat?ccctatctat?ttttgaagcc?ttcaat??????????????????????????????36

<210>62

<211>37

<212>DNA

<213>Gevo-284

<400>62

caagaggtcg?acatggattt?taatttaaca?agagaac?????????????????????????????37

<210>63

<211>39

<212>DNA

<213>Gevo-285

<400>63

caataaggat?ccttatctaa?aaatttttcc?tgaaataac???????????????????????????39

<210>64

<211>36

<212>DNA

<213>Gevo-286

<400>64

cgggaagtcg?acatgaataa?agcagattac?aagggc??????????????????????????36

<210>65

<211>36

<212>DNA

<213>Gevo-287

<400>65

gttcaaggat?ccttaattat?tagcagcttt?aacttg??????????????????????????36

<210>66

<211>38

<212>DNA

<213>Gevo-288

<400>66

caaaattgtc?gacatgaata?tagttgtttg?tttaaaac????????????????????????38

<210>67

<211>41

<212>DNA

<213>Gevo-289

<400>67

gttttaggat?ccttaaatat?agtgttcttc?ttttaatttt?g????????????????????41

<210>68

<211>37

<212>DNA

<213>Gevo-292

<400>68

caagaagtcg?acatgaaagt?tacaaatcaa?aaagaac?????????????????????????37

<210>69

<211>40

<212>DNA

<213>Gevo-293

<400>69

tcctatgcgg?ccgcttaaaa?tgattttata?tagatatcct??????????????????????40

<210>70

<211>35

<212>DNA

<213>Gevo-290

<400>70

aggaaagtcg?acatgaaagt?cacaacagta?aagga???????????????????????????35

<210>71

<211>40

<212>DNA

<213>Gevo-291

<400>71

atttaagcgg?ccgcttaagg?ttgtttttta?aaacaattta??????????????????????40

<210>72

<211>37

<212>DNA

<213>Gevo-294

<400>72

cataacgtcg?acatgctaag?ttttgattat?tcaatac????????????????????37

<210>73

<211>36

<212>DNA

<213>Gevo-247

<400>73

aattggatcc?ttaataagat?tttttaaata?tctcaa?????????????????????36

<210>74

<211>37

<212>DNA

<213>Gevo-295

<400>74

cataacgtcg?acatggttga?tttcgaatat?tcaatac????????????????????37

<210>75

<211>36

<212>DNA

<213>Gevo-159

<400>75

aattggatcc?ttacacagat?tttttgaata?tttgta?????????????????????36

<210>76

<211>35

<212>DNA

<213>Gevo-310

<400>76

gatcgagaat?tcatgaaaga?agttgtaata?gctag??????????????????????35

<210>77

<211>35

<212>DNA

<213>Gevo-309

<400>77

gttataggat?ccctagcact?tttctagcaa?tattg??????????????????????35

<210>78

<211>36

<212>DNA

<213>Gevo-296

<400>78

cggatagtcg?acatgaaaaa?ggtatgtgtt?ataggc?????????????????????36

<210>79

<211>38

<212>DNA

<213>Gevo-297

<400>79

tcccaaggat?ccttattttg?aataatcgta?gaaaccct????????????????38

<210>80

<211>35

<212>DNA

<213>Gevo-282

<400>80

tcctacgtcg?acatggaact?aaacaatgtc?atcct???????????????????35

<210>81

<211>36

<212>DNA

<213>Gevo-283

<400>81

taacttggat?ccctatctat?ttttgaagcc?ttcaat??????????????????36

<210>82

<211>37

<212>DNA

<213>Gevo-284

<400>82

caagaggtcg?acatggattt?taatttaaca?agagaac?????????????????37

<210>83

<211>37

<212>DNA

<213>Gevo-298

<400>83

gtaaagggat?ccttaactaa?aaatttttcc?tgaaatg?????????????????37

<210>84

<211>36

<212>DNA

<213>Gevo-286

<400>84

cgggaagtcg?acatgaataa?agcagattac?aagggc??????????????????36

<210>85

<211>36

<212>DNA

<213>Gevo-299

<400>85

gttcaaggat?ccttaattat?tagcagcttt?aacctg??????????????????36

<210>86

<211>38

<212>DNA

<213>Gevo-288

<400>86

caaaattgtc?gacatgaata?tagttgtttg?tttaaaac????????????????38

<210>87

<211>36

<212>DNA

<213>Gevo-300

<400>87

gactttggat?ccttaaatat?agtgttcttc?tttcag??????????????????????36

<210>88

<211>37

<212>DNA

<213>Gevo-292

<400>88

caagaagtcg?acatgaaagt?tacaaatcaa?aaagaac?????????????????????37

<210>89

<211>41

<212>DNA

<213>Gevo-301

<400>89

attttcggat?ccttaaaatg?attttatata?gatatctttt?a????????????????41

<210>90

<211>37

<212>DNA

<213>Gevo-302

<400>90

cttatagtcg?acatggattt?taacttaaca?gatattc?????????????????????37

<210>91

<211>36

<212>DNA

<213>Gevo-303

<400>91

ccgccaggat?ccttaacgta?acagagcacc?gccggt??????????????????????36

<210>92

<211>36

<212>DNA

<213>Gevo-304

<400>92

cggaaagtcg?acatggattt?agcagaatac?aaaggc??????????????????????36

<210>93

<211>34

<212>DNA

<213>Gevo-305

<400>93

ctttgtggat?ccttatgcaa?tgcctttctg?tttc????????????????????????34

<210>94

<211>37

<212>DNA

<213>Gevo-306

<400>94

caaactgaat?tcatggaaat?attggtatgt?gtcaaac????????????????37

<210>95

<211>35

<212>DNA

<213>Gevo-307

<400>95

accaacggat?ccttaaatga?ttttctgggc?aacca??????????????????35

<210>96

<211>34

<212>DNA

<213>Gevo-273

<400>96

gttacagtcg?acatgtctca?gaacgtttac?attg???????????????????34

<210>97

<211>35

<212>DNA

<213>Gevo-274

<400>97

gataacggat?cctcatatct?tttcaatgac?aatag??????????????????35

<210>98

<211>34

<212>DNA

<213>PflA_forw

<400>98

cattgaattc?atgtcagtta?ttggtcgcat?tcac???????????????????34

<210>99

<211>37

<212>DNA

<213>PflA_Rev

<400>99

cattgtcgac?ttagaacatt?accttatgac?cgtactg????????????????37

<210>100

<211>37

<212>DNA

<213>PflB_forw

<400>100

cattgaattc?atgtccgagc?ttaatgaaaa?gttagcc????????????????37

<210>101

<211>36

<212>DNA

<213>PflB_Rev

<400>101

cattgtcgac?ttacatagat?tgagtgaagg?tacgag?????????????????36

<210>102

<211>39

<212>DNA

<213>fdh1_forw

<400>102

cattgaattc?atgaagatcg?ttttagtctt?atatggtgc????????????????39

<210>103

<211>37

<212>DNA

<213>fdh1_rev

<400>103

cattgtcgac?ttatttctta?tcgtgtttac?cgtaagc??????????????????37

<210>104

<211>38

<212>DNA

＜213〉the K1ALD6_ right side 3

<400>104

gttaggatcc?ttaatccaac?ttgatcctga?cggccttg?????????????????38

<210>105

<211>43

<212>DNA

＜213〉a K1ALD6_ left side 5

<400>105

ccaagtcgac?atgtcctcta?caattgctga?gaaattgaac?ctc???????????43

<210>106

<211>40

<212>DNA

＜213〉the K1ACS1_ right side 3

<400>106

gttagcggcc?gcttataatt?tcacggaatc?gatcaagtgc???????????????40

<210>107

<211>37

<212>DNA

＜213〉a K1ACS1_ left side 5

<400>107

ccaagctagc?atgtctcctg?ctgttgatac?cgcttcc??????????????????37

<210>108

<211>49

<212>DNA

＜213〉the K1ACS2_ right side 3

<400>108

ggttggatcc?ttatttcttc?tgctgactga?aaaattgatt?ttctactgc?????49

<210>109

<211>35

<212>DNA

＜213〉a K1ACS2_ left side 5

<400>109

ccaagaattc?atgtcgtcgg?ataaattgca?taagg????????????????????35

<210>110

<211>26

<212>DNA

<213>Gevo-350

<400>110

cttaaattct?acttttatag?ttagtc????????????????????????????26

<210>111

<211>20

<212>DNA

<213>Gevo-352

<400>111

ccttcctttt?cggttagagc???????????????????????????????????20

<210>112

<211>31

<212>DNA

<213>Gevo-479

<400>112

catgccgtcg?acatgtcgcc?ctctgccgta?c??????????????????????31

<210>113

<211>38

<212>DNA

<213>Gevo-480

<400>113

gattaagcgg?ccgcttacaa?cttgaccgaa?tcaattag???????????????38

<210>114

<211>37

<212>DNA

<213>Gevo-483

<400>114

gatgaagtcg?acatgacaat?caaggaacat?aaagtag????????????????37

<210>115

<211>39

<212>DNA

<213>Gevo-484

<400>115

gttaaaggat?ccttatttct?ttttttgaga?gaaaaattg??????????????39

<210>116

<211>44

<212>DNA

<213>Gevo-606

<400>116

ttttgtcgac?actagtatgt?cagaacgttt?cccaaatgac?gtgg????????44

<210>117

<211>39

<212>DNA

<213>Gevo-607

<400>117

ttttctcgag?ttacgccaga?cgcgggttaa?ctttatctg????????????????39

<210>118

<211>40

<212>DNA

<213>Gevo-609

<400>118

ttttctcgag?ttacatcacc?agacggcgaa?tgtcagacag???????????????40

<210>119

<211>46

<212>DNA

<213>Gevo-610

<400>119

ttttgtcgac?actagtatga?gtactgaaat?caaaactcag?gtcgtg????????46

<210>120

<211>37

<212>DNA

<213>Gevo-611

<400>120

ttttctcgag?ttacttcttc?ttcgctttcg?ggttcgg??????????????????37

<210>121

<211>21

<212>DNA

<213>Gevo-616

<400>121

ctatttacca?ggctaaattc?c???????????????????????????????????21

<210>122

<211>22

<212>DNA

<213>Gevo-617

<400>122

tgaaggtaaa?aacatcgcgc?ac??????????????????????????????????22

<210>123

<211>23

<212>DNA

<213>Gevo-618

<400>123

cgtggcttcc?tgatcggcgg?tac?????????????????????????????????23

<210>124

<211>24

<212>DNA

<213>Gevo-619

<400>124

caccagcggc?tggcgtgaaa?gaag????????????????????????????????24

<210>125

<211>24

<212>DNA

<213>Gevo-620

<400>125

gaagtggaac?tgggccgcat?ccag???????????????????????????????????????????24

<210>126

<211>22

<212>DNA

<213>Gevo-621

<400>126

gacgtggttg?aaatgttcga?cc?????????????????????????????????????????????22

<210>127

<211>35

<212>DNA

<213>Gevo-637

<400>127

ttttgagctc?gccgatccca?ttaccgacat?ttggg???????????????????????????????35

<210>128

<211>96

<212>DNA

<213>Gevo-638

<400>128

aaagtcgaca?ccgatatacc?tgtatgtgtc?accaccaatg?tatctataag?tatccatgct????60

agccctaggt?ttatgtgatg?attgattgat?tgattg??????????????????????????????96

<210>129

<211>36

<212>DNA

<213>Gevo-639

<400>129

ttttctcgag?actagtatgt?ctgaaattac?tttggg??????????????????????????????36

<210>130

<211>36

<212>DNA

<213>Gevo-640

<400>130

ttttggatcc?ttattgctta?gcgttggtag?cagcag??????????????????????????????36

<210>131

<211>27

<212>DNA

<213>Gevo-641

<400>131

gttatcttgg?ctgatgcttg?ttgttcc????????????????????????????????????????27

<210>132

<211>41

<212>DNA

<213>Gevo-642

<400>132

ttttgtcgac?actagtatga?gtactgaaat?caaaactcag?g???????????????????????41

<210>133

<211>33

<212>DNA

<213>Gevo-643

<400>133

ccaagtcgac?atgactaagc?tacactttga?cac????????????????????????????????33

<210>134

<211>27

<212>DNA

<213>Gevo-644

<400>134

gtcggtaaga?gtgttgctgt?ggactcg???????????????????????????????????????27

<210>135

<211>42

<212>DNA

<213>Gevo-646

<400>135

ccaaggatcc?ttacaactta?attctgacag?cttttacttc?ag??????????????????????42

<210>136

<211>49

<212>DNA

<213>Gevo-653

<400>136

ttttgtcgac?actagtatgg?ctatcgaaat?caaagtaccg?gacatcggg???????????????49

<210>137

<211>25

<212>DNA

<213>Gevo-654

<400>137

cttccataga?agctttgtcg?ccttc?????????????????????????????????????????25

<210>138

<211>25

<212>DNA

<213>Gevo-655

<400>138

gtgcaatatc?atatagaagt?catcg?????????????????????????????????????????25

<210>139

<211>48

<212>DNA

<213>Gevo-656

<400>139

ttttctcgag?gctagcatgg?catcgtaccc?agagcacacc?attattgg????????????????48

<210>140

<211>40

<212>DNA

<213>Gevo-657

<400>140

ttttggatcc?tcacaatagc?atttccaaag?gattttcaat????????????????????????40

<210>141

<211>45

<212>DNA

<213>Gevo-658

<400>141

ttttctcgag?actagtatgg?tcatcatcgg?tggtggccct?gctgg??????????????????45

<210>142

<211>36

<212>DNA

<213>Gevo-659

<400>142

ttttggatcc?tcaacaatga?atagctttat?catagg????????????????????????????36

<210>143

<211>47

<212>DNA

<213>Gevo-660

<400>143

ttttctcgag?actagtatgg?caactttaaa?aacaactgat?aagaagg????????????????47

<210>144

<211>35

<212>DNA

<213>Gevo-661

<400>144

ttttagatct?ttaatcccta?gaggcaaaac?cttgc?????????????????????????????35

<210>145

<211>44

<212>DNA

<213>Gevo-662

<400>145

ttttctcgag?actagtatgg?cggaagaatt?ggaccgtgat?gatg???????????????????44

<210>146

<211>38

<212>DNA

<213>Gevo-663

<400>146

tttggatcct?tattcaattg?acaagacttc?tttgacag??????????????????????????38

<210>147

<211>48

<212>DNA

<213>Gevo-664

<400>147

ttttctcgag?actagtatgt?tacttgctgt?aaagacattt?tcaatgcc????????????????48

<210>148

<211>40

<212>DNA

<213>Gevo-665

<400>148

ttttggatcc?tcaaaatgat?tctaactccc?ttacgtaatc?????????????????????????40

<210>149

<211>27

<212>DNA

<213>Gevo-666

<400>149

ggtagaatta?ccaaggctga?cattgag???????????????????????????????????????27

<210>150

<211>21

<212>DNA

<213>Gevo-667

<400>150

cattggtgga?ggaatcatcg?g?????????????????????????????????????????????21

<210>151

<211>24

<212>DNA

<213>Gevo-668

<400>151

caccaataca?agaacgagga?cgcc??????????????????????????????????????????24

<210>152

<211>28

<212>DNA

<213>Gevo-669

<400>152

tggtacggtt?ccattccagg?gttaaagg??????????????????????????????????????28

<210>153

<211>25

<212>DNA

<213>Gevo-670

<400>153

gggtgatgtg?ctagcatacc?taggg?????????????????????????????????????????25

<210>154

<211>1197

<212>DNA

<213>ERG10

<400>154

atgtctcaga?acgtttacat?tgtatcgact?gccagaaccc?caattggttc?attccagggt???60

tctctatcct?ccaagacagc?agtggaattg?ggtgctgttg?ctttaaaagg?cgccttggct????120

aaggttccag?aattggatgc?atccaaggat?tttgacgaaa?ttatttttgg?taacgttctt????180

tctgccaatt?tgggccaagc?tccggccaga?caagttgctt?tggctgccgg?tttgagtaat????240

catatcgttg?caagcacagt?taacaaggtc?tgtgcatccg?ctatgaaggc?aatcattttg????300

ggtgctcaat?ccatcaaatg?tggtaatgct?gatgttgtcg?tagctggtgg?ttgtgaatct????360

atgactaacg?caccatacta?catgccagca?gcccgtgcgg?gtgccaaatt?tggccaaact????420

gttcttgttg?atggtgtcga?aagagatggg?ttgaacgatg?cgtacgatgg?tctagccatg????480

ggtgtacacg?cagaaaagtg?tgcccgtgat?tgggatatta?ctagagaaca?acaagacaat????540

tttgccatcg?aatcctacca?aaaatctcaa?aaatctcaaa?aggaaggtaa?attcgacaat????600

gaaattgtac?ctgttaccat?taagggattt?agaggtaagc?ctgatactca?agtcacgaag????660

gacgaggaac?ctgctagatt?acacgttgaa?aaattgagat?ctgcaaggac?tgttttccaa????720

aaagaaaacg?gtactgttac?tgccgctaac?gcttctccaa?tcaacgatgg?tgctgcagcc????780

gtcatcttgg?tttccgaaaa?agttttgaag?gaaaagaatt?tgaagccttt?ggctattatc????840

aaaggttggg?gtgaggccgc?tcatcaacca?gctgatttta?catgggctcc?atctcttgca????900

gttccaaagg?ctttgaaaca?tgctggcatc?gaagacatca?attctgttga?ttactttgaa????960

ttcaatgaag?ccttttcggt?tgtcggtttg?gtgaacacta?agattttgaa?gctagaccca???1020

tctaaggtta?atgtatatgg?tggtgctgtt?gctctaggtc?acccattggg?ttgttctggt???1080

gctagagtgg?ttgttacact?gctatccatc?ttacagcaag?aaggaggtaa?gatcggtgtt???1140

gccgccattt?gtaatggtgg?tggtggtgct?tcctctattg?tcattgaaaa?gatatga??????1197

<210>155

<211>849

<212>DNA

<213>Cb-hbd

<400>155

atgaaaaaga?tttttgtact?tggagcagga?acaatgggtg?ctggtatcgt?tcaagcattc?????60

gctcaaaaag?gttgtgaagt?aattgtaaga?gacataaagg?aagaatttgt?tgacagagga????120

atagctggaa?tcactaaagg?attagaaaag?caagttgcta?aaggaaaaat?gtctgaagaa????180

gataaagaag?ctatactttc?aagaatttca?ggaacaactg?atatgaaatt?agctgctgac????240

tgtgatttag?tagttgaagc?tgcaatcgaa?aacatgaaaa?ttaagaagga?aatcttcgct????300

gaattagatg?gaatttgtaa?gccagaagcg?attttagctt?caaacacttc?atctttatca????360

attactgaag?ttgcttcagc?tacaaagaga?cctgataaag?ttatcggaat?gcatttcttt????420

aatccagctc?cagtaatgaa?gcttgttgaa?attattaaag?gaatagctac?ttctcaagaa????480

acttttgatg?ctgttaagga?attatcagtt?gctattggaa?aagaaccagt?agaagttgca????540

gaagctccag?gattcgttgt?aaacagaata?ttaatcccaa?tgattaacga?agcttcattt????600

atcctacaag?aaggaatagc?ttcagttgaa?gatattgata?cagctatgaa?atatggtgct????660

aaccatccaa?tgggaccttt?agctttagga?gatcttattg?gattagacgt?ttgcttagct????720

atcatggatg?ttttattcac?tgaaacaggt?gataacaagt?acagagctag?cagcatatta????780

agaaaatatg?ttagagctgg?atggcttgga?agaaaatcag?gaaaaggatt?ctatgattat????840

tctaaataa????????????????????????????????????????????????????????????849

<210>156

<211>786

<212>DNA

<213>Cb-crt

<400>156

atggaattaa?aaaatgttat?tcttgaaaaa?gaagggcatt?tagctattgt?tacaatcaat?????60

agaccaaagg?cattaaatgc?attgaattca?gaaacactaa?aagatttaaa?tgttgtttta????120

gatgatttag?aagcagacaa?caatgtgtat?gcagttatag?ttacaggtgc?tggtgagaaa????180

tcttttgttg?ctggagcaga?tatttcagaa?atgaaagatc?ttaatgaaga?acaaggtaaa????240

gaatttggta?ttttaggaaa?caatgtcttc?agaagattag?aaaaattgga?taagccagtt????300

atcgcagcta?tatcaggatt?tgctcttggt?ggtggatgtg?aacttgctat?gtcatgtgac????360

ataagaatag?cttcagttaa?agctaaattt?ggtcaaccag?aagcaggact?tggaataact????420

ccaggatttg?gtggaactca?aagattagct?agaattgtag?ggccaggaaa?agctaaagaa????480

ttaatttata?cttgtgacct?tataaatgca?gaagaagctt?atagaatagg?tttagttaat????540

aaagtagttg?aattagaaaa?attgatggaa?gaagcaaaag?caatggctaa?caagattgca????600

gctaatgctc?caaaagcagt?tgcatattgt?aaagatgcta?tagacagagg?aatgcaagtt????660

gatatagatg?cagctatatt?aatagaagca?gaagactttg?gaaagtgctt?tgcaacagaa????720

gatcaaacag?aaggaatgac?tgcgttctta?gaaagaagag?cagaaaagaa?ttttcaaaat????780

aaataa???????????????????????????????????????????????????????????????786

<210>157

<211>1140

<212>DNA

<213>Cb-bcd

<400>157

atgaatttcc?aattaactag?agaacaacaa?ttagtacaac?aaatggttag?agaattcgca?????60

gtaaatgaag?ttaagccaat?agctgctgaa?atcgacgaat?cagaaagatt?ccctatggaa????120

aacgttgaaa?aaatggctaa?gcttaaaatg?atgggtatcc?cattttctaa?agaatttggt????180

ggagcaggcg?gagatgttct?ttcatatata?atatctgtgg?aagaattatc?aaaagtttgt????240

ggtactacag?gagttattct?ttcagcgcat?acatcattat?gtgcatcagt?aattaatgaa????300

aatggaacta?acgaacaaag?agcaaaatat?ttgccagatc?tttgtagtgg?taagaaaatc????360

ggtgctttcg?gattaacaga?accaggcgct?ggtacagatg?ctgcaggaca?acaaacaact????420

gctgtattag?aaggagacca?ttatgtatta?aatggttcaa?aaatcttcat?aacaaatggt????480

ggagttgctg?aaactttcat?aatatttgct?atgacagata?agagtcaagg?aacaaaagga????540

atttctgcat?tcatagtaga?aaagtcattc?ccaggattct?caataggaaa?attagaaaac????600

aagatgggga?tcagagcatc?ttcaactact?gagttagtta?tggaaaactg?tatagtacca???660

aaagaaaacc?tacttagcaa?agaaggtaag?ggatttggta?tagcaatgaa?aactcttgat???720

ggaggaagaa?ttggtatagc?tgctcaagct?ttaggtattg?cagaaggagc?ttttgaagaa???780

gctgttaact?atatgaaaga?aagaaaacaa?tttggtaaac?cattatcagc?attccaagga???840

ttacaatggt?atatagctga?aatggatgtt?aaaatccaag?ctgctaaata?cttagtatac???900

ctagctgcaa?caaagaagca?agctggtgag?ccttactcag?tggatgctgc?aagagctaaa???960

ttatttgcgg?cagatgttgc?aatggaagtt?acaactaaag?cagttcaaat?ctttggtgga??1020

tatggttaca?ctaaggaata?cccagtagaa?agaatgatga?gagatgctaa?aatatgcgaa??1080

atctacgaag?gaacttcaga?agttcaaaag?atggttatcg?caggaagcat?tttaagatag??1140

<210>158

<211>1140

<212>DNA

<213>Cb-etfA

<400>158

atgaatttcc?aattaactag?agaacaacaa?ttagtacaac?aaatggttag?agaattcgca????60

gtaaatgaag?ttaagccaat?agctgctgaa?atcgacgaat?cagaaagatt?ccctatggaa???120

aacgttgaaa?aaatggctaa?gcttaaaatg?atgggtatcc?cattttctaa?agaatttggt???180

ggagcaggcg?gagatgttct?ttcatatata?atatctgtgg?aagaattatc?aaaagtttgt???240

ggtactacag?gagttattct?ttcagcgcat?acatcattat?gtgcatcagt?aattaatgaa???300

aatggaacta?acgaacaaag?agcaaaatat?ttgccagatc?tttgtagtgg?taagaaaatc???360

ggtgctttcg?gattaacaga?accaggcgct?ggtacagatg?ctgcaggaca?acaaacaact???420

gctgtattag?aaggagacca?ttatgtatta?aatggttcaa?aaatcttcat?aacaaatggt???480

ggagttgctg?aaactttcat?aatatttgct?atgacagata?agagtcaagg?aacaaaagga???540

atttctgcat?tcatagtaga?aaagtcattc?ccaggattct?caataggaaa?attagaaaac???600

aagatgggga?tcagagcatc?ttcaactact?gagttagtta?tggaaaactg?tatagtacca???660

aaagaaaacc?tacttagcaa?agaaggtaag?ggatttggta?tagcaatgaa?aactcttgat???720

ggaggaagaa?ttggtatagc?tgctcaagct?ttaggtattg?cagaaggagc?ttttgaagaa???780

gctgttaact?atatgaaaga?aagaaaacaa?tttggtaaac?cattatcagc?attccaagga???840

ttacaatggt?atatagctga?aatggatgtt?aaaatccaag?ctgctaaata?cttagtatac???900

ctagctgcaa?caaagaagca?agctggtgag?ccttactcag?tggatgctgc?aagagctaaa???960

ttatttgcgg?cagatgttgc?aatggaagtt?acaactaaag?cagttcaaat?ctttggtgga??1020

tatggttaca?ctaaggaata?cccagtagaa?agaatgatga?gagatgctaa?aatatgcgaa??1080

atctacgaag?gaacttcaga?agttcaaaag?atggttatcg?caggaagcat?tttaagatag??1140

<210>159

<211>780

<212>DNA

<213>Cb-etfB

<400>159

atgaatatag?tagtttgtgt?aaaacaagtt?ccagatacta?cagcagtaaa?aattgatcct?????60

aaaactggta?cattaataag?agatggtgtt?ccatcaataa?tgaatccaga?ggataaacac????120

gctttagaag?gtgcattaca?attaaaagaa?aaagttggag?gaaaagttac?tgtaataagt????180

atgggacttc?caatggctaa?agcagtatta?agagaagcat?tatgtatggg?agctgatgaa????240

gctgtcctat?taacagatag?agcacttgga?ggagcagata?ctttagcaac?ttcaaaggca????300

cttgcaggag?taatagctaa?gttagattat?gatttggtat?ttgctggaag?acaagcaatt????360

gatggagata?ctgcacaagt?aggaccagaa?atagcagaac?atttaaacat?tccgcaagta????420

acttacgttc?aagacgttaa?agttgaagga?aatacattaa?tagtaaatag?agcactagaa????480

gatggacatc?aagtagtaga?agttaaaact?ccatgtctat?taactgcaat?cgaagaatta????540

aatgaaacta?gatatatgaa?tgttgtagat?atattcgaaa?cttcagatga?tgaaatcaaa????600

gttatgagcg?cagctgatat?agatgtagat?gtagctgaat?tagggcttaa?aggctcacct????660

acaaaggtta?agaagtcaat?gactaaggaa?gttaaaggtg?caggagaaat?cgtaagagaa????720

gcacctaaaa?atgcagcata?ctatgttgta?ggaaaattaa?aagaaaaaca?ctacatctaa????780

<210>160

<211>1167

<212>DNA

<213>Cb-adhA

<400>160

atggcacgtt?ttactttacc?aagagacatt?tatcatggag?aaggagcact?tgaggcactt?????60

aaaactttaa?aaggtaagaa?agctttctta?gtagttggtg?gcggatcaat?gaaaagattt????120

ggatttctta?aacaagttga?agattattta?aaagaagcag?gaatggaagt?agaattattt????180

gaaggtgttg?aaccagatcc?atcagtggaa?acagtaatga?aaggcgcaga?agctatgaga????240

aactttgagc?ctgattggat?agttgcaatg?ggtggaggat?caccaattga?tgctgcaaag????300

gctatgtgga?tattctacga?atacccagat?tttacttttg?aacaagcagt?tgttccattt????360

ggattaccag?accttagaca?aaaagctaag?tttgtagcta?ttccatcaac?aagcggtaca????420

gctacagaag?ttacagcatt?ctcagttatc?acaaattatt?cagaaaaaat?taaatatcct????480

ttagctgatt?ttaacataac?tccagatata?gcaatagttg?atccagcact?tgctcaaact????540

atgccaaaaa?ctttaacagc?tcatactgga?atggatgcat?taactcacgc?tatagaagca????600

tacactgcat?cacttcaatc?aaatttctca?gatccattag?caattaaagc?tgtagaaatg????660

gttcaagaaa?atttaatcaa?atcatttgaa?ggagataaag?aagctagaaa?tctaatgcat????720

gaagctcaat?gtttagctgg?aatggcattt?tctaatgcat?tacttggaat?agttcactca????780

atggctcata?aggttggtgc?tgtattccat?attcctcatg?gatgtgcaaa?tgctatattt????840

ttaccatatg?taattgagta?taacagaaca?aaatgcgaaa?atagatatgg?agatattgcg????900

agagccttaa?aattaaaagg?aaacaatgat?gccgagttaa?ctgattcatt?aattgaatta????960

attaatggat?taaatgataa?gttagagatt?cctcactcaa?tgaaagagta?tggagttact????1020

gaagaagatt?ttaaagctaa?tctttcattt?atcgctcata?acgcagtatt?agatgcatgc????1080

acaggatcaa?atcctagaga?aatagatgat?gctacaatgg?aaaaattatt?tgaatgcaca????1140

tactatggaa?ctaaagttaa?tttgtaa????????????????????????????????????????1167

<210>161

<211>1407

<212>DNA

<213>Cb-aldh

<400>161

atgaataaag?acacactaat?acctacaact?aaagatttaa?aagtaaaaac?aaatggtgaa?????60

aacattaatt?taaagaacta?caaggataat?tcttcatgtt?tcggagtatt?cgaaaatgtt????120

gaaaatgcta?taagcagcgc?tgtacacgca?caaaagatat?tatcccttca?ttatacaaaa????180

gagcaaagag?aaaaaatcat?aactgagata?agaaaggccg?cattacaaaa?taaagaggtc????240

ttggctacaa?tgattctaga?agaaacacat?atgggaagat?atgaggataa?aatattaaaa????300

catgaattgg?tagctaaata?tactcctggt?acagaagatt?taactactac?tgcttggtca????360

ggtgataatg?gtcttacagt?tgtagaaatg?tctccatatg?gtgttatagg?tgcaataact????420

ccttctacga?atccaactga?aactgtaata?tgtaatagca?taggcatgat?agctgctgga????480

aatgctgtag?tatttaacgg?acacccatgc?gctaaaaaat?gtgttgcctt?tgctgttgaa????540

atgataaata?aggcaattat?ttcatgtggc?ggtcctgaaa?atctagtaac?aactataaaa????600

aatccaacta?tggagtctct?agatgcaatt?attaagcatc?cttcaataaa?acttctttgc????660

ggaactgggg?gtccaggaat?ggtaaaaacc?ctcttaaatt?ctggtaagaa?agctataggt????720

gctggtgctg?gaaatccacc?agttattgta?gatgatactg?ctgatataga?aaaggctggt????780

aggagcatca?ttgaaggctg?ttcttttgat?aataatttac?cttgtattgc?agaaaaagaa????840

gtatttgttt?ttgagaatgt?tgcagatgat?ttaatatcta?acatgctaaa?aaataatgct????900

gtaattataa?atgaagatca?agtatcaaaa?ttaatagatt?tagtattaca?aaaaaataat????960

gaaactcaag?aatactttat?aaacaaaaaa?tgggtaggaa?aagatgcaaa?attattctta???1020

gatgaaatag?atgttgagtc?tccttcaaat?gttaaatgca?taatctgcga?agtaaatgca???1080

aatcatccat?ttgttatgac?agaactcatg?atgccaatat?tgccaattgt?aagagttaaa???1140

gatatagatg?aagctattaa?atatgcaaag?atagcagaac?aaaatagaaa?acatagtgcc???1200

tatatttatt?ctaaaaatat?agacaaccta?aatagatttg?aaagagaaat?agatactact???1260

atttttgtaa?agaatgctaa?atcttttgct?ggtgttggtt?atgaagcaga?aggatttaca???1320

actttcacta?ttgctggatc?tactggtgag?ggaataacct?ctgcaaggaa?ttttacaaga???1380

caaagaagat?gtgtacttgc?cggctaa???????????????????????????????????????1407

<210>162

<211>1179

<212>DNA

<213>Ca-thl

atgaaagaag?ttgtaatagc?tagtgcagta?agaacagcga?ttggatctta?tggaaagtct?????60

cttaaggatg?taccagcagt?agatttagga?gctacagcta?taaaggaagc?agttaaaaaa????120

gcaggaataa?aaccagagga?tgttaatgaa?gtcattttag?gaaatgttct?tcaagcaggt????180

ttaggacaga?atccagcaag?acaggcatct?tttaaagcag?gattaccagt?tgaaattcca????240

gctatgacta?ttaataaggt?ttgtggttca?ggacttagaa?cagttagctt?agcagcacaa????300

attataaaag?caggagatgc?tgacgtaata?atagcaggtg?gtatggaaaa?tatgtctaga????360

gctccttact?tagcgaataa?cgctagatgg?ggatatagaa?tgggaaacgc?taaatttgtt????420

gatgaaatga?tcactgacgg?attgtgggat?gcatttaatg?attaccacat?gggaataaca????480

gcagaaaaca?tagctgagag?atggaacatt?tcaagagaag?aacaagatga?gtttgctctt????540

gcatcacaaa?aaaaagctga?agaagctata?aaatcaggtc?aatttaaaga?tgaaatagtt????600

cctgtagtaa?ttaaaggcag?aaagggagaa?actgtagttg?atacagatga?gcaccctaga????660

tttggatcaa?ctatagaagg?acttgcaaaa?ttaaaacctg?ccttcaaaaa?agatggaaca????720

gttacagctg?gtaatgcatc?aggattaaat?gactgtgcag?cagtacttgt?aatcatgagt????780

gcagaaaaag?ctaaagagct?tggagtaaaa?ccacttgcta?agatagtttc?ttatggttca????840

gcaggagttg?acccagcaat?aatgggatat?ggacctttct?atgcaacaaa?agcagctatt????900

gaaaaagcag?gttggacagt?tgatgaatta?gatttaatag?aatcaaatga?agcttttgca????960

gctcaaagtt?tagcagtagc?aaaagattta?aaatttgata?tgaataaagt?aaatgtaaat???1020

ggaggagcta?ttgcccttgg?tcatccaatt?ggagcatcag?gtgcaagaat?actcgttact???1080

cttgtacacg?caatgcaaaa?aagagatgca?aaaaaaggct?tagcaacttt?atgtataggt???1140

ggcggacaag?gaacagcaat?attgctagaa?aagtgctag??????????????????????????1179

<210>163

<211>849

<212>DNA

<213>Ca-hbd

<400>163

atgaaaaagg?tatgtgttat?aggtgcaggt?actatgggtt?caggaattgc?tcaggcattt?????60

gcagctaaag?gatttgaagt?agtattaaga?gatattaaag?atgaatttgt?tgatagagga????120

ttagatttta?tcaataaaaa?tctttctaaa?ttagttaaaa?aaggaaagat?agaagaagct????180

actaaagttg?aaatcttaac?tagaatttcc?ggaacagttg?accttaatat?ggcagctgat????240

tgcgatttag?ttatagaagc?agctgttgaa?agaatggata?ttaaaaagca?gatttttgct????300

gacttagaca?atatatgcaa?gccagaaaca?attcttgcat?caaatacatc?atcactttca????360

ataacagaag?tggcatcagc?aactaaaaga?cctgataagg?ttataggtat?gcatttcttt????420

aatccagctc?ctgttatgaa?gcttgtagag?gtaataagag?gaatagctac?atcacaagaa????480

acttttgatg?cagttaaaga?gacatctata?gcaataggaa?aagatcctgt?agaagtagca????540

gaagcaccag?gatttgttgt?aaatagaata?ttaataccaa?tgattaatga?agcagttggt????600

atattagcag?aaggaatagc?ttcagtagaa?gacatagata?aagctatgaa?acttggagct????660

aatcacccaa?tgggaccatt?agaattaggt?gattttatag?gtcttgatat?atgtcttgct????720

ataatggatg?ttttatactc?agaaactgga?gattctaagt?atagaccaca?tacattactt????780

aagaagtatg?taagagcagg?atggcttgga?agaaaatcag?gaaaaggttt?ctacgattat????840

tcaaaataa????????????????????????????????????????????????????????????849

<210>164

<211>786

<212>DNA

<213>Ca-crt

<400>164

atggaactaa?acaatgtcat?ccttgaaaag?gaaggtaaag?ttgctgtagt?taccattaac?????60

agacctaaag?cattaaatgc?gttaaatagt?gatacactaa?aagaaatgga?ttatgttata????120

ggtgaaattg?aaaatgatag?cgaagtactt?gcagtaattt?taactggagc?aggagaaaaa????180

tcatttgtag?caggagcaga?tatttctgag?atgaaggaaa?tgaataccat?tgaaggtaga????240

aaattcggga?tacttggaaa?taaagtgttt?agaagattag?aacttcttga?aaagcctgta????300

atagcagctg?ttaatggttt?tgctttagga?ggcggatgcg?aaatagctat?gtcttgtgat????360

ataagaatag?cttcaagcaa?cgcaagattt?ggtcaaccag?aagtaggtct?cggaataaca????420

cctggttttg?gtggtacaca?aagactttca?agattagttg?gaatgggcat?ggcaaagcag????480

cttatattta?ctgcacaaaa?tataaaggca?gatgaagcat?taagaatcgg?acttgtaaat????540

aaggtagtag?aacctagtga?attaatgaat?acagcaaaag?aaattgcaaa?caaaattgtg????600

agcaatgctc?cagtagctgt?taagttaagc?aaacaggcta?ttaatagagg?aatgcagtgt????660

gatattgata?ctgctttagc?atttgaatca?gaagcatttg?gagaatgctt?ttcaacagag????720

gatcaaaagg?atgcaatgac?agctttcata?gagaaaagaa?aaattgaagg?cttcaaaaat????780

agatag???????????????????????????????????????????????????????????????786

<210>165

<211>1140

<212>DNA

<213>Ca-bcd

<400>165

atggatttta?atttaacaag?agaacaagaa?ttagtaagac?agatggttag?agaatttgct?????60

gaaaatgaag?ttaaacctat?agcagcagaa?attgatgaaa?cagaaagatt?tccaatggaa????120

aatgtaaaga?aaatgggtca?gtatggtatg?atgggaattc?cattttcaaa?agagtatggt????180

ggcgcaggtg?gagatgtatt?atcttatata?atcgccgttg?aggaattatc?aaaggtttgc????240

ggtactacag?gagttattct?ttcagcacat?acatcacttt?gtgcttcatt?aataaatgaa????300

catggtacag?aagaacaaaa?acaaaaatat?ttagtacctt?tagctaaagg?tgaaaaaata????360

ggtgcttatg?gattgactga?gccaaatgca?ggaacagatt?ctggagcaca?acaaacagta????420

gctgtacttg?aaggagatca?ttatgtaatt?aatggttcaa?aaatattcat?aactaatgga????480

ggagttgcag?atacttttgt?tatatttgca?atgactgaca?gaactaaagg?aacaaaaggt????540

atatcagcat?ttataataga?aaaaggcttc?aaaggtttct?ctattggtaa?agttgaacaa????600

aagcttggaa?taagagcttc?atcaacaact?gaacttgtat?ttgaagatat?gatagtacca????660

gtagaaaaca?tgattggtaa?agaaggaaaa?ggcttcccta?tagcaatgaa?aactcttgat????720

ggaggaagaa?ttggtatagc?agctcaagct?ttaggtatag?ctgaaggtgc?tttcaacgaa????780

gcaagagctt?acatgaagga?gagaaaacaa?tttggaagaa?gccttgacaa?attccaaggt????840

cttgcatgga?tgatggcaga?tatggatgta?gctatagaat?cagctagata?tttagtatat????900

aaagcagcat?atcttaaaca?agcaggactt?ccatacacag?ttgatgctgc?aagagctaag????960

cttcatgctg?caaatgtagc?aatggatgta?acaactaagg?cagtacaatt?atttggtgga???1020

tacggatata?caaaagatta?tccagttgaa?agaatgatga?gagatgctaa?gataactgaa???1080

atatatgaag?gaacttcaga?agttcagaaa?ttagttattt?caggaaaaat?ttttagataa???1140

<210>166

<211>1011

<212>DNA

<213>Ca-etfA

<400>166

atgaataaag?cagattacaa?gggcgtatgg?gtgtttgctg?aacaaagaga?cggagaatta?????60

caaaaggtat?cattggaatt?attaggtaaa?ggtaaggaaa?tggctgagaa?attaggcgtt????120

gaattaacag?ctgttttact?tggacataat?actgaaaaaa?tgtcaaagga?tttattatct????180

catggagcag?ataaggtttt?agcagcagat?aatgaacttt?tagcacattt?ttcaacagat????240

ggatatgcta?aagttatatg?tgatttagtt?aatgaaagaa?agccagaaat?attattcata????300

ggagctactt?tcataggaag?agatttagga?ccaagaatag?cagcaagact?ttctactggt????360

ttaactgctg?attgtacatc?acttgacata?gatgtagaaa?atagagattt?attggctaca????420

agaccagcgt?ttggtggaaa?tttgatagct?acaatagttt?gttcagacca?cagaccacaa????480

atggctacag?taagacctgg?tgtgtttgaa?aaattacctg?ttaatgatgc?aaatgtttct????540

gatgataaaa?tagaaaaagt?tgcaattaaa?ttaacagcat?cagacataag?aacaaaagtt????600

tcaaaagttg?ttaagcttgc?taaagatatt?gcagatatcg?gagaagctaa?ggtattagtt????660

gctggtggta?gaggagttgg?aagcaaagaa?aactttgaaa?aacttgaaga?gttagcaagt????720

ttacttggtg?gaacaatagc?cgcttcaaga?gcagcaatag?aaaaagaatg?ggttgataag????780

gaccttcaag?taggtcaaac?tggtaaaact?gtaagaccaa?ctctttatat?tgcatgtggt????840

atatcaggag?ctatccagca?tttagcaggt?atgcaagatt?cagattacat?aattgctata????900

aataaagatg?tagaagcccc?aataatgaag?gtagcagatt?tggctatagt?tggtgatgta????960

aataaagttg?taccagaatt?aatagctcaa?gttaaagctg?ctaataatta?a????????????1011

<210>167

<211>780

<212>DNA

<213>Ca-etfB

<400>167

atgaatatag?ttgtttgttt?aaaacaagtt?ccagatacag?cggaagttag?aatagatcca?????60

gttaagggaa?cacttataag?agaaggagtt?ccatcaataa?taaatccaga?tgataaaaac????120

gcacttgagg?aagctttagt?attaaaagat?aattatggtg?cacatgtaac?agttataagt????180

atgggacctc?cacaagctaa?aaatgcttta?gtagaagctt?tggctatggg?tgctgatgaa????240

gctgtacttt?taacagatag?agcatttgga?ggagcagata?cacttgcgac?ttcacataca????300

attgcagcag?gaattaagaa?gctaaaatat?gatatagttt?ttgctggaag?gcaggctata????360

gatggagata?cagctcaggt?tggaccagaa?atagctgagc?atcttggaat?acctcaagta????420

acttatgttg?agaaagttga?agttgatgga?gatactttaa?agattagaaa?agcttgggaa????480

gatggatatg?aagttgttga?agttaagaca?ccagttcttt?taacagcaat?taaagaatta????540

aatgttccaa?gatatatgag?tgtagaaaaa?atattcggag?catttgataa?agaagtaaaa????600

atgtggactg?ccgatgatat?agatgtagat?aaggctaatt?taggtcttaa?aggttcacca????660

actaaagtta?agaagtcatc?aactaaagaa?gttaaaggac?agggagaagt?tattgataag????720

cctgttaagg?aagcagctgc?atatgttgtc?tcaaaattaa?aagaagaaca?ctatatttaa????780

<210>168

<211>2577

<212>DNA

<213>Ca-adhE2

<400>168

atgaaagtta?caaatcaaaa?agaactaaaa?caaaagctaa?atgaattgag?agaagcgcaa?????60

aagaagtttg?caacctatac?tcaagagcaa?gttgataaaa?tttttaaaca?atgtgccata????120

gccgcagcta?aagaaagaat?aaacttagct?aaattagcag?tagaagaaac?aggaataggt????180

cttgtagaag?ataaaattat?aaaaaatcat?tttgcagcag?aatatatata?caataaatat????240

aaaaatgaaa?aaacttgtgg?cataatagac?catgacgatt?ctttaggcat?aacaaaggtt????300

gctgaaccaa?ttggaattgt?tgcagccata?gttcctacta?ctaatccaac?ttccacagca????360

attttcaaat?cattaatttc?tttaaaaaca?agaaacgcaa?tattcttttc?accacatcca????420

cgtgcaaaaa?aatctacaat?tgctgcagca?aaattaattt?tagatgcagc?tgttaaagca????480

ggagcaccta?aaaatataat?aggctggata?gatgagccat?caatagaact?ttctcaagat????540

ttgatgagtg?aagctgatat?aatattagca?acaggaggtc?cttcaatggt?taaagcggcc????600

tattcatctg?gaaaacctgc?aattggtgtt?ggagcaggaa?atacaccagc?aataatagat????660

gagagtgcag?atatagatat?ggcagtaagc?tccataattt?tatcaaagac?ttatgacaat????720

ggagtaatat?gcgcttctga?acaatcaata?ttagttatga?attcaatata?cgaaaaagtt????780

aaagaggaat?ttgtaaaacg?aggatcatat?atactcaatc?aaaatgaaat?agctaaaata????840

aaagaaacta?tgtttaaaaa?tggagctatt?aatgctgaca?tagttggaaa?atctgcttat????900

ataattgcta?aaatggcagg?aattgaagtt?cctcaaacta?caaagatact?tataggcgaa????960

gtacaatctg?ttgaaaaaag?cgagctgttc?tcacatgaaa?aactatcacc?agtacttgca????1020

atgtataaag?ttaaggattt?tgatgaagct?ctaaaaaagg?cacaaaggct?aatagaatta????1080

ggtggaagtg?gacacacgtc?atctttatat?atagattcac?aaaacaataa?ggataaagtt????1140

aaagaatttg?gattagcaat?gaaaacttca?aggacattta?ttaacatgcc?ttcttcacag????1200

ggagcaagcg?gagatttata?caattttgcg?atagcaccat?catttactct?tggatgcggc????1260

acttggggag?gaaactctgt?atcgcaaaat?gtagagccta?aacatttatt?aaatattaaa????1320

agtgttgctg?aaagaaggga?aaatatgctt?tggtttaaag?tgccacaaaa?aatatatttt????1380

aaatatggat?gtcttagatt?tgcattaaaa?gaattaaaag?atatgaataa?gaaaagagcc????1440

tttatagtaa?cagataaaga?tctttttaaa?cttggatatg?ttaataaaat?aacaaaggta????1500

ctagatgaga?tagatattaa?atacagtata?tttacagata?ttaaatctga?tccaactatt????1560

gattcagtaa?aaaaaggtgc?taaagaaatg?cttaactttg?aacctgatac?tataatctct????1620

attggtggtg?gatcgccaat?ggatgcagca?aaggttatgc?acttgttata?tgaatatcca????1680

gaagcagaaa?ttgaaaatct?agctataaac?tttatggata?taagaaagag?aatatgcaat????1740

ttccctaaat?taggtacaaa?ggcgatttca?gtagctattc?ctacaactgc?tggtaccggt????1800

tcagaggcaa?caccttttgc?agttataact?aatgatgaaa?caggaatgaa?atacccttta????1860

acttcttatg?aattgacccc?aaacatggca?ataatagata?ctgaattaat?gttaaatatg????1920

cctagaaaat?taacagcagc?aactggaata?gatgcattag?ttcatgctat?agaagcatat????1980

gtttcggtta?tggctacgga?ttatactgat?gaattagcct?taagagcaat?aaaaatgata????2040

tttaaatatt?tgcctagagc?ctataaaaat?gggactaacg?acattgaagc?aagagaaaaa????2100

atggcacatg?cctctaatat?tgcggggatg?gcatttgcaa?atgctttctt?aggtgtatgc????2160

cattcaatgg?ctcataaact?tggggcaatg?catcacgttc?cacatggaat?tgcttgtgct????2220

gtattaatag?aagaagttat?taaatataac?gctacagact?gtccaacaaa?gcaaacagca????2280

ttccctcaat?ataaatctcc?taatgctaag?agaaaatatg?ctgaaattgc?agagtatttg????2340

aatttaaagg?gtactagcga?taccgaaaag?gtaacagcct?taatagaagc?tatttcaaag????2400

ttaaagatag?atttgagtat?tccacaaaat?ataagtgccg?ctggaataaa?taaaaaagat????2460

ttttataata?cgctagataa?aatgtcagag?cttgcttttg?atgaccaatg?tacaacagct????2520

aatcctaggt?atccacttat?aagtgaactt?aaggatatct?atataaaatc?attttaa???????2577

<210>169

<211>2589

<212>DNA

<213>Ca-aad

<400>169

atgaaagtca?caacagtaaa?ggaattagat?gaaaaactca?aggtaattaa?agaagctcaa?????60

aaaaaattct?cttgttactc?gcaagaaatg?gttgatgaaa?tctttagaaa?tgcagcaatg????120

gcagcaatcg?acgcaaggat?agagctagca?aaagcagctg?ttttggaaac?cggtatgggc????180

ttagttgaag?acaaggttat?aaaaaatcat?tttgcaggcg?aatacatcta?taacaaatat????240

aaggatgaaa?aaacctgcgg?tataattgaa?cgaaatgaac?cctacggaat?tacaaaaata?????300

gcagaaccta?taggagttgt?agctgctata?atccctgtaa?caaaccccac?atcaacaaca?????360

atatttaaat?ccttaatatc?ccttaaaact?agaaatggaa?ttttcttttc?gcctcaccca?????420

agggcaaaaa?aatccacaat?actagcagct?aaaacaatac?ttgatgcagc?cgttaagagt?????480

ggtgccccgg?aaaatataat?aggttggata?gatgaacctt?caattgaact?aactcaatat?????540

ttaatgcaaa?aagcagatat?aacccttgca?actggtggtc?cctcactagt?taaatctgct?????600

tattcttccg?gaaaaccagc?aataggtgtt?ggtccgggta?acaccccagt?aataattgat?????660

gaatctgctc?atataaaaat?ggcagtaagt?tcaattatat?tatccaaaac?ctatgataat?????720

ggtgttatat?gtgcttctga?acaatctgta?atagtcttaa?aatccatata?taacaaggta?????780

aaagatgagt?tccaagaaag?aggagcttat?ataataaaga?aaaacgaatt?ggataaagtc?????840

cgtgaagtga?tttttaaaga?tggatccgta?aaccctaaaa?tagtcggaca?gtcagcttat?????900

actatagcag?ctatggctgg?cataaaagta?cctaaaacca?caagaatatt?aataggagaa?????960

gttacctcct?taggtgaaga?agaacctttt?gcccacgaaa?aactatctcc?tgttttggct????1020

atgtatgagg?ctgacaattt?tgatgatgct?ttaaaaaaag?cagtaactct?aataaactta????1080

ggaggcctcg?gccatacctc?aggaatatat?gcagatgaaa?taaaagcacg?agataaaata????1140

gatagattta?gtagtgccat?gaaaaccgta?agaacctttg?taaatatccc?aacctcacaa????1200

ggtgcaagtg?gagatctata?taattttaga?ataccacctt?ctttcacgct?tggctgcgga????1260

ttttggggag?gaaattctgt?ttccgagaat?gttggtccaa?aacatctttt?gaatattaaa????1320

accgtagctg?aaaggagaga?aaacatgctt?tggtttagag?ttccacataa?agtatatttt????1380

aagttcggtt?gtcttcaatt?tgctttaaaa?gatttaaaag?atctaaagaa?aaaaagagcc????1440

tttatagtta?ctgatagtga?cccctataat?ttaaactatg?ttgattcaat?aataaaaata????1500

cttgagcacc?tagatattga?ttttaaagta?tttaataagg?ttggaagaga?agctgatctt????1560

aaaaccataa?aaaaagcaac?tgaagaaatg?tcctccttta?tgccagacac?tataatagct????1620

ttaggtggta?cccctgaaat?gagctctgca?aagctaatgt?gggtactata?tgaacatcca????1680

gaagtaaaat?ttgaagatct?tgcaataaaa?tttatggaca?taagaaagag?aatatatact????1740

ttcccaaaac?tcggtaaaaa?ggctatgtta?gttgcaatta?caacttctgc?tggttccggt????1800

tctgaggtta?ctccttttgc?tttagtaact?gacaataaca?ctggaaataa?gtacatgtta????1860

gcagattatg?aaatgacacc?aaatatggca?attgtagatg?cagaacttat?gatgaaaatg????1920

ccaaagggat?taaccgctta?ttcaggtata?gatgcactag?taaatagtat?agaagcatac????1980

acatccgtat?atgcttcaga?atacacaaac?ggactagcac?tagaggcaat?acgattaata????2040

tttaaatatt?tgcctgaggc?ttacaaaaac?ggaagaacca?atgaaaaagc?aagagagaaa????2100

atggctcacg?cttcaactat?ggcaggtatg?gcatccgcta?atgcatttct?aggtctatgt????2160

cattccatgg?caataaaatt?aagttcagaa?cacaatattc?ctagtggcat?tgccaatgca????2220

ttactaatag?aagaagtaat?aaaatttaac?gcagttgata?atcctgtaaa?acaagcccct????2280

tgcccacaat?ataagtatcc?aaacaccata?tttagatatg?ctcgaattgc?agattatata????2340

aagcttggag?gaaatactga?tgaggaaaag?gtagatctct?taattaacaa?aatacatgaa????2400

ctaaaaaaag?ctttaaatat?accaacttca?ataaaggatg?caggtgtttt?ggaggaaaac????2460

ttctattcct?cccttgatag?aatatctgaa?cttgcactag?atgatcaatg?cacaggcgct????2520

aatcctagat?ttcctcttac?aagtgagata?aaagaaatgt?atataaattg?ttttaaaaaa????2580

caaccttaa????????????????????????????????????????????????????????????2589

<210>170

<211>1167

<212>DNA

<213>Ca-bdhA

<400>170

atgctaagtt?ttgattattc?aataccaact?aaagtttttt?ttggaaaagg?aaaaatagac?????60

gtaattggag?aagaaattaa?gaaatatggc?tcaagagtgc?ttatagttta?tggcggagga????120

agtataaaaa?ggaacggtat?atatgataga?gcaacagcta?tattaaaaga?aaacaatata????180

gctttctatg?aactttcagg?agtagagcca?aatcctagga?taacaacagt?aaaaaaaggc????240

atagaaatat?gtagagaaaa?taatgtggat?ttagtattag?caataggggg?aggaagtgca????300

atagactgtt?ctaaggtaat?tgcagctgga?gtttattatg?atggcgatac?atgggacatg????360

gttaaagatc?catctaaaat?aactaaagtt?cttccaattg?caagtatact?tactctttca????420

gcaacagggt?ctgaaatgga?tcaaattgca?gtaatttcaa?atatggagac?taatgaaaag????480

cttggagtag?gacatgatga?tatgagacct?aaattttcag?tgttagatcc?tacatatact????540

tttacagtac?ctaaaaatca?aacagcagcg?ggaacagctg?acattatgag?tcacaccttt????600

gaatcttact?ttagtggtgt?tgaaggtgct?tatgtgcagg?acggtatagc?agaagcaatc????660

ttaagaacat?gtataaagta?tggaaaaata?gcaatggaga?agactgatga?ttacgaggct????720

agagctaatt?tgatgtgggc?ttcaagttta?gctataaatg?gtctattatc?acttggtaag????780

gatagaaaat?ggagttgtca?tcctatggaa?cacgagttaa?gtgcatatta?tgatataaca????840

catggtgtag?gacttgcaat?tttaacacct?aattggatgg?aatatattct?aaatgacgat????900

acacttcata?aatttgtttc?ttatggaata?aatgtttggg?gaatagacaa?gaacaaagat????960

aactatgaaa?tagcacgaga?ggctattaaa?aatacgagag?aatactttaa?ttcattgggt???1020

attccttcaa?agcttagaga?agttggaata?ggaaaagata?aactagaact?aatggcaaag???1080

caagctgtta?gaaattctgg?aggaacaata?ggaagtttaa?gaccaataaa?tgcagaggat???1140

gttcttgaga?tatttaaaaa?atcttat???????????????????????????????????????1167

<210>171

<211>1173

<212>DNA

<213>Ca-bdhB

<400>171

atggttgatt?tcgaatattc?aataccaact?agaatttttt?tcggtaaaga?taagataaat?????60

gtacttggaa?gagagcttaa?aaaatatggt?tctaaagtgc?ttatagttta?tggtggagga???120

agtataaaga?gaaatggaat?atatgataaa?gctgtaagta?tacttgaaaa?aaacagtatt???180

aaattttatg?aacttgcagg?agtagagcca?aatccaagag?taactacagt?tgaaaaagga???240

gttaaaatat?gtagagaaaa?tggagttgaa?gtagtactag?ctataggtgg?aggaagtgca???300

atagattgcg?caaaggttat?agcagcagca?tgtgaatatg?atggaaatcc?atgggatatt???360

gtgttagatg?gctcaaaaat?aaaaagggtg?cttcctatag?ctagtatatt?aaccattgct???420

gcaacaggat?cagaaatgga?tacgtgggca?gtaataaata?atatggatac?aaacgaaaaa???480

ctaattgcgg?cacatccaga?tatggctcct?aagttttcta?tattagatcc?aacgtatacg???540

tataccgtac?ctaccaatca?aacagcagca?ggaacagctg?atattatgag?tcatatattt???600

gaggtgtatt?ttagtaatac?aaaaacagca?tatttgcagg?atagaatggc?agaagcgtta???660

ttaagaactt?gtattaaata?tggaggaata?gctcttgaga?agccggatga?ttatgaggca???720

agagccaatc?taatgtgggc?ttcaagtctt?gcgataaatg?gacttttaac?atatggtaaa???780

gacactaatt?ggagtgtaca?cttaatggaa?catgaattaa?gtgcttatta?cgacataaca???840

cacggcgtag?ggcttgcaat?tttaacacct?aattggatgg?agtatatttt?aaataatgat???900

acagtgtaca?agtttgttga?atatggtgta?aatgtttggg?gaatagacaa?agaaaaaaat???960

cactatgaca?tagcacatca?agcaatacaa?aaaacaagag?attactttgt?aaatgtacta??1020

ggtttaccat?ctagactgag?agatgttgga?attgaagaag?aaaaattgga?cataatggca??1080

aaggaatcag?taaagcttac?aggaggaacc?ataggaaacc?taagaccagt?aaacgcctcc??1140

gaagtcctac?aaatattcaa?aaaatctgtg?taa???????????????????????????????1173

<210>172

<211>48

<212>DNA

＜213〉AU1 label

<400>172

atggatactt?atagatacat?tggtggtgac?acatacaggt?atatcggt?????????????????48

<210>173

<211>33

<212>DNA

＜213〉HA label

<400>173

atgtacccat?acgatgttcc?tgactatgcg?ggt?????????????????????????????????33

<210>174

<211>33

<212>DNA

＜213〉myc label

<400>174

atggaacaaa?aactcatctc?agaagaagat?ggt?????????????????????????????????33

<210>175

<211>403

<212>DNA

＜213〉TEF1 promotor

<400>175

catagcttca?aaatgtttct?actccttttt?tactcttcca?gattttctcg?gactccgcgc???60

atcgccgtac?cacttcaaaa?cacccaagca?cagcatacta?aatttcccct?ctttcttcct??120

ctagggtgtc?gttaattacc?cgtactaaag?gtttggaaaa?gaaaaaagag?accgcctcgt??180

ttctttttct?tcgtcgaaaa?aggcaataaa?aatttttatc?acgtttcttt?ttcttgaaaa??240

tttttttttt?gatttttttc?tctttcgatg?acctcccatt?gatatttaag?ttaataaacg??300

gtcttcaatt?tctcaagttt?cagtttcatt?tttcttgttc?tattacaact?ttttttactt??360

cttgctcatt?agaaagaaag?catagcaatc?taatctaagt?ttt????????????????????403

<210>176

<211>650

<212>DNA

＜213〉TDH3 promotor

<400>176

agtttatcat?tatcaatact?cgccatttca?aagaatacgt?aaataattaa?tagtagtgat???60

tttcctaact?ttatttagtc?aaaaaattag?ccttttaatt?ctgctgtaac?ccgtacatgc??120

ccaaaatagg?gggcgggtta?cacagaatat?ataacatcgt?aggtgtctgg?gtgaacagtt??180

tattcctggc?atccactaaa?tataatggag?cccgcttttt?aagctggcat?ccagaaaaaa??240

aaagaatccc?agcaccaaaa?tattgttttc?ttcaccaacc?atcagttcat?aggtccattc??300

tcttagcgca?actacagaga?acaggggcac?aaacaggcaa?aaaacgggca?caacctcaat??360

ggagtgatgc?aacctgcctg?gagtaaatga?tgacacaagg?caattgaccc?acgcatgtat??420

ctatctcatt?ttcttacacc?ttctattacc?ttctgctctc?tctgatttgg?aaaaagctga??480

aaaaaaaggt?tgaaaccagt?tccctgaaat?tattccccta?cttgactaat?aagtatataa??540

agacggtagg?tattgattgt?aattctgtaa?atctatttct?taaacttctt?aaattctact??600

tttatagtta?gtcttttttt?tagttttaaa?acaccagaac?ttagtttcga?????????????650

<210>177

<211>493

<212>DNA

＜213〉MET3 promotor

<400>177

tttagtacta?acagagactt?ttgtcacaac?tacatataag?tgtacaaata?tagtacagat???60

atgacacact?tgtagcgcca?acgcgcatcc?tacggattgc?tgacagaaaa?aaaggtcacg??120

tgaccagaaa?agtcacgtgt?aattttgtaa?ctcaccgcat?tctagcggtc?cctgtcgtgc??180

acactgcact?caacaccata?aaccttagca?acctccaaag?gaaatcaccg?tataacaaag??240

ccacagtttt?acaacttagt?ctcttatgaa?gttacttacc?aatgagaaat?agaggctctt??300

tctcgagaaa?tatgaatatg?gatatatata?tatatatata?tatatatata?tatatatatg??360

taaacttggt?tcttttttag?cttgtgatct?ctagcttggg?tctctctctg?tcgtaacagt??420

tgtgatatcg?tttcttaaca?attgaaaagg?aactaagaaa?gtataataat?aacaagaata??480

aagtataatt?aac?????????????????????????????????????????????????????493

<210>178

<211>461

<212>DNA

＜213〉CUP1 promotor

<400>178

gccgatccca?ttaccgacat?ttgggcgcta?tacgtgcata?tgttcatgta?tgtatctgta???60

tttaaaacac?ttttgtatta?tttttcctca?tatatgtgta?taggtttata?cggatgattt??120

aattattact?tcaccaccct?ttatttcagg?ctgatatctt?agccttgtta?ctagttagaa??180

aaagacattt?ttgctgtcag?tcactgtcaa?gagattcttt?tgctggcatt?tcttctagaa??240

gcaaaaagag?cgatgcgtct?tttccgctga?accgttccag?caaaaaagac?taccaacgca??300

atatggattg?tcagaatcat?ataaaagaga?agcaaataac?tccttgtctt?gtatcaattg??360

cattataata?tcttcttgtt?agtgcaatat?catatagaag?tcatcgaaat?agatattaag??420

aaaaacaaac?tgtacaatca?atcaatcaat?catcacataa?a??????????????????????461

<210>179

<211>1197

<212>DNA

<213>Ca-ter

<400>179

atgatagtaa?aagcaaagtt?tgtaaaagga?tttatcagag?atgtacatcc?ttatggttgc???60

agaagggaag?tactaaatca?aatagattat?tgtaagaagg?ctattgggtt?taggggacca??120

aagaaggttt?taattgttgg?agcctcatct?gggtttggtc?ttgctactag?aatttcagtt??180

gcatttggag?gtccagaagc?tcacacaatt?ggagtatcct?atgaaacagg?agctacagat??240

agaagaatag?gaacagcggg?atggtataat?aacatatttt?ttaaagaatt?tgctaaaaaa??300

aaaggattag?ttgcaaaaaa?cttcattgag?gatgcctttt?ctaatgaaac?caaagataaa??360

gttattaagt?atataaagga?tgaatttggt?aaaatagatt?tatttgttta?tagtttagct??420

gcgcctagga?gaaaggacta?taaaactgga?aatgtttata?cttcaagaat?aaaaacaatt??480

ttaggagatt?ttgagggacc?gactattgat?gttgaaagag?acgagattac?tttaaaaaag??540

gttagtagtg?ctagcattga?agaaattgaa?gaaactagaa?aggtaatggg?tggagaggat??600

tggcaagagt?ggtgtgaaga?gctgctttat?gaagattgtt?tttcggataa?agcaactacc??660

atagcatact?cgtatatagg?atccccaaga?acctacaaga?tatatagaga?aggtactata??720

ggaatagcta?aaaaggatct?tgaagataag?gctaagctta?taaatgaaaa?acttaacaga??780

gttataggtg?gtagagcctt?tgtgtctgtg?aataaagcat?tagttacaaa?agcaagtgca??840

tatattccaa?cttttcctct?ttatgcagct?attttatata?aggtcatgaa?agaaaaaaat??900

attcatgaaa?attgtattat?gcaaattgag?agaatgtttt?ctgaaaaaat?atattcaaat??960

gaaaaaatac?aatttgatga?caagggaaga?ttaaggatgg?acgatttaga?gcttagaaaa?1020

gacgttcaag?acgaagttga?tagaatatgg?agtaatatta?ctcctgaaaa?ttttaaggaa????1080

ttatctgatt?ataagggata?caaaaaagaa?ttcatgaact?taaacggttt?tgatctagat????1140

ggggttgatt?atagtaaaga?cctggatata?gaattattaa?gaaaattaga?accttaa???????1197

<210>180

<211>1194

<212>DNA

<213>Ah-ter

<400>180

atgatcatta?aaccgaaagt?tcgtggcttc?atttgtacca?ccactcatcc?ggttggctgt?????60

gaagctaatg?tacgccgcca?gatcgcgtat?accaaagcaa?aaggcactat?cgaaaacggc????120

cctaagaaag?tgctggtgat?tggtgcgagc?accggttacg?gtctggcgtc?ccgcattgca????180

gcggcgttcg?gtagcggcgc?cgcgaccctg?ggtgttttct?tcgaaaaagc?gggctccgaa????240

actaaaaccg?cgaccgcagg?ttggtacaac?tctgccgcgt?ttgacaaagc?cgccaaagag????300

gctggcctgt?atgcgaaatc?tattaacggt?gacgcgttca?gcaacgaatg?ccgtgctaaa????360

gtgatcgaac?tgatcaaaca?ggatctgggc?caaattgatc?tggttgttta?ttctctggcc????420

tccccggttc?gtaaactgcc?ggataccggc?gaagttgtgc?gcagcgctct?gaaacctatt????480

ggtgaagtgt?acaccacgac?cgcaattgat?actaataagg?accagattat?caccgcaacc????540

gtcgagccgg?ccaacgagga?agagatccag?aataccatca?ctgtgatggg?cggtcaagac????600

tgggaactgt?ggatggcagc?actgcgcgac?gcaggtgttc?tggcagacgg?tgcaaagagc????660

gtcgcttact?cttacatcgg?cactgacctg?acttggccga?tctactggca?tggcaccctg????720

ggtcgcgcga?aagaggatct?ggatcgcgca?gcggcagcga?tccgcggtga?tctggccggt????780

aagggcggta?ctgcgcacgt?tgccgttctg?aaatccgtgg?tcacccaggc?atcttctgca????840

atcccggtga?tgccgctgta?tatttctatg?gcctttaaaa?tcatgaaaga?gaagggtatc????900

cacgaaggct?gtatggagca?agtggaccgc?atgatgcgta?ctcgcctgta?cgcggcggac????960

atggcactgg?atgaccaggc?gcgtatccgt?atggacgatt?gggaactgcg?tgaagatgtt???1020

cagcagactt?gccgtgatct?gtggccgtcc?attacctccg?aaaacctgtg?cgagctgacc???1080

gattacactg?gttacaaaca?ggaatttctg?cgtctgttcg?gtttcggtct?ggaagaagta???1140

gactacgatg?cagacgttaa?cccggacgtt?aaatttgatg?ttgtcgaact?gtga?????????1194

<210>181

<211>1218

<212>DNA

<213>Eg-ter

<400>181

atggccatgt?tcaccactac?cgccaaggtt?attcagccga?aaatccgtgg?ttttatctgt?????60

acgaccaccc?acccgattgg?ctgtgaaaaa?cgcgtgcagg?aagaaattgc?ttacgcacgt????120

gcacatccac?cgaccagccc?gggtccgaaa?cgtgtcctgg?tcatcggctg?ttccactggc????180

tacggcctgt?ctactcgtat?caccgcagct?ttcggctatc?aggcggctac?tctgggcgtg????240

ttcctggctg?gtccgccgac?taaaggtcgc?ccggctgcgg?ccggttggta?taacaccgta????300

gctttcgaaa?aagcggccct?ggaagccggt?ctgtatgccc?gctccctgaa?cggtgacgct????360

tttgactcta?ctaccaaagc?acgcaccgtg?gaagctatca?aacgtgacct?gggcaccgtt????420

gacctggtgg?tttatagcat?tgcagctccg?aaacgtaccg?atccggctac?cggcgtgctg????480

cacaaagcgt?gtctgaaacc?gatcggtgcg?acctacacca?accgtacggt?aaatactgac????540

aaagctgaag?ttacggacgt?gtccatcgaa?ccggcgagcc?cagaagaaat?tgcagacact????600

gtgaaagtaa?tgggtggcga?agactgggaa?ctgtggattc?aggctctgtc?tgaagccggc????660

gttctggcag?aaggcgcgaa?aaccgtcgca?tactcttata?tcggtccgga?gatgacctgg????720

ccggtgtact?ggtccggcac?cattggtgaa?gccaaaaagg?atgttgaaaa?agccgctaaa????780

cgtattaccc?agcagtacgg?ctgtccggca?tacccggttg?tggcaaaagc?actggtgacg????840

caggcatcct?ccgcgatccc?ggtcgtcccg?ctgtatattt?gtctgctgta?ccgtgtaatg????900

aaagaaaaag?gcactcacga?aggttgcatc?gaacaaatgg?tgcgtctgct?gaccacgaaa????960

ctgtacccgg?aaaacggtgc?cccgatcgtt?gatgaagcgg?gccgtgttcg?tgtggacgat???1020

tgggaaatgg?cagaagacgt?tcagcaagcc?gttaaagacc?tgtggagcca?ggtgagcacg???1080

gcaaacctga?aagatatttc?cgacttcgcc?ggttaccaaa?ccgagttcct?gcgcctgttt???1140

ggttttggta?tcgatggcgt?ggactatgac?cagccggttg?acgtagaggc?agacctgccg???1200

agcgcagctc?agcagtaa?????????????????????????????????????????????????1218

<210>182

<211>1344

<212>DNA

<213>Sc-ccr

<400>182

atgaccgtga?aagacattct?ggacgctatt?caatctaaag?acgccacttc?cgcggatttc?????60

gcagctctgc?aactgccgga?gtcctaccgt?gccatcaccg?ttcacaaaga?tgaaactgaa????120

atgttcgcgg?gtctggaaac?tcgtgacaaa?gatccacgta?aatccattca?cctggacgaa????180

gttccagtgc?cggaactggg?tccgggcgaa?gccctggtgg?cagttatggc?aagctccgtt????240

aactacaact?ctgtatggac?gtctatcttt?gaaccggtaa?gcaccttcgc?cttcctggaa????300

cgctacggca?aactgtctcc?gctgaccaaa?cgtcatgatc?tgccatacca?catcatcggt????360

tctgacctgg?caggcgtcgt?cctgcgtacc?ggccctggtg?ttaacgcctg?gcagccgggt????420

gacgaagtcg?ttgcccattg?cctgtctgtt?gaactggaat?cccctgatgg?ccatgatgac????480

accatgctgg?acccggagca?gcgtatttgg?ggcttcgaaa?ctaactttgg?tggtctggct????540

gagattgctc?tggtgaagac?taaccagctg?atgccgaaac?caaaacacct?gacttgggaa????600

gaagccgcgg?ctccgggcct?ggtcaacagc?actgcgtatc?gtcagctggt?ttctcgtaac????660

ggtgctgcta?tgaaacaggg?tgataacgtt?ctgatctggg?gcgcgtccgg?tggtctgggc????720

tcttacgcga?cccagttcgc?actggccggt?ggcgcgaatc?cgatctgcgt?tgttagctct????780

ccgcagaaag?ctgaaatttg?tcgttctatg?ggcgcagaag?cgatcattga?tcgcaacgca?????840

gagggctaca?aattttggaa?agacgaacat?acccaggacc?ctaaggaatg?gaagcgtttc?????900

ggcaaacgta?tccgcgaact?gactggtggt?gaagacattg?atatcgtttt?tgaacaccct?????960

ggtcgtgaga?cttttggtgc?gtctgtatac?gttacccgca?agggcggtac?gatcaccacc????1020

tgtgcatcta?cctctggcta?catgcatgag?tatgataacc?gttacctgtg?gatgtccctg????1080

aaacgtatca?tcggctctca?ctttgctaac?tatcgcgaag?cctatgaggc?aaaccgtctg????1140

atcgctaaag?gcaaaattca?tccgactctg?tctaaaacct?attccctgga?ggaaactggc????1200

caggcggcct?acgacgtaca?ccgtaacctg?caccagggca?aagttggcgt?tctgtgcctg????1260

gctccggaag?aaggtctggg?tgttcgtgac?gctgaaatgc?gtgctcagca?cattgacgcg????1320

attaaccgtt?tccgtaatgt?gtga???????????????????????????????????????????1344

<210>183

<211>36

<212>DNA

<213>Gevo-345

<400>183

atgtttgtcg?acatgatagt?aaaagcaaag?tttgta???????????????????????????????36

<210>184

<211>41

<212>DNA

<213>Gevo-346

<400>184

cttaatgcgg?ccgcttaagg?ttctaatttt?cttaataatt?c?????????????????????????41

<210>185

<211>35

<212>DNA

<213>Gevo-343

<400>185

gcttgagtcg?acatgatcat?taaaccgaaa?gttcg????????????????????????????????35

<210>186

<211>37

<212>DNA

<213>Gevo-344

<400>186

atttaaggat?cctcacagtt?cgacaacatc?aaattta??????????????????????????????37

<210>187

<211>32

<212>DNA

<213>Gevo-347

<400>187

catcacgtcg?acatggccat?gt?tcaccact?ac??????????????????????????????????32

<210>188

<211>31

<212>DNA

<213>Gevo-348

<400>188

ctcgcgggat?ccttactgct?gagctgcgct?c???????????????????????????????31

<210>189

<211>33

<212>DNA

<213>Gevo-341

<400>189

gtcttagtcg?acatgaccgt?gaaagacatt?ctg?????????????????????????????33

<210>190

<211>34

<212>DNA

<213>Gevo-342

<400>190

attggcggat?cctcacacat?tacggaaacg?gtta????????????????????????????34

Claims (64)

1. metabolic engineering yeast, its can the metabolism carbon source to generate propyl carbinol, at least a approach is configured to generate the kytoplasm acetyl that another kytoplasm acetyl-the CoA amount increases-CoA amount that generates with respect to wild-type yeast, and at least a heterologous gene is encoded and expression can utilize NADH acetyl-CoA to be changed at least a enzyme of the pathways metabolism of propyl carbinol.

2. the yeast of claim 1, wherein said at least a heterologous gene is encoded individually and is expressed and can utilize NADH acetyl-CoA to be changed at least a enzyme of the pathways metabolism of propyl carbinol.

3. the yeast of claim 1 is encoded also to express to the wherein said at least a heterologous gene and at least a unartificial yeast assortment of genes and can be utilized NADH acetyl-CoA to be changed at least a enzyme of the pathways metabolism of propyl carbinol.

4. the yeast of claim 1, wherein said yeast are crossed the expression pyruvic carboxylase and are generated to improve kytoplasm acetyl-CoA.

5. the yeast of claim 4, wherein said pyruvic carboxylase is coded by genes of brewing yeast PDC1.

6. the yeast of claim 4, wherein said pyruvic carboxylase is by at least a coded among genes of brewing yeast PDC1, PDC5 and the PDC6.

7. the yeast of claim 1, wherein said yeast are crossed the expression aldehyde dehydrogenase and are generated to improve kytoplasm acetyl-CoA.

8. the yeast of claim 7, wherein said aldehyde dehydrogenase is coded by genes of brewing yeast ALD6.

9. the yeast of claim 7, wherein said aldehyde dehydrogenase is coded by Kluyveromyces lactis gene A LD6.

10. the yeast of claim 1, wherein said yeast are crossed the expression Acetyl-CoA synthetase and are generated to improve kytoplasm acetyl-CoA.

11. the yeast of claim 10, wherein said Acetyl-CoA synthetase is by at least a coded among genes of brewing yeast ACS1 and the genes of brewing yeast ACS2.

12. the yeast of claim 10, wherein said Acetyl-CoA synthetase is by at least a coded among Kluyveromyces lactis gene A CS1 and the Kluyveromyces lactis gene A CS2.

13. the yeast of claim 1, wherein said yeast are crossed expression aldehyde dehydrogenase and Acetyl-CoA synthetase, and the two generates to improve kytoplasm acetyl-CoA.

14. the yeast of claim 13, wherein said aldehyde dehydrogenase is coded by genes of brewing yeast ALD6, and described Acetyl-CoA synthetase is by at least a coded among genes of brewing yeast ACS1 and the genes of brewing yeast ACS2.

15. the yeast of claim 13, wherein said aldehyde dehydrogenase is coded by Kluyveromyces lactis gene A LD6, and described Acetyl-CoA synthetase is by at least a coded among Kluyveromyces lactis gene A CS1 and the Kluyveromyces lactis gene A CS2.

16. the yeast of claim 13, wherein said yeast are crossed the expression pyruvic carboxylase and are generated to improve kytoplasm acetyl-CoA.

17. the yeast of claim 16, wherein said pyruvic carboxylase is by at least a coded among PDC1, PDC5 and the PDC6, aldehyde dehydrogenase is coded by genes of brewing yeast ALD6, and described Acetyl-CoA synthetase is by at least a coded among genes of brewing yeast ACS1 and the genes of brewing yeast ACS2.

18. the yeast of claim 16, wherein said pyruvic carboxylase is coded by Kluyveromyces lactis gene PDC1, aldehyde dehydrogenase is coded by Kluyveromyces lactis gene A LD6, and described Acetyl-CoA synthetase is by at least a coded among Kluyveromyces lactis gene A CS1 and the Kluyveromyces lactis gene A CS2.

19. the yeast of claim 1, wherein said yeast are crossed the expression pyruvic oxidase and are generated to improve kytoplasm acetyl-CoA.

20. the yeast of claim 19, wherein said yeast are crossed expression and are generated to improve kytoplasm acetyl-CoA by bacillus coli gene aceE, aceF, the coded pyruvic oxidase of lpdA.

21. the yeast of claim 20, wherein the PDC activity be reduce with eliminate one of.

22. the yeast of claim 19, wherein said yeast are crossed expression and are generated to improve kytoplasm acetyl-CoA by the coded pyruvic oxidase of genes of brewing yeast PDA1, the PDB1, PDX1, LAT1, the LPD1 that have deleted the terminal Mitochondrially targeted signal of N-.

23. the yeast of claim 22, wherein the PDC activity be reduce with eliminate one of.

24. the yeast of claim 23, wherein said yeast are (1) genotype pdc2 Δs, and the yeast saccharomyces cerevisiae of one of (2) genotype pdc1 Δ, genotype pdc5 Δ and genotype pdc6 Δ.

25. the yeast of claim 23, wherein said yeast are the Kluyveromyces lactis of genotype pdc1 Δ.

26. the yeast of claim 1, wherein said yeast are crossed expression pyruvic acid formic acid lyase and hydrogenlyase, and the two generates to improve kytoplasm acetyl-CoA.

27. the yeast of claim 26, wherein said yeast are crossed expression by bacillus coli gene pflA and the coded pyruvic acid formic acid lyase of bacillus coli gene pflB, and generate to improve kytoplasm acetyl-CoA with Candida boidinii gene FDH1 combination.

28. the yeast of claim 27, wherein the PDC activity be reduce with eliminate one of.

29. the yeast of claim 27, wherein said yeast are (1) genotype pdc2 Δs, and 2) yeast saccharomyces cerevisiae of one of genotype pdc1 Δ, genotype pdc5 Δ and genotype pdc6 Δ.

30. the yeast of claim 27, wherein said yeast are the Kluyveromyces lactis of genotype pdc1 Δ.

31. the yeast of claim 1, at least a molecular evolution that carried out is to strengthen by its coded proteinic enzymic activity in the wherein said at least a heterologous gene.

32. the yeast of claim 1, the gene of wherein at least a other coding alcoholdehydrogenase is inactivated, and makes active fully the reduction to generate with respect to wild-type of alcoholdehydrogenase improve kytoplasm acetyl-CoA and generate.

33. the yeast of claim 32, wherein said yeast is a yeast saccharomyces cerevisiae, and described alcoholdehydrogenase is coded by ADH1.

34. the yeast of claim 32, wherein said yeast is a Kluyveromyces lactis, and its described alcoholdehydrogenase is coded by ADH1.

35. the yeast of claim 32, wherein said yeast is a yeast saccharomyces cerevisiae, and described alcoholdehydrogenase is coded by ADH1, ADH2, ADH3 and ADH4.

36. the yeast of claim 32, wherein said yeast is a Kluyveromyces lactis, and described alcoholdehydrogenase is coded by ADHI, ADHII, ADHIII and ADHIV.

37. the yeast of claim 1, wherein said yeast are from the species with one of subordinate: yeast belong, moral restrain yeast belong, Pichia, Hansenula, the mould genus of Western alpine yarrow, Aspergillus, genus kluyveromyces, pipe capsule yeast belong, Schizosaccharomyces, mycocandida, Trichosporon, Yamadazyma, spore torulopsis and genera cryptococcus are arranged.

38. the yeast of claim 1, wherein said approach provides equilibrated NADH to generate when generating propyl carbinol and consumes in the metabolism carbon source.

39. a method of producing propyl carbinol, this method comprises:

(a) provide following metabolic engineering yeast, its can the metabolism carbon source to generate propyl carbinol, at least a approach is configured to generate the kytoplasm acetyl that another kytoplasm acetyl-the CoA amount increases-CoA amount that generates with respect to wild-type yeast, and at least a heterologous gene is encoded and expression can utilize NADH acetyl-CoA to be changed at least a enzyme of the pathways metabolism of propyl carbinol; And

(b) cultivate this metabolic engineering yeast, the time of cultivation and condition are in order to generate propyl carbinol.

40. a method of using yeast to produce propyl carbinol, this method comprises:

(a) the metabolic engineering yeast generates to improve kytoplasm acetyl-CoA;

(b) the metabolic engineering yeast to be to express the pathways metabolism that carbon source is changed into propyl carbinol, wherein this approach need at least a for this yeast non-natural enzyme, wherein step (a) and (b) can be with arbitrary order enforcement; And

(c) cultivate this yeast, the time of cultivation and condition are in order to generate the propyl carbinol of recyclable amount.

41. a method of using yeast to produce propyl carbinol, this method comprises:

(a) cultivate the metabolic engineering yeast, the time of cultivation and condition are in order to generate the yeast cell biomass but do not activate propyl carbinol and generate; And

(b) change culture condition in another section period, the time of cultivation and condition are in order to generate the propyl carbinol of recyclable amount.

42. a metabolic engineering yeast, it can the metabolism carbon source also generate acetyl-CoA amount of measuring increase with respect to kytoplasm acetyl-CoA that wild-type yeast generated.

43. the yeast of claim 42, wherein said yeast are crossed expression pyruvic carboxylase, aldehyde dehydrogenase and Acetyl-CoA synthetase and are generated to improve kytoplasm acetyl-CoA.

44. the yeast of claim 42, wherein said pyruvic carboxylase is by at least a coded among genes of brewing yeast PDC1, PDC5 and the PDC6, aldehyde dehydrogenase is coded by genes of brewing yeast ALD6, and Acetyl-CoA synthetase is by at least a coded among genes of brewing yeast ACS1 and the ACS2.

45. the yeast of claim 44, the deactivation of wherein said alcoholdehydrogenase by deletion genes of brewing yeast ADH1.

46. the yeast of claim 42, wherein said yeast is a genus kluyveromyces, described pyruvic carboxylase is coded by Kluyveromyces lactis gene KIPDC1, aldehyde dehydrogenase is coded by Kluyveromyces lactis gene KIALD6, and Acetyl-CoA synthetase is by at least a coded among Kluyveromyces lactis gene KIACS1 and the KIACS2.

47. the yeast of claim 46, the deactivation of wherein said alcoholdehydrogenase by deletion Kluyveromyces lactis gene A DH1.

48. the yeast of claim 42, wherein said yeast are crossed the expression pyruvic oxidase and are generated to improve kytoplasm acetyl-CoA.

49. the yeast of claim 48, wherein said yeast are crossed expression and are generated to improve kytoplasm acetyl-CoA by bacillus coli gene aceE, bacillus coli gene aceF and the coded pyruvic oxidase of bacillus coli gene lpdA.

50. the yeast of claim 49, wherein the PDC activity be reduce with eliminate one of.

51. the yeast of claim 49, wherein said yeast are (1) genotype pdc2 Δs, and the yeast saccharomyces cerevisiae of one of (2) genotype pdc1 Δ, genotype pdc5 Δ and genotype pdc6 Δ.

52. the yeast of claim 49, wherein said yeast are the Kluyveromyces lactis of genotype pdc1 Δ.

53. the yeast of claim 48, wherein said yeast are crossed expression and are generated to improve kytoplasm acetyl-CoA by the coded pyruvic oxidase of genes of brewing yeast PDA1, the PDB1, PDX1, LAT1 and the LPD1 that have deleted the terminal Mitochondrially targeted signal of N-.

54. the yeast of claim 53, wherein the PDC activity be reduce with eliminate one of.

55. the yeast of claim 53, wherein said yeast are (1) genotype pdc2 Δs, and the yeast saccharomyces cerevisiae of one of (2) genotype pdc1 Δ, genotype pdc5 Δ and genotype pdc6 Δ.

56. the yeast of claim 53, wherein said yeast are the Kluyveromyces lactis of genotype pdc1 Δ.

57. the yeast of claim 42, wherein said yeast are crossed expression pyruvic acid formic acid lyase and hydrogenlyase, and the two generates to improve kytoplasm acetyl-CoA.

58. the yeast of claim 57, wherein said yeast are crossed expression by the coded pyruvic acid formic acid lyase of bacillus coli gene pflA, pflB, and generate to improve kytoplasm acetyl-CoA with Candida boidinii gene FDH1 combination.

59. the yeast of claim 58, wherein the PDC activity be reduce with eliminate one of.

60. the yeast of claim 59, wherein said yeast are (1) genotype pdc2 Δs, and the yeast saccharomyces cerevisiae of one of (2) genotype pdc1 Δ, genotype pdc5 Δ and genotype pdc6 Δ.

61. the yeast of claim 59, wherein said yeast are the Kluyveromyces lactis of genotype pdc1.

62. the yeast of claim 42, wherein at least a gene have carried out molecular evolution to strengthen by its coded proteinic enzymic activity.

63. a method that improves the zymic metabolic activity, this method comprise that yeast generates the kytoplasm acetyl that another kytoplasm acetyl-the CoA amount increases-CoA amount with respect to wild-type yeast generated.

64. a metabolic engineering yeast, it has at least a approach, and this approach is configured to generate the kytoplasm acetyl that another kytoplasm acetyl-the CoA amount increases-CoA amount that generates with respect to wild-type yeast.

Images