KR102306725B1 - Genetically engineered yeast having acetoin producing ability and method for producing acetoin using the same - Google Patents

Abstract

The present invention relates to a genetically engineered yeast having an acetoin-producing ability, and an acetoin production method using the same. In the genetically engineered yeast having an acetoin-producing ability and the yeast excellent in an acetoin-producing ability, i) an alcohol dehydrogenase (ADH) gene, a glycerol-3-phosphate dehydrogenase (GPD) gene, and a 2,3-butanediol dehydrogenase (BDH) gene are deleted, ii) an alsS gene, an alsD gene, and a noxE gene are introduced, and iii) EMP46, PEP7, SUR1, and HXK2 are mutated. When acetoin is produced by using the same, a large amount of acetoin can be produced from the same amount of glucose. Therefore, the genetically engineered yeast and evolved yeast having acetoin-producing ability according to the present invention can be useful to produce acetoin at high yield.

Description

Genetically engineered yeast having acetoin-producing ability and acetoin production method using the same

The present invention relates to a genetically engineered yeast having acetoin-producing ability and a method for producing acetoin using the same.

Acetoin (acetoin) is a perfume with a buttery scent and is widely used in food, cosmetics, cigarettes, detergents, etc., as well as acts as an attractant of pests and can be used as an insect repellent. This acetoin is included in 30 platform chemicals that can be produced from biomass determined by the U.S. Department of Energy due to the advantages of various uses and mass production.

Currently, most of acetoin is produced by chemical synthesis, but in the cosmetic field and food field, preference for natural fragrance is increasing, so the acetoin production method through a biological process is attracting attention. Recently, research on a method for producing acetoin using microorganisms is being actively conducted. For example, a method for producing acetoin using recombinant Enterobactera erogenes and recombinant Bacillus subtilis has been developed (Korean Patent Application No. 10-2016-0006582 and Korean Patent Application No. 2015-0081821) like). However, it is mainly carried out based on bacterial strains, and most of them have a problem in that they have pathogenicity or lack of resistance to acidic conditions, osmotic pressure, high concentration of glucose or high concentration of acetoin, so that the production yield is low.

Accordingly, there is a need for a method for producing acetoin in high yield using microorganisms recognized as GRAS (Generally Recognized As Safe) by the US FDA.

Korean Patent Application No. 10-2016-0006582 Korean Patent Application No. 10-2015-0081821

Accordingly, the present inventors studied to develop a genetically engineered yeast that produces acetoin from glucose in a high yield, and as a result, i) alcohol dehydrogenase, glycerol-3-phosphate dehydrogenase) and 2,3-butanediol dehydrogenase (2,3-butanediol dehydrogenase) activity is decreased, ii) acetolactate synthase, acetolactate decarboxylase and NADH The present invention was completed by confirming that the activity of NADH oxidase is increased, and iii) yeast in which EMP46, PEP7, SUR1 and/or HXK2 is mutated produces acetoin in high yield.

In order to achieve the above object, one aspect of the present invention, compared to the parent strain, i) alcohol dehydrogenase (alcohol dehydrogenase), glycerol-3-phosphate dehydrogenase (glycerol-3-phosphate dehydrogenase) and 2,3-butane Diol dehydrogenase (2,3-butanediol dehydrogenase) activity is reduced, ii) acetolactate synthase (acetolactate synthase), acetolactate decarboxylase (acetolactate decarboxylase) and NADH oxidase (NADH oxidase) activity is increased, and iii) any one selected from the group consisting of EMP46, PEP7, SUR1, HXK2 and combinations thereof is mutated, to provide a genetically engineered yeast.

Another aspect of the present invention comprises the steps of: i) culturing the genetically engineered yeast; And ii) provides a method for producing acetoin comprising the step of obtaining acetoin produced from the yeast.

Another aspect of the present invention is i) a gene encoding an alcohol dehydrogenase of wild-type yeast, a gene encoding a glycerol-3-phosphate dehydrogenase, a gene encoding 2,3-butanediol dehydrogenase, and combinations thereof Deleting any one selected from the group consisting of; ii) any one exogenous gene selected from the group consisting of a gene encoding acetolactate synthase, a gene encoding acetolactate decarboxylase, a gene encoding NADH oxidase, and combinations thereof introducing; And iii) it provides a method for producing a yeast excellent in acetoin-producing ability, comprising the step of culturing the yeast at least 15 times in an acetoin-added medium.

Another aspect of the present invention provides a yeast excellent in acetoin-producing ability prepared by the above method.

Another aspect of the present invention, i) culturing the yeast excellent in the acetoin-producing ability; And ii) provides a method for producing acetoin comprising the step of obtaining acetoin produced from the yeast.

The genetically engineered yeast having acetoin-producing ability and yeast having excellent acetoin-producing ability according to the present invention are i) ADH (alcohol dehydrogenase) gene, GPD (glycerol-3-phosphate dehydrogenase) gene, and BDH (2,3-butanediol) dehydrogenase) gene is deleted, ii) alsS gene, alsD gene and noxE gene are introduced, iii) EMP46, PEP7, SUR1 and HXK2 are mutated. Able to produce positive amounts of acetoin. Therefore, the genetically engineered yeast and evolved yeast having acetoin-producing ability according to the present invention can be usefully used to produce acetoin in high yield.

1 is a diagram schematically showing an acetoin production pathway and a competitive pathway according to an embodiment.
2 is a photograph taken of culturing the JHY902D strain and the JHY903 strain according to an embodiment in YPD medium or YPD medium containing acetoin at a concentration of 18 g/L.
Figure 3a is a graph showing the growth rate of JHY902A strain, D1 to D3 strain, JHY902D strain and JHY903 strain according to an embodiment.
Figure 3b is a graph showing the glucose concentration in the culture of JHY902A strain, D1 to D3 strain, JHY902D strain and JHY903 strain according to an embodiment.
Figure 4 is a graph showing the production of acetoin and 2,3-butanediol of JHY902A strain, D1 to D3 strain, JHY902D strain and JHY903 strain according to an embodiment.
Figure 5a is a graph showing the growth rate of the strain JHY903 and JHY903-1 to JHY903-9 according to an embodiment.
Figure 5b is a graph showing the glucose concentration in the culture solution of the strain JHY903 and JHY903-1 to JHY903-9 according to an embodiment.
6 is a graph showing the production of acetoin and 2,3-butanediol of JHY903 strains and JHY903-1 to JHY903-9 strains according to an embodiment.
7 shows the results of analyzing the stereoselectivity and stereospecificity of the enzymes ARA1, YPR1 and YMR226C for racemic acetoin (3R/S-acetoin) by gas chromatography (GC).
Figure 8a is a graph showing the growth rate of JHY903 strain, JHY903 [ARA1] strain and JHY903 [YPR1] strain according to an embodiment.
Figure 8b is a graph showing the glucose concentration in the culture of JHY903 strain, JHY903 [ARA1] strain and JHY903 [YPR1] strain in one embodiment.
Figure 9a is a graph showing the production of 2,3-butanediol of JHY903 strain, JHY903 [ARA1] strain and JHY903 [YPR1] strain according to an embodiment.
Figure 9b is a graph showing the production of acetoin in JHY903 strain, JHY903 [ARA1] strain and JHY903 [YPR1] strain according to an embodiment.
Figure 10a is a graph showing the growth rate of strain JHY903, strain JHY903-4, strain JHY903-45 and strain JHY903-459 according to an embodiment.
Figure 10b is a graph showing the glucose concentration in the culture of JHY903 strain, JHY903-4 strain, JHY903-45 strain and JHY903-459 strain according to an embodiment.
11 is a graph showing the production of acetoin and 2,3-butanediol of strain JHY903, strain JHY903-4, strain JHY903-45 and strain JHY903-459 according to an embodiment.

One aspect of the present invention, compared to the parent strain i) alcohol dehydrogenase (alcohol dehydrogenase), glycerol-3-phosphate dehydrogenase (glycerol-3-phosphate dehydrogenase) and 2,3-butanediol dehydrogenase (2,3) -butanediol dehydrogenase) activity is decreased, ii) acetolactate synthase, acetolactate decarboxylase and NADH oxidase activity are increased, iii) EMP46, Provided is a genetically engineered yeast in which any one selected from the group consisting of PEP7, SUR1, HXK2 and combinations thereof is mutated.

The term "acetoin" used in the present invention is used interchangeably with 3-hydroxybutanone or acetyl methyl carbinol, and the molecular formula of _{C 4} H ₈ O ₂ It may mean a compound having a. The acetoin may include (R)-acetoin.

As used herein, the term “alcohol dehydrogenase” may refer to an enzyme that promotes interconversion between alcohol and aldehyde or ketone by oxidation of NADH. The alcohol dehydrogenase may include an enzyme having a similar activity, even if the name of the enzyme is different, for example, ADH1, ADH2, ADH3, ADH4, ADH5, ADH6, ADH7 or SFA1. Preferably, the alcohol dehydrogenase may be any one selected from the group consisting of ADH1, ADH2, ADH3, ADH4, ADH5, and combinations thereof.

Specifically, the alcohol dehydrogenase is about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more of the amino acid sequence represented by SEQ ID NO: 99, 101, 103, 105, or 107 , at least about 92%, at least about 95%, at least about 97%, at least about 98%, or at least about 99% sequence homology.

The term "glycerol-3-phosphate dehydrogenase" used in the present invention may refer to an enzyme that promotes conversion to glycerol-3-phosphate (G3P). The glycerol-3-phosphate dehydrogenase may include an enzyme having a similar activity, even if the name of the enzyme is different, and may be any one selected from the group consisting of GPD1, GPD2, and combinations thereof.

In addition, the glycerol-3-phosphate dehydrogenase is about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 92% of the amino acid sequence represented by SEQ ID NO: 109 or 111 or more, about 95% or more, about 97% or more, about 98% or more, or about 99% or more sequence homology.

The term "homology" used in the present invention means the degree of agreement with a given polynucleotide sequence and may be expressed as a percentage. In this case, the homologous sequence having the same or similar activity to the given polynucleotide sequence is expressed as "% homology". For example, using standard software that calculates parameters such as score, identity and similarity, specifically BLAST 2.0, or by Southern hybridization experiments under defined stringent conditions. It can be confirmed by comparing , and an appropriate hybridization condition defined can be determined by a method well known to those skilled in the art.

The term "2,3-butanediol dehydrogenase" used in the present invention may refer to an enzyme that uses acetoin, NADH, and H + as substrates to produce 2,3-butanediol and NAD +, It belongs to the oxidoreductase family. The 2,3-butanediol dehydrogenase may include an enzyme having a similar activity (eg, isoenzyme or homolog) even if the name of the enzyme is different, for example, BDH1, BDH2 from Saccharomyces cerevisiae, BDH99::67 from Paenibacillus polymyxa, Bacillus subtilis, Enterococcus faecium Enterococcus durans Mycobacteria It may be a 2,3-butanediol dehydrogenase derived from Mycobacterium sp. Lactobacillus lactis.

In addition, the 2,3-butanediol dehydrogenase is about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 92% of the amino acid sequence represented by SEQ ID NO: 113. or more, about 95% or more, about 97% or more, about 98% or more, or about 99% or more sequence homology.

As used herein, the term "decrease in activity" means expressing a smaller amount of an enzyme or polypeptide compared to the parent strain (eg, a strain or cell that is not genetically engineered), or the activity of the enzyme or polypeptide is reduced. It may mean inactivated or inactivated (inactivation). In addition, the strain with reduced activity may have a genetic modification that reduces the activity of one or more enzymes or polypeptides compared to a strain without the genetic modification.

Genetic modifications that reduce the activity of the enzyme or polypeptide include 1) deletion of a part or all of a gene encoding the enzyme or polypeptide, 2) modification of an expression control sequence to decrease the expression of the gene, 3) the enzyme or Modification of the above gene sequence on the chromosome or 4) a combination thereof so that the activity of the polypeptide is weakened.

The method of deleting part or all of the gene encoding the enzyme or polypeptide may be performed by, for example, transforming a cassette for gene deletion into a parent strain using a Cre/loxP recombination system, and chromosomal insertion in yeast. It can be performed by replacing a gene encoding an endogenous target protein in a chromosome with a gene or a marker gene in which some nucleic acid sequences have been deleted through the vector. The method of modifying the expression control sequence is performed by inducing mutation in the expression control sequence by deletion, insertion, non-conservative or conservative substitution, or a combination thereof, of a nucleic acid sequence to further weaken the activity of the expression control sequence, or It can be carried out by replacing it with a nucleic acid sequence having activity. The expression control sequence includes a promoter, an operator sequence, a sequence encoding a ribosome binding site, and a sequence controlling the termination of transcription and translation. In addition, the method of modifying a nucleotide sequence on a chromosome encoding the enzyme or polypeptide induces sequence mutation by deletion, insertion, non-conservative or conservative substitution of the nucleotide sequence, or a combination thereof to further weaken the activity of the protein. It can be carried out by replacing the nucleotide sequence with an improved nucleotide sequence to have weaker activity.

In the yeast, a gene encoding alcohol dehydrogenase, a gene encoding glycerol-3-phosphate dehydrogenase, and a gene encoding 2,3-butanediol dehydrogenase may be deleted.

The gene encoding the alcohol dehydrogenase is about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% of the nucleotide sequence represented by SEQ ID NO: 100, 102, 104, 106, or 108 or more, about 92% or more, about 95% or more, about 97% or more, about 98% or more, or about 99% or more sequence homology.

The gene encoding the glycerol-3-phosphate dehydrogenase is about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about the nucleotide sequence represented by SEQ ID NO: 110 or 112. It may have 92% or more, about 95% or more, about 97% or more, about 98% or more, or about 99% or more sequence homology.

The gene encoding the 2,3-butanediol dehydrogenase is about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about the nucleotide sequence represented by SEQ ID NO: 114. It may have 92% or more, about 95% or more, about 97% or more, about 98% or more, or about 99% or more sequence homology.

The genes encoding the alcohol dehydrogenase include adh1 gene (SEQ ID NO: 100), adh2 gene (SEQ ID NO: 102), adh3 gene (SEQ ID NO: 104), adh4 gene (SEQ ID NO: 106), adh5 gene (SEQ ID NO: 108) And it may be any one selected from the group consisting of combinations thereof. The gene encoding the glycerol-3-phosphate dehydrogenase may be any one selected from the group consisting of a gpd1 gene (SEQ ID NO: 110), a gpd2 gene (SEQ ID NO: 112), and combinations thereof. The gene encoding the 2,3-butanediol dehydrogenase may be a bdh1 gene (SEQ ID NO: 114).

In addition, the yeast is selected from the group consisting of a gene encoding acetolactate synthase, a gene encoding acetolactate decarboxylase, a gene encoding NADH oxidase, and combinations thereof. gene) may be included.

The term "acetolactate synthase" used in the present invention is used interchangeably with acetohydroxy acid synthase (AHAS), and regulates the biosynthetic pathway of branched chain amino acids such as leucine, valine and isoleucine. As the enzyme, it may be an enzyme that synthesizes one molecule of carbon dioxide and acetolactate from two molecules of pyruvic acid, respectively. The acetolactate synthase may include an enzyme (eg, isoenzyme or homologue) having a similar activity even if the name of the enzyme is different, for example, by the alsS gene derived from Bacillus subtilis. acetolactate synthase encoded by acetolactate synthase, acetolactate synthase I encoded by the ilvB gene or ilvN gene from E. coli, acetolactate synthase II encoded by the ilvGMEDA gene from E. coli, or ilvI or ilvH from E. coli acetolactate synthase III encoded by the gene. In addition, in addition to Escherichia coli, Saccharomyces cerevisiae, Bacillus anthracis, Haemophilus influenzae (Haemophilusinfluenzae), Salmonella typhimurium (Salmonella Typhimurium), Thermotaga maritima (Thermotogamaritima), Corynebacterium glue It may be an acetolactate synthase derived from Corynebacterium glutamicum, Mycobacterium tuberculosis, or Streptomyces cinnamonensis. Additionally, plant-derived acetolactate synthase may be derived from Arabidopsisthhaliana, Gossypiumhirsutum, Helianthus annuus, or Brassicanapus.

In addition, the acetolactate synthase is about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 92% or more, about 95% or more of the amino acid sequence represented by SEQ ID NO: 32. % or greater, about 97% or greater, about 98% or greater, or about 99% or greater sequence homology.

As used herein, the term “acetolactate decarboxylase” may refer to an enzyme that produces acetoin by removing carbon dioxide from acetolactate. The acetolactate decarboxylase may include an enzyme having a similar activity (eg, isoenzyme or homologue) even if the name of the enzyme is different, for example, alsD from Bacillus subtilis, lacto aldB from Bacillus delbrueckii subsp. lactis, Brevibacillus brevis, Enterobacter aerogenes, Leuconostoc lactis, Saccharomyces cerevisiae It may be acetolactate decarboxylase from Staphylococcus aureus.

In addition, the acetolactate decarboxylase is about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 92% or more, about It may be a polypeptide having at least 95%, at least about 97%, at least about 98%, or at least about 99% sequence homology.

An acetoin production pathway of genetically engineered yeast capable of effectively producing acetoin will be described with reference to FIG. 1 . 1 is a diagram schematically showing an acetoin production pathway and a competitive pathway according to one embodiment.

As shown in FIG. 1 , the yeast according to one embodiment has increased activity of acetolactate synthase and acetolactate decarboxylase compared to the parent strain, and thus can effectively produce acetoin. In addition, in the production of acetoin, in order to suppress the production of by-products and further enhance the production of acetoin, the yeast may be one in which the competitive metabolic pathway of the acetoin production pathway is additionally blocked. The competitive metabolic pathway may be an ethanol and glycerol synthesis metabolic pathway, as shown in FIG. 1 , and the competitive metabolic pathway may be achieved by reducing the activity of alcohol dehydrogenase or glycerol-3-phosphate dehydrogenase. In addition, it is possible to further increase the production of acetoin by eliminating the metabolic pathway that converts acetoin to 2,3-butanediol. In addition, a process for reducing cofactor imbalance may be additionally performed. As shown in the following reaction, yeast produces two molecules of NADH by consuming two molecules of NAD+ while producing two molecules of pyruvic acid from glucose through glycolysis. Accordingly, NADH (excess) and NAD+ (shortage) phenomena may occur in the acetoin synthesis pathway.

By increasing the activity of NADH oxidase to oxidize NADH, the imbalance of cofactors (NAD+/NADH) can be resolved. The yeast may include an exogenous gene encoding NADH oxidase.

The term "NADH oxidase" used in the present invention may refer to an enzyme mediating a reaction for producing water and NAD+ using oxygen and NADH as substrates. The NADH oxidase may include an enzyme having a similar activity (eg, isoenzyme or homologue) even if the name of the enzyme is different, for example, nox1, nox3, nox4, Lactococcus lactis-derived enzyme It may include noxE, and, in addition, Enterococcus genus, Lactobacillus genus, Disulfovibriosp. , Clostridium sp. It may be an NADH oxidase derived from Streptococcus genus.

In addition, the NADH oxidase is about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 92% or more, about 95% or more of the amino acid sequence represented by SEQ ID NO: 46. , at least about 97%, at least about 98%, or at least about 99% sequence homology.

As used herein, the term "increase in activity" refers to the activity of a polypeptide or enzyme of the same type as compared to a polypeptide or enzyme not or possessed by the parent strain (eg, a non-genetically engineered strain or cell). It may mean to have a higher activity than this, or to express a higher amount of a polypeptide or enzyme of the same type. In addition, the strain with increased activity may have a genetic modification that increases the activity of one or more enzymes or polypeptides compared to a strain without the genetic modification.

The genetic modification to increase the activity of the enzyme or polypeptide is 1) modification of the expression control sequence to increase the expression of the gene, 2) modification of the gene sequence on the chromosome to increase the activity of the enzyme or polypeptide, 3) the above It can be carried out by additionally introducing a gene encoding an enzyme or a polypeptide or 4) a combination thereof.

The acetolactate synthase may be encoded by the alsS gene, the alsS gene may be one comprising a nucleotide sequence shown in SEQ ID NO: 31. The acetolactate dicarboxylate raised may be encoded by the gene alsD, alsD the gene may be one comprising a nucleotide sequence shown in SEQ ID NO: 33. The NADH oxidase may be encoded by the gene noxE, noxE the gene may be the one that includes the nucleotide sequence shown in SEQ ID NO: 45.

As used herein, the term “exogenous” may mean that a referenced molecule or referenced activity is introduced into a host cell. The molecule may be introduced, for example, as non-chromosomal genetic material such as a plasmid or the introduction of an encoding nucleic acid into the host genetic material, such as by insertion into a host chromosome. With respect to the expression of an encoding nucleic acid, the term "exogenous" indicates that the encoding nucleic acid is introduced into an organism in an expressible form. With respect to biosynthetic activity, the term “exogenous” refers to an activity introduced into a host parent cell.

The exogenous gene may be expressed in an amount sufficient to increase the activity of the mentioned enzyme compared to the parent strain in the yeast. Homologs of the exogenous gene encoding acetolactate synthase, the exogenous gene encoding acetolactate decarboxylase, and the exogenous gene encoding NADH oxidase are derived from different microorganisms, but the proteins they encode It may refer to a gene encoding a protein exhibiting an activity similar to

The exogenous gene encoding the acetolactate synthase has about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 92% or more of the amino acid sequence represented by SEQ ID NO: 32. , which encodes an amino acid sequence having sequence homology of about 95% or more, about 97% or more, about 98% or more, or about 99% or more. The exogenous gene encoding the acetolactate synthase has about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 92% of the nucleotide sequence represented by SEQ ID NO: 31, respectively. It may have a sequence homology of at least about 95%, at least about 97%, at least about 98%, or at least about 99%.

The exogenous gene encoding the exogenous gene encoding the acetolactate decarboxylase is about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% of the amino acid sequence represented by SEQ ID NO: 34, respectively. % or more, about 92% or more, about 95% or more, about 97% or more, about 98% or more, or about 99% or more of sequence homology. The exogenous gene encoding the exogenous gene encoding the acetolactate decarboxylase is about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% of the nucleotide sequence represented by SEQ ID NO: 33, respectively. % or more, about 92% or more, about 95% or more, about 97% or more, about 98% or more, or about 99% or more sequence homology.

The exogenous gene encoding the NADH oxidase is about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 92% or more, about the amino acid sequence represented by SEQ ID NO: 46. It may encode an amino acid sequence having at least 95%, at least about 97%, at least about 98%, or at least about 99% sequence homology. The exogenous gene encoding the NADH oxidase is about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 92% or more, It may have at least about 95%, at least about 97%, at least about 98%, or at least about 99% sequence homology.

Such an exogenous gene may be changed to a sequence with codons suitable for expression in a microorganism, or a sequence with optimized codons. This codon change may be appropriately made within a range in which the amino acid sequence of the protein does not change.

The exogenous gene may be introduced into the parent strain through an expression vector. In addition, the exogenous gene may be introduced into the parent strain in the form of a linear polynucleotide. In addition, the exogenous gene may be expressed from an expression vector (eg, a plasmid) in a strain. In addition, the exogenous gene may be expressed by being inserted into a genetic material (eg, a chromosome) in a strain for stable expression. The vector may include an origin of replication, a promoter, a gene encoding the enzyme, and a terminator. The replication initiation point may include a yeast autonomous replication sequence (ARS). The yeast self-replicating sequence may be stabilized by a yeast centromeric sequence (CEN). The promoter may be selected from the group consisting of TDH3 promoter, TEF promoter, and FBA1 promoter. The terminator may be selected from the group consisting of CYC1, GPM1, and FBA1. The vector may further include a selection marker.

The yeast may include a single gene, a plurality of genes, for example, 2 to 10 copy numbers. The yeast is, for example, 1 to 10, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 2 to 10, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, or 2 to 3 copies of the gene encoding the enzyme. When the yeast comprises a plurality of genes, each gene may be a copy of the same gene or may include copies of two or more different genes. The multiple copies of the exogenous gene may be comprised at the same locus, or at multiple loci, within the genome of the host cell.

As used herein, the term "EMP46" refers to a protein involved in glycoprotein secretion, nuclear structure, and gene silencing, and may have a sequence disclosed in NCBI Reference Sequence: NC_001144.5. The EMP46 may have an amino acid sequence represented by SEQ ID NO: 115. In addition, the EMP46 may be encoded by the nucleotide sequence represented by SEQ ID NO: 116. The EMP46 mutation may be one in which leucine, which is the 160th amino acid in the amino acid sequence shown in SEQ ID NO: 115, is substituted with phenylalanine. In addition, the EMP46 mutation may be one in which guanine, which is the 480th base, of the nucleotide sequence represented by SEQ ID NO: 116 is substituted with thymine.

As used herein, the term “PEP7” refers to a protein involved in vacuole separation and liquid protein secretion, and may have a sequence disclosed in NCBI Reference Sequence: NC_001136.10. The PEP7 may have an amino acid sequence represented by SEQ ID NO: 117. In addition, the PEP7 may be encoded by the nucleotide sequence represented by SEQ ID NO: 118. The PEP7 mutation may be one in which glutamine, which is the 169th amino acid in the amino acid sequence shown in SEQ ID NO: 117, is substituted with lysine. In addition, the PEP7 mutation may be one in which cytosine, which is the 505th base in the nucleotide sequence represented by SEQ ID NO: 118, is substituted with adenine.

The term "SUR1" used in the present invention is a protein involved in the synthesis of mannosyl phosphoryl inositol ceramide, and catalyzes the reaction of adding mannosyl to phosphoryl inositol ceramide. The SUR1 may have a sequence disclosed in NCBI Reference Sequence: NC_001148.4. The SUR1 may have an amino acid sequence represented by SEQ ID NO: 119. In addition, the SUR1 may be encoded by the nucleotide sequence represented by SEQ ID NO: 120. The SUR1 mutation may be one in which histidine, which is the 176th amino acid in the amino acid sequence shown in SEQ ID NO: 119, is substituted with tyrosine. The SUR1 mutation may be one in which cytosine, which is the 526th base in the nucleotide sequence represented by SEQ ID NO: 120, is substituted with thymine.

As used herein, the term "HXK2" refers to a reaction in which phosphorylation of hexoses such as D-glucose and D-fructose to hexose 6-phosphate (D-glucose 6-phosphate and D-fructose 6-phosphate, respectively). As the protein involved, it may be one having the sequence disclosed in NCBI Reference Sequence: NC_001139.9. The HXK2 may have an amino acid sequence represented by SEQ ID NO: 121. In addition, the HXK2 may be encoded by the nucleotide sequence shown in SEQ ID NO: 122. The HXK2 mutation may be one in which guanine, which is the 754th base in the nucleotide sequence shown in SEQ ID NO: 122, is substituted with thymine.

The yeast may be a My process (Saccharomyces), inclusive Vero My process (Kluyveromyces), Pichia (Pichia), Hanse Cronulla (Hansenula), my process to Xi Kosaka (Zygosaccharomyce s) or Candida (Candida) spp a saccharide . In addition, the yeast may be a Saccharomyces genus strain. The Saccharomyces sp. strain is Saccharomyces cerevisiae ( S. scerevisiae ), Saccharomyces bayanus ( S. bayanus ), Saccharomyces paradoxus ( S. paradoxus ), Saccharomyces It may be a non-catheter (S. mikatae), and a saccharide as MY-ku laundry process Havre swallow any one selected from the group consisting of (S. kudriavzevii).

The yeast is arabinose dehydrogenase (ARA1), NADP-dependent aldo-keto reductase (NADPH-dependent aldo-keto reductase, YPR1), NADP-dependent 3-hydroxy acid dehydrogenase (NADP-dependent 3- The activity of any one selected from the group consisting of hydroxy acid dehydrogenase, YMR226C) and combinations thereof may be reduced. Specifically, the yeast is selected from the group consisting of a gene encoding arabinose dehydrogenase, a gene encoding NADP-dependent aldo-kedo reductase, a gene encoding NADP-dependent 3-hydroxy acid dehydrogenase, and combinations thereof. Any one selected may be deleted.

The term "arabinose dehydrogenase" used in the present invention may refer to an enzyme involved in catalyzing the oxidation of D-arabinose, L-xylose, L-fucose and L-galactose in the presence of NADP+, for example For example, it may be ARA1. The arabinose dehydrogenase may have a sequence described in NCBI Reference Sequence: NC_001134.8. The arabinose dehydrogenase is It may be one encoded by the ara1 gene including the nucleotide sequence represented by SEQ ID NO: 124.

As used herein, the term "NADP-dependent aldo-kedo reductase" is an enzyme capable of reducing various substrates including 2-methylbutylaldehyde, and can be rapidly induced by osmotic and oxidative stress. For example, the NADP-dependent aldo-kedo reductase may be GTR3, YJR096W or YPR1. The NADP-dependent aldo-kedo reductase may have the sequence described in NCBI Reference Sequence: NC_001136.10. The NADP-dependent aldo-kedo reductase is It may be one encoded by the ypr1 gene including the nucleotide sequence represented by SEQ ID NO: 126.

The term "NADP-dependent 3-hydroxy acid dehydrogenase" used in the present invention may be a NADP-dependent dehydrogenase having a broad substrate specificity acting on 3-hydroxy acids, for example, NRE1, IRC24, ENV9 or YMR226C. The NADP-dependent 3-hydroxy acid dehydrogenase may have a sequence described in NCBI Reference Sequence: NC_001145.3. The NADP-dependent 3-hydroxy acid dehydrogenase may be encoded by the ymr226c gene including the nucleotide sequence shown in SEQ ID NO: 128.

Another aspect of the present invention comprises the steps of: i) culturing the genetically engineered yeast; And ii) provides a method for producing acetoin comprising the step of obtaining acetoin produced from the yeast.

In the method for producing the acetoin, refer to the description of the genetically engineered yeast described above for the yeast.

The term "cultivation" used in the present invention may refer to a series of actions in which the yeast is grown in an appropriately artificially controlled environmental condition in order to produce acetoin from the yeast. The method of culturing the cells in the present invention can be performed using a method widely known in the art. Specifically, the culture may be continuously cultured in a batch process or injection batch or repeated fed batch process. The medium used for culture may include one or more substrates that can be metabolized to acetoin, for example, in a conventional medium containing an appropriate carbon source, nitrogen source, amino acid, vitamin, etc. under aerobic conditions at temperature, pH It is necessary to meet the requirements of a particular strain in an appropriate manner while adjusting the etc.

As a carbon source that can be used, glucose is used as the main carbon source, and in addition, sugars and carbohydrates such as xylose, sucrose, lactose, fructose, maltose, starch, cellulose, soybean oil, sunflower oil, castor oil, coconut oil, etc. Oils and fats, fatty acids such as palmitic acid, stearic acid and linoleic acid, alcohols such as glycerol, ethanol, and organic acids such as acetic acid may be included.

The culture may be characterized by culturing yeast in the presence of glucose.

These substances can be used individually or as a mixture. Examples of the nitrogen source that can be used include inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, anmonium carbonate, and ammonium nitrate; Amino acids such as glutamic acid, methionine, and glutamine, and organic nitrogen sources such as peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steep liquor, casein hydrolyzate, fish or its degradation products, defatted soybean cake or its degradation products, etc. can These nitrogen sources may be used alone or in combination. The medium may contain monopotassium phosphate, dipotassium phosphate and the corresponding sodium-containing salt as phosphorus. The phosphorus that may be used includes potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salt. In addition, as the inorganic compound, sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, calcium carbonate, and the like may be used. Finally, in addition to the above substances, essential growth substances such as amino acids and vitamins can be used.

Typically, yeast can be grown in a suitable medium at a temperature ranging from about 20°C to about 37°C. In the present invention, the growth medium is, for example, a broth containing yeast nitrogen base, ammonium sulfate, and dextrose as a carbon/energy source, or most strains of Saccharomyces cerevisiae. It may be a commercially prepared conventional medium such as YPD medium in which peptone, yeast extract and dextrose are blended in an optimal ratio for growth. Other defined or synthetic growth media may be used, and media suitable for the growth of specific microorganisms are known to those skilled in the art of microbiology or fermentation science.

Acetoin produced by the yeast can be isolated from the culture medium using a method known in the art. This separation method may be centrifugation, filtration, ion exchange chromatography or crystallization. For example, the culture may be centrifuged at low speed to remove biomass, and the obtained supernatant may be separated through ion exchange chromatography.

Another aspect of the present invention is i) a gene encoding an alcohol dehydrogenase of wild-type yeast, a gene encoding a glycerol-3-phosphate dehydrogenase, a gene encoding 2,3-butanediol dehydrogenase, and combinations thereof Deleting any one selected from the group consisting of; ii) introducing any one exogenous gene selected from the group consisting of a gene encoding acetolactate synthase, a gene encoding acetolactate decarboxylase, a gene encoding NADH oxidase, and combinations thereof; And iii) it provides a method for producing a yeast excellent in acetoin-producing ability comprising the step of culturing the yeast at least 15 times in an acetoin-added medium.

Specifically, the method comprises deleting a gene encoding an alcohol dehydrogenase of wild-type yeast, a gene encoding a glycerol-3-phosphate dehydrogenase, and a gene encoding a 2,3-butanediol dehydrogenase in step i). and a gene encoding acetolactate synthase, a gene encoding acetolactate decarboxylase and a gene encoding NADH oxidase may be introduced in step ii).

The gene encoding the alcohol dehydrogenase, the gene encoding the glycerol-3-phosphate dehydrogenase and the gene encoding the 2,3-butanediol dehydrogenase and the method for deleting them are the same as described above in the genetically engineered yeast. do.

The medium to which acetoin is added may be characterized in that at least 4 g/L of acetoin is added. The acetoin-added medium may be gradually increased from a concentration of 4 g/L to a concentration of 16.5 g/L.

The exogenous gene encoding acetolactate synthase, the exogenous gene encoding acetolactate decarboxylase and the exogenous gene encoding NADH oxidase and the method for introducing them are the same as described above in the genetically engineered yeast.

The number of passages in step ii) may be at least 15 times or more, preferably, the number of passages in step ii) is 15 times, 17 times, 19 times, 21 times, 23 times, 25 times, 27 times or 29 or more.

In one embodiment of the present invention, the gene encoding alcohol dehydrogenase, the gene encoding glycerol-3-phosphate dehydrogenase, and the gene encoding 2,3-butanediol dehydrogenase are deleted in yeast (JHY605) After introducing an exogenous gene encoding acetolactate synthase, an exogenous gene encoding acetolactate decarboxylase and an exogenous gene encoding NADH oxidase, it was transferred to a medium supplemented with acetoin at a concentration of 4 g/L. When the OD value becomes about 1.0 at a wavelength of 600 nm by culturing at 37 ° C. temperature and 200 rpm conditions, the step of subculturing the yeast strain can be repeated 19 times or more. In this case, the acetoin-added medium may be gradually increased from a concentration of 4 g/L to a concentration of 16.5 g/L.

After step ii), the yeast consists of a gene encoding arabinose dehydrogenase, a gene encoding NADP-dependent aldo-kedo reductase, a gene encoding NADP-dependent 3-hydroxy acid dehydrogenase, and combinations thereof. It may further comprise the step of deleting any one selected from the group.

The gene encoding arabinose dehydrogenase, the gene encoding NADP-dependent aldo-kedo reductase, the gene encoding NADP-dependent 3-hydroxy acid dehydrogenase, and methods for deleting them are described above in genetically engineered yeast. same as bar

Another aspect of the present invention provides a yeast excellent in acetoin-producing ability prepared by the above method.

Another aspect of the present invention, i) culturing the yeast excellent in the acetoin-producing ability; And ii) provides a method for producing acetoin comprising the step of obtaining acetoin produced from the yeast. The culture method and the method for obtaining acetoin are the same as those described above in the method for producing acetoin using genetically engineered yeast.

Hereinafter, the present invention will be described in more detail by way of Examples. However, the following examples are only for illustrating the present invention, and the scope of the present invention is not limited thereto.

Preparation Example 1. Genetically engineered Saccharomyces cerevisiae having acetoin-producing ability ( S. cerevisiae ) strain production

S. cerevisiae produces ethanol as a major metabolite, and the genes involved in this are six alcohol dehydrogenases (ADH1, ADH2, ADH3, ADH4, ADH5, SFA1) using NADH as a cofactor and alcohol dehydrogenase using NADPH. (ADH6, ADH7) has been reported to exist. When the adh1, adh2, adh3, adh4 and adh5 genes are removed to reduce the production of ethanol, a major metabolite, a cofactor imbalance occurs. It is produced as a major metabolite. In addition, acetoin is converted to 2,3-butanediol by 2,3-butanediol dehydrogenase (BDH1). Therefore, the dehydrogenase, glycerol-3-phosphate dehydrogenase, and 2,3-butanediol dehydrogenase that block all synthesis pathways of ethanol, glycerol, and 2,3-butanediol are deficient strains (adh1-5Δ gpd1,2Δ, bdh1Δ) to produce acetoin (Fig. 1).

The strains prepared through adaptive evolution and transformation using strains deficient in the dehydrogenase, glycerol-3-phosphate dehydrogenase, and 2,3-butanediol dehydrogenase are summarized in Table 1 below.

strain name genotype JHY605 CEN.PK2-1c adh1β::loxP adh2β::loxP adh3β::loxP adh4β::loxP adh5β::loxP gpd1β::loxP gpd2β::loxP JHY901 CEN.PK2-1c adh1β::loxP adh2β::loxP adh3β::loxP adh4β::loxP adh5β::loxP gpd1β::loxP gpd2β::loxP bdh1::loxP JHY902A JHY901 adh2β:: P _TDH3 -alsS- T _CYC1 , P _TEF -alsD- T _GPM1 , P _FBA1 -noxE- T _FBA1 JHY902D JHY901 delta:: P _TDH3 -alsS- T _CYC1 , P _TEF -alsD- T _GPM1 , P _FBA1 -noxE- T _FBA1 JHY903 Evolved strain from JHY902D JHY903-1 JHY903 bdh2△ JHY903-2 JHY903 gre3△ JHY903-3 JHY903yjr096w △ JHY903-4 JHY903 ara1△ JHY903-5 JHY903 ypr1△ JHY903-6 JHY903 nre1△ JHY903-7 JHY903 irc24△ JHY903-8 JHY903 env9△ JHY903-9 JHY903 ymr226c△ JHY903-45 JHY903 ara1△ ypr1△ JHY903-49 JHY903 ara1△ ymr226c△ JHY903-459 JHY903 ara1△ ypr1△ ymr226c△

In addition, the plasmids used to construct the strains listed in Table 1 are shown in Table 2 below.

plasmid characteristic p413G CEN/ARS plasmid, HIS3 , P _TDH3 , T _CYC1 p414G CEN/ARS plasmid, TRP1 , P _TDH3 , T _CYC1 p416G CEN/ARS plasmid, URA3 , P _TDH3 , T _CYC1 pUG27 Plasmid containing loxP - URA3 - loxP deletion cassette pSH63 Plasmid containing the Cre-recombinase under the control of GAL1 promoter; TRP1 ADH2-SDN Plasmid containing P _TDH3 - alsS -T _CYC1 , P _TEF - alsD -T _GPM1 , P _FBA1 - noxE -T _FBA1 , and loxp-HIS-loxp flanked by 300 bp of up- and downstream of ADH2 Delta-SDN Plasmid containing P _TDH3 - alsS -T _CYC1 , P _TEF - alsD -T _GPM1 , P _FBA1 - noxE -T _FBA1 , and loxp-HIS-loxp flanked by YARCdelta4-1 and 2 p413G-alsS p413G containing P _TDH3 - alsS -T _CYC1 p414G-alsD p414G containing P _TDH3 - alsD -T _CYC1 p416F-noxE p416F containing P _FBA1 - noxE -T _CYC1 p416G-ARA1 p416G containing P _TDH3 - ARA1 -T _CYC1 p416G-YPR1 p416G containing P _TDH3 - YPR1 -T _CYC1 p416G-YMR226C p416G containing P _TDH3 - YMR226C -T _CYC1 pET-28b(+) Km ^R , His ₆ -tagged protein expression vector pET-ARA1-His Ara1-His ₆ expression plasmid pET-YPR1-His Ypr1-His ₆ expression plasmid pET-YMR226C-His Ymr226c-His ₆ expression plasmid pET-alsS-His AlsS-His ₆ expression plasmid p413-Cas9-ARA1 target gRNA CEN/ARS plasmid, HIS3 , P _TDH3 -CAS9- T _TPI1 , P _SNR52 - ARA1 gRNA-T _SUP4 p413-Cas9-BDH2 target gRNA CEN/ARS plasmid, HIS3 , P _TDH3 -CAS9- T _TPI1 , P _SNR52 - BDH2 gRNA-T _SUP4 p413-Cas9-YPR1 target gRNA CEN/ARS plasmid, HIS3 , P _TDH3 -CAS9- T _TPI1 , P _SNR52 - YPR1 gRNA-T _SUP4 p413-Cas9-GRE3 target gRNA CEN/ARS plasmid, HIS3 , P _TDH3 -CAS9- T _TPI1 , P _SNR52 - GRE3 gRNA-T _SUP4 p413-Cas9-YJR096W target gRNA CEN/ARS plasmid, HIS3 , P _TDH3 -CAS9- T _TPI1 , P _SNR52 - YJR096W gRNA-T _SUP4 p413-Cas9-YMR226C target gRNA CEN/ARS plasmid, HIS3 , P _TDH3 -CAS9- T _TPI1 , P _SNR52 - YMR226C gRNA-T _SUP4 p413-Cas9-NRE1 target gRNA CEN/ARS plasmid, HIS3 , P _TDH3 -CAS9- T _TPI1 , P _SNR52 - NRE1 gRNA-T _SUP4 p413-Cas9-IRC24 target gRNA CEN/ARS plasmid, HIS3 , P _TDH3 -CAS9- T _TPI1 , P _SNR52 - IRC24 gRNA-T _SUP4 p413-Cas9-ENV9 target gRNA CEN/ARS plasmid, HIS3 , P _TDH3 -CAS9- T _TPI1 , P _SNR52 - ENV9 gRNA-T _SUP4

Example 1. adh gene( adh1, adh2, adh3, adh4, adh5 ) and gpd gene( gpd1, gpd2 ) is missing S. cerevisiae Strain (JHY605) production

S. cerevisiae strains lacking adh genes ( adh1, adh2, adh3, adh4, adh5 ) and gpd genes ( gpd1, gpd2 ) were prepared using the Cre/loxP recombination system. The cassette for gene deletion was PCR using pUG27 (plasmid containing loxP-his5+-loxP deletion cassette, Euroscarf, Germany) or pUG72 (plasmid containing loxP-URA3-loxP deletion cassette, Euroscarf, Germany) plasmid as a template obtained through amplification. As a primer set for constructing the gene deletion cassette, SEQ ID NOs: 1 and 2 ( adh1 ), SEQ ID NOs: 3 and 4 ( adh2 ), SEQ ID NOs: 5 and 6 ( adh3 ), SEQ ID NOs: 7 and 8 ( adh 4), SEQ ID NO: Combinations of 9 and 10 ( adh5 ), SEQ ID NOs: 11 and 12 ( gpd1 ), and SEQ ID NOs: 13 and 14 ( gpd2 ) were used to delete the corresponding gene, respectively.

S. cerevisiae strain CEN. To delete adh genes ( adh1, adh2, adh3, adh4, adh5 ) and gpd genes ( gpd1, gpd2 ) in PK2-1C ( MATaura3-52trp1-289 leu2-3,112 his3β1 MAL2-8C SUC2 ) (Euroscarf, Germany), The cassette for the gene deletion was introduced using a chemical transformation method using lithium acetate. Then, the transformed S. cerevisiae strain was cultured in SC medium (20 g/L glucose, 6.7 g/L YNB, appropriate amino acid addition), and S. cerevisiae strains lacking each gene were selected.

At this time, as a primer for confirmation, SEQ ID NOs: 15 and 16 ( adh1 ), SEQ ID NOs: 17 and 18 ( adh2 ), SEQ ID NOs: 19 and 20 ( adh3 ), SEQ ID NOs: 21 and 22 ( adh4 ), SEQ ID NOs: 23 and 24 ( adh5 ), SEQ ID NOs: 25 and 26 ( gpd1 ), and SEQ ID NOs: 27 and 28 ( gpd2 ) were used in combination to confirm the deletion of the gene.

pSH63 (TRP1, Cre recombinase) expressing Cre recombinase in order to remove the selectable markers possessed by the S. cerevisiae strain lacking the adh genes ( adh1, adh2, adh3, adh4, adh5 ) and gpd genes ( gpd1, gpd2) Under the control of GAL1 promoter, Euroscarf, Germany), the adh gene ( adh1, adh2, adh3, adh4, adh5 ) and gpd gene ( gpd1, gpd2 ) from which the selectable marker gene was removed S. cerevisiae The strain was named "JHY605" strain (CEN.PK2-1C adh1β::loxP adh2β::loxP adh3β::loxP adh4β::loxP adh5β::loxP gpd1β::loxP gpd2β::loxP ).

Example 2. adh gene( adh1, adh2, adh3, adh4, adh5 ), gpd gene( gpd1, gpd2 ) and the BDH gene ( bdh1 ) is missing S. cerevisiae Strain (JHY901) production

In addition to the JHY605 strain prepared in Example 1, a JHY901 strain in which the bdh1 gene was deleted was prepared. The JHY901 strain was produced using the Cre/loxp recombination system and was performed in the same manner as in Example 1. At this time, the cassette for the bdh1 gene deletion was obtained through PCR amplification using the pUG27 plasmid as a template, and a combination of SEQ ID NOs: 29 and 30 was used as a primer set for the production of the bdh1 gene deletion cassette.

Example 3. adh gene( adh1, adh2, adh3, adh4, adh5 ), gpd gene( gpd1, gpd2 ) and bdh gene( bdh1 ) is missing, alsD, alsD and noxE gene introduced S. cerevisiae Strain (JHY902A/ JHY902D) production

As acetolactate synthase, alsS (base sequence of SEQ ID NO: 31, amino acid sequence of SEQ ID NO: 32) derived from Bacillus subtilis, and acetolactate decarboxylase derived from Bacillus subtilis alsD ( The nucleotide sequence of SEQ ID NO: 33 and the amino acid sequence of SEQ ID NO: 34) were used, respectively. Specifically, the alsS gene and the alsD gene were obtained through PCR (alsS gene: primer sets of SEQ ID NOs: 35 and 36, alsD gene: primer sets of SEQ ID NOs: 37 and 38) using Bacillus subtilis genomic DNA as a template. As the NADH oxidase, Lactococcus lactis-derived noxE gene (base sequence of SEQ ID NO: 45, amino acid sequence of SEQ ID NO: 46) was used. The noxE gene was obtained through PCR (primer sets of SEQ ID NOs: 47 and 48) using the genomic DNA of Lactococcus lactis as a template. In order to insert a gene cassette through homologous recombination, ADH2-1 (upper 301 bp, SEQ ID NO: 129), ADH2-2 (lower 302 bp, SEQ ID NO: 130) and YARCdelta4 of the ADH2 gene YARCdelta4-1 (167 bp, sequence No. 131) and YARCdelta4-2 (170 bp, SEQ ID NO: 132) using S. cerevisiae genomic DNA as a template, PCR (upper 302 bp: primer set of SEQ ID NOs: 133 and 134, lower 301 bp: SEQ ID NO: 135 and 136 primer set) (YARCdelta4-1: primer set of SEQ ID NOs: 137 and 138, YARCdelta4-2: primer set of SEQ ID NO: 139 and 140).

To overexpress the alsS gene and the alsD gene, the TDH3 promoter (SEQ ID NO: 39) and the TEF1 promoter (SEQ ID NO: 40) and the CYC1 terminator (SEQ ID NO: 41) and GPM1 terminator (SEQ ID NO: 42) were used, respectively. TDH3 promoter and TEF1 promoter were obtained by treating p414GPD and p414TEF vectors (Mumberg et al., 1995) with SacI and SpeI restriction enzymes, respectively. The GPM1 terminator was obtained through PCR (GPM1 terminator: primer sets of SEQ ID NOs: 43 and 44) using S. cerevisiae genomic DNA as a template. The obtained promoter fragment was cloned using SacI and SpeI restriction enzymes, and the terminator fragment was cloned using XhoI and KpnI restriction enzymes. In addition, in order to overexpress the noxE gene, the FBA1 promoter (SEQ ID NO: 49) and the FBA1 terminator (SEQ ID NO: 50) were used. The FBA1 promoter and terminator were obtained through PCR (promoter: primer sets of SEQ ID NOs: 51 and 52, terminator: primer sets of SEQ ID NOs: 53 and 54) using S. cerevisiae genomic DNA as a template. Thereafter, the alsS gene and the noxE and alsD genes were cloned using BamHI and XhoI restriction enzymes, and the resulting vectors were respectively p414_P _TDH3 - alsS -T _CYC1 , p414_P _{TEF1 -} alsD -T _GPM1 and p414_P _FBA1 - noxE -T _FBA1 named as

Finally, in order to express all the necessary genes using a single vector, a PCR product having a 'promoter-gene-terminator' using the primer sets of SEQ ID NOs: 55 and 56, respectively, as a template for each of the three types of vectors cloned above got This PCR product has an MluI restriction enzyme sequence at the 5' end and an AscI-NotI-MluI sequence at the 3' end. The p413G plasmid vector ( HIS3 , P _TDH3, T _CYC1 ) (Mumberg et al., 1995) was treated with BssHII restriction enzyme and the PCR product P _{TEF1 -} alsD- T _GPM1 was treated with MluI restriction enzyme and cloned into p413-D vector. has been obtained. In order to additionally insert sequences necessary for homologous recombination into the vector at both ends of the gene cassette, PCR products including the ampicillin resistance gene (Amp R) and bacterial replication origin (pUG ori) from 413GPD and ADH2-1 (or YARCdelta4-1), ADH2-2 (or YARCdelta4-2) was ligated through overlap PCR (a total of three fragments) and inserted into p413-D vector treated with SacI and NotI restriction enzymes. In addition, for additional cloning, the AscI and NotI restriction enzyme sites of the vector and the MluI and NotI restriction enzyme sites of the PCR product were used. Since AscI and MluI form the same sticky end, they can adhere to each other, and after adhesion, they are no longer recognized by each enzyme. Cloning is possible. Through this method, P _TDH3 - alsS -T _CYC1 and P _FBA1 - noxE -T _FBA1 were sequentially cloned, and finally ADH2-SDN vector or delta-SDN vector was constructed.

Then, in the defective adh2 gene locus of the JHY901 strain prepared in Example 2, an acetoin -related gene insertion cassette I consisting of the alsS gene, alsD gene and noxE gene was obtained from the ADH2-SDN plasmid by treatment with SwaI restriction enzyme and then transformed and the HIS3 selection marker was removed using pSH63 expressing Cre recombinase to prepare JHY902A strain. In the case of strain JHY902A, inhibition of cell growth and glucose uptake was observed, which was caused by the accumulation of acetaldehyde in cells due to lack of expression of acetolactate decarboxylase (alsS), which competes with pyruvate decarboxylase. was observed to be

Accordingly, in order to control the expression level of alsS through a multi-copy integration that targets hundreds of delta sequences present in the S. cerevisiae strain, the alsS gene, alsD gene and noxE with delta sequences added to both ends of the cassette Acetoin-related gene insertion cassette II containing the gene was obtained from Delta-SDN plasmid by treatment with SwaI restriction enzyme, and then transformed into JHY901 strain. After randomly selecting four transformants (D1-D4), the HIS3 selection marker was removed using pSH63 expressing Cre recombinase. As a result, it was confirmed that cell growth and glucose uptake inhibition were recovered in the D4 strain, and this was named "JHY902D" strain.

Example 4. Acetoin production improved through adaptive evolution S. cerevisiae Selection of strain (JHY903)

The JHY902D strain prepared in Example 3 was subcultured 19 times consecutively in YPD medium in which the acetoin concentration was gradually increased from the 4 g/L concentration to the 16.5 g/L concentration, and the resistance to acetoin and the acetoin-producing ability were The enhanced JHY903 strain was selected. Thereafter, the JHY902D strain and the JHY903 strain were cultured in YPD medium and YPD medium supplemented with 18 g/L of acetoin, respectively, to confirm the growth level.

As a result, it was confirmed that the JHY903 strain forms colonies even in the acetoin-added medium, whereas the JHY902D strain does not form colonies well (FIG. 2).

The JHY903 strain and the JHY902A strain, D1 to D3 strain and JHY902D strain prepared in Example 3 were compared to acetoin resistance and acetoin production ability. The acetoin production medium is YPD5 (50 g/L glucose, 10 g/L yeast extract, 20 g/L bacto-peptone) or YPD10 (100 g/L glucose, 10 g/L yeast extract, 20 g/L bacto). -peptone) was used. Cell culture was performed at 30° C. at 170 rpm using a shaker incubator. As for the culture conditions for producing acetoin, in the case of an experiment using YPD5 medium, the initial inoculation cell concentration was _{fixed at OD 600} = 0.5, and 10 ml medium was performed in a 100 ml Erlenmeyer flask. In addition, in the case of the experiment using the YPD10 medium, the initial inoculation cell concentration was OD ₆₀₀ = 5, and 10 ml medium was used in a 100 ml Erlenmeyer flask.

The resistance to the acetoin was confirmed by measuring the cell concentration in the acetoin production medium in which the acetoin concentration was increased over time, and the production of metabolites including the acetoin was confirmed by the following method. In order to analyze metabolites, 800 μl of the culture medium of each strain was centrifuged to obtain a supernatant, which was filtered through a 0.22 μm filter to perform HPLC analysis. In this case, UltiMate 3000 HPLC system (Thermo fishers scientific) was used, and a BioRad Aminex HPX-87H column and a refractive index detector (RI detector) were used. As the mobile phase, 5 mM sulfuric acid was used, the flow rate was 0.6 ml/min, and the temperature was set to 60°C.

As a result, as shown in FIGS. 3A and 3B , the cell concentration and glucose consumption of the JHY902D strain and the JHY903 strain were significantly increased than that of the JHY902A strain and the D1 to D3 strains. In particular, the cell concentration of the JHY903 strain was increased more than twice that of the JHY902A strain and the D1 to D3 strains.

In addition, as shown in FIG. 4 , it was confirmed that the acetoin production of the JHY902D strain and the JHY903 strain was significantly increased. In particular, in the case of the JHY902D strain, the production of 2,3-butanediol as well as acetoin increased, whereas in the case of the JHY903 strain, the acetoin production increased by about 24.5%, but the production of by-products such as 2,3-butanediol decreased. did.

Example 5 Adaptive Evolution S. cerevisiae Gene mutation analysis of strain (JHY903)

The genome of the adapted-evolved JHY903 strain selected in Example 4 was requested to Macrogen and the nucleotide sequence was analyzed. As a result, it was confirmed that a total of four genes in the genome were mutated, and information on the mutated genes is shown in Table 3 below.

gene type of mutation amino acid changes EMP46 Missense(TTG->TTT) 160Leu->Phe PEP7 Missense (CAA->AAA) 169Gln->Lys SUR1 Missense(CAT->TAT) 176His->Tyr HXK2 Nonsense(GGA->TGA) 252Gly->Stop

Example 6. arabinose dehydrogenase (ARA1) gene or 2,3-butanediol production related gene addition deletion strain (JHY903-X) production

JHY903 strain produced R-acetoin type as a major acetoin stereoisomer by two enzymes (AlsS, AlsD) reaction. On the other hand, 2,3-butanediol dehydrogenase (BDH1) has (R)-stereospecific alcohol reductase activity for acetoin, and arabinose dehydrogenase (ARA1) has (S)-stereospecific for acetoin. It is known to have red alcohol reductase activity. Accordingly, in order to reduce the production of 2,3-butanediol, the bdh2 gene, the gre3 gene , the yjr096w gene, the ara1 gene, the ypr1 gene , the nre1 gene, the irc24 gene, the env9 gene or the ymr226c gene were further deleted in the JHY903 strain.

As a method of deleting a gene, a Coex413-Cas9-target gene gRNA plasmid was used as the CRISPR/Cas9 system. At this time, the Coex413-Cas9-target gene gRNA plasmid was performed in the same manner as in Example 3 for preparing the p413-SDN plasmid, and the primer sets described in Table 4 below were used for each defective gene.

sequence information SEQ ID NO: ARA1 target gRNA F TACGAATGGCTCTGTCTCGT GTTTTAGAGCTAGAAATAGC 57 ARA1 target gRNA R ACGAGACAGAGCCATTCGTA GATCATTTATTCTTTCACTGC 58 BDH2 target gRNA F AAGGTAGTTGTCGAGCCCAC GTTTTAGAGCTAGAAATAGC 59 BDH2 target gRNA R GTGGGCTCGACAACTACCTT GATCATTTATTCTTTCACTGC 60 YPR1 target gRNA F ATGCAAGAGTTGCCAAAGAC GTTTTAGAGCTAGAAATAGC 61 YPR1 target gRNA R GTCTTTGGCAACTCTTGCAT GATCATTTATTCTTTCACTGC 62 GRE3 target gRNA F TGGTTGTAGAATCAAGCCCG GTTTTAGAGCTAGAAATAGC 63 GRE3 target gRNA R CGGGCTTGATTCTACAACCA GATCATTTATTCTTTCACTGCG 64 YJR096W target gRNA F AGAAGCGGTTGATGAAGGAT GTTTTAGAGCTAGAAATAGC 65 YJR096W target gRNA R ATCCTTCATCAACCGCTTCT GATCATTTATTCTTTCACTGC 66 YMR226C target gRNA F AGTTAGATACAGAGGTAACG GTTTTAGAGCTAGAAATAGC 67 YMR226C target gRNA R CGTTACCTCTGTATCTAACT GATCATTTATTCTTTCACTGC 68 NRE1 target gRNA F AGGTATCGGTAAGTCCATCG GTTTTAGAGCTAGAAATAGC 69 NRE1 target gRNA R CGATGGACTTACCGATACCT GATCATTTATTCTTTCACTGCG 70 IRC24 target gRNA F CGTCTACGGCGTAGCAAGAA GTTTTAGAGCTAGAAATAGC 71 IRC24 target gRNA R TTCTTGCTACGCCGTAGAC GGATCATTTATTCTTTCACTGCG 72 ENV9 target gRNA F AGGAAGATTGCTGTAGTAAC GTTTTAGAGCTAGAAATAGC 73 ENV9 target gRNA R GTTACTACAGCAATCTTCCT GATCATTTATCTTTCACTGCG 74

Thereafter, each corresponding gene deletion was confirmed using the primers and PCR of Table 5 below.

sequence information SEQ ID NO: ARA1 upstream F GCCTCCACCTTAACATCTTA 75 ARA1 downstream R ACGTACGGCGAATGATTATA 76 BDH2 upstream F GCATTGGTTAGCTCAGATAT 77 BDH2 downstream R CTGCCCCACTTTTAT ATGTC 78 YPR1 upstream F AGCCTATTTGGAAAAGACTG 79 YPR1 downstream R CAGTAGAAGCGCAACTAGTA 80 GRE3 upstream F TGTTTCCCAATTGTTGCTGG 81 GRE3 downstream R TTGGGACCGCTTTGCTCTCT 82 YJR096W upstream F TTGTCCTTATTTGAGGCTCC 83 YJR096W downstream R ATTGCGCTTATCTTTTGGCA 84 YMR226C upstream F AACACTCGACCAGAACGATC 85 YMR226C downstream R CAGCCTAGTTTAGCCAAATC 86 NRE1 upstream F TCAATATCTCCGCTACAACG 87 NRE1 downstream R GATGTAATGTGACGGCAGCC 88 IRC24 upstream F TTCTTGTCAACAGGTGCTAG 89 IRC24 downstream R TTACCGATACCTCTGGAAAC 90 ENV9 upstream F ATTGAGCCACAGGTCTTTCG 91 ENV9 downstream R AGATCCAAGCCTGATAGACC 92

The used Coex413-Cas9-target gene gRNA plasmid was removed by culturing in YPD medium for about 18 hours. The further deficient JHY903-1 to JHY903-9 strains were cultured in YPD (50 g/L glucose, 10 g/L yeast extract, 20 g/L bacto-peptone). In the case of the strain containing the Coex413-Cas9-target gene gRNA plasmid, it was cultured in SC-HWU medium (with amino acids added except for 20 g/L glucose, 6.7 g/L YNB, histidine, tryptophan, and uracil). In the case of an experiment using YPD5 or SC-HWU medium as the culture conditions for producing acetoin, the initial inoculation cell concentration was _{fixed at OD 600} = 0.5, and 10 ml medium was used in a 100 ml Erlenmeyer flask. Cell concentration, acetoin production and 2,3-butanediol production were measured in the same manner as in Example 4. As a result, among the JHY903-1 to JHY903-9 strains, the cell concentrations of the JHY903-2 strain and the JHY903-6 strain were reduced compared to the JHY903 strain, and there was no significant difference in glucose uptake ( FIGS. 5A and 5B ). In addition, with respect to metabolite production, in the case of the JHY903-4 strain, the production of 2,3-butanediol decreased by about 36.1%, but the production of acetoin did not increase significantly compared to the JHY903 strain. In addition, in the case of the JHY903-4 strain, 2,3-butanediol was still produced despite the removal of the ara1 gene, and through this, it was found that the enzyme involved in the production of 2,3-butanediol is still present in the cell. could In addition, it was confirmed that the production of 2,3-butanediol was reduced in JHY903-2, JHY903-5 and JHY 903-7 strains. Among these strains, in the case of the JHY903-5 strain, the production of acetoin was significantly increased compared to the JHY903 strain (FIG. 6).

Example 7. in vitro Confirmation of reactivity to acetoin of ARA1, YPR1 and YMR226C enzymes through analysis

In order to directly check whether ARA1, YPR1 or YMR226C enzymes reduce acetoin to 2,3-butanediol, _{each protein tagged with His 6} at the C-terminus was purified through Ni-NTA system and then subjected to race Mixed acetoin (3R/S-acetoin) and cofactors (NADH or NADPH) were added and reacted, and then the reactants were analyzed.

At this time, _{to purify the ARA1, YPR1 or YMR226C enzyme having His 6} bound to the C-terminus, Escherichia coli Rogetta gami2 (DE3) was used. To construct a plasmid loaded with each enzyme and _{a gene encoding His 6} , each gene was secured using the primers shown in Table 6 below, and then in the pET-28b vector treated with restriction enzymes of NcoI and NotI. By combining each gene treated with the same restriction enzyme, pET-YPR1-His and pET-YMR226C-His plasmids loaded with a gene encoding YPR1 or YMR226C enzyme _{with His 6 attached to the C-terminus were constructed. PscI and NotI} A pET-ARA1-His plasmid was prepared by binding the ARA1 gene treated with a restriction enzyme.

sequence information SEQ ID NO: ARA1 for pET PscI F TCGA ACATGT CCTCTTCTTCAGTAGCCTCAAC 93 ARA1 for pET NotI R TCGA GCGGCCGC ATACTTTAAATTGTCCAAGTTTGG 94 YPR1 for pET NcoI F TCGA CCATGG GTCCTGCTACGTTAAAGAATTC 95 YPR1 for pET NotI R TCGA GCGGCCG C TTGGAAAATTGGGAAGGATC 96 YMR226C for pET NcoI F TCGA CCATGG GTTCCCAAGGTAGAAAAGCTGC 97 YMR226C for pET NotI R TCGA GCGGCCGC TCCACGGAAGATATGATGAG 98

E. coli transformed with each of the pET-ARA1-His, pET-YPR1-His and pET-YMR226C-His plasmids was inoculated into an LB medium containing 30 μg/ml kanamycin and 20 μg/ml chloramphenicol and then OD at 37°C. _{Incubate until 600} values reached 0.8-1.0. Protein expression was induced by incubation at 30° C. for 5 hours after addition of 1 mM IPTG. After collecting the cells by centrifugation, a binding buffer containing 0.1% protease inhibitor cocktail and 1 mM PMSF (phenylmethylsulfonyl fluoride) (50 mM Tris-HCl (pH 8.0), 100 mM NaCl, 5 mM imidazole, 0.1 mM EDTA) Cells were lysed (20 sec-on, 10 sec-off cycle for 30 minutes) using an ultrasonic disruptor after suspension. After obtaining the supernatant through centrifugation, it was reacted with HisPur Ni-NTA resin at 4°C for 1 hour and 30 minutes. Thereafter, the Ni-NTA resin was washed with a wash buffer (40 mM Tris-HCl (pH 8.0), 500 mM NaCl, 50 mM imidazole, 0.1 mM EDTA) and loaded onto an Econo-Pac® chromatography column. After each enzyme was eluted using an elution buffer (50 mM Tris-HCl (pH 8.0), 50 mM NaCl, 300 mM imidazole, 0.1 mM EDTA), protein purity was confirmed using 1.2% SDS-PAGE. After confirming the purity, only parts with high purity were collected and buffer exchange was performed with the reaction buffer using a Microcon-30kDa Centrifugal Filter Unit. In order to confirm the stereospecificity and selectivity of the ARA1, YPR1 and YMR226C enzymes obtained above, 50 mM Tris-HCl containing 125 μM of each enzyme, 5 mM (R/S)-acetoin, 5 mM NADH or NADPH ( pH 7.0) in the buffer for about 24 hours.

In order to analyze metabolites, 800 μl of the culture medium was centrifuged to obtain a supernatant, which was filtered through a 0.22 μm filter to perform HPLC analysis. UltiMate 3000 HPLC system (Thermo fishers scientific) was used, and BioRad Aminex HPX-87H column and refractive index detector (RI detector) were used. Then, in order to analyze the stereoisomers of acetoin and 2,3-butanediol, the HPLC analysis sample was extracted after mixing 1:1 with ethyl acetate, and GC (gas chromatography) analysis was performed. For the GC analysis, FID (275° C.) of 450-GC (Bruker) equipped with a β-DEXTM 120 column and helium were used as carrier gases (25 ml/min). The temperature of the column was maintained at 75° C. for 8 minutes, increased by 2° C. per minute to reach 85° C., and then maintained for 14 minutes.

As a result, it was confirmed that ARA1, YPR1, and YMR226C enzymes convert acetoin to 2,3-butanediol (FIG. 7). Specifically, as a result of analyzing the reaction products of the ARA1 enzyme and the YPR1 enzyme, respectively, S,S-2,3-butanediol and meso-2,3-butanediol were produced from racemic acetoin, which It means that it has (S)-stereospecific alcohol-forming reductase activity for acetoin. In addition, it was confirmed that the YPR1 enzyme had high stereoselectivity for 3R-acetoin. On the other hand, the YMR226C enzyme had (S)-alcohol-forming reductase activity as well as (R)-alcohol-forming reductase activity and high stereoselectivity for 3S-acetoin. ARA1 enzyme and YPR1 enzyme used both NADH and NADPH as coenzymes, but YMR226C enzyme reduced acetoin to 2,3-butanediol by selectively using only NADPH.

Example 8. Metabolite-producing ability of strains overexpressed with arabinose dehydrogenase (ARA1) or NADP-dependent aldo-kedo reductase (YPR1)

In Example 7, when the ARA1 enzyme and the YPR1 enzyme, each of which confirmed the reduction effect of 2,3-butanediol, were overexpressed in the JHY903 strain, to determine whether the production of meso-2,3-butanediol was increased, ARA1 After the enzyme and YPR1 enzyme were overexpressed in the JHY903 strain, respectively, the cell concentration, acetoin production and 2,3-butanediol production of the strain were measured in the same manner as in Example 4.

First, in order to prepare a plasmid that overexpresses ARA1 and YPR1 enzymes in yeast cells, each gene was secured using the primers shown in Table 7 below. Thereafter, the ARA1 gene treated with NheI and BamHI restriction enzymes was ligated to the p416G vector treated with SpeI and BamHI restriction enzymes to construct p416G-ARA1. In addition, the YPR1 gene treated with the same restriction enzymes was bound to the p416G vector treated with SpeI and XhoI to construct p416G-YPR1.

sequence information SEQ ID NO: ARA1 for pRS NheI F TCGA GCTAGC ATGTCTTCTTCAGTAGCCTC 99 ARA1 for pRS BamH1 R TCGA GGATCC TTAATACTTTAAATTGTCCAAGTTTG 100 YPR1 for pRS SpeI F TCGA ACTAGT ATGCCTGCTACGTTAAAGAA 101 YPR1 for pRS XhoI R TCGA CTCGAG TCATTGGAAAATTGGGAAGGA 102

p413G ( HIS3 , P _{TDH3 ,} T _CYC1 ) (Mumberg et al., 1995), p414G ( TRP1 , P _TDH3, T _CYC1 ) (Mumberg et al., 1995) using a chemical transformation method using lithium acetate. ), and p416G ( URA3 , P _{TDH3 ,} T _CYC1 ) (Mumberg et al., 1995) plasmids were added to prepare JHY903 [EV] strain. In addition, the JHY903 strain containing p413G, p414G, and p416G-ARA1 plasmids to overexpress the ARA1 enzyme (hereinafter, JHY903 [ARA1]) and the JHY903 strain containing the p413G, p414G, and p416G-YPR1 plasmids to overexpress the YPR1 enzyme (hereinafter referred to as JHY903 strain) JHY903 [YPR1]) was prepared.

As a result, there was no significant difference in the cell concentration and glucose consumption of JHY903 [ARA1] and JHY903 [YPR1] compared to the JHY903 strain ( FIGS. 8a and 8b ). On the other hand, compared with the JHY903 strain, the production of 2,3-butanediol of the JHY903 [ARA1] strain and the JHY903 [YPR1] strain was significantly increased (FIG. 9a). In addition, it was confirmed that not only the JHY903 strain, but also the JHY903 [ARA1] strain and the JHY903 [YPR1] strain showed high acetoin production (Fig. 9b).

Example 9. Confirmation of the final strain in which the meso-2,3-butanediol production pathway is blocked and its acetoin-producing ability

By removing the ypr1 gene and the ymr226c gene, the Meso-2,3-butanediol production pathway was blocked to produce a strain with increased acetoin-producing ability. First, in order to reduce the production of Meso-2,3-butanediol, a strain (JHY903-45) in which the ara1 gene and the ypr1 gene were further deleted was prepared, and furthermore, the ara1 gene, the ypr1 gene and the ymr226c gene were further deleted. A strain (JHY903-459) was prepared. At this time, the JHY903 strain, the JHY903-4 strain, the JHY903-45 strain and the JHY903-459 strain were further deleted genes in the same manner as in Example 6, and the cell concentration and acetoin and meso- in the same manner as in Example 4 The production of 2,3-butanediol was confirmed. The production of metabolites of the strains is summarized in Table 8 below.

strain Description Fermentation time(h) Products(g/L) Productivity
of acetoin
(g/(L h)) Yield
of acetoin
(g/g glucose) Glycerol Ethanol R,R/S,S-2,3-BDO Meso-2,3-BDO Acetoin Batch flask fermentation JHY903 Evolved strain of adh1-5β gpd1,2β bdh1β delta::alsS-alsD-noxE 28 0.26±0.00 0.06±0.00 0.07±0.02 0.51±0.06 22.70±0.05 0.811±0.002 0.447±0.000 JHY903-4 JHY903 ara1β 28 0.30±0.07 0.05±0.00 0.09±0.00 0.36±0.02 22.77±0.07 0.813±0.003 0.448±0.000 JHY903-45 JHY903 ara1β ypr1β 28 0.25±0.00 0.06±0.00 0.08±0.00 0.19±0.00 23.04±0.05 0.823±0.002 0.454±0.000 JHY903-459 JHY903 ara1β ypr1β ymr226cβ 28 0.34±0.08 0.05±0.00 0.11±0.00 0.19±0.01 24.76±0.13 0.884±0.005 0.488±0.001

As a result, in the case of the JHY903-45 strain, the production of meso-2,3-butanediol was reduced by about 47.2% compared to the JHY903 strain. In the case of the JHY903-459 strain, it was confirmed that about 24.76 g/L of acetoin was produced from 50.73 g/L of glucose, which was an efficiency of about 99.78% of the theoretical production yield ( FIGS. 10a, 10b and 11 ). ).

<110> Seoul National University R&DB Foundation <120> GENETICALLY ENGINEERED YEAST HAVING ACETOIN PRODUCING ABILITY AND METHOD FOR PRODUCTING ACETOIN USING THE SAME <130> FPD/202003-0098 <160> 140 <170> KoPatentIn 3.0 <210> 1 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> F primer for ADH1 deletion <400> 1 ttcaagctat accaagcata caatcaacta tctcatatac acagctgaag cttcgtacgc 60 60 <210> 2 <211> 59 <212> DNA <213> Artificial Sequence <220> <223> R primer for ADH1 deletion <400> 2 cttatttaat aataaaaatc ataaatcata agaaattcgc gcataggcca ctagtggat 59 <210> 3 <211> 59 <212> DNA <213> Artificial Sequence <220> <223> F primer for ADH2 deletion <400> 3 tacaatcaac tatcaactat taactatatc gtaatacaca cagctgaagc ttcgtacgc 59 <210> 4 <211> 59 <212> DNA <213> Artificial Sequence <220> <223> R primer for ADH2 deletion <400> 4 ataatgaaaa ctataaatcg taaagacata agagatccgc gcataggcca ctagtggat 59 <210> 5 <211> 59 <212> DNA <213> Artificial Sequence <220> <223> F primer for ADH3 deletion <400> 5 gttaaaacta ggaatagtat agtcataagt taacaccatc cagctgaagc ttcgtacgc 59 <210> 6 <211> 59 <212> DNA <213> Artificial Sequence <220> <223> R primer for ADH3 deletion <400> 6 acaaagactt tcataaaaag tttgggtgcg taacacgcta gcataggcca ctagtggat 59 <210> 7 <211> 59 <212> DNA <213> Artificial Sequence <220> <223> F primer for ADH4 deletion <400> 7 caagtttaca tttgcaacaa ctaatagtca aataagaaaa cagctgaagc ttcgtacgc 59 <210> 8 <211> 59 <212> DNA <213> Artificial Sequence <220> <223> R primer for ADH4 deletion <400> 8 gcacacgcat aattgacgtt tatgagttcg ttcgattttt gcataggcca ctagtggat 59 <210> 9 <211> 59 <212> DNA <213> Artificial Sequence <220> <223> F primer for ADH5 deletion <400> 9 agaaaattat ttaactacat atctacaaaa tcaaagcatc cagctgaagc ttcgtacgc 59 <210> 10 <211> 59 <212> DNA <213> Artificial Sequence <220> <223> R primer for ADH5 deletion <400> 10 taaaaagtaa aaatatattc atcaaattcg ttacaaaaga gcataggcca ctagtggat 59 <210> 11 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> F primer for GPD1 deletion <400> 11 cacccccccc ctccacaaac acaaatattg ataatataaa gcagctgaag cttcgtacgc 60 60 <210> 12 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> R primer for GPD1 deletion <400> 12 aagtggggga aagtatgata tgttatcttt ctccaataaa tgcataggcc actagtggat 60 60 <210> 13 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> F primer for GPD2 deletion <400> 13 tctctttccc tttccttttc cttcgctccc cttccttatc acagctgaag cttcgtacgc 60 60 <210> 14 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> R primer for GPD2 deletion <400> 14 ggcaacagga aagatcagag ggggaggggg ggggagagtg tgcataggcc actagtggat 60 60 <210> 15 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> F primer for identification of ADH1 deletion <400> 15 caccatatcc gcaatgac 18 <210> 16 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> R primer for identification of ADH1 deletion <400> 16 gtgttgtcct ctgaggac 18 <210> 17 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> F primer for identification of ADH2 deletion <400> 17 accgggcatc tccaactt 18 <210> 18 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> R primer for identification of ADH2 deletion <400> 18 ccatgtctac agtttagagg 20 <210> 19 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> F primer for identification of ADH3 deletion <400> 19 atgagcagca gccattttg 19 <210> 20 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> R primer for identification of ADH3 deletion <400> 20 tgatggtgat aatgtctctc a 21 <210> 21 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> F primer for identification of ADH4 deletion <400> 21 aagaactagt ttttagttcg cg 22 <210> 22 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> R primer for identification of ADH4 deletion <400> 22 agaacttccg ttcttctttt 20 <210> 23 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> F primer for identification of ADH5 deletion <400> 23 ctgctatctg cttgtagaag 20 <210> 24 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> R primer for identification of ADH5 deletion <400> 24 gaaacgtttg tataggttgt 20 <210> 25 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> F primer for identification of GPD1 deletion <400> 25 cgccttgctt ctctcccctt 20 <210> 26 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> R primer for identification of GPD1 deletion <400> 26 ccgacagcct ctgaatgagt 20 <210> 27 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> F primer for identification of GPD2 deletion <400> 27 tacggaccta ttgccattgt 20 <210> 28 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> R primer for identification of GPD2 deletion <400> 28 ttaagggcta tagataacag 20 <210> 29 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> F primer for BDH1 deletion <400> 29 gatttgctca cgctactttg 20 <210> 30 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> R primer for BDH1 deletion <400> 30 gccatgcttt gttttagacg 20 <210> 31 <211> 1713 <212> DNA <213> Artificial Sequence <220> <223> Bacillus subtilis alsS <400> 31 atgacaaaag caacaaaaga acaaaaatcc cttgtgaaaa acagaggggc ggagcttgtt 60 gttgattgct tagtggagca aggtgtcaca catgtatttg gcattccagg tgcaaaaatt 120 gatgcggtat ttgacgcttt acaagataaa ggacctgaaa ttatcgttgc ccggcacgaa 180 caaaacgcag cattcatggc ccaagcagtc ggccgtttaa ctggaaaacc gggagtcgtg 240 ttagtcacat caggaccggg tgcctctaac ttggcaacag gcctgctgac agcgaacact 300 gaaggagacc ctgtcgttgc gcttgctgga aacgtgatcc gtgcagatcg tttaaaacgg 360 acacatcaat ctttggataa tgcggcgcta ttccagccga ttacaaaata cagtgtagaa 420 gttcaagatg taaaaaatat accggaagct gttacaaatg catttaggat agcgtcagca 480 gggcaggctg gggccgcttt tgtgagcttt ccgcaagatg ttgtgaatga agtcacaaat 540 acgaaaaacg tgcgtgctgt tgcagcgcca aaactcggtc ctgcagcaga tgatgcaatc 600 agtgcggcca tagcaaaaat ccaaacagca aaacttcctg tcgttttggt cggcatgaaa 660 ggcggaagac cggaagcaat taaagcggtt cgcaagcttt tgaaaaaggt tcagcttcca 720 tttgttgaaa catatcaagc tgccggtacc ctttctagag atttagagga tcaatatttt 780 ggccgtatcg gtttgttccg caaccagcct ggcgatttac tgctagagca ggcagatgtt 840 gttctgacga tcggctatga cccgattgaa tatgatccga aattctggaa tatcaatgga 900 gaccggacaa ttatccattt agacgagatt atcgctgaca ttgatcatgc ttaccagcct 960 gatcttgaat tgatcggtga cattccgtcc acgatcaatc atatcgaaca cgatgctgtg 1020 aaagtggaat ttgcagagcg tgagcagaaa atcctttctg atttaaaaca atatatgcat 1080 gaaggtgagc aggtgcctgc agattggaaa tcagacagag cgcaccctct tgaaatcgtt 1140 aaagagttgc gtaatgcagt cgatgatcat gttacagtaa cttgcgatat cggttcgcac 1200 gccatttgga tgtcacgtta tttccgcagc tacgagccgt taacattaat gatcagtaac 1260 ggtatgcaaa cactcggcgt tgcgcttcct tgggcaatcg gcgcttcatt ggtgaaaccg 1320 ggagaaaaag tggtttctgt ctctggtgac ggcggtttct tattctcagc aatggaatta 1380 gagacagcag ttcgactaaa agcaccaatt gtacacattg tatggaacga cagcacatat 1440 gacatggttg cattccagca attgaaaaaa tataaccgta catctgcggt cgatttcgga 1500 aatatcgata tcgtgaaata tgcggaaagc ttcggagcaa ctggcttgcg cgtagaatca 1560 ccagaccagc tggcagatgt tctgcgtcaa ggcatgaacg ctgaaggtcc tgtcatcatc 1620 gatgtcccgg ttgactacag tgataacatt aatttagcaa gtgacaagct tccgaaagaa 1680 ttcggggaac tcatgaaaac gaaagctctc tag 1713 <210> 32 <211> 570 <212> PRT <213> Artificial Sequence <220> <223> Bacillus subtilis alsS <400> 32 Met Thr Lys Ala Thr Lys Glu Gln Lys Ser Leu Val Lys Asn Arg Gly 1 5 10 15 Ala Glu Leu Val Val Asp Cys Leu Val Glu Gln Gly Val Thr His Val 20 25 30 Phe Gly Ile Pro Gly Ala Lys Ile Asp Ala Val Phe Asp Ala Leu Gln 35 40 45 Asp Lys Gly Pro Glu Ile Ile Val Ala Arg His Glu Gln Asn Ala Ala 50 55 60 Phe Met Ala Gln Ala Val Gly Arg Leu Thr Gly Lys Pro Gly Val Val 65 70 75 80 Leu Val Thr Ser Gly Pro Gly Ala Ser Asn Leu Ala Thr Gly Leu Leu 85 90 95 Thr Ala Asn Thr Glu Gly Asp Pro Val Val Ala Leu Ala Gly Asn Val 100 105 110 Ile Arg Ala Asp Arg Leu Lys Arg Thr His Gln Ser Leu Asp Asn Ala 115 120 125 Ala Leu Phe Gln Pro Ile Thr Lys Tyr Ser Val Glu Val Gln Asp Val 130 135 140 Lys Asn Ile Pro Glu Ala Val Thr Asn Ala Phe Arg Ile Ala Ser Ala 145 150 155 160 Gly Gln Ala Gly Ala Ala Phe Val Ser Phe Pro Gln Asp Val Val Asn 165 170 175 Glu Val Thr Asn Thr Lys Asn Val Arg Ala Val Ala Ala Pro Lys Leu 180 185 190 Gly Pro Ala Ala Asp Asp Ala Ile Ser Ala Ala Ile Ala Lys Ile Gln 195 200 205 Thr Ala Lys Leu Pro Val Val Leu Val Gly Met Lys Gly Gly Arg Pro 210 215 220 Glu Ala Ile Lys Ala Val Arg Lys Leu Leu Lys Lys Val Gln Leu Pro 225 230 235 240 Phe Val Glu Thr Tyr Gln Ala Ala Gly Thr Leu Ser Arg Asp Leu Glu 245 250 255 Asp Gln Tyr Phe Gly Arg Ile Gly Leu Phe Arg Asn Gln Pro Gly Asp 260 265 270 Leu Leu Leu Glu Gln Ala Asp Val Val Leu Thr Ile Gly Tyr Asp Pro 275 280 285 Ile Glu Tyr Asp Pro Lys Phe Trp Asn Ile Asn Gly Asp Arg Thr Ile 290 295 300 Ile His Leu Asp Glu Ile Ile Ala Asp Ile Asp His Ala Tyr Gln Pro 305 310 315 320 Asp Leu Glu Leu Ile Gly Asp Ile Pro Ser Thr Ile Asn His Ile Glu 325 330 335 His Asp Ala Val Lys Val Glu Phe Ala Glu Arg Glu Gln Lys Ile Leu 340 345 350 Ser Asp Leu Lys Gln Tyr Met His Glu Gly Glu Gln Val Pro Ala Asp 355 360 365 Trp Lys Ser Asp Arg Ala His Pro Leu Glu Ile Val Lys Glu Leu Arg 370 375 380 Asn Ala Val Asp Asp His Val Thr Val Thr Cys Asp Ile Gly Ser His 385 390 395 400 Ala Ile Trp Met Ser Arg Tyr Phe Arg Ser Tyr Glu Pro Leu Thr Leu 405 410 415 Met Ile Ser Asn Gly Met Gln Thr Leu Gly Val Ala Leu Pro Trp Ala 420 425 430 Ile Gly Ala Ser Leu Val Lys Pro Gly Glu Lys Val Val Ser Val Ser 435 440 445 Gly Asp Gly Gly Phe Leu Phe Ser Ala Met Glu Leu Glu Thr Ala Val 450 455 460 Arg Leu Lys Ala Pro Ile Val His Ile Val Trp Asn Asp Ser Thr Tyr 465 470 475 480 Asp Met Val Ala Phe Gln Gln Leu Lys Lys Tyr Asn Arg Thr Ser Ala 485 490 495 Val Asp Phe Gly Asn Ile Asp Ile Val Lys Tyr Ala Glu Ser Phe Gly 500 505 510 Ala Thr Gly Leu Arg Val Glu Ser Pro Asp Gln Leu Ala Asp Val Leu 515 520 525 Arg Gln Gly Met Asn Ala Glu Gly Pro Val Ile Ile Asp Val Pro Val 530 535 540 Asp Tyr Ser Asp Asn Ile Asn Leu Ala Ser Asp Lys Leu Pro Lys Glu 545 550 555 560 Phe Gly Glu Leu Met Lys Thr Lys Ala Leu 565 570 <210> 33 <211> 768 <212> DNA <213> Artificial Sequence <220> <223> Bacillus subtilis alsD <400> 33 atgaaacgag aaagcaacat tcaagtgctc agccgtggtc aaaaagatca gcctgtgagc 60 cagatttatc aagtatcaac aatgacttct ctattagacg gagtatatga cggagatttt 120 gaactgtcag agattccgaa atatggagac ttcggtatcg gaacctttaa caagcttgac 180 ggagagctga ttgggtttga cggcgaattt taccgtcttc gctcagacgg aaccgcgaca 240 ccggtccaaa atggagaccg ttcaccgttc tgttcattta cgttctttac accggacatg 300 acgcacaaaa ttgatgcgaa aatgacacgc gaagactttg aaaaagagat caacagcatg 360 ctgccaagca gaaacttatt ttatgcaatt cgcattgacg gattgtttaa aaaggtgcag 420 acaagaacag tagaacttca agaaaaacct tacgtgccaa tggttgaagc ggtcaaaaca 480 cagccgattt tcaacttcga caacgtgaga ggaacgattg taggtttctt gacaccagct 540 tatgcaaacg gaatcgccgt ttctggctat cacctgcact tcattgacga aggacgcaat 600 tcaggcggac acgtttttga ctatgtgctt gaggattgca cggttacgat ttctcaaaaa 660 atgaacatga atctcagact tccgaacaca gcggatttct ttaatgcgaa tctggataac 720 cctgattttg cgaaagatat cgaaacaact gaaggaagcc ctgaataa 768 <210> 34 <211> 255 <212> PRT <213> Artificial Sequence <220> <223> Bacillus subtilis alsD <400> 34 Met Lys Arg Glu Ser Asn Ile Gln Val Leu Ser Arg Gly Gln Lys Asp 1 5 10 15 Gln Pro Val Ser Gln Ile Tyr Gln Val Ser Thr Met Thr Ser Leu Leu 20 25 30 Asp Gly Val Tyr Asp Gly Asp Phe Glu Leu Ser Glu Ile Pro Lys Tyr 35 40 45 Gly Asp Phe Gly Ile Gly Thr Phe Asn Lys Leu Asp Gly Glu Leu Ile 50 55 60 Gly Phe Asp Gly Glu Phe Tyr Arg Leu Arg Ser Asp Gly Thr Ala Thr 65 70 75 80 Pro Val Gln Asn Gly Asp Arg Ser Pro Phe Cys Ser Phe Thr Phe Phe 85 90 95 Thr Pro Asp Met Thr His Lys Ile Asp Ala Lys Met Thr Arg Glu Asp 100 105 110 Phe Glu Lys Glu Ile Asn Ser Met Leu Pro Ser Arg Asn Leu Phe Tyr 115 120 125 Ala Ile Arg Ile Asp Gly Leu Phe Lys Lys Val Gln Thr Arg Thr Val 130 135 140 Glu Leu Gln Glu Lys Pro Tyr Val Pro Met Val Glu Ala Val Lys Thr 145 150 155 160 Gln Pro Ile Phe Asn Phe Asp Asn Val Arg Gly Thr Ile Val Gly Phe 165 170 175 Leu Thr Pro Ala Tyr Ala Asn Gly Ile Ala Val Ser Gly Tyr His Leu 180 185 190 His Phe Ile Asp Glu Gly Arg Asn Ser Gly Gly His Val Phe Asp Tyr 195 200 205 Val Leu Glu Asp Cys Thr Val Thr Ile Ser Gln Lys Met Asn Met Asn 210 215 220 Leu Arg Leu Pro Asn Thr Ala Asp Phe Phe Asn Ala Asn Leu Asp Asn 225 230 235 240 Pro Asp Phe Ala Lys Asp Ile Glu Thr Thr Glu Gly Ser Pro Glu 245 250 255 <210> 35 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> F primer for alsS <400> 35 ctgaggatcc atgacaaaag caacaaaaga ac 32 <210> 36 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> R primer for alsS <400> 36 ctgactcgag ctagagagct ttcgttttca 30 <210> 37 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> F primer for alsD <400> 37 ctgaggatcc atgaaacgag aaagcaacat 30 <210> 38 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> R primer for alsD <400> 38 ctgactcgag ttattcaggg cttccttcag 30 <210> 39 <211> 644 <212> DNA <213> Artificial Sequence <220> <223> TDH3 promoter <400> 39 tcattatcaa tactcgccat ttcaaagaat acgtaaataa ttaatagtag tgattttcct 60 aactttattt agtcaaaaaa ttagcctttt aattctgctg taacccgtac atgcccaaaa 120 tagggggcgg gttacacaga atatataaca tcgtaggtgt ctgggtgaac agtttattcc 180 tggcatccac taaatataat ggagcccgct ttttaagctg gcatccagaa aaaaaaagaa 240 tcccagcacc aaaatattgt tttcttcacc aaccatcagt tcataggtcc attctcttag 300 cgcaactaca gagaacaggg gcacaaacag gcaaaaaacg ggcacaacct caatggagtg 360 atgcaacctg cctggagtaa atgatgacac aaggcaattg acccacgcat gtatctatct 420 cattttctta caccttctat taccttctgc tctctctgat ttggaaaaag ctgaaaaaaa 480 aggttgaaac cagttccctg aaattattcc cctacttgac taataagtat ataaagacgg 540 taggtattga ttgtaattct gtaaatctat ttcttaaact tcttaaattc tacttttata 600 gttagtcttt tttttagttt taaaacacca gaacttagtt tcga 644 <210> 40 <211> 401 <212> DNA <213> Artificial Sequence <220> <223> TEF1 promoter <400> 40 atagcttcaa aatgtttcta ctcctttttt actcttccag attttctcgg actccgcgca 60 tcgccgtacc acttcaaaac acccaagcac agcatactaa atttcccctc tttcttcctc 120 tagggtgtcg ttaattaccc gtactaaagg tttggaaaag aaaaaagaga ccgcctcgtt 180 tctttttctt cgtcgaaaaa ggcaataaaa atttttatca cgtttctttt tcttgaaaat 240 tttttttttg atttttttt ctttcgatga cctcccattg atatttaagt taataaacgg 300 tcttcaattt ctcaagtttc agtttcattt ttcttgttct attacaactt tttttacttc 360 ttgctcatta gaaagaaagc atagcaatct aatctaagtt t 401 <210> 41 <211> 248 <212> DNA <213> Artificial Sequence <220> <223> CYC1 terminator <400> 41 tcatgtaatt agttatgtca cgcttacatt cacgccctcc ccccacatcc gctctaaccg 60 aaaaggaagg agttagacaa cctgaagtct aggtccctat ttattttttt atagttatgt 120 tagtattaag aacgttattt atatttcaaa tttttctttt ttttctgtac agacgcgtgt 180 acgcatgtaa cattatactg aaaaccttgc ttgagaaggt tttgggacgc tcgaaggctt 240 taatttgc 248 <210> 42 <211> 401 <212> DNA <213> Artificial Sequence <220> <223> GPM1 terminator <400> 42 gtctgaagaa tgaatgattt gatgatttct ttttccctcc atttttctta ctgaatatat 60 caatgatata gacttgtata gtttattatt tcaaattaag tagctatata tagtcaagat 120 aacgtttgtt tgacacgatt acattattcg tcgacatctt ttttcagcct gtcgtggtag 180 caatttgagg agtattatta attgaatagg ttcattttgc gctcgcataa acagttttcg 240 tcagggacag tatgttggaa tgagtggtaa ttaatggtga catgacatgt tatagcaata 300 accttgatgt ttacatcgta gtttaatgta caccccgcga attcgttcaa gtaggagtgc 360 accaattgca aagggaaaag ctgaatgggc agttcgaata g 401 <210> 43 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> F primer for GPM1 terminator <400> 43 gtcactcgag gtctgaagaa tgaatgattt g 31 <210> 44 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> R primer for GPM1 terminator <400> 44 gtcaggtacc tattcgaact gcccattca 29 <210> 45 <211> 1341 <212> DNA <213> Artificial Sequence <220> <223> Lactococcus lactis noxE <400> 45 atgaaaatcg tagttatcgg tacgaaccac gcaggcattg ctacagcaaa tacattaatt 60 gatcgatatc caggccatga gattgttatg attgaccgta acagtaatat gagttacttg 120 gggtgtggga cagctatttg ggtcggaaga caaattgaaa aaccagatga gctgttttat 180 gccaaagcag aagattttga aaaaaaggga gtaaagatat taacagaaac agaagtttca 240 gaaattgact tactaataa aatgatttat gccaagtcaa aaactggaga aaagattaca 300 gaaagttatg ataaactcgt tctggcaaca ggttcacgtc caattattcc taacttgcca 360 ggaaaagatc ttaaaggcat tcatttttta aaactttttc aagaagggca agccattgac 420 gaagagtttg ctaagaatga tgtgaaacgg attgctgtga ttggtgctgg ttatattggg 480 acagaaattg ctgaagctgc caaacgtcgt ggaaaagaag tcctactttt tgatgcagaa 540 agtacttcac ttgcttcata ttatgatgaa gagtttgcta aagggatgga tgaaaatctt 600 gcccaacatg gaattgaact ccattttggg gaattagctc aagagtttaa ggcaaatgaa 660 aaaggtcatg tatcacagat tgtaactaat aaatcaactt atgatgttga cctcgttatt 720 aattgtattg gctttacagc caatagtgca ttggctggtg aacatttaga aacctttaaa 780 aatggagcaa tcaaagtgga taaacatcaa caaagtagtg acccagatgt ttctgctgta 840 ggagatgttg ccacaatcta ttctaatgct ttacaagact tcacctacat tgcccttgcc 900 tcaaacgctg ttcgctcagg gattgttgct ggtcataata ttggaggaaa atcaatagag 960 tctgttggtg tacaaggttc taatggaatc tctatttttg gttacaatat gacttctacg 1020 ggcttgtcgg ttaaagctgc gaaaaaaatc ggcctagaag tttcatttag tgattttgaa 1080 gataagcaaa aagcatggtt ccttcatgaa aataatgata gtgtgaaaat tcgtatcgtt 1140 tatgaaacaa aaaatcgcag aattattggt gctcaacttg ctagcaagag tgaaataatt 1200 gcaggaaata ttaatatgtt tagtttagct attcaagaaa agaaaacgat tgatgaatta 1260 gccttacttg atttattctt cttaccacac ttcaatagtc catataatta catgactgtt 1320 gcagctttaa atgcaaaata a 1341 <210> 46 <211> 446 <212> PRT <213> Artificial Sequence <220> <223> Lactococcus lactis noxE <400> 46 Met Lys Ile Val Val Ile Gly Thr Asn His Ala Gly Ile Ala Thr Ala 1 5 10 15 Asn Thr Leu Ile Asp Arg Tyr Pro Gly His Glu Ile Val Met Ile Asp 20 25 30 Arg Asn Ser Asn Met Ser Tyr Leu Gly Cys Gly Thr Ala Ile Trp Val 35 40 45 Gly Arg Gln Ile Glu Lys Pro Asp Glu Leu Phe Tyr Ala Lys Ala Glu 50 55 60 Asp Phe Glu Lys Lys Gly Val Lys Ile Leu Thr Glu Thr Glu Val Ser 65 70 75 80 Glu Ile Asp Phe Thr Asn Lys Met Ile Tyr Ala Lys Ser Lys Thr Gly 85 90 95 Glu Lys Ile Thr Glu Ser Tyr Asp Lys Leu Val Leu Ala Thr Gly Ser 100 105 110 Arg Pro Ile Ile Pro Asn Leu Pro Gly Lys Asp Leu Lys Gly Ile His 115 120 125 Phe Leu Lys Leu Phe Gln Glu Gly Gln Ala Ile Asp Glu Glu Phe Ala 130 135 140 Lys Asn Asp Val Lys Arg Ile Ala Val Ile Gly Ala Gly Tyr Ile Gly 145 150 155 160 Thr Glu Ile Ala Glu Ala Ala Lys Arg Arg Gly Lys Glu Val Leu Leu 165 170 175 Phe Asp Ala Glu Ser Thr Ser Leu Ala Ser Tyr Tyr Asp Glu Glu Phe 180 185 190 Ala Lys Gly Met Asp Glu Asn Leu Ala Gln His Gly Ile Glu Leu His 195 200 205 Phe Gly Glu Leu Ala Gln Glu Phe Lys Ala Asn Glu Lys Gly His Val 210 215 220 Ser Gln Ile Val Thr Asn Lys Ser Thr Tyr Asp Val Asp Leu Val Ile 225 230 235 240 Asn Cys Ile Gly Phe Thr Ala Asn Ser Ala Leu Ala Gly Glu His Leu 245 250 255 Glu Thr Phe Lys Asn Gly Ala Ile Lys Val Asp Lys His Gln Gln Ser 260 265 270 Ser Asp Pro Asp Val Ser Ala Val Gly Asp Val Ala Thr Ile Tyr Ser 275 280 285 Asn Ala Leu Gln Asp Phe Thr Tyr Ile Ala Leu Ala Ser Asn Ala Val 290 295 300 Arg Ser Gly Ile Val Ala Gly His Asn Ile Gly Gly Lys Ser Ile Glu 305 310 315 320 Ser Val Gly Val Gln Gly Ser Asn Gly Ile Ser Ile Phe Gly Tyr Asn 325 330 335 Met Thr Ser Thr Gly Leu Ser Val Lys Ala Ala Lys Lys Ile Gly Leu 340 345 350 Glu Val Ser Phe Ser Asp Phe Glu Asp Lys Gln Lys Ala Trp Phe Leu 355 360 365 His Glu Asn Asn Asp Ser Val Lys Ile Arg Ile Val Tyr Glu Thr Lys 370 375 380 Asn Arg Arg Ile Ile Gly Ala Gln Leu Ala Ser Lys Ser Glu Ile Ile 385 390 395 400 Ala Gly Asn Ile Asn Met Phe Ser Leu Ala Ile Gln Glu Lys Lys Thr 405 410 415 Ile Asp Glu Leu Ala Leu Leu Asp Leu Phe Phe Leu Pro His Phe Asn 420 425 430 Ser Pro Tyr Asn Tyr Met Thr Val Ala Ala Leu Asn Ala Lys 435 440 445 <210> 47 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> F primer for noxE <400> 47 gactaagctt atgaaaatcg tagttatcgg t 31 <210> 48 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> R primer for noxE <400> 48 gactctcgag ttattttgca tttaaagctg ca 32 <210> 49 <211> 821 <212> DNA <213> Artificial Sequence <220> <223> FBA1 porter <400> 49 atccaactgg caccgctggc ttgaacaaca ataccagcct tccaacttct gtaaataacg 60 gcggtacgcc agtgccacca gtaccgttac ctttcggtat acctcctttc cccatgtttc 120 caatgccctt catgcctcca acggctacta tcacaaatcc tcatcaagct gacgcaagcc 180 ctaagaaatg aataacaata ctgacagtac taaataattg cctacttggc ttcacatacg 240 ttgcatacgt cgatatagat aataatgata atgacagcag gattatcgta atacgtaata 300 gttgaaaatc tcaaaaatgt gtgggtcatt acgtaaataa tgataggaat gggattcttc 360 tatttttcct ttttccattc tagcagccgt cgggaaaacg tggcatcctc tctttcgggc 420 tcaattggag tcacgctgcc gtgagcatcc tctctttcca tatctaacaa ctgagcacgt 480 aaccaatgga aaagcatgag cttagcgttg ctccaaaaaa gtattggatg gttaatacca 540 tttgtctgtt ctcttctgac tttgactcct caaaaaaaaa aaatctacaa tcaacagatc 600 gcttcaatta cgccctcaca aaaacttttt tccttcttct tcgccccacgt taaattttat 660 ccctcatgtt gtctaacgga tttctgcact tgatttatta taaaaagaca aagacataat 720 acttctctat caatttcagt tattgttctt ccttgcgtta ttcttctgtt cttctttttc 780 ttttgtcata tataaccata accaagtaat acatattcaa a 821 <210> 50 <211> 202 <212> DNA <213> Artificial Sequence <220> <223> FBA1 terminator <400> 50 gttaattcaa attaattgat atagtttttt aatgagtatt gaatctgttt agaaataatg 60 gaatattatt tttatttatt tatttatatt attggtcggc tcttttcttc tgaaggtcaa 120 tgacaaaatg atatgaagga aataatgatt tctaaaattt tacaacgtaa gatattttta 180 caaaagccta gctcatcttt tg 202 <210> 51 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> F primer for FBA1 promoter <400> 51 gtcagagctc atccaactgg caccgctg 28 <210> 52 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> R primer for FBA1 promoter <400> 52 gtcagagctc atccaactgg caccgctg 28 <210> 53 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> F primer for FBA1 terminator <400> 53 gtcactcgag taagttaatt caaattaatt gatatag 37 <210> 54 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> R primer for FBA1 terminator <400> 54 gtcaggtacc caaaagatga gctaggcttt 30 <210> 55 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Universal F primer <400> 55 gactacgcgt ggaacaaaag ctggagctc 29 <210> 56 <211> 49 <212> DNA <213> Artificial Sequence <220> <223> Universal R primer <400> 56 gactacgcgt gcggccgcta atggcgcgcc atagggcgaa ttgggtacc 49 <210> 57 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> ARA1 target gRNA F <400> 57 tacgaatggc tctgtctcgt gttttagagc tagaaatagc 40 <210> 58 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> ARA1 target gRNA R <400> 58 acgagacaga gccattcgta gatcatttat ctttcactgc 40 <210> 59 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> BDH2 target gRNA F <400> 59 aaggtagttg tcgagcccac gttttagagc tagaaatagc 40 <210> 60 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> BDH2 target gRNA R <400> 60 gtgggctcga caactacctt gatcatttat ctttcactgc 40 <210> 61 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> YPR1 target gRNA F <400> 61 atgcaagagt tgccaaagac gttttagagc tagaaatagc 40 <210> 62 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> YPR1 target gRNA R <400> 62 gtctttggca actcttgcat gatcatttat ctttcactgc 40 <210> 63 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> GRE3 target gRNA F <400> 63 tggttgtaga atcaagcccg gttttagagc tagaaatagc 40 <210> 64 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> GRE3 target gRNA R <400> 64 cgggcttgat tctacaacca gatcatttat ctttcactgc g 41 <210> 65 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> YJR096W target gRNA F <400> 65 agaagcggtt gatgaaggat gttttagagc tagaaatagc 40 <210> 66 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> YJR096W target gRNA R <400> 66 atccttcatc aaccgcttct gatcatttat ctttcactgc 40 <210> 67 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> YMR226C target gRNA F <400> 67 agttagatac agaggtaacg gttttagagc tagaaatagc 40 <210> 68 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> YMR226C target gRNA R <400> 68 cgttacctct gtatctaact gatcatttat ctttcactgc 40 <210> 69 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> NRE1 target gRNA F <400> 69 aggtatcggt aagtccatcg gttttagagc tagaaatagc 40 <210> 70 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> NRE1 target gRNA R <400> 70 cgatggactt accgatacct gatcatttat ctttcactgc g 41 <210> 71 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> IRC24 target gRNA F <400> 71 cgtctacggc gtagcaagaa gttttagagc tagaaatagc 40 <210> 72 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> IRC24 target gRNA R <400> 72 ttcttgctac gccgtagacg gatcatttat ctttcactgc g 41 <210> 73 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> ENV9 target gRNA F <400> 73 aggaagattg ctgtagtaac gttttagagc tagaaatagc 40 <210> 74 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> ENV9 target gRNA R <400> 74 gttactacag caatcttcct gatcatttat ctttcactgc g 41 <210> 75 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ARA1 upstream F <400> 75 gcctccacct taacatctta 20 <210> 76 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ARA1 downstream R <400> 76 acgtacggcg aatgattata 20 <210> 77 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> BDH2 upstream F <400> 77 gcattggtta gctcagatat 20 <210> 78 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> BDH2 downstream R <400> 78 ctgccccact tttatatgtc 20 <210> 79 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> YPR1 upstream F <400> 79 agcctatttg gaaaagactg 20 <210> 80 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> YPR1 downstream R <400> 80 cagtagaagc gcaactagta 20 <210> 81 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GRE3 upstream F <400> 81 tgtttcccaa ttgttgctgg 20 <210> 82 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GRE3 downstream R <400> 82 ttgggaccgc tttgctctct 20 <210> 83 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> YJR096W upstream F <400> 83 ttgtccttat ttgaggctcc 20 <210> 84 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> YJR096W downstream R <400> 84 attgcgctta tcttttggca 20 <210> 85 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> YMR226C upstream F <400> 85 aacactcgac cagaacgatc 20 <210> 86 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> YMR226C downstream R <400> 86 cagcctagtt tagccaaatc 20 <210> 87 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> NRE1 upstream F <400> 87 tcaatatctc cgctacaacg 20 <210> 88 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> NRE1 downstream R <400> 88 gatgtaatgt gacggcagcc 20 <210> 89 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> IRC24 upstream F <400> 89 ttcttgtcaa caggtgctag 20 <210> 90 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> IRC24 downstream R <400> 90 ttaccgatac ctctggaaac 20 <210> 91 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ENV9 upstream F <400> 91 attgagccac aggtctttcg 20 <210> 92 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ENV9 downstream R <400> 92 agatccaagc ctgatagacc 20 <210> 93 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> ARA1 for pET PscI F <400> 93 tcgaacatgt cctcttcttc agtagcctca ac 32 <210> 94 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> ARA1 for pET NotI R <400> 94 tcgagcggcc gcatacttta aattgtccaa gtttgg 36 <210> 95 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> YPR1 for pET NcoI F <400> 95 tcgaccatgg gtcctgctac gttaaagaat tc 32 <210> 96 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> YPR1 for pET NotI R <400> 96 tcgagcggcc gcttggaaaa ttgggaagga tc 32 <210> 97 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> YMR226C for pET NcoI F <400> 97 tcgaccatgg gttcccaagg tagaaaagct gc 32 <210> 98 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> YMR226C for pET NotI R <400> 98 tcgagcggcc gctccacgga agatatgatg ag 32 <210> 99 <211> 348 <212> PRT <213> Artificial Sequence <220> <223> Saccharomyces cerevisiae ADH1 <400> 99 Met Ser Ile Pro Glu Thr Gln Lys Gly Val Ile Phe Tyr Glu Ser His 1 5 10 15 Gly Lys Leu Glu Tyr Lys Asp Ile Pro Val Pro Lys Pro Lys Ala Asn 20 25 30 Glu Leu Leu Ile Asn Val Lys Tyr Ser Gly Val Cys His Thr Asp Leu 35 40 45 His Ala Trp His Gly Asp Trp Pro Leu Pro Val Lys Leu Pro Leu Val 50 55 60 Gly Gly His Glu Gly Ala Gly Val Val Val Gly Met Gly Glu Asn Val 65 70 75 80 Lys Gly Trp Lys Ile Gly Asp Tyr Ala Gly Ile Lys Trp Leu Asn Gly 85 90 95 Ser Cys Met Ala Cys Glu Tyr Cys Glu Leu Gly Asn Glu Ser Asn Cys 100 105 110 Pro His Ala Asp Leu Ser Gly Tyr Thr His Asp Gly Ser Phe Gln Gln 115 120 125 Tyr Ala Thr Ala Asp Ala Val Gln Ala Ala His Ile Pro Gln Gly Thr 130 135 140 Asp Leu Ala Gln Val Ala Pro Ile Leu Cys Ala Gly Ile Thr Val Tyr 145 150 155 160 Lys Ala Leu Lys Ser Ala Asn Leu Met Ala Gly His Trp Val Ala Ile 165 170 175 Ser Gly Ala Ala Gly Gly Leu Gly Ser Leu Ala Val Gln Tyr Ala Lys 180 185 190 Ala Met Gly Tyr Arg Val Leu Gly Ile Asp Gly Gly Glu Gly Lys Glu 195 200 205 Glu Leu Phe Arg Ser Ile Gly Gly Glu Val Phe Ile Asp Phe Thr Lys 210 215 220 Glu Lys Asp Ile Val Gly Ala Val Leu Lys Ala Thr Asp Gly Gly Ala 225 230 235 240 His Gly Val Ile Asn Val Ser Val Ser Glu Ala Ala Ile Glu Ala Ser 245 250 255 Thr Arg Tyr Val Arg Ala Asn Gly Thr Thr Val Leu Val Gly Met Pro 260 265 270 Ala Gly Ala Lys Cys Cys Ser Asp Val Phe Asn Gln Val Val Lys Ser 275 280 285 Ile Ser Ile Val Gly Ser Tyr Val Gly Asn Arg Ala Asp Thr Arg Glu 290 295 300 Ala Leu Asp Phe Phe Ala Arg Gly Leu Val Lys Ser Pro Ile Lys Val 305 310 315 320 Val Gly Leu Ser Thr Leu Pro Glu Ile Tyr Glu Lys Met Glu Lys Gly 325 330 335 Gln Ile Val Gly Arg Tyr Val Val Asp Thr Ser Lys 340 345 <210> 100 <211> 1047 <212> DNA <213> Artificial Sequence <220> <223> Saccharomyces cerevisiae ADH1 <400> 100 atgtctatcc cagaaactca aaaaggtgtt atcttctacg aatcccacgg taagttggaa 60 tacaaagata ttccagttcc aaagccaaag gccaacgaat tgttgatcaa cgttaaatac 120 tctggtgtct gtcacactga cttgcacgct tggcacggtg actggccatt gccagttaag 180 ctaccattag tcggtggtca cgaaggtgcc ggtgtcgttg tcggcatggg tgaaaacgtt 240 aagggctgga agatcggtga ctacgccggt atcaaatggt tgaacggttc ttgtatggcc 300 tgtgaatact gtgaattggg taacgaatcc aactgtcctc acgctgactt gtctggttac 360 acccacgacg gttctttcca acaatacgct accgctgacg ctgttcaagc cgctcacatt 420 cctcaaggta ccgacttggc ccaagtcgcc cccatcttgt gtgctggtat caccgtctac 480 aaggctttga agtctgctaa cttgatggcc ggtcactggg ttgctatctc cggtgctgct 540 ggtggtctag gttctttggc tgttcaatac gccaaggcta tgggttacag agtcttgggt 600 attgacggtg gtgaaggtaa ggaagaatta ttcagatcca tcggtggtga agtcttcatt 660 gacttcacta aggaaaagga cattgtcggt gctgttctaa aggccactga cggtggtgct 720 cacggtgtca tcaacgtttc cgtttccgaa gccgctattg aagcttctac cagatacgtt 780 agagctaacg gtaccaccgt tttggtcggt atgccagctg gtgccaagtg ttgttctgat 840 gtcttcaacc aagtcgtcaa gtccatctct attgttggtt cttacgtcgg taacagagct 900 gacaccagag aagctttgga cttcttcgcc agaggtttgg tcaagtctcc aatcaaggtt 960 gtcggcttgt ctaccttgcc agaaatttac gaaaagatgg aaaagggtca aatcgttggt 1020 agatacgttg ttgacacttc taaataa 1047 <210> 101 <211> 348 <212> PRT <213> Artificial Sequence <220> <223> Saccharomyces cerevisiae ADH2 <400> 101 Met Ser Ile Pro Glu Thr Gln Lys Ala Ile Ile Phe Tyr Glu Ser Asn 1 5 10 15 Gly Lys Leu Glu His Lys Asp Ile Pro Val Pro Lys Pro Lys Pro Asn 20 25 30 Glu Leu Leu Ile Asn Val Lys Tyr Ser Gly Val Cys His Thr Asp Leu 35 40 45 His Ala Trp His Gly Asp Trp Pro Leu Pro Thr Lys Leu Pro Leu Val 50 55 60 Gly Gly His Glu Gly Ala Gly Val Val Val Gly Met Gly Glu Asn Val 65 70 75 80 Lys Gly Trp Lys Ile Gly Asp Tyr Ala Gly Ile Lys Trp Leu Asn Gly 85 90 95 Ser Cys Met Ala Cys Glu Tyr Cys Glu Leu Gly Asn Glu Ser Asn Cys 100 105 110 Pro His Ala Asp Leu Ser Gly Tyr Thr His Asp Gly Ser Phe Gln Glu 115 120 125 Tyr Ala Thr Ala Asp Ala Val Gln Ala Ala His Ile Pro Gln Gly Thr 130 135 140 Asp Leu Ala Glu Val Ala Pro Ile Leu Cys Ala Gly Ile Thr Val Tyr 145 150 155 160 Lys Ala Leu Lys Ser Ala Asn Leu Arg Ala Gly His Trp Ala Ala Ile 165 170 175 Ser Gly Ala Ala Gly Gly Leu Gly Ser Leu Ala Val Gln Tyr Ala Lys 180 185 190 Ala Met Gly Tyr Arg Val Leu Gly Ile Asp Gly Gly Pro Gly Lys Glu 195 200 205 Glu Leu Phe Thr Ser Leu Gly Gly Glu Val Phe Ile Asp Phe Thr Lys 210 215 220 Glu Lys Asp Ile Val Ser Ala Val Val Lys Ala Thr Asn Gly Gly Ala 225 230 235 240 His Gly Ile Ile Asn Val Ser Val Ser Glu Ala Ala Ile Glu Ala Ser 245 250 255 Thr Arg Tyr Cys Arg Ala Asn Gly Thr Val Val Leu Val Gly Leu Pro 260 265 270 Ala Gly Ala Lys Cys Ser Ser Asp Val Phe Asn His Val Val Lys Ser 275 280 285 Ile Ser Ile Val Gly Ser Tyr Val Gly Asn Arg Ala Asp Thr Arg Glu 290 295 300 Ala Leu Asp Phe Phe Ala Arg Gly Leu Val Lys Ser Pro Ile Lys Val 305 310 315 320 Val Gly Leu Ser Ser Leu Pro Glu Ile Tyr Glu Lys Met Glu Lys Gly 325 330 335 Gln Ile Ala Gly Arg Tyr Val Val Asp Thr Ser Lys 340 345 <210> 102 <211> 1047 <212> DNA <213> Artificial Sequence <220> <223> Saccharomyces cerevisiae ADH2 <400> 102 atgtctattc cagaaactca aaaagccatt atcttctacg aatccaacgg caagttggag 60 cataaggata tcccagttcc aaagccaaag cccaacgaat tgttaatcaa cgtcaagtac 120 tctggtgtct gccacaccga tttgcacgct tggcatggtg actggccatt gccaactaag 180 ttaccattag ttggtggtca cgaaggtgcc ggtgtcgttg tcggcatggg tgaaaacgtt 240 aagggctgga agatcggtga ctacgccggt atcaaatggt tgaacggttc ttgtatggcc 300 tgtgaatact gtgaattggg taacgaatcc aactgtcctc acgctgactt gtctggttac 360 acccacgacg gttctttcca agaatacgct accgctgacg ctgttcaagc cgctcacatt 420 cctcaaggta ctgacttggc tgaagtcgcg ccaatcttgt gtgctggtat caccgtatac 480 aaggctttga agtctgccaa cttgagagca ggccactggg cggccatttc tggtgctgct 540 ggtggtctag gttctttggc tgttcaatat gctaaggcga tgggttacag agtcttaggt 600 attgatggtg gtccaggaaa ggaagaattg tttacctcgc tcggtggtga agtattcatc 660 gacttcacca aagagaagga cattgttagc gcagtcgtta aggctaccaa cggcggtgcc 720 cacggtatca tcaatgtttc cgtttccgaa gccgctatcg aagcttctac cagatactgt 780 agggcgaacg gtactgttgt cttggttggt ttgccagccg gtgcaaagtg ctcctctgat 840 gtcttcaacc acgttgtcaa gtctatctcc attgtcggct cttacgtggg gaacagagct 900 gataccagag aagccttaga tttctttgcc agaggtctag tcaagtctcc aataaaggta 960 gttggcttat ccagtttacc agaaatttac gaaaagatgg agaagggcca aattgctggt 1020 agatacgttg ttgacacttc taaataa 1047 <210> 103 <211> 375 <212> PRT <213> Artificial Sequence <220> <223> Saccharomyces cerevisiae ADH3 <400> 103 Met Leu Arg Thr Ser Thr Leu Phe Thr Arg Arg Val Gln Pro Ser Leu 1 5 10 15 Phe Ser Arg Asn Ile Leu Arg Leu Gln Ser Thr Ala Ala Ile Pro Lys 20 25 30 Thr Gln Lys Gly Val Ile Phe Tyr Glu Asn Lys Gly Lys Leu His Tyr 35 40 45 Lys Asp Ile Pro Val Pro Glu Pro Lys Pro Asn Glu Ile Leu Ile Asn 50 55 60 Val Lys Tyr Ser Gly Val Cys His Thr Asp Leu His Ala Trp His Gly 65 70 75 80 Asp Trp Pro Leu Pro Val Lys Leu Pro Leu Val Gly Gly His Glu Gly 85 90 95 Ala Gly Val Val Val Lys Leu Gly Ser Asn Val Lys Gly Trp Lys Val 100 105 110 Gly Asp Leu Ala Gly Ile Lys Trp Leu Asn Gly Ser Cys Met Thr Cys 115 120 125 Glu Phe Cys Glu Ser Gly His Glu Ser Asn Cys Pro Asp Ala Asp Leu 130 135 140 Ser Gly Tyr Thr His Asp Gly Ser Phe Gln Gln Phe Ala Thr Ala Asp 145 150 155 160 Ala Ile Gln Ala Ala Lys Ile Gln Gln Gly Thr Asp Leu Ala Glu Val 165 170 175 Ala Pro Ile Leu Cys Ala Gly Val Thr Val Tyr Lys Ala Leu Lys Glu 180 185 190 Ala Asp Leu Lys Ala Gly Asp Trp Val Ala Ile Ser Gly Ala Ala Gly 195 200 205 Gly Leu Gly Ser Leu Ala Val Gln Tyr Ala Thr Ala Met Gly Tyr Arg 210 215 220 Val Leu Gly Ile Asp Ala Gly Glu Glu Lys Glu Lys Leu Phe Lys Lys 225 230 235 240 Leu Gly Gly Glu Val Phe Ile Asp Phe Thr Lys Thr Lys Asn Met Val 245 250 255 Ser Asp Ile Gln Glu Ala Thr Lys Gly Gly Pro His Gly Val Ile Asn 260 265 270 Val Ser Val Ser Glu Ala Ala Ile Ser Leu Ser Thr Glu Tyr Val Arg 275 280 285 Pro Cys Gly Thr Val Val Leu Val Gly Leu Pro Ala Asn Ala Tyr Val 290 295 300 Lys Ser Glu Val Phe Ser His Val Val Lys Ser Ile Asn Ile Lys Gly 305 310 315 320 Ser Tyr Val Gly Asn Arg Ala Asp Thr Arg Glu Ala Leu Asp Phe Phe 325 330 335 Ser Arg Gly Leu Ile Lys Ser Pro Ile Lys Ile Val Gly Leu Ser Glu 340 345 350 Leu Pro Lys Val Tyr Asp Leu Met Glu Lys Gly Lys Ile Leu Gly Arg 355 360 365 Tyr Val Val Asp Thr Ser Lys 370 375 <210> 104 <211> 1128 <212> DNA <213> Artificial Sequence <220> <223> Saccharomyces cerevisiae ADH3 <400> 104 atgttgagaa cgtcaacatt gttcaccagg cgtgtccaac caagcctatt ttctagaaac 60 attcttagat tgcaatccac agctgcaatc cctaagactc aaaaaggtgt catcttttat 120 gagaataagg ggaagctgca ttacaaagat atccctgtcc ccgagcctaa gccaaatgaa 180 attttaatca acgttaaata ttctggtgta tgtcacaccg atttacatgc ttggcacggc 240 gattggccat tacctgttaa actaccatta gtaggtggtc atgaaggtgc tggtgtagtt 300 gtcaaactag gttccaatgt caagggctgg aaagtcggtg atttagcagg tatcaaatgg 360 ctgaacggtt cttgtatgac atgcgaattc tgtgaatcag gtcatgaatc aaattgtcca 420 gatgctgatt tatctggtta cactcatgat ggttctttcc aacaatttgc gaccgctgat 480 gctattcaag ccgccaaaat tcaacagggt accgacttgg ccgaagtagc cccaatatta 540 tgtgctggtg ttactgtata taaagcacta aaagaggcag acttgaaagc tggtgactgg 600 gttgccatct ctggtgctgc aggtggcttg ggttccttgg ccgttcaata tgcaactgcg 660 atgggttaca gagttctagg tattgatgca ggtgaggaaa aggaaaaact tttcaagaaa 720 ttggggggtg aagtattcat cgactttact aaaacaaaga atatggtttc tgacattcaa 780 gaagctacca aaggtggccc tcatggtgtc attaacgttt ccgtttctga agccgctatt 840 tctctatcta cggaatatgt tagaccatgt ggtaccgtcg ttttggttgg tttgcccgct 900 aacgcctacg ttaaatcaga ggtattctct catgtggtga agtccatcaa tatcaagggt 960 tcttatgttg gtaacagagc tgatacgaga gaagccttag acttctttag cagaggtttg 1020 atcaaatcac caatcaaaat tgttggatta tctgaattac caaaggttta tgacttgatg 1080 gaaaagggca agattttggg tagatacgtc gtcgatacta gtaaataa 1128 <210> 105 <211> 382 <212> PRT <213> Artificial Sequence <220> <223> Saccharomyces cerevisiae ADH4 <400> 105 Met Ser Ser Val Thr Gly Phe Tyr Ile Pro Ile Ser Phe Phe Gly 1 5 10 15 Glu Gly Ala Leu Glu Glu Thr Ala Asp Tyr Ile Lys Asn Lys Asp Tyr 20 25 30 Lys Lys Ala Leu Ile Val Thr Asp Pro Gly Ile Ala Ala Ile Gly Leu 35 40 45 Ser Gly Arg Val Gln Lys Met Leu Glu Glu Arg Gly Leu Asn Val Ala 50 55 60 Ile Tyr Asp Lys Thr Gln Pro Asn Pro Asn Ile Ala Asn Val Thr Ala 65 70 75 80 Gly Leu Lys Val Leu Lys Glu Glu Asn Ser Glu Ile Val Val Ser Ile 85 90 95 Gly Gly Gly Ser Ala His Asp Asn Ala Lys Ala Ile Ala Leu Leu Ala 100 105 110 Thr Asn Gly Gly Glu Ile Gly Asp Tyr Glu Gly Val Asn Gln Ser Lys 115 120 125 Lys Ala Ala Leu Pro Leu Phe Ala Ile Asn Thr Thr Ala Gly Thr Ala 130 135 140 Ser Glu Met Thr Arg Phe Thr Ile Ile Ser Asn Glu Glu Lys Lys Ile 145 150 155 160 Lys Met Ala Ile Ile Asp Asn Asn Val Thr Pro Ala Val Ala Val Asn 165 170 175 Asp Pro Ser Thr Met Phe Gly Leu Pro Pro Ala Leu Thr Ala Ala Thr 180 185 190 Gly Leu Asp Ala Leu Thr His Cys Ile Glu Ala Tyr Val Ser Thr Ala 195 200 205 Ser Asn Pro Ile Thr Asp Ala Cys Ala Leu Lys Gly Ile Asp Leu Ile 210 215 220 Asn Glu Ser Leu Val Ala Ala Tyr Lys Asp Gly Lys Asp Lys Lys Ala 225 230 235 240 Arg Thr Asp Met Cys Tyr Ala Glu Tyr Leu Ala Gly Met Ala Phe Asn 245 250 255 Asn Ala Ser Leu Gly Tyr Val His Ala Leu Ala His Gln Leu Gly Gly 260 265 270 Phe Tyr His Leu Pro His Gly Val Cys Asn Ala Val Leu Leu Pro His 275 280 285 Val Gln Glu Ala Asn Met Gln Cys Pro Lys Ala Lys Lys Arg Leu Gly 290 295 300 Glu Ile Ala Leu His Cys Gly Ala Ser Gln Glu Asp Pro Glu Glu Thr 305 310 315 320 Ile Lys Ala Leu His Val Leu Asn Arg Thr Met Asn Ile Pro Arg Asn 325 330 335 Leu Lys Asp Leu Gly Val Lys Thr Glu Asp Phe Asp Ile Leu Ala Glu 340 345 350 His Ala Met His Asp Ala Cys His Leu Thr Asn Pro Val Gln Phe Thr 355 360 365 Lys Glu Gln Val Val Ala Ile Ile Lys Lys Ala Tyr Glu Tyr 370 375 380 <210> 106 <211> 1149 <212> DNA <213> Artificial Sequence <220> <223> Saccharomyces cerevisiae ADH4 <400> 106 atgtcttccg ttactgggtt ttacattcca ccaatctctt tctttggtga aggtgcttta 60 gaagaaaccg ctgattacat caaaaacaag gattacaaaa aggctttgat cgttactgat 120 cctggtattg cagctattgg tctctccggt agagtccaaa agatgttgga agaacgtggc 180 ttaaacgttg ctatctatga caaaactcaa ccaaacccaa atattgccaa tgtcacagct 240 ggtttgaagg ttttgaagga agaaaactct gaaattgtcg tttccattgg tggtggttct 300 gctcacgaca atgctaaggc cattgcttta ttggctacta acggtgggga aattggagat 360 tatgaaggtg tcaaccaatc taagaaggct gctttaccgc tatttgccat caacactact 420 gctggtactg cttccgagat gaccagattc actattatct ctaatgaaga aaagaaaatc 480 aagatggcca tcattgacaa caacgtcact ccagctgttg ctgtcaacga cccatctacc 540 atgtttggtt tgccacctgc tttgactgct gctactggtc tagatgcttt gactcactgt 600 atcgaagctt acgtttccac cgcctctaac ccaatcaccg atgcttgtgc tttgaagggt 660 attgatttga tcaatgaaag cttggtcgcc gcatacaaag acggtaaaga caagaaggcc 720 agaactgata tgtgttacgc agaatacttg gcaggtatgg ctttcaacaa tgcttctcta 780 ggttatgttc atgcccttgc tcatcaactt ggtggtttct accacttgcc tcatggtgtt 840 tgtaacgctg tcttgttgcc tcatgttcaa gaggccaaca tgcaatgtcc aaaggccaag 900 aagagattag gtgaaattgc cttgcattgc ggtgcttctc aagaagatcc agaagaaacc 960 atcaaggctt tgcacgtttt aaacagaacc atgaacattc caagaaactt gaaagactta 1020 ggtgttaaaa ccgaagattt tgacattttg gctgaacacg ccatgcatga tgcctgccat 1080 ttgactaacc cagttcaatt caccaaagaa caagtggttg ccattatcaa gaaagcctat 1140 gaatattaa 1149 <210> 107 <211> 351 <212> PRT <213> Artificial Sequence <220> <223> Saccharomyces cerevisiae ADH5 <400> 107 Met Pro Ser Gln Val Ile Pro Glu Lys Gln Lys Ala Ile Val Phe Tyr 1 5 10 15 Glu Thr Asp Gly Lys Leu Glu Tyr Lys Asp Val Thr Val Pro Glu Pro 20 25 30 Lys Pro Asn Glu Ile Leu Val His Val Lys Tyr Ser Gly Val Cys His 35 40 45 Ser Asp Leu His Ala Trp His Gly Asp Trp Pro Phe Gln Leu Lys Phe 50 55 60 Pro Leu Ile Gly Gly His Glu Gly Ala Gly Val Val Val Lys Leu Gly 65 70 75 80 Ser Asn Val Lys Gly Trp Lys Val Gly Asp Phe Ala Gly Ile Lys Trp 85 90 95 Leu Asn Gly Thr Cys Met Ser Cys Glu Tyr Cys Glu Val Gly Asn Glu 100 105 110 Ser Gln Cys Pro Tyr Leu Asp Gly Thr Gly Phe Thr His Asp Gly Thr 115 120 125 Phe Gln Glu Tyr Ala Thr Ala Asp Ala Val Gln Ala Ala His Ile Pro 130 135 140 Pro Asn Val Asn Leu Ala Glu Val Ala Pro Ile Leu Cys Ala Gly Ile 145 150 155 160 Thr Val Tyr Lys Ala Leu Lys Arg Ala Asn Val Ile Pro Gly Gln Trp 165 170 175 Val Thr Ile Ser Gly Ala Cys Gly Gly Leu Gly Ser Leu Ala Ile Gln 180 185 190 Tyr Ala Leu Ala Met Gly Tyr Arg Val Ile Gly Ile Asp Gly Gly Asn 195 200 205 Ala Lys Arg Lys Leu Phe Glu Gln Leu Gly Gly Glu Ile Phe Ile Asp 210 215 220 Phe Thr Glu Glu Lys Asp Ile Val Gly Ala Ile Ile Lys Ala Thr Asn 225 230 235 240 Gly Gly Ser His Gly Val Ile Asn Val Ser Val Ser Glu Ala Ala Ile 245 250 255 Glu Ala Ser Thr Arg Tyr Cys Arg Pro Asn Gly Thr Val Val Leu Val 260 265 270 Gly Met Pro Ala His Ala Tyr Cys Asn Ser Asp Val Phe Asn Gln Val 275 280 285 Val Lys Ser Ile Ser Ile Val Gly Ser Cys Val Gly Asn Arg Ala Asp 290 295 300 Thr Arg Glu Ala Leu Asp Phe Phe Ala Arg Gly Leu Ile Lys Ser Pro 305 310 315 320 Ile His Leu Ala Gly Leu Ser Asp Val Pro Glu Ile Phe Ala Lys Met 325 330 335 Glu Lys Gly Glu Ile Val Gly Arg Tyr Val Val Glu Thr Ser Lys 340 345 350 <210> 108 <211> 1056 <212> DNA <213> Artificial Sequence <220> <223> Saccharomyces cerevisiae ADH5 <400> 108 atgccttcgc aagtcattcc tgaaaaacaa aaggctattg tcttttatga gacagatgga 60 aaattggaat ataaagacgt cacagttccg gaacctaagc ctaacgaaat tttagtccac 120 gttaaatatt ctggtgtttg tcatagtgac ttgcacgcgt ggcacggtga ttggccattt 180 caattgaaat ttccattaat cggtggtcac gaaggtgctg gtgttgttgt taagttggga 240 tctaacgtta agggctggaa agtcggtgat tttgcaggta taaaatggtt gaatgggact 300 tgcatgtcct gtgaatattg tgaagtaggt aatgaatctc aatgtcctta tttggatggt 360 actggcttca cacatgatgg tacttttcaa gaatacgcaa ctgccgatgc cgttcaagct 420 gcccatattc caccaaacgt caatcttgct gaagttgccc caatcttgtg tgcaggtatc 480 actgtttata aggcgttgaa aagagccaat gtgataccag gccaatgggt cactatatcc 540 ggtgcatgcg gtggcttggg ttctctggca atccaatacg cccttgctat gggttacagg 600 gtcattggta tcgatggtgg taatgccaag cgaaagttat ttgaacaatt aggcggagaa 660 atattcatcg atttcacgga agaaaaagac attgttggtg ctataataaa ggccactaat 720 ggcggttctc atggagttat taatgtgtct gtttctgaag cagctatcga ggcttctacg 780 aggtattgta ggcccaatgg tactgtcgtc ctggttggta tgccagctca tgcttactgc 840 aattccgatg ttttcaatca agttgtaaaa tcaatctcca tcgttggatc ttgtgttgga 900 aatagagctg atacaaggga ggctttagat ttcttcgcca gaggtttgat caaatctccg 960 atccacttag ctggcctatc ggatgttcct gaaatttttg caaagatgga gaagggtgaa 1020 attgttggta gatatgttgt tgagacttct aaatga 1056 <210> 109 <211> 391 <212> PRT <213> Artificial Sequence <220> <223> Saccharomyces cerevisiae GPD1 <400> 109 Met Ser Ala Ala Ala Asp Arg Leu Asn Leu Thr Ser Gly His Leu Asn 1 5 10 15 Ala Gly Arg Lys Arg Ser Ser Ser Ser Val Ser Leu Lys Ala Ala Glu 20 25 30 Lys Pro Phe Lys Val Thr Val Ile Gly Ser Gly Asn Trp Gly Thr Thr 35 40 45 Ile Ala Lys Val Val Ala Glu Asn Cys Lys Gly Tyr Pro Glu Val Phe 50 55 60 Ala Pro Ile Val Gln Met Trp Val Phe Glu Glu Glu Ile Asn Gly Glu 65 70 75 80 Lys Leu Thr Glu Ile Ile Asn Thr Arg His Gln Asn Val Lys Tyr Leu 85 90 95 Pro Gly Ile Thr Leu Pro Asp Asn Leu Val Ala Asn Pro Asp Leu Ile 100 105 110 Asp Ser Val Lys Asp Val Asp Ile Ile Val Phe Asn Ile Pro His Gln 115 120 125 Phe Leu Pro Arg Ile Cys Ser Gln Leu Lys Gly His Val Asp Ser His 130 135 140 Val Arg Ala Ile Ser Cys Leu Lys Gly Phe Glu Val Gly Ala Lys Gly 145 150 155 160 Val Gln Leu Leu Ser Ser Tyr Ile Thr Glu Glu Leu Gly Ile Gln Cys 165 170 175 Gly Ala Leu Ser Gly Ala Asn Ile Ala Thr Glu Val Ala Gln Glu His 180 185 190 Trp Ser Glu Thr Thr Val Ala Tyr His Ile Pro Lys Asp Phe Arg Gly 195 200 205 Glu Gly Lys Asp Val Asp His Lys Val Leu Lys Ala Leu Phe His Arg 210 215 220 Pro Tyr Phe His Val Ser Val Ile Glu Asp Val Ala Gly Ile Ser Ile 225 230 235 240 Cys Gly Ala Leu Lys Asn Val Val Ala Leu Gly Cys Gly Phe Val Glu 245 250 255 Gly Leu Gly Trp Gly Asn Asn Ala Ser Ala Ala Ile Gln Arg Val Gly 260 265 270 Leu Gly Glu Ile Ile Arg Phe Gly Gln Met Phe Phe Pro Glu Ser Arg 275 280 285 Glu Glu Thr Tyr Tyr Gln Glu Ser Ala Gly Val Ala Asp Leu Ile Thr 290 295 300 Thr Cys Ala Gly Gly Arg Asn Val Lys Val Ala Arg Leu Met Ala Thr 305 310 315 320 Ser Gly Lys Asp Ala Trp Glu Cys Glu Lys Glu Leu Leu Asn Gly Gln 325 330 335 Ser Ala Gln Gly Leu Ile Thr Cys Lys Glu Val His Glu Trp Leu Glu 340 345 350 Thr Cys Gly Ser Val Glu Asp Phe Pro Leu Phe Glu Ala Val Tyr Gln 355 360 365 Ile Val Tyr Asn Asn Tyr Pro Met Lys Asn Leu Pro Asp Met Ile Glu 370 375 380 Glu Leu Asp Leu His Glu Asp 385 390 <210> 110 <211> 1176 <212> DNA <213> Artificial Sequence <220> <223> Saccharomyces cerevisiae GPD1 <400> 110 atgtctgctg ctgctgatag attaaactta acttccggcc acttgaatgc tggtagaaag 60 agaagttcct cttctgtttc tttgaaggct gccgaaaagc ctttcaaggt tactgtgatt 120 ggatctggta actggggtac tactattgcc aaggtggttg ccgaaaattg taagggatac 180 ccagaagttt tcgctccaat agtacaaatg tgggtgttcg aagaagagat caatggtgaa 240 aaattgactg aaatcataaa tactagacat caaaacgtga aatacttgcc tggcatcact 300 ctacccgaca atttggttgc taatccagac ttgattgatt cagtcaagga tgtcgacatc 360 atcgttttca acattccaca tcaatttttg ccccgtatct gtagccaatt gaaaggtcat 420 gttgattcac acgtcagagc tatctcctgt ctaaagggtt ttgaagttgg tgctaaaggt 480 gtccaattgc tatcctctta catcactgag gaactaggta ttcaatgtgg tgctctatct 540 ggtgctaaca ttgccaccga agtcgctcaa gaacactggt ctgaaacaac agttgcttac 600 cacattccaa aggatttcag aggcgagggc aaggacgtcg accataaggt tctaaaggcc 660 ttgttccaca gaccttactt ccacgttagt gtcatcgaag atgttgctgg tatctccatc 720 tgtggtgctt tgaagaacgt tgttgcctta ggttgtggtt tcgtcgaagg tctaggctgg 780 ggtaacaacg cttctgctgc catccaaaga gtcggtttgg gtgagatcat cagattcggt 840 caaatgtttt tcccagaatc tagagaagaa acatactacc aagagtctgc tggtgttgct 900 gatttgatca ccacctgcgc tggtggtaga aacgtcaagg ttgctaggct aatggctact 960 tctggtaagg acgcctggga atgtgaaaag gagttgttga atggccaatc cgctcaaggt 1020 ttaattacct gcaaagaagt tcacgaatgg ttggaaacat gtggctctgt cgaagacttc 1080 ccattatttg aagccgtata ccaaatcgtt tacaacaact acccaatgaa gaacctgccg 1140 gacatgattg aagaattaga tctacatgaa gattag 1176 <210> 111 <211> 440 <212> PRT <213> Artificial Sequence <220> <223> Saccharomyces cerevisiae GPD2 <400> 111 Met Leu Ala Val Arg Arg Leu Thr Arg Tyr Thr Phe Leu Lys Arg Thr 1 5 10 15 His Pro Val Leu Tyr Thr Arg Arg Ala Tyr Lys Ile Leu Pro Ser Arg 20 25 30 Ser Thr Phe Leu Arg Arg Ser Leu Leu Gln Thr Gln Leu His Ser Lys 35 40 45 Met Thr Ala His Thr Asn Ile Lys Gln His Lys His Cys His Glu Asp 50 55 60 His Pro Ile Arg Arg Ser Asp Ser Ala Val Ser Ile Val His Leu Lys 65 70 75 80 Arg Ala Pro Phe Lys Val Thr Val Ile Gly Ser Gly Asn Trp Gly Thr 85 90 95 Thr Ile Ala Lys Val Ile Ala Glu Asn Thr Glu Leu His Ser His Ile 100 105 110 Phe Glu Pro Glu Val Arg Met Trp Val Phe Asp Glu Lys Ile Gly Asp 115 120 125 Glu Asn Leu Thr Asp Ile Ile Asn Thr Arg His Gln Asn Val Lys Tyr 130 135 140 Leu Pro Asn Ile Asp Leu Pro His Asn Leu Val Ala Asp Pro Asp Leu 145 150 155 160 Leu His Ser Ile Lys Gly Ala Asp Ile Leu Val Phe Asn Ile Pro His 165 170 175 Gln Phe Leu Pro Asn Ile Val Lys Gln Leu Gln Gly His Val Ala Pro 180 185 190 His Val Arg Ala Ile Ser Cys Leu Lys Gly Phe Glu Leu Gly Ser Lys 195 200 205 Gly Val Gln Leu Leu Ser Ser Tyr Val Thr Asp Glu Leu Gly Ile Gln 210 215 220 Cys Gly Ala Leu Ser Gly Ala Asn Leu Ala Pro Glu Val Ala Lys Glu 225 230 235 240 His Trp Ser Glu Thr Thr Val Ala Tyr Gln Leu Pro Lys Asp Tyr Gln 245 250 255 Gly Asp Gly Lys Asp Val Asp His Lys Ile Leu Lys Leu Leu Phe His 260 265 270 Arg Pro Tyr Phe His Val Asn Val Ile Asp Asp Val Ala Gly Ile Ser 275 280 285 Ile Ala Gly Ala Leu Lys Asn Val Val Ala Leu Ala Cys Gly Phe Val 290 295 300 Glu Gly Met Gly Trp Gly Asn Asn Ala Ser Ala Ala Ile Gln Arg Leu 305 310 315 320 Gly Leu Gly Glu Ile Ile Lys Phe Gly Arg Met Phe Phe Pro Glu Ser 325 330 335 Lys Val Glu Thr Tyr Tyr Gln Glu Ser Ala Gly Val Ala Asp Leu Ile 340 345 350 Thr Thr Cys Ser Gly Gly Arg Asn Val Lys Val Ala Thr Tyr Met Ala 355 360 365 Lys Thr Gly Lys Ser Ala Leu Glu Ala Glu Lys Glu Leu Leu Asn Gly 370 375 380 Gln Ser Ala Gln Gly Ile Ile Thr Cys Arg Glu Val His Glu Trp Leu 385 390 395 400 Gln Thr Cys Glu Leu Thr Gln Glu Phe Pro Leu Phe Glu Ala Val Tyr 405 410 415 Gln Ile Val Tyr Asn Asn Val Arg Met Glu Asp Leu Pro Glu Met Ile 420 425 430 Glu Glu Leu Asp Ile Asp Asp Glu 435 440 <210> 112 <211> 1323 <212> DNA <213> Artificial Sequence <220> <223> Saccharomyces cerevisiae GPD2 <400> 112 atgcttgctg tcagaagatt aacaagatac acatcctta agcgaacgca tccggtgtta 60 tatactcgtc gtgcatataa aattttgcct tcaagatcta ctttcctaag aagatcatta 120 ttacaaacac aactgcactc aaagatgact gctcatacta atatcaaaca gcacaaacac 180 tgtcatgagg accatcctat cagaagatcg gactctgccg tgtcaattgt acatttgaaa 240 cgtgcgccct tcaaggttac agtgattggt tctggtaact gggggaccac catcgccaaa 300 gtcattgcgg aaaacacaga attgcattcc catatcttcg agccagaggt gagaatgtgg 360 gtttttgatg aaaagatcgg cgacgaaaat ctgacggata tcataaatac aagacaccag 420 aacgttaaat atctacccaa tattgacctg ccccataatc tagtggccga tcctgatctt 480 ttacactcca tcaagggtgc tgacatcctt gttttcaaca tccctcatca atttttacca 540 aacatagtca aacaattgca aggccacgtg gcccctcatg taagggccat ctcgtgtcta 600 aaagggttcg agttgggctc caagggtgtg caattgctat cctcctatgt tactgatgag 660 ttaggaatcc aatgtggcgc actatctggt gcaaacttgg caccggaagt ggccaaggag 720 cattggtccg aaaccaccgt ggcttaccaa ctaccaaagg attatcaagg tgatggcaag 780 gatgtagatc ataagatttt gaaattgctg ttccacagac cttacttcca cgtcaatgtc 840 atcgatgatg ttgctggtat atccattgcc ggtgccttga agaacgtcgt ggcacttgca 900 tgtggtttcg tagaaggtat gggatggggt aacaatgcct ccgcagccat tcaaaggctg 960 ggtttaggtg aaattatcaa gttcggtaga atgtttttcc cagaatccaa agtcgagacc 1020 tactatcaag aatccgctgg tgttgcagat ctgatcacca cctgctcagg cggtagaaac 1080 gtcaaggttg ccacatacat ggccaagacc ggtaagtcag ccttggaagc agaaaaggaa 1140 ttgcttaacg gtcaatccgc ccaagggata atcacatgca gagaagttca cgagtggcta 1200 caaacatgtg agttgaccca agaattccca ttattcgagg cagtctacca gatagtctac 1260 aacaacgtcc gcatggaaga cctaccggag atgattgaag agctagacat cgatgacgaa 1320 tag 1323 <210> 113 <211> 382 <212> PRT <213> Artificial Sequence <220> <223> Saccharomyces cerevisiae BDH1 <400> 113 Met Arg Ala Leu Ala Tyr Phe Lys Lys Gly Asp Ile His Phe Thr Asn 1 5 10 15 Asp Ile Pro Arg Pro Glu Ile Gln Thr Asp Asp Glu Val Ile Ile Asp 20 25 30 Val Ser Trp Cys Gly Ile Cys Gly Ser Asp Leu His Glu Tyr Leu Asp 35 40 45 Gly Pro Ile Phe Met Pro Lys Asp Gly Glu Cys His Lys Leu Ser Asn 50 55 60 Ala Ala Leu Pro Leu Ala Met Gly His Glu Met Ser Gly Ile Val Ser 65 70 75 80 Lys Val Gly Pro Lys Val Thr Lys Val Lys Val Gly Asp His Val Val 85 90 95 Val Asp Ala Ala Ser Ser Cys Ala Asp Leu His Cys Trp Pro His Ser 100 105 110 Lys Phe Tyr Asn Ser Lys Pro Cys Asp Ala Cys Gln Arg Gly Ser Glu 115 120 125 Asn Leu Cys Thr His Ala Gly Phe Val Gly Leu Gly Val Ile Ser Gly 130 135 140 Gly Phe Ala Glu Gln Val Val Val Ser Gln His His Ile Ile Pro Val 145 150 155 160 Pro Lys Glu Ile Pro Leu Asp Val Ala Ala Leu Val Glu Pro Leu Ser 165 170 175 Val Thr Trp His Ala Val Lys Ile Ser Gly Phe Lys Lys Gly Ser Ser 180 185 190 Ala Leu Val Leu Gly Ala Gly Pro Ile Gly Leu Cys Thr Ile Leu Val 195 200 205 Leu Lys Gly Met Gly Ala Ser Lys Ile Val Val Ser Glu Ile Ala Glu 210 215 220 Arg Arg Ile Glu Met Ala Lys Lys Leu Gly Val Glu Val Phe Asn Pro 225 230 235 240 Ser Lys His Gly His Lys Ser Ile Glu Ile Leu Arg Gly Leu Thr Lys 245 250 255 Ser His Asp Gly Phe Asp Tyr Ser Tyr Asp Cys Ser Gly Ile Gln Val 260 265 270 Thr Phe Glu Thr Ser Leu Lys Ala Leu Thr Phe Lys Gly Thr Ala Thr 275 280 285 Asn Ile Ala Val Trp Gly Pro Lys Pro Val Pro Phe Gln Pro Met Asp 290 295 300 Val Thr Leu Gln Glu Lys Val Met Thr Gly Ser Ile Gly Tyr Val Val 305 310 315 320 Glu Asp Phe Glu Glu Val Val Arg Ala Ile His Asn Gly Asp Ile Ala 325 330 335 Met Glu Asp Cys Lys Gln Leu Ile Thr Gly Lys Gln Arg Ile Glu Asp 340 345 350 Gly Trp Glu Lys Gly Phe Gln Glu Leu Met Asp His Lys Glu Ser Asn 355 360 365 Val Lys Ile Leu Leu Thr Pro Asn Asn His Gly Glu Met Lys 370 375 380 <210> 114 <211> 1149 <212> DNA <213> Artificial Sequence <220> <223> Saccharomyces cerevisiae BDH1 <400> 114 atgagagctt tggcatattt caagaagggt gatattcact tcactaatga tatccctagg 60 ccagaaatcc aaaccgacga tgaggttatt atcgacgtct cttggtgtgg gatttgtggc 120 tcggatcttc acgagtactt ggatggtcca atcttcatgc ctaaagatgg agagtgccat 180 aaattatcca acgctgcttt acctctggca atgggccatg agatgtcagg aattgtttcc 240 aaggttggtc ctaaagtgac aaaggtgaag gttggcgacc acgtggtcgt tgatgctgcc 300 agcagttgtg cggacctgca ttgctggcca cactccaaat tttacaattc caaaccatgt 360 gatgcttgtc agaggggcag tgaaaatcta tgtacccacg ccggttttgt aggactaggt 420 gtgatcagtg gtggctttgc tgaacaagtc gtagtctctc aacatcacat tatcccggtt 480 ccaaaggaaa ttcctctaga tgtggctgct ttagttgagc ctctttctgt cacctggcat 540 gctgttaaga tttctggttt caaaaaaggc agttcagcct tggttcttgg tgcaggtccc 600 attgggttgt gtaccatttt ggtacttaag ggaatggggg ctagtaaaat tgtagtgtct 660 gaaattgcag agagaagaat agaaatggcc aagaaactgg gcgttgaggt gttcaatccc 720 tccaagcacg gtcataaatc tatagagata ctacgtggtt tgaccaagag ccatgatggg 780 tttgattaca gttatgattg ttctggtatt caagttactt tcgaaacctc tttgaaggca 840 ttaacattca aggggacagc caccaacatt gcagtttggg gtccaaaacc tgtcccattc 900 caaccaatgg atgtgactct ccaagagaaa gttatgactg gttcgatcgg ctatgttgtc 960 gaagacttcg aagaagttgt tcgtgccatc cacaacggag acatcgccat ggaagattgt 1020 aagcaactaa tcactggtaa gcaaaggatt gaggacggtt gggaaaaggg attccaagag 1080 ttgatggatc acaaggaatc caacgttaag attctattga cgcctaacaa tcacggtgaa 1140 atgaagtaa 1149 <210> 115 <211> 444 <212> PRT <213> Artificial Sequence <220> <223> Saccharomyces cerevisiae EMP46 <400> 115 Met Thr Thr Arg Lys Thr Ala Ser Ser Leu Gln Leu Leu Gly Lys Ile 1 5 10 15 Thr Gly Thr Lys Ala Gly Thr Lys Gln Lys Lys Met Asn Phe Ile Asn 20 25 30 Gly Leu Ile Trp Leu Tyr Met Cys Val Trp Met Val His Gly Lys Val 35 40 45 Thr Gln Lys Asp Glu Leu Lys Trp Asn Lys Gly Tyr Ser Leu Pro Asn 50 55 60 Leu Leu Glu Val Thr Asp Gln Gln Lys Glu Leu Ser Gln Trp Thr Leu 65 70 75 80 Gly Asp Lys Val Lys Leu Glu Glu Gly Arg Phe Val Leu Thr Pro Gly 85 90 95 Lys Asn Thr Lys Gly Ser Leu Trp Leu Lys Pro Glu Tyr Ser Ile Lys 100 105 110 Asp Ala Met Thr Ile Glu Trp Thr Phe Arg Ser Phe Gly Phe Arg Gly 115 120 125 Ser Thr Lys Gly Gly Leu Ala Phe Trp Leu Lys Gln Gly Asn Glu Gly 130 135 140 Asp Ser Thr Glu Leu Phe Gly Gly Ser Ser Lys Lys Phe Asn Gly Leu 145 150 155 160 Met Ile Leu Leu Arg Leu Asp Asp Lys Leu Gly Glu Ser Val Thr Ala 165 170 175 Tyr Leu Asn Asp Gly Thr Lys Asp Leu Asp Ile Glu Ser Ser Pro Tyr 180 185 190 Phe Ala Ser Cys Leu Phe Gln Tyr Gln Asp Ser Met Val Pro Ser Thr 195 200 205 Leu Arg Leu Thr Tyr Asn Pro Leu Asp Asn His Leu Leu Lys Leu Gln 210 215 220 Met Asp Asn Arg Val Cys Phe Gln Thr Arg Lys Val Lys Phe Met Gly 225 230 235 240 Ser Ser Pro Phe Arg Ile Gly Thr Ser Ala Ile Asn Asp Ala Ser Lys 245 250 255 Glu Ser Phe Glu Ile Leu Lys Met Lys Leu Tyr Asp Gly Val Ile Glu 260 265 270 Asp Ser Leu Ile Pro Asn Val Asn Pro Met Gly Gln Pro Arg Val Val 275 280 285 Thr Lys Val Ile Asn Ser Gln Thr Gly Glu Glu Ser Phe Arg Glu Lys 290 295 300 Met Pro Phe Ser Asp Lys Glu Glu Ser Ile Thr Ser Asn Glu Leu Phe 305 310 315 320 Glu Lys Met Asn Lys Leu Glu Gly Lys Ile Met Ala Asn Asp Ile Asp 325 330 335 Pro Leu Leu Arg Lys Met Asn Lys Ile Val Glu Asn Glu Arg Glu Leu 340 345 350 Ile Gln Arg Leu Arg Pro Leu Leu Asp Leu Lys Lys Thr Ala Ile Ser 355 360 365 Asp Asp Ser Phe Gln Asp Phe Leu Ser Met Asn Ala Asn Leu Asp Arg 370 375 380 Leu Ile Lys Glu Gln Glu Lys Ile Arg Gln Asp Ala Lys Leu Tyr Gly 385 390 395 400 Lys Gln Thr Lys Gly His Asp Glu Ile Phe Ser Lys Ile Ser Val Trp 405 410 415 Leu Ala Leu Leu Ile Phe Ile Met Ile Thr Leu Ala Tyr Tyr Met Phe 420 425 430 Arg Ile Asn Gln Asp Ile Lys Lys Val Lys Leu Leu 435 440 <210> 116 <211> 1335 <212> DNA <213> Artificial Sequence <220> <223> Saccharomyces cerevisiae EMP46 <400> 116 atgactacaa gaaagacagc ttcctctctt cagcttttag ggaaaatcac aggaacaaaa 60 gctggaacta agcaaaagaa gatgaatttc attaatggac tgatatggtt atatatgtgt 120 gtgtggatgg tacacggaaa agtgacgcag aaggatgaat tgaaatggaa taaaggatac 180 tcgctaccaa atttgctaga agtgacagat cagcaaaaag aactttcaca atggactttg 240 ggtgacaaag taaaacttga agaagggagg tttgttttaa ctcctggaaa gaacacaaag 300 ggttcacttt ggttgaaacc tgaatattca ataaaggatg caatgacaat agagtggacg 360 tttagaagtt tcgggttcag aggcagcaca aaggggggtc ttgcattttg gctgaagcaa 420 ggaaatgagg gagatagtac cgagttattt ggtggaagtt cgaagaagtt taatggtttg 480 atgatattgt tacgattaga cgataagttg ggagagagcg tgacagcgta tttgaatgac 540 ggaacaaaag atcttgatat tgaatcctca ccgtactttg cgtcatgtct gttccaatac 600 caggattcca tggtaccatc aacattaaga ttgacttaca atccactaga taatcacttg 660 ttaaagttgc aaatggacaa cagagtgtgt ttccagacaa ggaaagttaa atttatgggc 720 agcagcccat ttaggattgg aacaagtgct atcaacgatg catccaaaga atcgtttgaa 780 atcttgaaaa tgaagcttta tgacggagtt atagaggatt cgctaattcc taacgtgaat 840 cctatgggac aacccagagt ggttactaaa gtgatcaatt ctcaaactgg tgaagagagt 900 ttcagggaaa agatgccatt ttctgataag gaagaaagta taacgagtaa cgagcttttc 960 gaaaagatga acaagttgga ggggaaaatc atggcaaatg atatcgatcc attactccgc 1020 aagatgaaca agattgtgga gaatgaacgt gaactgattc aacgtttaag accactgtta 1080 gatctgaaga aaacagccat aagtgacgat agtttccaag attttctttc gatgaacgca 1140 aacctggaca gattgataaa agaacaagaa aaaattcgac aagatgccaa gctgtatggc 1200 aagcagacca aaggtcatga tgagatattt tccaaaataa gtgtatggtt ggcactgctg 1260 attttcatta tgatcacatt ggcgtactac atgtttagaa ttaaccaaga catcaagaag 1320 gtcaaacttc tgtaa 1335 <210> 117 <211> 515 <212> PRT <213> Artificial Sequence <220> <223> Saccharomyces cerevisiae PEP7 <400> 117 Met Asp Leu Glu Asn Val Ser Cys Pro Ile Cys Leu Arg Lys Phe Asp 1 5 10 15 Asn Leu Gln Ala Leu Asn Ala His Leu Asp Val Glu His Gly Phe Asn 20 25 30 Asp Asn Glu Asp Ser Leu Gly Ser Asn Asp Ser Arg Leu Val Asn Gly 35 40 45 Lys Gln Lys Lys Ala Arg Ser Val Asp Ser Ser Ala Gln Lys Leu Lys 50 55 60 Arg Ser His Trp Glu Lys Phe Lys Lys Gly Lys Ser Cys Cys His Thr 65 70 75 80 Cys Gly Arg Thr Leu Asn Asn Asn Ile Gly Ala Ile Asn Cys Arg Lys 85 90 95 Cys Gly Lys Leu Tyr Cys Arg Arg His Leu Pro Asn Met Ile Lys Leu 100 105 110 Asn Leu Ser Ala Gln Tyr Asp Pro Arg Asn Gly Lys Trp Tyr Asn Cys 115 120 125 Cys His Asp Cys Phe Val Thr Lys Pro Gly Tyr Asn Asp Tyr Gly Glu 130 135 140 Val Ile Asp Leu Thr Pro Glu Phe Phe Lys Val Arg Asn Ile Lys Arg 145 150 155 160 Glu Asp Lys Asn Leu Arg Leu Leu Gln Leu Glu Asn Arg Phe Val Arg 165 170 175 Leu Val Asp Gly Leu Ile Thr Leu Tyr Asn Thr Tyr Ser Arg Ser Ile 180 185 190 Ile His Asn Leu Lys Met Asn Ser Glu Met Ser Lys Leu Glu Arg Thr 195 200 205 Val Thr Pro Trp Arg Asp Asp Arg Ser Val Leu Phe Cys Asn Ile Cys 210 215 220 Ser Glu Pro Phe Gly Leu Leu Leu Arg Lys His His Cys Arg Leu Cys 225 230 235 240 Gly Met Val Val Cys Asp Asp Ala Asn Arg Asn Cys Ser Asn Glu Ile 245 250 255 Ser Ile Gly Tyr Leu Met Ser Ala Ala Ser Asp Leu Pro Phe Glu Tyr 260 265 270 Asn Ile Gln Lys Asp Asp Leu Leu His Ile Pro Ile Ser Ile Arg Leu 275 280 285 Cys Ser His Cys Ile Asp Met Leu Phe Ile Gly Arg Lys Phe Asn Lys 290 295 300 Asp Val Arg Met Pro Leu Ser Gly Ile Phe Ala Lys Tyr Asp Ser Met 305 310 315 320 Gln Asn Ile Ser Lys Val Ile Asp Ser Leu Leu Pro Ile Phe Glu Asp 325 330 335 Ser Leu Asn Ser Leu Lys Val Glu Thr Ala Lys Asp Ser Glu Asn Thr 340 345 350 Leu Asp Pro Lys Asn Leu Asn Asp Leu Ala Arg Leu Arg His Lys Leu 355 360 365 Leu Asn Ser Phe Asn Leu Tyr Asn Thr Leu Thr Arg Gln Leu Leu Ser 370 375 380 Val Glu Pro Gln Ser His Leu Glu Arg Gln Leu Gln Asn Ser Ile Lys 385 390 395 400 Ile Ala Ser Ala Ala Tyr Ile Asn Glu Lys Ile Leu Pro Leu Lys Ser 405 410 415 Leu Pro Ala Ile Leu Asn Pro Glu Gly His Lys Thr Asn Glu Asp Gly 420 425 430 Gln Lys Ala Glu Pro Glu Val Lys Lys Leu Ser Gln Leu Met Ile Glu 435 440 445 Asn Leu Thr Ile Lys Glu Val Lys Glu Leu Arg Glu Glu Leu Met Val 450 455 460 Leu Lys Glu Gln Ser Tyr Leu Ile Glu Ser Thr Ile Gln Asp Tyr Lys 465 470 475 480 Lys Gln Arg Arg Leu Glu Glu Ile Val Thr Leu Asn Lys Asn Leu Glu 485 490 495 Glu Leu His Ser Arg Ile His Thr Val Gln Ser Lys Leu Gly Asp His 500 505 510 Gly Phe Asn 515 <210> 118 <211> 1548 <212> DNA <213> Artificial Sequence <220> <223> Saccharomyces cerevisiae PEP7 <400> 118 atggatcttg aaaatgtttc atgtcccatt tgtctaagga agtttgataa cttacaagca 60 ctaaatgcac atttagatgt tgagcatggg tttaacgata atgaagattc actcggttcc 120 aatgacagtc gtttagtcaa tggtaaacaa aaaaaggcca gatctgttga tagcagtgcg 180 caaaagttga aaagaagcca ttgggaaaaa ttcaaaaaag ggaagagctg ctgtcaccaca 240 tgtggaagga ctttgaataa caatattggc gccattaatt gtaggaaatg tggtaaactg 300 tattgcagaa ggcatcttcc taatatgatt aaacttaatc tttccgcaca gtatgacccc 360 agaaacggga aatggtacaa ttgctgccat gattgtttcg tcacaaaacc tggctataac 420 gattatggcg aagtaataga tttaacacca gaattcttta aggtacgaaa tataaaaaga 480 gaggacaaaa acttaaggct attacaatta gaaaatcggt ttgtccgtct agttgatggt 540 ctgattacac tctataacac atattctaga tccattattc ataatttgaa gatgaatagt 600 gaaatgtcca agttagaacg tacagttact ccatggagag atgatagaag tgtactcttt 660 tgtaacatat gttccgaacc atttgggcta tactaagga agcatcactg cagattatgt 720 ggtatggttg tttgtgatga tgcaaacagg aactgctcaa acgaaataag cataggttat 780 ttaatgtctg cagcgtcaga tctgccattc gaatacaata tacagaagga tgatttactt 840 catattccta tatccataag attgtgctcg cactgtatcg atatgctatt tattggcagg 900 aaatttaaca aggacgttag aatgccgcta agtggaatat ttgctaaata tgatagcatg 960 caaaatatat ccaaagtaat tgatagtctc ctgcccattt ttgaagactc attgaatagc 1020 ctgaaggtgg agactgccaa ggattctgaa aatacacttg atccaaagaa tctgaatgat 1080 ctcgctcgat taagacataa attactcaat tcttttaact tgtataatac actaacaaga 1140 cagctcttaa gtgtagaacc tcaaagtcat ctagagagac aacttcaaaa ttcgatcaag 1200 atagcttccg ctgcatacat aaacgaaaaa atcctaccgt tgaagtcgct tccggcaatt 1260 ttgaacccag agggccataa aacgaatgaa gatgggcaaa aagctgaacc agaggtaaag 1320 aaattatcgc aactaatgat cgaaaacttg accataaaag aagtgaaaga gctgagagaa 1380 gaattgatgg tcctaaagga acaaagctac cttattgagt ccacaattca ggactataag 1440 aagcagcgta gattggagga gatcgtcaca cttaataaga acttagaaga attgcactca 1500 agaatacata ccgttcaatc gaagctgggt gaccatgggt ttaattaa 1548 <210> 119 <211> 382 <212> PRT <213> Artificial Sequence <220> <223> Saccharomyces cerevisiae SUR1 <400> 119 Met Arg Lys Glu Leu Lys Tyr Leu Ile Cys Phe Asn Ile Leu Leu Leu 1 5 10 15 Leu Ser Ile Ile Tyr Tyr Thr Phe Asp Leu Leu Thr Leu Cys Ile Asp 20 25 30 Asp Thr Val Lys Asp Ala Ile Leu Glu Glu Asp Leu Asn Pro Asp Ala 35 40 45 Pro Pro Lys Pro Gln Leu Ile Pro Lys Ile Ile His Gln Thr Tyr Lys 50 55 60 Thr Glu Asp Ile Pro Glu His Trp Lys Glu Gly Arg Gln Lys Cys Leu 65 70 75 80 Asp Leu His Pro Asp Tyr Lys Tyr Ile Leu Trp Thr Asp Glu Met Ala 85 90 95 Tyr Glu Phe Ile Lys Glu Glu Tyr Pro Trp Phe Leu Asp Thr Phe Glu 100 105 110 Asn Tyr Lys Tyr Pro Ile Glu Arg Ala Asp Ala Ile Arg Tyr Phe Ile 115 120 125 Leu Ser His Tyr Gly Gly Val Tyr Ile Asp Leu Asp Asp Gly Cys Glu 130 135 140 Arg Lys Leu Asp Pro Leu Leu Ala Phe Pro Ala Phe Leu Arg Lys Thr 145 150 155 160 Ser Pro Leu Gly Val Ser Asn Asp Val Met Gly Ser Val Pro Arg His 165 170 175 Pro Phe Phe Leu Lys Ala Leu Lys Ser Leu Lys His Tyr Asp Lys Tyr 180 185 190 Trp Phe Ile Pro Tyr Met Thr Ile Met Gly Ser Thr Gly Pro Leu Phe 195 200 205 Leu Ser Val Ile Trp Lys Gln Tyr Lys Arg Trp Arg Ile Pro Lys Asn 210 215 220 Gly Thr Val Arg Ile Leu Gln Pro Ala Tyr Tyr Lys Met His Ser Tyr 225 230 235 240 Ser Phe Phe Ser Ile Thr Lys Gly Ser Ser Trp His Leu Asp Asp Ala 245 250 255 Lys Leu Met Lys Ala Leu Glu Asn His Ile Leu Ser Cys Val Val Thr 260 265 270 Gly Phe Ile Phe Gly Phe Phe Ile Leu Tyr Gly Glu Phe Thr Phe Tyr 275 280 285 Cys Trp Leu Cys Ser Lys Asn Phe Ser Asn Leu Thr Lys Asn Trp Lys 290 295 300 Leu Asn Ala Ile Lys Val Arg Phe Val Thr Ile Leu Asn Ser Leu Gly 305 310 315 320 Leu Arg Leu Lys Leu Ser Lys Ser Thr Ser Asp Thr Ala Ser Ala Thr 325 330 335 Leu Leu Ala Arg Gln Gln Lys Arg Leu Arg Lys Asp Ser Asn Thr Asn 340 345 350 Ile Val Leu Leu Lys Ser Ser Arg Lys Ser Asp Val Tyr Asp Leu Glu 355 360 365 Lys Asn Asp Ser Ser Lys Tyr Ser Leu Gly Asn Asn Ser Ser 370 375 380 <210> 120 <211> 1149 <212> DNA <213> Artificial Sequence <220> <223> Saccharomyces cerevisiae SUR1 <400> 120 atgagaaaag aattaaaata ccttatctgc ttcaacatac tcctcctgtt atctataata 60 tactacactt tcgatttgct aacgttgtgt attgacgata ctgttaaaga tgctatactt 120 gaggaagact taaatccaga tgcacctcca aagcctcaac taatacctaa aatcatacat 180 cagacttata aaacggaaga catccctgag cactggaaag agggtagaca aaaatgtctc 240 gatctacatc cagattacaa gtacatccta tggacggacg agatggccta tgagtttata 300 aaggaagaat acccgtggtt tctcgatact tttgagaact acaaataccc catagaacgt 360 gccgatgcca ttcgttactt tatcctgtcc cattatggtg gtgtatacat cgatttagat 420 gacggctgcg aaaggaaact agatcctttg ttagctttcc cggccttctt aagaaagact 480 tcacctttag gtgtctcaaa cgatgtcatg ggttctgtgc ctagacatcc cttcttttta 540 aaggctttaa agtctttgaa acactatgac aagtactggt ttatccctta catgactatt 600 atggggtcta ctggtccgtt gtttttaagt gttatttgga agcagtacaa aagatggcgc 660 atacctaaga acgggacagt aagaatccta caacctgctt actacaagat gcatagttat 720 tcatttttct ccattactaa aggctcttca tggcatttag atgatgcaaa attgatgaaa 780 gcactagaaa accacatctt atcctgcgtc gttaccggat ttatttttgg cttctttatc 840 ctatatggtg aattcacgtt ctattgctgg ttgtgttcca agaatttcag taatctaacc 900 aaaaattgga aacttaatgc tataaaagta agatttgtca caattctaaa ttcattaggc 960 ttaagactaa agttgagtaa aagtaccagt gatactgcca gtgccacttt gctggcaagg 1020 cagcagaaac gattgaggaa agattccaat acaaacatag tgttactaaa atcttcaagg 1080 aaaagtgatg tttacgattt agaaaagaat gattcttcta agtactcact gggaaataac 1140 agctcgtaa 1149 <210> 121 <211> 486 <212> PRT <213> Artificial Sequence <220> <223> Saccharomyces cerevisiae HXK2 <400> 121 Met Val His Leu Gly Pro Lys Lys Pro Gln Ala Arg Lys Gly Ser Met 1 5 10 15 Ala Asp Val Pro Lys Glu Leu Met Gln Gln Ile Glu Asn Phe Glu Lys 20 25 30 Ile Phe Thr Val Pro Thr Glu Thr Leu Gln Ala Val Thr Lys His Phe 35 40 45 Ile Ser Glu Leu Glu Lys Gly Leu Ser Lys Lys Gly Gly Asn Ile Pro 50 55 60 Met Ile Pro Gly Trp Val Met Asp Phe Pro Thr Gly Lys Glu Ser Gly 65 70 75 80 Asp Phe Leu Ala Ile Asp Leu Gly Gly Thr Asn Leu Arg Val Val Leu 85 90 95 Val Lys Leu Gly Gly Asp Arg Thr Phe Asp Thr Thr Gln Ser Lys Tyr 100 105 110 Arg Leu Pro Asp Ala Met Arg Thr Thr Gln Asn Pro Asp Glu Leu Trp 115 120 125 Glu Phe Ile Ala Asp Ser Leu Lys Ala Phe Ile Asp Glu Gln Phe Pro 130 135 140 Gln Gly Ile Ser Glu Pro Ile Pro Leu Gly Phe Thr Phe Ser Phe Pro 145 150 155 160 Ala Ser Gln Asn Lys Ile Asn Glu Gly Ile Leu Gln Arg Trp Thr Lys 165 170 175 Gly Phe Asp Ile Pro Asn Ile Glu Asn His Asp Val Val Pro Met Leu 180 185 190 Gln Lys Gln Ile Thr Lys Arg Asn Ile Pro Ile Glu Val Val Ala Leu 195 200 205 Ile Asn Asp Thr Thr Gly Thr Leu Val Ala Ser Tyr Tyr Thr Asp Pro 210 215 220 Glu Thr Lys Met Gly Val Ile Phe Gly Thr Gly Val Asn Gly Ala Tyr 225 230 235 240 Tyr Asp Val Cys Ser Asp Ile Glu Lys Leu Gln Gly Lys Leu Ser Asp 245 250 255 Asp Ile Pro Pro Ser Ala Pro Met Ala Ile Asn Cys Glu Tyr Gly Ser 260 265 270 Phe Asp Asn Glu His Val Val Leu Pro Arg Thr Lys Tyr Asp Ile Thr 275 280 285 Ile Asp Glu Glu Ser Pro Arg Pro Gly Gln Gln Thr Phe Glu Lys Met 290 295 300 Ser Ser Gly Tyr Tyr Leu Gly Glu Ile Leu Arg Leu Ala Leu Met Asp 305 310 315 320 Met Tyr Lys Gln Gly Phe Ile Phe Lys Asn Gln Asp Leu Ser Lys Phe 325 330 335 Asp Lys Pro Phe Val Met Asp Thr Ser Tyr Pro Ala Arg Ile Glu Glu 340 345 350 Asp Pro Phe Glu Asn Leu Glu Asp Thr Asp Asp Leu Phe Gln Asn Glu 355 360 365 Phe Gly Ile Asn Thr Thr Val Gln Glu Arg Lys Leu Ile Arg Arg Leu 370 375 380 Ser Glu Leu Ile Gly Ala Arg Ala Ala Arg Leu Ser Val Cys Gly Ile 385 390 395 400 Ala Ala Ile Cys Gln Lys Arg Gly Tyr Lys Thr Gly His Ile Ala Ala 405 410 415 Asp Gly Ser Val Tyr Asn Arg Tyr Pro Gly Phe Lys Glu Lys Ala Ala 420 425 430 Asn Ala Leu Lys Asp Ile Tyr Gly Trp Thr Gln Thr Ser Leu Asp Asp 435 440 445 Tyr Pro Ile Lys Ile Val Pro Ala Glu Asp Gly Ser Gly Ala Gly Ala 450 455 460 Ala Val Ile Ala Ala Leu Ala Gln Lys Arg Ile Ala Glu Gly Lys Ser 465 470 475 480 Val Gly Ile Ile Gly Ala 485 <210> 122 <211> 1461 <212> DNA <213> Artificial Sequence <220> <223> Saccharomyces cerevisiae HXK2 <400> 122 atggttcatt taggtccaaa aaaaccacaa gccagaaagg gttccatggc cgatgtgcca 60 aaggaattga tgcaacaaat tgagaatttt gaaaaaattt tcactgttcc aactgaaact 120 ttacaagccg ttaccaagca cttcatttcc gaattggaaa agggtttgtc caagaagggt 180 ggtaacattc caatgattcc aggttgggtt atggatttcc caactggtaa ggaatccggt 240 gatttcttgg ccattgattt gggtggtacc aacttgagag ttgtcttagt caagttgggc 300 ggtgaccgta cctttgacac cactcaatct aagtacagat taccagatgc tatgagaact 360 actcaaaatc cagacgaatt gtgggaattt attgccgact ctttgaaagc ttttattgat 420 gagcaattcc cacaaggtat ctctgagcca attccattgg gtttcacctt ttctttccca 480 gcttctcaaa acaaaatcaa tgaaggtatc ttgcaaagat ggactaaagg ttttgatatt 540 ccaaacattg aaaaccacga tgttgttcca atgttgcaaa agcaaatcac taagaggaat 600 atcccaattg aagttgttgc tttgataaac gacactaccg gtactttggt tgcttcttac 660 tacactgacc cagaaactaa gatgggtgtt atcttcggta ctggtgtcaa tggtgcttac 720 tacgatgttt gttccgatat cgaaaagcta caaggaaaac tatctgatga cattccacca 780 tctgctccaa tggccatcaa ctgtgaatac ggttccttcg ataatgaaca tgtcgttttg 840 ccaagaacta aatacgatat caccattgat gaagaatctc caagaccagg ccaacaaacc 900 tttgaaaaaa tgtcttctgg ttactactta ggtgaaattt tgcgtttggc cttgatggac 960 atgtacaaac aaggtttcat cttcaagaac caagacttgt ctaagttcga caagcctttc 1020 gtcatggaca cttcttaccc agccagaatc gaggaagatc cattcgagaa cctagaagat 1080 accgatgact tgttccaaaa tgagttcggt atcaacacta ctgttcaaga acgtaaattg 1140 atcagacgtt tatctgaatt gattggtgct agagctgcta gattgtccgt ttgtggtatt 1200 gctgctatct gtcaaaagag aggttacaag accggtcaca tcgctgcaga cggttccgtt 1260 tacaacagat acccaggttt caaagaaaag gctgccaatg ctttgaagga catttacggc 1320 tggactcaaa cctcactaga cgactaccca atcaagattg ttcctgctga agatggttcc 1380 ggtgctggtg ccgctgttat tgctgctttg gcccaaaaaa gaattgctga aggtaagtcc 1440 gttggtatca tcggtgctta a 1461 <210> 123 <211> 344 <212> PRT <213> Artificial Sequence <220> <223> Saccharomyces cerevisiae ARA1 <400> 123 Met Ser Ser Ser Val Ala Ser Thr Glu Asn Ile Val Glu Asn Met Leu 1 5 10 15 His Pro Lys Thr Thr Glu Ile Tyr Phe Ser Leu Asn Asn Gly Val Arg 20 25 30 Ile Pro Ala Leu Gly Leu Gly Thr Ala Asn Pro His Glu Lys Leu Ala 35 40 45 Glu Thr Lys Gln Ala Val Lys Ala Ala Ile Lys Ala Gly Tyr Arg His 50 55 60 Ile Asp Thr Ala Trp Ala Tyr Glu Thr Glu Pro Phe Val Gly Glu Ala 65 70 75 80 Ile Lys Glu Leu Leu Glu Asp Gly Ser Ile Lys Arg Glu Asp Leu Phe 85 90 95 Ile Thr Thr Lys Val Trp Pro Val Leu Trp Asp Glu Val Asp Arg Ser 100 105 110 Leu Asn Glu Ser Leu Lys Ala Leu Gly Leu Glu Tyr Val Asp Leu Leu 115 120 125 Leu Gln His Trp Pro Leu Cys Phe Glu Lys Ile Lys Asp Pro Lys Gly 130 135 140 Ile Ser Gly Leu Val Lys Thr Pro Val Asp Asp Ser Gly Lys Thr Met 145 150 155 160 Tyr Ala Ala Asp Gly Asp Tyr Leu Glu Thr Tyr Lys Gln Leu Glu Lys 165 170 175 Ile Tyr Leu Asp Pro Asn Asp His Arg Val Arg Ala Ile Gly Val Ser 180 185 190 Asn Phe Ser Ile Glu Tyr Leu Glu Arg Leu Ile Lys Glu Cys Arg Val 195 200 205 Lys Pro Thr Val Asn Gln Val Glu Thr His Pro His Leu Pro Gln Met 210 215 220 Glu Leu Arg Lys Phe Cys Phe Met His Asp Ile Leu Leu Thr Ala Tyr 225 230 235 240 Ser Pro Leu Gly Ser His Gly Ala Pro Asn Leu Lys Ile Pro Leu Val 245 250 255 Lys Lys Leu Ala Glu Lys Tyr Asn Val Thr Gly Asn Asp Leu Leu Ile 260 265 270 Ser Tyr His Ile Arg Gln Gly Thr Ile Val Ile Pro Arg Ser Leu Asn 275 280 285 Pro Val Arg Ile Ser Ser Ser Ile Glu Phe Ala Ser Leu Thr Lys Asp 290 295 300 Glu Leu Gln Glu Leu Asn Asp Phe Gly Glu Lys Tyr Pro Val Arg Phe 305 310 315 320 Ile Asp Glu Pro Phe Ala Ala Ile Leu Pro Glu Phe Thr Gly Asn Gly 325 330 335 Pro Asn Leu Asp Asn Leu Lys Tyr 340 <210> 124 <211> 1035 <212> DNA <213> Artificial Sequence <220> <223> Saccharomyces cerevisiae ARA1 <400> 124 atgtcttctt cagtagcctc aaccgaaaac atagtcgaaa atatgttgca tccaaagact 60 acagaaatat acttttcact caacaatggt gttcgtatcc cagcactggg tttggggaca 120 gcaaatcctc acgaaaagtt agctgaaaca aaacaagccg taaaagctgc aatcaaagct 180 ggatacaggc acattgatac tgcttgggcc tacgagacag agccattcgt aggtgaagcc 240 atcaaggagt tattagaaga tggatctatc aaaagggagg atcttttcat aaccacaaaa 300 gtgtggccgg ttctatggga cgaagtggac agatcattga atgaatcttt gaaagcttta 360 ggcttggaat acgtcgactt gctcttgcaa cattggccgc tatgttttga aaagattaag 420 gaccctaagg ggatcagcgg actggtgaag actccggttg atgattctgg aaaaacaatg 480 tatgctgccg acggtgacta tttagaaact tacaagcaat tggaaaaaat ttaccttgat 540 cctaacgatc atcgtgtgag agccattggt gtctcaaatt tttccattga gtatttggaa 600 cgtctcatta aggaatgcag agttaagcca acggtgaacc aagtggaaac tcaccctcac 660 ttaccacaaa tggaactaag aaagttctgc tttatgcacg acattctgtt aacagcatac 720 tcaccattag gttcccatgg cgcaccaaac ttgaaaatcc cactagtgaa aaagcttgcc 780 gaaaagtaca atgtcacagg aaatgacttg ctaatttctt accatattag acaaggcact 840 atcgtaattc cgagatcctt gaatccagtt aggatttcct cgagtattga attcgcatct 900 ttgacaaagg atgaattaca agagttgaac gacttcggtg aaaaataccc agtgagattc 960 atcgatgagc catttgcagc catccttcca gagtttactg gtaacggacc aaacttggac 1020 aatttaaagt attaa 1035 <210> 125 <211> 312 <212> PRT <213> Artificial Sequence <220> <223> Saccharomyces cerevisiae YPR1 <400> 125 Met Pro Ala Thr Leu Lys Asn Ser Ser Ala Thr Leu Lys Leu Asn Thr 1 5 10 15 Gly Ala Ser Ile Pro Val Leu Gly Phe Gly Thr Trp Arg Ser Val Asp 20 25 30 Asn Asn Gly Tyr His Ser Val Ile Ala Ala Leu Lys Ala Gly Tyr Arg 35 40 45 His Ile Asp Ala Ala Ala Ile Tyr Leu Asn Glu Glu Glu Val Gly Arg 50 55 60 Ala Ile Lys Asp Ser Gly Val Pro Arg Glu Glu Ile Phe Ile Thr Thr 65 70 75 80 Lys Leu Trp Gly Thr Glu Gln Arg Asp Pro Glu Ala Ala Leu Asn Lys 85 90 95 Ser Leu Lys Arg Leu Gly Leu Asp Tyr Val Asp Leu Tyr Leu Met His 100 105 110 Trp Pro Val Pro Leu Lys Thr Asp Arg Val Thr Asp Gly Asn Val Leu 115 120 125 Cys Ile Pro Thr Leu Glu Asp Gly Thr Val Asp Ile Asp Thr Lys Glu 130 135 140 Trp Asn Phe Ile Lys Thr Trp Glu Leu Met Gln Glu Leu Pro Lys Thr 145 150 155 160 Gly Lys Thr Lys Ala Val Gly Val Ser Asn Phe Ser Ile Asn Asn Ile 165 170 175 Lys Glu Leu Leu Glu Ser Pro Asn Asn Lys Val Val Pro Ala Thr Asn 180 185 190 Gln Ile Glu Ile His Pro Leu Leu Pro Gln Asp Glu Leu Ile Ala Phe 195 200 205 Cys Lys Glu Lys Gly Ile Val Val Glu Ala Tyr Ser Pro Phe Gly Ser 210 215 220 Ala Asn Ala Pro Leu Leu Lys Glu Gln Ala Ile Ile Asp Met Ala Lys 225 230 235 240 Lys His Gly Val Glu Pro Ala Gln Leu Ile Ile Ser Trp Ser Ile Gln 245 250 255 Arg Gly Tyr Val Val Leu Ala Lys Ser Val Asn Pro Glu Arg Ile Val 260 265 270 Ser Asn Phe Lys Ile Phe Thr Leu Pro Glu Asp Asp Phe Lys Thr Ile 275 280 285 Ser Asn Leu Ser Lys Val His Gly Thr Lys Arg Val Val Asp Met Lys 290 295 300 Trp Gly Ser Phe Pro Ile Phe Gln 305 310 <210> 126 <211> 939 <212> DNA <213> Artificial Sequence <220> <223> Saccharomyces cerevisiae YPR1 <400> 126 atgcctgcta cgttaaagaa ttcttctgct acattaaaac taaatactgg tgcctccatt 60 ccagtgttgg gtttcggcac ttggcgttcc gttgacaata acggttacca ttctgtaatt 120 gcagctttga aagctggata cagacacatt gatgctgcgg ctatctattt gaatgaagaa 180 gaagttggca gggctatttaa agattccgga gtccctcgtg aggaaatttt tattactact 240 aagctttggg gtacggaaca acgtgatccg gaagctgctc taaacaagtc tttgaaaaga 300 ctaggcttgg attatgttga cctatatctg atgcattggc cagtgccttt gaaaaccgac 360 agagttactg atggtaacgt tctgtgcatt ccaacattag aagatggcac tgttgacatc 420 gatactaagg aatggaattt tatcaagacg tgggagttga tgcaagagtt gccaaagacg 480 ggcaaaacta aagccgttgg tgtctctaat ttttctatta acaacattaa agaattatta 540 gaatctccaa ataacaaggt ggtaccagct actaatcaaa ttgaaattca tccattgcta 600 ccacaagacg aattgattgc cttttgtaag gaaaagggta ttgttgttga agcctactca 660 ccatttggga gtgctaatgc tcctttacta aaagagcaag caattattga tatggctaaa 720 aagcacggcg ttgagccagc acagcttatt atcagttgga gtattcaaag aggctacgtt 780 gttctggcca aatcggttaa tcctgaaaga attgtatcca attttaagat tttcactctg 840 cctgaggatg atttcaagac tattagtaac ctatccaaag tgcatggtac aaagagagtc 900 gttgatatga agtggggatc cttcccaatt ttccaatga 939 <210> 127 <211> 267 <212> PRT <213> Artificial Sequence <220> <223> Saccharomyces cerevisiae YMR226C <400> 127 Met Ser Gln Gly Arg Lys Ala Ala Glu Arg Leu Ala Lys Lys Thr Val 1 5 10 15 Leu Ile Thr Gly Ala Ser Ala Gly Ile Gly Lys Ala Thr Ala Leu Glu 20 25 30 Tyr Leu Glu Ala Ser Asn Gly Asp Met Lys Leu Ile Leu Ala Ala Arg 35 40 45 Arg Leu Glu Lys Leu Glu Glu Leu Lys Lys Thr Ile Asp Gln Glu Phe 50 55 60 Pro Asn Ala Lys Val His Val Ala Gln Leu Asp Ile Thr Gln Ala Glu 65 70 75 80 Lys Ile Lys Pro Phe Ile Glu Asn Leu Pro Gln Glu Phe Lys Asp Ile 85 90 95 Asp Ile Leu Val Asn Asn Ala Gly Lys Ala Leu Gly Ser Asp Arg Val 100 105 110 Gly Gln Ile Ala Thr Glu Asp Ile Gln Asp Val Phe Asp Thr Asn Val 115 120 125 Thr Ala Leu Ile Asn Ile Thr Gln Ala Val Leu Pro Ile Phe Gln Ala 130 135 140 Lys Asn Ser Gly Asp Ile Val Asn Leu Gly Ser Ile Ala Gly Arg Asp 145 150 155 160 Ala Tyr Pro Thr Gly Ser Ile Tyr Cys Ala Ser Lys Phe Ala Val Gly 165 170 175 Ala Phe Thr Asp Ser Leu Arg Lys Glu Leu Ile Asn Thr Lys Ile Arg 180 185 190 Val Ile Leu Ile Ala Pro Gly Leu Val Glu Thr Glu Phe Ser Leu Val 195 200 205 Arg Tyr Arg Gly Asn Glu Glu Gln Ala Lys Asn Val Tyr Lys Asp Thr 210 215 220 Thr Pro Leu Met Ala Asp Asp Val Ala Asp Leu Ile Val Tyr Ala Thr 225 230 235 240 Ser Arg Lys Gln Asn Thr Val Ile Ala Asp Thr Leu Ile Phe Pro Thr 245 250 255 Asn Gln Ala Ser Pro His His Ile Phe Arg Gly 260 265 <210> 128 <211> 804 <212> DNA <213> Artificial Sequence <220> <223> Saccharomyces cerevisiae YMR226C <400> 128 atgtcccaag gtagaaaagc tgcagaaaga ttggctaaga agactgtcct cattacaggt 60 gcatctgctg gtattggtaa ggcgaccgca ttagagtact tggaggcatc caatggtgat 120 atgaaactga tcttggctgc tagaagatta gaaaagctcg aggaattgaa gaagaccatt 180 gatcaagagt ttccaaacgc aaaagttcat gtggcccagc tggatatcac tcaagcagaa 240 aaaatcaagc ccttcattga aaacttgcca caagagttca aggatattga cattctggtg 300 aacaatgccg gaaaggctct tggcagtgac cgtgtgggcc agatcgcaac ggaggatatc 360 caggacgtgt ttgacaccaa cgtcacggct ttaatcaata tcacacaagc tgtactgccc 420 atattccaag ccaagaattc aggagatatt gtaaatttgg gttcaatcgc tggcagagac 480 gcatacccaa caggttctat ctattgtgcc tctaagtttg ccgtgggggc gttcactgat 540 agtttgagaa aggagctcat caacactaaa attagagtca ttctaattgc accagggcta 600 gtcgagactg aattttcact agttagatac agaggtaacg aggaacaagc caagaatgtt 660 tacaaggata ctaccccatt gatggctgat gacgtggctg atctgatcgt ctatgcaact 720 tccagaaaac aaaatactgt aattgcagac actttaatct ttccaacaaa ccaagcgtca 780 cctcatcata tcttccgtgg ataa 804 <210> 129 <211> 301 <212> DNA <213> Artificial Sequence <220> <223> ADH2-1 <400> 129 ccacttcacg agactgatct cctctgccgg aacaccgggc atctccaact tataagttgg 60 agaaataaga gaatttcaga ttgagagaat gaaaaaaaaa aaaaaaaaaa aggcagagga 120 gagcatagaa atggggttca ctttttggta aagctatagc atgcctatca catataaata 180 gagtgccagt agcgactttt ttcacactcg aaatactctt actactgctc tcttgttgtt 240 tttatcactt cttgtttctt cttggtaaat agaatatcaa gctacaaaaa gcatacaatc 300 a 301 <210> 130 <211> 302 <212> DNA <213> Artificial Sequence <220> <223> ADH2-2 <400> 130 gcggatctct tatgtcttta cgatttatag ttttcattat caagtatgcc tatattagta 60 tatagcatct ttagatgaca gtgttcgaag tttcacgaat aaaagataat attctacttt 120 ttgctcccac cgcgtttgct agcacgagtg aacaccatcc ctcgcctgtg agttgtaccc 180 attcctctaa actgtagaca tggtagcttc agcagtgttc gttatgtacg gcatcctcca 240 acaaacagtc ggttatagtt tgtcctgctc ctctgaatcg tctccctcga tatttctcat 300 tt 302 <210> 131 <211> 167 <212> DNA <213> Artificial Sequence <220> <223> YARCdelta4-1 <400> 131 tgttggaata gaaatcaact atcatctact aactagtatt tacattacta gtatattatc 60 atatacggtg ttagaagatg acgcaaatga tgagaaatag tcatctaaat tagtggaagc 120 tgaaacgcaa ggattgataa tgtaatagga tcaatgaata taaacat 167 <210> 132 <211> 170 <212> DNA <213> Artificial Sequence <220> <223> YARCdelta4-2 <400> 132 ataaaacgga atgaggaata atcgtaatat tagtatgtag aaatatagat tccattttga 60 ggattcctat atcctcgagg agaacttcta gtatattctg tatacctaat attatagcct 120 ttatcaacaa tggaatccca acaattatct caacattcac ccatttctca 170 <210> 133 <211> 88 <212> DNA <213> Artificial Sequence <220> <223> F primer for ADH2-1 <400> 133 tttccccgaa aagtgcattt aaatccactt cacgagactg atcttttccc cgaaaagtgc 60 atttaaatcc acttcaggag actgatct 88 <210> 134 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> R primer for ADH2-1 <400> 134 tcgagcggcc gcgggccctg attgtatgct ttttgtagct tg 42 <210> 135 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> F primer for ADH2-2 <400> 135 ctgagagctc ttaattaagc ggatctctta tgtctttacg 40 <210> 136 <211> 47 <212> DNA <213> Artificial Sequence <220> <223> R primer for ADH2-2 <400> 136 catgttcttt cctgcgattt aaataaatga gaaatatcga gggagac 47 <210> 137 <211> 51 <212> DNA <213> Artificial Sequence <220> <223> F primer for YARCdelta4-1 <400> 137 cacaatttccc cgaaaagtgc atttaaattg ttggaataga aatcaactat c 51 <210> 138 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> R primer for YARCdelta4-1 <400> 138 atagcggccg catgtttata ttcattgatc ctattaca 38 <210> 139 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> F primer for YARCdelta4-2 <400> 139 atagagctca taaaacggaa tgaggaataa tcg 33 <210> 140 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> R primer for YARCdelta4-2 <400> 140 tgagaaatgg gtgaatgttg ag 22

Claims (22)

Compared to the parent strain,
i) alcohol dehydrogenase, glycerol-3-phosphate dehydrogenase and 2,3-butanediol dehydrogenase activity is reduced,
ii) increases the activity of acetolactate synthase, acetolactate decarboxylase and NADH oxidase;
iii) genetically engineered yeast in which EMP46, PEP7, SUR1 and HXK2 are mutated. According to claim 1,
The alcohol dehydrogenase is any one selected from the group consisting of ADH1, ADH2, ADH3, ADH4, ADH5, and combinations thereof, genetically engineered yeast. According to claim 1,
The genetically engineered yeast, wherein the glycerol-3-phosphate dehydrogenase is any one selected from the group consisting of GPD1, GPD2, and combinations thereof. According to claim 1,
The yeast is any one exogenous gene selected from the group consisting of a gene encoding acetolactate synthase, a gene encoding acetolactate decarboxylase, a gene encoding NADH oxidase, and combinations thereof A genetically engineered yeast comprising a. According to claim 1,
The EMP46 mutation is a genetically engineered yeast wherein guanine, which is the 480th base of the nucleotide sequence represented by SEQ ID NO: 116, is substituted with thymine. According to claim 1,
The PEP7 mutation is a genetically engineered yeast wherein cytosine, which is the 505th base in the nucleotide sequence represented by SEQ ID NO: 118, is substituted with adenine. According to claim 1,
The SUR1 mutation is a genetically engineered yeast wherein cytosine, which is the 526th base of the nucleotide sequence shown in SEQ ID NO: 120, is substituted with thymine. According to claim 1,
The HXK2 mutation is a genetically engineered yeast wherein guanine, which is the 754th base, of the nucleotide sequence shown in SEQ ID NO: 122 is substituted with thymine. According to claim 1,
The yeast is arabinose dehydrogenase, NADP-dependent aldo-keto reductase (NADPH-dependent aldo-keto reductase), NADP-dependent 3-hydroxy acid dehydrogenase (NADP-dependent 3-hydroxy acid dehydrogenase) And a genetically engineered yeast with reduced activity of any one selected from the group consisting of combinations thereof. 10. The method of claim 9,
The yeast is selected from the group consisting of a gene encoding arabinose dehydrogenase, a gene encoding NADP-dependent aldo-kedo reductase, a gene encoding NADP-dependent 3-hydroxy acid dehydrogenase, and combinations thereof. A genetically engineered yeast, wherein one is deleted. 11. The method of claim 10,
The arabinose dehydrogenase is The genetically engineered yeast, which is encoded by the ara1 gene comprising the nucleotide sequence shown in SEQ ID NO: 124. 11. The method of claim 10,
The NADP-dependent aldo-kedo reductase is A genetically engineered yeast that is encoded by the ypr1 gene comprising the nucleotide sequence represented by SEQ ID NO: 126. 11. The method of claim 10,
The NADP-dependent 3-hydroxy acid dehydrogenase is encoded by the ymr226c gene comprising the nucleotide sequence shown in SEQ ID NO: 128, the genetically engineered yeast. According to claim 1,
The yeast cell is a Saccharomyces genus strain, the genetically engineered yeast. 15. The method of claim 14,
The Saccharomyces sp. strain is Saccharomyces cerevisiae ( S. scerevisiae ), Saccharomyces bayanus ( S. bayanus ), Saccharomyces paradoxus ( S. paradoxus ), Saccharomyces US category (S. mikatae), and a saccharide as MY-ku laundry process Havre lots (S. kudriavzevii) any one of a will, a genetically manipulate the yeast is selected from the group consisting of oil. i) culturing the genetically engineered yeast of any one of claims 1-15; and
ii) A method for producing acetoin, comprising the step of obtaining acetoin produced from the yeast. 17. The method of claim 16,
The culturing method for producing acetoin, characterized in that culturing yeast in the presence of glucose. i) deleting a gene encoding an alcohol dehydrogenase of wild-type yeast, a gene encoding a glycerol-3-phosphate dehydrogenase and a gene encoding a 2,3-butanediol dehydrogenase;
ii) introducing a gene encoding acetolactate synthase, a gene encoding acetolactate decarboxylase and a gene encoding NADH oxidase; and
iii) subculturing the yeast in an acetoin-added medium at least 15 times,
A method for producing the yeast having excellent acetoin-producing ability according to claim 1. 19. The method of claim 18,
The method to which the acetoin is added medium is characterized in that at least 4 g / L concentration of acetoin is added. 19. The method of claim 18,
After step ii), the yeast consists of a gene encoding arabinose dehydrogenase, a gene encoding NADP-dependent aldo-kedo reductase, a gene encoding NADP-dependent 3-hydroxy acid dehydrogenase, and combinations thereof. The method further comprising the step of deleting any one selected from the group. A yeast having excellent acetoin-producing ability, prepared by the method of any one of claims 18 to 20. i) culturing the yeast having excellent acetoin-producing ability of claim 21; and
ii) A method for producing acetoin, comprising the step of obtaining acetoin produced from the yeast.

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