KR102136841B1 - Recombinant Yeast Producing Ethanol from Xylose and Method for Producing Ethanol by Using the Recombinant Yeast - Google Patents

Abstract

The present invention relates to a recombinant yeast having high ethanol production capacity from xylose, and more specifically, Spadaspora room passalidarum) derived xylitol dehydrogenase (XDH: a gene encoding xylitol dehydrogenase) and xylose reductase (XR: gene coding for xylose reductase) and Pichia styryl blood tooth (Pichia stipitis) derived from the xylene rule Rocca or kinase (XK : xylulokinase) is a recombinant yeast having the ability to produce ethanol from xylose into which a gene encoding is introduced, and a method for producing ethanol using the recombinant yeast.
Recombinant yeast according to the present invention in producing ethanol from cellulose-based biomass rich in xylose, does not accumulate much by-product xylitol, can produce ethanol in high yield, and thus produces cellulose ethanol (Cellulosic ethanol: CE). It can be very useful.

Description

Recombinant Yeast Producing Ethanol from Xylose and Method for Producing Ethanol by Using the Recombinant Yeast}

The present invention relates to a recombinant yeast having high ethanol production capacity from xylose, and more specifically, a gene encoding xylitol dehydrogenase (XDH: xylitol dehydrogenase) derived from Spathaspora passalidarum and xylose reductase ( XR: xylose reductase (XR) gene and Pichia stipitis- derived xylulokinase (XK: xylulokinase)-encoded gene is introduced from xylose, which is a recombinant yeast having ethanol production capacity and It relates to a method for producing ethanol using the recombinant yeast.

As interest in biofuels and renewable energy increases, research to use cellulose ethanol (CE) as a next-generation fuel has been actively conducted, but technical difficulties such as pre-treatment technology, saccharification enzyme, and fermentation strain discovery It is emerging as an element.

In particular, unlike traditional ethanol fermentation, which uses hexasaccharide glucose (Glucose) as a carbon source, cellulose ethanol is produced using xylose (Xylose, wood sugar) as a carbon source, thereby producing ethanol with high efficiency using xylose. Development of a strain that can be desperately needed

The representative ethanol fermentation strain is Yeast, which is represented by Saccharomyces cerevisiae . Yeast is excellent in productivity and yield and has strong resistance to ethanol, so it is traditionally used in the ethanol industry and fermentation industry. It has been a strain. However, there is a limitation that xylose, a pentose sugar, cannot be used as a carbon source. Therefore, by introducing genes involved in the metabolic pathway of xylose into S. cerevisiae using metabolic engineering, development of S. cerevisiae capable of fermenting ethanol using xylose as a carbon source has been made.

For example, xyl1 , a gene encoding xylose reductase from Pichia stipitis , xyl2 , a gene encoding xylitol dehydrogenase, and By introducing xyl3 , a gene encoding xylulokinase, into S. cerevisiae , S. cerevisiae can produce ethanol using xylose (US 5,789,210B). However, when using the strain, the productivity or yield did not reach the commercialization level (productivity 1 g/l/h, yield 0.45 g/g or more). This low productivity and yield are known to be caused by cofactor imbalance caused by NADPH-dependent xylose reductase (XR) and NAD-dependent xylose dehydrogenase (XDH).

Thus, the present inventors, as a result of diligent efforts to increase ethanol productivity in a transformed yeast strain having ethanol production capacity using xylose, surprisingly, xylitol dehydrogenase (XDH: xylitol dehydrogenase) derived from Spathaspora passalidarum The gene encoding xyl2, the xylose reductase encoding gene xyl1, and Recombinant yeast in which the gene xyl3 encoding xylulokinase (XK) derived from Pichia stipitis is introduced, particularly in the case of the S. cerevisiae strain, xyl1 , xyl2 derived from Pichia stipitis And it was confirmed that the xyl3 shows superior ethanol production capacity and yield compared to the existing results using S. cerevisiae introduced, and completed the present invention.

An object of the present invention is to provide a recombinant yeast having the ability to effectively convert xylose derived from cellulose-based biomass to ethanol for production in order to produce cellulose ethanol (CE).

Another object of the present invention is to provide a method for producing ethanol using recombinant yeast having the ability to effectively convert the xylose to ethanol.

In order to achieve the above object, the present invention is a spadaspora passaridarum ( Spathaspora passalidarum ) Derived Xylitol dehydrogenase-encoding gene, xylose reductase-encoding gene, and Pichia stipitis- derived xylulurokinase -encoding gene are introduced, and recombination with ethanol production capacity from xylose Provides yeast.

In addition, the present invention (a) culturing the yeast in a xylose-containing medium to produce ethanol; And (b) provides a method for producing ethanol from xylose comprising the step of obtaining the produced ethanol.

The yeast strain of the present invention can produce ethanol from xylose-rich cellulose-based biomass, and does not accumulate much by-product xylitol, and can produce ethanol with high yield, thereby producing biofuel using cellulose ethanol (CE). It can be very useful.

1 shows a recombinant vector containing a gene encoding xylose reductase (XR): (A) PsXR; (B) SpXR ^CO .
2 shows a recombinant vector containing a gene encoding xylitol dehydrogenase (XDH): (A) PsXDH; (B) SpXDH.
Figure 3 shows a recombinant vector containing a gene (PsXK) encoding xylulurokinase (XK).
Figure 4 shows the result of culturing the recombinant yeast of the present invention under anaerobic conditions, reducing by-product xylitol production and improving ethanol production capacity.

In one aspect, the present invention is a Spathaspora passalidarum Derived Xylitol dehydrogenase-encoding gene, xylose reductase-encoding gene, and Pichia stipitis- derived xylulurokinase -encoding gene are introduced, and recombination with ethanol production capacity from xylose It is about yeast.

Xylose is a biomass that is present in waste wood, etc. in the form of polymer xylan, and is a monosaccharide of 5 carbons that can be metabolized as a useful product by various organisms.It is a two-step pentos phosphate pathway (Pentose phosphate pathway (PPP).

However, Saccharomyces cerevisiae , a yeast mainly used in conventional ethanol fermentation, is a gene xyl1 and xylitol dehydrogenase (xylitol dehydrogenase) encoding xylose reductase (XR) in terms of xylose metabolism. The xyl2 gene encoding dehydrogenase (XDH) was not present, so xylose could not be used as a metabolism or carbon source.

Xylose is reduced to xylitol by xylose reductase (XR) using NADH or NADPH as a cofactor, and xylitol is oxidized to xylulose by xylitol dehydrogenase (XDH) using NAD+ as a cofactor, and additionally xylized It is converted to xylulose-5-phosphate by xylulokinase (XK) introduced into cellulose, and metabolism proceeds through a pentose phosphate cycle. Xylulurokinase (XK) is an enzyme present in yeast, but if XR and XDH are introduced into the strain without overexpression, ethanol can be produced from xylose, but yield and productivity are significantly lower.

Unless otherwise defined in the present invention, xyl1 is a gene encoding xylose reductase, xyl2 is a gene encoding xylitol dehydrogenase, xyl3 is a gene encoding xylulurokinase , and XR is xylose reduction. The enzyme, XDH means xylitol dehydrogenase, and XK means xylulurokinase.

In one aspect of the present invention, by overexpressing the xylitol dehydrogenase of Spathaspora passalidarum derived from yeast (XDH) SpXDH and Pichia stipitis derived from xylose the reductase (XR) PsXR and Pichia stipitis derived xylene rule Rocca or kinase (XK) PsXK , Byproduct xylitol production efficiency was reduced.

In another aspect of the present invention, by overexpressing the xylitol dehydrogenase of Spathaspora passalidarum derived from yeast (XDH) SpXDH and Spathaspora passalidarum derived from xylose the reductase (XR) SpXR and Pichia stipitis derived xylene rule Rocca or kinase (XK) PsXK , Reduced by-product xylitol production and dramatically increased ethanol production efficiency.

In the present invention, the gene encoding xylitol dehydrogenase ( xyl2 ) is characterized by being derived from Pichia stipitis or Spathaspora passalidarum .

In the present invention, the xylitol dehydrogenase preferably has the amino acid sequence of SEQ ID NO: 7 or SEQ ID NO: 5. SEQ ID NO: 7 refers to the amino acid sequence of xylitol dehydrogenase (PsXDH) derived from Pichia stipitis , and SEQ ID NO: 5 refers to the amino acid sequence of xylitol dehydrogenase (SpXDH) derived from Spathaspora passalidarum .

Preferably, it has a nucleotide sequence of gene PsXyl2 SEQ ID NO: 8 encoding Pichia stipitis- derived xylitol dehydrogenase or gene SpXyl2 SEQ ID NO: 6 encoding Spathaspora passalidarum- derived xylitol dehydrogenase. The base sequence of SEQ ID NO: 8 or 6 is a sequence of a wild type gene encoding xylitol dehydrogenase derived from P. stipitis or S. passalidarum .

In the present invention, the gene encoding xylose reductase ( xyl1 ) is characterized by being derived from Pichia stipitis or Spathaspora passalidarum , and the gene encoding xylulurokinase ( xyl3 ) is also derived from Pichia stipitis . do.

In the present invention, the xylose reductase preferably has the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3. SEQ ID NO: 1 refers to the amino acid sequence of xylitol dehydrogenase (SpXR) from Spathaspora passalidarum , and SEQ ID NO: 3 refers to the amino acid sequence of xylitol dehydrogenase (PsXR) from Pichia stipitis .

In addition, the gene encoding the xylose reductase derived from Pichia stipitis ( Psxyl1 ) is preferably characterized by having the nucleotide sequence of SEQ ID NO: 4, the gene encoding the xylose reductase derived from Spathaspora passalidarum ( Spxyl1 ) sequence The base sequence of No. 2 is a sequence obtained by codon optimization so as to be optimally expressed in S. cerevisiae .

In the present invention, SEQ ID NO: 9 of the xylulokinase refers to the amino acid sequence of xylulurokinase (PsXK) derived from Pichia stipitis .

In addition, the gene ( Psxyl3 ) encoding xylulurokinase derived from Pichia stipitis is preferably characterized by having a nucleotide sequence of SEQ ID NO: 10, and is a natural type encoding xylulurokinase derived from P. stipitise ( wild type) is the sequence of the gene.

Sequence numbers and definitions of the enzymes and base sequences encoding them in the present invention are as shown in Table 1.

Sequence number and definition of each enzyme and base sequence encoding it in the present invention order
number Display Justice One SpXR Xylose from Spathaspora passalidarum
Reductase amino acid sequence 2 Spxyl1 ^CO It encodes SpXR and expresses it in recombinant yeast.
Sequence sequence optimized for codon 3 PsXR Xylose reductase from Pichia stipitis
Amino acid sequence 4 Psxyl1 The base sequence of the gene encoding PsXR 5 SpXDH Spathaspora derived from passalidarum
Xylitol dehydrogenase amino acid sequence 6 Spxyl2 Base sequence encoding SpXDH 7 PsXDH Xylitol dehydrogenase from Pichia stipitis
Amino acid sequence 8 Psxyl2 Base sequence encoding PsXDH 9 PsXK Xylulurokinase from Pichia stipitis
Amino acid sequence 10 Psxyl3 Base sequence encoding PsXK

In the present invention, the term “vector” refers to a DNA preparation containing a DNA sequence operably linked to a suitable regulatory sequence capable of expressing DNA in a suitable host, in particular recombinant yeast. The vector can be a plasmid, phage particle, or simply a potential genomic insert. Once transformed into a suitable host, the vector can replicate and function independently of the host genome, or in some cases can be integrated into the genome itself. As plasmids are the most commonly used form of current vectors, “plasmid” and “vector” are sometimes used interchangeably in the context of the present invention. However, the present invention includes other forms of vectors having functions equivalent to those known or known in the art.

The expression “expression control sequence” refers to a DNA sequence essential for the expression of a coding sequence operably linked in a particular host organism. Such regulatory sequences include promoters for conducting transcription, any operator sequences for regulating such transcription, sequences encoding suitable mRNA ribosomal binding sites, and sequences that regulate termination of transcription and translation. For example, regulatory sequences suitable for prokaryotes include promoters, optionally operator sequences, and ribosome binding sites. Eukaryotic cells include promoters, polyadenylation signals and enhancers. In the plasmid, the factor that most influences the amount of gene expression is the promoter.

In order to express the DNA sequence of the present invention, any of a wide variety of expression control sequences can be used in the vector. Examples of useful expression control sequences include, for example, lac system, trp system, TAC or TRC system, T3 and T7 promoters, major operator and promoter regions of phage lambda, regulatory regions of fd coded proteins, 3-phosphoglycerol Promoters for rate kinase or other glycolytic enzymes, promoters of the phosphatase, for example Pho5, a promoter of the yeast alpha-crossing system and a composition known to regulate expression of genes in prokaryotic or eukaryotic cells or their viruses Other sequences of induction and several combinations thereof.

A nucleic acid is “operably linked” when placed in a functional relationship with other nucleic acid sequences. This may be a gene and regulatory sequence(s) linked to the appropriate molecule (eg, a transcriptional activation protein) in a manner that allows for gene expression when bound to the regulatory sequence(s). For example, DNA for a pre-sequence or secretory leader is operably linked to DNA for a polypeptide when expressed as a shear protein that participates in the secretion of the polypeptide; A promoter or enhancer is operably linked to a coding sequence when it affects transcription of the sequence; Or the ribosome binding site is operably linked to the coding sequence if it affects the transcription of the sequence; Alternatively, the ribosome binding site is operably linked to the coding sequence when arranged to facilitate translation. Generally, “operably linked” means that the linked DNA sequence is in contact, and in the case of a secretory leader, is in contact and is present in the reading frame. However, the enhancer does not need to be in contact. Linking these sequences is accomplished by ligation (linking) at convenient restriction enzyme sites. If such a site does not exist, a synthetic oligonucleotide adapter or linker according to a conventional method is used.

As used herein, the term “expression vector” is usually a recombinant carrier into which a heterologous DNA fragment is inserted, and generally refers to a double-stranded DNA fragment. Here, the heterologous DNA refers to heterologous DNA, which is DNA not naturally found in the host cell. Once expressed in a host cell, the expression vector can replicate independent of the host chromosomal DNA and several copies of the vector and its inserted (heterologous) DNA can be produced.

As is well known in the art, in order to increase the expression level of a gene transformed in a host cell, the gene must be operably linked to transcriptional and translational expression control sequences that function in the selected expression host. Preferably, the expression control sequence and the corresponding gene are included in one expression vector including a bacterial selection marker and a replication origin. When the expression host is a eukaryotic cell, the expression vector must further include an expression marker useful in the eukaryotic expression host.

As used herein, the term “transformation” means that DNA is introduced into a host so that the DNA can be cloned as an extrachromosomal factor or by chromosomal integration. As used herein, the term “transfection” means that the expression vector is received by the host cell, whether or not any coding sequence is actually expressed.

Of course, it should be understood that not all vectors and expression control sequences are equally functional in expressing the DNA sequence of the present invention. Likewise, not all hosts function the same for the same expression system. However, those skilled in the art can make appropriate selection among various vectors, expression control sequences and hosts without departing from the scope of the present invention without undue experimental burden. For example, when choosing a vector, the host must be considered, because the vector must be cloned in it. The number of copies of the vector, the ability to control the number of copies, and the expression of other proteins encoded by the vector, such as antibiotic markers, should also be considered. In selecting an expression control sequence, several factors must be considered. For example, relative strength of the sequence, controllability and compatibility with the DNA sequence of the present invention should be considered, particularly with regard to possible secondary structures. A single-cell host can select from the host a selected vector, the toxicity of the product encoded by the DNA sequence of the invention, the secretion properties, the ability to accurately fold the protein, culture and fermentation requirements, and the product encoded by the DNA sequence of the invention It should be selected considering factors such as ease of purification. Within the scope of these parameters, those skilled in the art can select various vector/expression control sequences/host combinations capable of expressing the DNA sequence of the present invention in fermentation or in large-scale animal culture.

In another aspect, the present invention comprises the steps of (a) culturing the recombinant yeast in a xylose-containing medium to produce ethanol; And (b) obtaining the produced ethanol.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as limited by these examples.

Example 1: xyl1 , xyl2 or xyl3 Construction of recombinant vector containing gene

1.1 Gene encoding xylose reductase (XR) ( xyl1 Construction of recombinant vector containing)

To produce a vector containing a gene encoding a xylose reductase, the pRS426 TEF vector, a Saccharomyces cerevisiae expression vector having a TEF promoter of S. cerevisiae and a CYC1 terminator (Mumberg D. et al., Gene 156:119, 1995) was used as the backbone.

Spxyl1 ^CO is obtained from S. passalidarum- derived Spxyl1 gene having a nucleotide sequence of SEQ ID NO: 2 using a program jointly developed by Bionia and KAIST to obtain a nucleotide sequence (SEQ ID NO: 2) optimized in S. cerevisiae , It was synthesized and used (Bionia, Korea).

As the xylose reductase (XR) gene to be introduced into the pRS426 TEF vector, Psxyl1 derived from Pichia stipitis was used.

Psxyl1, the P. stipitis P. stipitis strain is to a genomic DNA template obtained in (ATCC 58785), and the product was amplified by performing PCR under TaOpt (optimal annealing temperature) of 57 ℃ using primers.

Forward primer PsXR:

5'-GATCGGATCCATGCCTTCTATTAAGTTGAA-3' (SEQ ID NO: 11)

Reverse primer PsXR:

5'-TCGACTCGAGTTAGACGAAGATAGGAATCT-3' (SEQ ID NO: 12)

Synthesized Spxyl1 ^CO gene of S. passalidarum and Psxyl1 fragment of amplified P. stipitis were ligated to pRS426 TEF vector digested with Bam HI and Xho I, respectively, to pRS426 TEF PsXR( xyl1 ) and pRS426 TEF SpXR ^CO ( xyl1 ), respectively. It was produced (Fig. 1A, B).

1.2 Xylitol dehydrogenase (XDH) gene ( xyl2 Construction of recombinant vector containing)

To construct a vector containing the xylitol dehydrogenase gene, the backbone of the pRS424 TEF vector (Mumberg D. et al., Gene 156:119, 1995), an S. cerevisiae expression vector having a TEF promoter of S. cerevisiae and a CYC1 terminator, It was used as.

S. passalidarum of Spxyl2 was amplified by performing PCR under the (optimal annealing temperature) of 58 to TaOpt using the genomic DNA as a template obtained in S. passalidarum strain (ATCC MYA-43455), primers.

Forward primer SpXDH:

5-GATCGGATCCATGGTTGCTAACCCATCATTAGTT-3 (SEQ ID NO: 13)

Reverse primer SpXDH:

5-TCGACTCGAGTTATAATGGACCATCAATCAAACA-3 (SEQ ID NO: 14)

P. stipitis- derived Psxyl2 was amplified by performing PCR under TaOpt (optimal annealing temperature) at 60° C. using the following primers as a genomic DNA template obtained from the P. stipitis strain ( ATCC 58785 ) .

Forward primer PsXDH:

5'-GATCGGATCCATGACTGCTAACCCTTCCTT-3' (SEQ ID NO: 15)

Reverse primer PsXDH:

5'-TCGACTCGAGTTACTCAGGGCCGTCAATGA-3' (SEQ ID NO: 16)

Spxyl2 fragments of amplified S. passalidarum and Psxyl2 fragments of amplified P. stipitis were ligated to pRS424 TEF vectors cut with BamHI and XhoI, respectively, to prepare pRS424 TEF SpXDH ( xyl2 ) and pRS424 TEF PsXDH ( xyl2 ), respectively . (Figure 2 A, B).

1.3 Xylulokinase (XK) gene ( xyl3 Construction of recombinant vector containing)

In order to produce a vector containing kinase genes in xylene rule, the TEF promoter and the S. cerevisiae CYC1 of S. cerevisiae expression vector pRS425 having the TEF vector terminator (Mumberg D. et al, Gene 156 :. 119, 1995) Was used as the backbone.

Psxyl3 of P. stipitis was amplified by performing PCR under the P. stipitis strain (ATCC 58785) genomic DNA as a template by, for TaOpt (optimal annealing temperature) of 57 ℃ using primers obtained from.

Forward primer PsXK:

5'-GATCGGATCCATGACCACTACCCCATTTGA-3' (SEQ ID NO: 17)

Reverse primer PsXK:

5'-TCGACTCGAGTTAGTGTTTCAATTCACTTT-3' (SEQ ID NO: 18)

The amplified Ps xyl3 fragment of P. stipitis was ligated after cutting the pRS425 TEF vector with Bam HI and Xho I to prepare pRS425 TEF PsXK ( xyl3 ) (FIG. 3).

Example 2: Production of ethanol from xylose using recombinant yeast

The Saccharomyces cerevisiae CEN-PK2-1D (euroscarf 30000D) was transformed with a recombinant vector containing xylose reductase (XR), xylitol dehydrogenase (XDH) and xylulurokinase (XK) genes prepared in Example 1, respectively. As shown in Table 2, a recombinant yeast strain capable of producing ethanol from xylose was prepared, and ethanol and xylitol production amounts of each strain were checked according to the type of the introduced gene.

Recombinant yeast experimental group Experimental group Introduced gene SET Recombinant vector introduced Control PsXR/PsXDH/PsXK pRS426 TEF PsXR( xyl1 ), pRS424 TEF PsXDH( xyl2 ), pRS425 TEF PsXK( xyl3 ) One PsXR/SpXDH/PsXK pRS426 TEF PsXR( xyl1 ), pRS424 TEF SpXDH( xyl2 ), pRS425 TEF PsXK( xyl3 ) 2 SpXR ^co /PsXDH/PsXK pRS426 TEF SpXR ^CO ( xyl1 ), pRS424 TEF PsXDH( xyl2 ), pRS425 TEF PsXK( xyl3 ) 3 SpXR ^co /SpXDH/PsXK pRS426 TEF SpXR ^CO ( xyl1 ), pRS424 TEF SpXDH( xyl2 ), pRS425 TEF PsXK( xyl3 )

Media type and culture conditions Seed culture (16 hours) YNB w/o ura, leu, trp (2% glucose)
5ml, 30℃, 200rpm Pre-culture (48 hours) YNB w/o ura, leu, trp (2% glucose)
100ml in 500ml flask, 30℃, 200rpm Main culture (120 hours) YP (4% xylose)
-Microaerobic: 100ml in 500ml flask, 30℃, 80rpm
-Anaerobic: 100ml in 300ml serum bottle, 30℃, 150rpm

YNB: Yeast Nitrogen Base (6.7 g/L) (Sigma, St. Louis, USA)

YP: Yeast Extract (10 g/L), Peptone (20 g/L) (Difco, Lab., Detroit, MI, USA)

Table 4 and FIG. 4 show the types of the recombinant vectors introduced for each strain and the ethanol and xylitol production amounts of the strains into which the recombinant vectors were introduced.

As a result, the recombinant S. cerevisiae sets 1 to 3 are xyl1 and xyl2 derived from Pichia stipitis as a control. And xyl3 introduced S. cerevisiae showed significantly higher ethanol production capacity and lower xylitol production capacity .

Since the specific parts of the present invention have been described in detail above, for those skilled in the art, this specific technique is only an example of a preferred implementation, and the scope of the present invention is not limited thereby. It will be obvious. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

<110> SK Innovation Co., Ltd. SK Energy Co., LTD. <120> Recombinant Yeast Producing Ethanol from Xylose and Method for Producing Ethanol by Using the Recombinant Yeast <130> P13-B121 <160> 18 <170> KopatentIn 2.0 <210> 1 <211> 317 <212> PRT <213> Spathaspora passalidarum <400> 1 Met Ser Phe Lys Leu Ser Ser Gly Tyr Glu Met Pro Lys Ile Gly Phe 1 5 10 15 Gly Thr Trp Lys Met Asp Lys Ala Thr Ile Pro Gln Gln Ile Tyr Asp 20 25 30 Ala Ile Lys Gly Gly Ile Arg Ser Phe Asp Gly Ala Glu Asp Tyr Gly 35 40 45 Asn Glu Lys Glu Val Gly Leu Gly Tyr Lys Lys Ala Ile Glu Asp Gly 50 55 60 Leu Val Lys Arg Glu Asp Leu Phe Ile Thr Ser Lys Leu Trp Asn Asn 65 70 75 80 Phe His Asp Pro Lys Asn Val Glu Lys Ala Leu Asp Arg Thr Leu Ala 85 90 95 Asp Leu Gln Leu Asp Tyr Val Asp Leu Phe Leu Ile His Phe Pro Ile 100 105 110 Ala Phe Lys Phe Val Pro Leu Glu Glu Arg Tyr Pro Pro Cys Phe Tyr 115 120 125 Cys Gly Asp Gly Asp Asn Phe His Tyr Glu Asp Val Pro Leu Leu Glu 130 135 140 Thr Trp Lys Ala Leu Glu Ala Leu Val Lys Lys Gly Lys Ile Arg Ser 145 150 155 160 Leu Gly Val Ser Asn Phe Thr Gly Ala Leu Leu Leu Asp Leu Leu Arg 165 170 175 Gly Ser Thr Ile Lys Pro Ala Val Leu Gln Val Glu His His Pro Tyr 180 185 190 Leu Gln Gln Pro Arg Leu Ile Glu Phe Ala Gln Lys Gln Gly Leu Val 195 200 205 Val Thr Ala Tyr Ser Ser Phe Gly Pro Gln Ser Phe Thr Glu Leu Asn 210 215 220 Gln Asn Arg Ala Asn Asn Thr Pro Arg Leu Phe Asp His Glu Val Ile 225 230 235 240 Lys Lys Ile Ala Ala Arg Arg Gly Arg Thr Pro Ala Gln Val Ile Leu 245 250 255 Arg Trp Ala Thr Gln Arg Asn Val Val Ile Ile Pro Lys Ser Asp Thr 260 265 270 Pro Glu Arg Leu Val Glu Asn Leu Ala Val Phe Asp Phe Asp Leu Thr 275 280 285 Glu Glu Asp Phe Lys Glu Ile Ala Ala Leu Asp Ala Asn Leu Arg Phe 290 295 300 Asn Asp Pro Trp Asp Trp Asp His Ile Pro Ile Phe Val 305 310 315 <210> 2 <211> 951 <212> DNA <213> Spathaspora passalidarum <400> 2 atgtctttta aatatcttca ggttatgaaa tgccaaaaat cggttttggt acttggaaga 60 tggacaaggc caccattcct cagcaaattt acgatgctat caagggtggt atcagatcat 120 tcgatggtgc tgaagattac ggtaacgaaa aggaagtggt cttggttaca agaaggctat 180 tgaagacggt cttgttaaga gagaagatct tttcattacc tccaagttat ggaataactt 240 tcatgaccca aagaatgtgg aaaaggcttt agacagaact ttagctgatt tacaattaga 300 tacgtcgact tatttttaat tcatttccca attgctttca agtttgttcc attagaagaa 360 agatacccac cttgcttcta ctgtggtgat ggtgacaact tccattatga agatgtccca 420 ttattggaaa cctggaaggc tttagaagcc ttggttaaga agggtaagat tagatcactt 480 ggtgtttcta acttcactgg tgctttgttg ttggatttac ttagaggttc taccattaag 540 ccagctgttt tgcaagtcga acatcatcca tacttgcaac aaccaagatt aattgaattt 600 gctcaaaagc aaggtcttgt tgtcactgct tactcttcat ttggtcctca atctttcact 660 gaattgaacc aaaacagagc taacaacacc ccaagattgt ttgaccacga agttatcaag 720 aagattgctg ctagaagggg cagaactcca gctcaagtta tcttaagatg ggccacccaa 780 agaaatgtcg tgattattcc aaaatccgat actccagaaa gattggtcga aaacttggct 840 gtctttgact ttgacttaac tgaagaagat ttcaaagaaa ttgccgcctt ggacgctaat 900 ttgagattta atgacccatg ggactgggac catattccaa tctttgttta a 951 <210> 3 <211> 318 <212> PRT <213> Pichia stipitis <400> 3 Met Pro Ser Ile Lys Leu Asn Ser Gly Tyr Asp Met Pro Ala Val Gly 1 5 10 15 Phe Gly Cys Trp Lys Val Asp Val Asp Thr Cys Ser Glu Gln Ile Tyr 20 25 30 Arg Ala Ile Lys Thr Gly Tyr Arg Leu Phe Asp Gly Ala Glu Asp Tyr 35 40 45 Ala Asn Glu Lys Leu Val Gly Ala Gly Val Lys Lys Ala Ile Asp Glu 50 55 60 Gly Ile Val Lys Arg Glu Asp Leu Phe Leu Thr Ser Lys Leu Trp Asn 65 70 75 80 Asn Tyr His His Pro Asp Asn Val Glu Lys Ala Leu Asn Arg Thr Leu 85 90 95 Ser Asp Leu Gln Val Asp Tyr Val Asp Leu Phe Leu Ile His Phe Pro 100 105 110 Val Thr Phe Lys Phe Val Pro Leu Glu Glu Lys Tyr Pro Pro Gly Phe 115 120 125 Tyr Cys Gly Lys Gly Asp Asn Phe Asp Tyr Glu Asp Val Pro Ile Leu 130 135 140 Glu Thr Trp Lys Ala Leu Glu Lys Leu Val Lys Ala Gly Lys Ile Arg 145 150 155 160 Ser Ile Gly Val Ser Asn Phe Pro Gly Ala Leu Leu Leu Asp Leu Leu 165 170 175 Arg Gly Ala Thr Ile Lys Pro Ser Val Leu Gln Val Glu His His Pro 180 185 190 Tyr Leu Gln Gln Pro Arg Leu Ile Glu Phe Ala Gln Ser Arg Gly Ile 195 200 205 Ala Val Thr Ala Tyr Ser Ser Phe Gly Pro Gln Ser Phe Val Glu Leu 210 215 220 Asn Gln Gly Arg Ala Leu Asn Thr Ser Pro Leu Phe Glu Asn Glu Thr 225 230 235 240 Ile Lys Ala Ile Ala Ala Lys His Gly Lys Ser Pro Ala Gln Val Leu 245 250 255 Leu Arg Trp Ser Ser Gln Arg Gly Ile Ala Ile Ile Pro Lys Ser Asn 260 265 270 Thr Val Pro Arg Leu Leu Glu Asn Lys Asp Val Asn Ser Phe Asp Leu 275 280 285 Asp Glu Gln Asp Phe Ala Asp Ile Ala Lys Leu Asp Ile Asn Leu Arg 290 295 300 Phe Asn Asp Pro Trp Asp Trp Asp Lys Ile Pro Ile Phe Val 305 310 315 <210> 4 <211> 957 <212> DNA <213> Pichia stipitis <400> 4 atgccttcta ttaagttgaa ctctggttac gacatgccag ccgtcggttt cggctgttgg 60 aaagtcgacg tcgacacctg ttctgaacag atctaccgtg ctatcaagac cggttacaga 120 ttgttcgacg gtgccgaaga ttacgccaac gaaaagttag ttggtgccgg tgtcaagaag 180 gccattgacg aaggtatcgt caagcgtgaa gacttgttcc ttacctccaa gttgtggaac 240 aactaccacc acccagacaa cgtcgaaaag gccttgaaca gaaccctttc tgacttgcaa 300 gttgactacg ttgacttgtt cttgatccac ttcccagtca ccttcaagtt cgttccatta 360 gaagaaaagt acccaccagg attctactgt ggtaagggtg acaacttcga ctacgaagat 420 gttccaattt tagagacctg gaaggctctt gaaaagttgg tcaaggccgg taagatcaga 480 tctatcggtg tttctaactt cccaggtgct ttgctcttgg acttgttgag aggtgctacc 540 atcaagccat ctgtcttgca agttgaacac cacccatact tacaacaacc aagattgatc 600 gaattcgctc aatcccgtgg tattgctgtc accgcttact cttcgttcgg tcctcaatct 660 ttcgttgaat tgaaccaagg tagagctttg aacacttctc cattgttcga gaacgaaact 720 atcaaggcta tcgctgctaa gcacggtaag tctccagctc aagtcttgtt gagatggtct 780 tcccaaagag gcattgccat cattccaaag tccaacactg tcccaagatt gttggaaaac 840 aaggacgtca acagcttcga cttggacgaa caagatttcg ctgacattgc caagttggac 900 atcaacttga gattcaacga cccatgggac tgggacaaga ttcctatctt cgtctaa 957 <210> 5 <211> 362 <212> PRT <213> Spathaspora passalidarum <400> 5 Met Val Ala Asn Pro Ser Leu Val Leu Lys Lys Ile Asp Glu Ile Val 1 5 10 15 Phe Glu Asn Gln Glu Ala Pro Thr Ile Thr Glu Pro Thr Asp Val Ile 20 25 30 Val Gln Val Lys Lys Thr Gly Ile Cys Gly Ser Asp Ile His Phe Tyr 35 40 45 Gln His Gly Lys Ile Gly Asn Tyr Ile Leu Thr Lys Pro Met Val Leu 50 55 60 Gly His Glu Ser Ala Gly Val Val Thr Glu Val Gly Pro Gly Val Lys 65 70 75 80 Tyr Leu Arg Val Gly Asp Asn Val Ala Ile Glu Pro Gly Val Pro Ser 85 90 95 Arg Phe Ser Asp Ala Tyr Lys Ser Gly Arg Tyr Asn Leu Cys Pro His 100 105 110 Met Arg Phe Ala Ala Thr Pro Ser Thr Lys Asp Glu Pro Asn Pro Pro 115 120 125 Gly Thr Leu Cys Lys Tyr Phe Lys Ser Pro Glu Asp Phe Leu Val Lys 130 135 140 Leu Pro Asp His Val Ser Leu Glu Leu Gly Ala Met Val Glu Pro Leu 145 150 155 160 Ser Val Gly Val His Ala Cys Lys Ile Gly Lys Val Lys Phe Gly Asp 165 170 175 Thr Val Ala Val Phe Gly Ala Gly Pro Val Gly Leu Leu Thr Ala Ala 180 185 190 Thr Ala Lys Thr Phe Gly Ala Ala Lys Val Ile Ile Ile Asp Val Phe 195 200 205 Asp Asn Lys Leu Gln Met Ala Lys Asp Ile Gly Val Val Thr His Thr 210 215 220 Phe Asn Ser Lys Thr Asp Gly Asp Tyr Asn Asp Leu Ile Lys His Phe 225 230 235 240 Gly Gly Gln Pro Asn Val Val Leu Glu Cys Thr Gly Ala Asp Pro Cys 245 250 255 Val Gly Met Gly Val Asn Ile Cys Ala Pro Gly Gly Thr Phe Ile Gln 260 265 270 Val Gly Asn Ala Ala Ala Pro Val Lys Phe Pro Ile Thr Gln Phe Ala 275 280 285 Met Lys Glu Leu Thr Leu Tyr Gly Ser Phe Arg Tyr Gly Phe Gly Asp 290 295 300 Tyr Gln Asp Ala Val Asn Ile Phe Asp Ala Asn Tyr Lys Asn Gly Lys 305 310 315 320 Asp Lys Ala Pro Ile Asp Phe Glu Arg Leu Ile Thr His Arg Phe Lys 325 330 335 Phe Asp Asp Ala Ile Lys Ala Tyr Asp Leu Val Arg Ser Gly Cys Gly 340 345 350 Ala Val Lys Cys Leu Ile Asp Gly Pro Glu 355 360 <210> 6 <211> 1094 <212> DNA <213> Spathaspora passalidarum <400> 6 atggttgcta acccatcatt agttcttaac aagattgacg acatcacctt cgaaacctac 60 gaagccccag aaattgtcga accaaccgac gttattgtcg aagttaaaaa gactggtatc 120 tgtggttctg atatccacta ctatgcccac ggtaagattg gtaactttat cttgaccaag 180 ccaatggttt tgggtcacga atctgccggt gttgtttccc aagttggtaa gggtgtcaag 240 cacttgaagg ttggtgacag agttgccatt gaaccaggta ttccatccag attatccgac 300 gcttacaagt ctggtcacta caacttgtgt cctcacatgt gttttgctgc cactccaaac 360 tccactgaag gtgaaccaaa cccaccaggt accttgtgta aatatttcaa gtccccagaa 420 gatttcttgg ttaagttgcc agaacacgtc tccttggaat tgggtgccat ggttgaacca 480 ttgtctgtcg gtgtccacgc ctccaagtta ggtaaggtta ctttcggtga caatgttgcc 540 gttttcggtg ctggtccagt tggtttattg gctgctgcca ccgccaagac ctttggtgct 600 gccagagtca ttgtcattga tatctttgac aacaagttac aaatggccaa ggacattggt 660 gctgccactc acaccttcaa ctccaagact ggtggtgatt acaaggactt gattgctgcc 720 tttgacggtg ttgaaccaaa tgttattttg gaatgtaccg gtgctgaacc atgtattgcc 780 atgggtgtcc aaattgctgc tccaggtggt agatttgtcc aagttggtaa tgctggtgcc 840 gctgtcaagt tcccaattac tgaatttgct actaaggaat tgaccttatt cggttctttc 900 agatatggtt acggtgacta ccaaactgcc gttaacattt tcgatgccaa ctacaagaat 960 ggtaaggaca aggctccaat tgactttgaa caattgatta ccacagattc aagtttgacg 1020 atgccatcaa ggcttacgac ttggttagag ccggttctgg tgccgtcaag tgtttgattg 1080 atggtccatt ataa 1094 <210> 7 <211> 363 <212> PRT <213> Pichia stipitis <400> 7 Met Thr Ala Asn Pro Ser Leu Val Leu Asn Lys Ile Asp Asp Ile Ser 1 5 10 15 Phe Glu Thr Tyr Asp Ala Pro Glu Ile Ser Glu Pro Thr Asp Val Leu 20 25 30 Val Gln Val Lys Lys Thr Gly Ile Cys Gly Ser Asp Ile His Phe Tyr 35 40 45 Ala His Gly Arg Ile Gly Asn Phe Val Leu Thr Lys Pro Met Val Leu 50 55 60 Gly His Glu Ser Ala Gly Thr Val Val Gln Val Gly Lys Gly Val Thr 65 70 75 80 Ser Leu Lys Val Gly Asp Asn Val Ala Ile Glu Pro Gly Ile Pro Ser 85 90 95 Arg Phe Ser Asp Glu Tyr Lys Ser Gly His Tyr Asn Leu Cys Pro His 100 105 110 Met Ala Phe Ala Ala Thr Pro Asn Ser Lys Glu Gly Glu Pro Asn Pro 115 120 125 Pro Gly Thr Leu Cys Lys Tyr Phe Lys Ser Pro Glu Asp Phe Leu Val 130 135 140 Lys Leu Pro Asp His Val Ser Leu Glu Leu Gly Ala Leu Val Glu Pro 145 150 155 160 Leu Ser Val Gly Val His Ala Ser Lys Leu Gly Ser Val Ala Phe Gly 165 170 175 Asp Tyr Val Ala Val Phe Gly Ala Gly Pro Val Gly Leu Leu Ala Ala 180 185 190 Ala Val Ala Lys Thr Phe Gly Ala Lys Gly Val Ile Val Val Asp Ile 195 200 205 Phe Asp Asn Lys Leu Lys Met Ala Lys Asp Ile Gly Ala Ala Thr His 210 215 220 Thr Phe Asn Ser Lys Thr Gly Gly Ser Glu Glu Leu Ile Lys Ala Phe 225 230 235 240 Gly Gly Asn Val Pro Asn Val Val Leu Glu Cys Thr Gly Ala Glu Pro 245 250 255 Cys Ile Lys Leu Gly Val Asp Ala Ile Ala Pro Gly Gly Arg Phe Val 260 265 270 Gln Val Gly Asn Ala Ala Gly Pro Val Ser Phe Pro Ile Thr Val Phe 275 280 285 Ala Met Lys Glu Leu Thr Leu Phe Gly Ser Phe Arg Tyr Gly Phe Asn 290 295 300 Asp Tyr Lys Thr Ala Val Gly Ile Phe Asp Thr Asn Tyr Gln Asn Gly 305 310 315 320 Arg Glu Asn Ala Pro Ile Asp Phe Glu Gln Leu Ile Thr His Arg Tyr 325 330 335 Lys Phe Lys Asp Ala Ile Glu Ala Tyr Asp Leu Val Arg Ala Gly Lys 340 345 350 Gly Ala Val Lys Cys Leu Ile Asp Gly Pro Glu 355 360 <210> 8 <211> 1095 <212> DNA <213> Pichia stipitis <400> 8 atggtagcta atccctcatt agtacttaat aaaattgatg acattacatt cgaaacttat 60 gaagcaccag aaattgtgga gcctacagat gtaatagtgg aagtaaaaaa aacaggcata 120 tgtggatctg atatacatta ttatgctcat ggaaaaatag gtaactttat cttgaccaag 180 ccaatggttc taggacacga aagtgcaggt gttgtttccc aggttggtaa aggtgtcaaa 240 catttgaagg ttggagacag agtagcaatt gagccaggta ttccttcacg tttatctgat 300 gcttataagt ctggtcatta caacttgtgt cctcatatgt gctttgctgc tactccaaac 360 tccactgagg gtgaaccaaa cccccctggt acattgtgta aatatttcaa aagtccagaa 420 gatttcctcg ttaaattgcc cgaacacgtc tcattggaac taggtgccat ggttgaacca 480 ttgtctgttg gtgtccacgc gtcgaagtta ggtaaggtaa cttttggtga taacgttgcg 540 gttttcggtg ctggtccagt tggtctattg gctgctgcca ctgccaaaac ctttggagct 600 gcaagagtga tcgtgattga tatctttgac aataaattac aaatggccaa ggacattggt 660 gctgctacgc atacatttaa ttccaagacg ggtggagatt ataaagattt aatcgcagca 720 tttgatggtg ttgaacctaa tgttatactt gaatgtaccg gtgcggaacc ttgtatagcc 780 atgggagtgc aaatagcagc tccaggtggg agatttgtcc aagttggtaa tgctggtgcc 840 gctgtcaaat tcccaattac tgaatttgct actaaggagt tgaccttatt tggttctttt 900 agatatggtt acggtgatta ccaaactgcc gtgaatattt ttgatgcaaa ctacaaaaat 960 ggtaaagata aagctccaat tgacttcgaa caattgatta cacatagatt caagtttgac 1020 gatgcaatca aggcttacga cttggttagg gccggtagcg gtgctgtaaa atgccttatt 1080 gatggcccat tataa 1095 <210> 9 <211> 623 <212> PRT <213> Pichia stipitis <400> 9 Met Thr Thr Thr Pro Phe Asp Ala Pro Asp Lys Leu Phe Leu Gly Phe 1 5 10 15 Asp Leu Ser Thr Gln Gln Leu Lys Ile Ile Val Thr Asp Glu Asn Leu 20 25 30 Ala Ala Leu Lys Thr Tyr Asn Val Glu Phe Asp Ser Ile Asn Ser Ser 35 40 45 Val Gln Lys Gly Val Ile Ala Ile Asn Asp Glu Ile Ser Lys Gly Ala 50 55 60 Ile Ile Ser Pro Val Tyr Met Trp Leu Asp Ala Leu Asp His Val Phe 65 70 75 80 Glu Asp Met Lys Lys Asp Gly Phe Pro Phe Asn Lys Val Val Gly Ile 85 90 95 Ser Gly Ser Cys Gln Gln His Gly Ser Val Tyr Trp Ser Arg Thr Ala 100 105 110 Glu Lys Val Leu Ser Glu Leu Asp Ala Glu Ser Ser Leu Ser Ser Gln 115 120 125 Met Arg Ser Ala Phe Thr Phe Lys His Ala Pro Asn Trp Gln Asp His 130 135 140 Ser Thr Gly Lys Glu Leu Glu Glu Phe Glu Arg Val Ile Gly Ala Asp 145 150 155 160 Ala Leu Ala Asp Ile Ser Gly Ser Arg Ala His Tyr Arg Phe Thr Gly 165 170 175 Leu Gln Ile Arg Lys Leu Ser Thr Arg Phe Lys Pro Glu Lys Tyr Asn 180 185 190 Arg Thr Ala Arg Ile Ser Leu Val Ser Ser Phe Val Ala Ser Val Leu 195 200 205 Leu Gly Arg Ile Thr Ser Ile Glu Glu Ala Asp Ala Cys Gly Met Asn 210 215 220 Leu Tyr Asp Ile Glu Lys Arg Glu Phe Asn Glu Glu Leu Leu Ala Ile 225 230 235 240 Ala Ala Gly Val His Pro Glu Leu Asp Gly Val Glu Gln Asp Gly Glu 245 250 255 Ile Tyr Arg Ala Gly Ile Asn Glu Leu Lys Arg Lys Leu Gly Pro Val 260 265 270 Lys Pro Ile Thr Tyr Glu Ser Glu Gly Asp Ile Ala Ser Tyr Phe Val 275 280 285 Thr Arg Tyr Gly Phe Asn Pro Asp Cys Lys Ile Tyr Ser Phe Thr Gly 290 295 300 Asp Asn Leu Ala Thr Ile Ile Ser Leu Pro Leu Ala Pro Asn Asp Ala 305 310 315 320 Leu Ile Ser Leu Gly Thr Ser Thr Thr Val Leu Ile Ile Thr Lys Asn 325 330 335 Tyr Ala Pro Ser Ser Gln Tyr His Leu Phe Lys His Pro Thr Met Pro 340 345 350 Asp His Tyr Met Gly Met Ile Cys Tyr Cys Asn Gly Ser Leu Ala Arg 355 360 365 Glu Lys Val Arg Asp Glu Val Asn Glu Lys Phe Asn Val Glu Asp Lys 370 375 380 Lys Ser Trp Asp Lys Phe Asn Glu Ile Leu Asp Lys Ser Thr Asp Phe 385 390 395 400 Asn Asn Lys Leu Gly Ile Tyr Phe Pro Leu Gly Glu Ile Val Pro Asn 405 410 415 Ala Ala Ala Gln Ile Lys Arg Ser Val Leu Asn Ser Lys Asn Glu Ile 420 425 430 Val Asp Val Glu Leu Gly Asp Lys Asn Trp Gln Pro Glu Asp Asp Val 435 440 445 Ser Ser Ile Val Glu Ser Gln Thr Leu Ser Cys Arg Leu Arg Thr Gly 450 455 460 Pro Met Leu Ser Lys Ser Gly Asp Ser Ser Ala Ser Ser Ser Ala Ser 465 470 475 480 Pro Gln Pro Glu Gly Asp Gly Thr Asp Leu His Lys Val Tyr Gln Asp 485 490 495 Leu Val Lys Lys Phe Gly Asp Leu Tyr Thr Asp Gly Lys Lys Gln Thr 500 505 510 Phe Glu Ser Leu Thr Ala Arg Pro Asn Arg Cys Tyr Tyr Val Gly Gly 515 520 525 Ala Ser Asn Asn Gly Ser Ile Ile Arg Lys Met Gly Ser Ile Leu Ala 530 535 540 Pro Val Asn Gly Asn Tyr Lys Val Asp Ile Pro Asn Ala Cys Ala Leu 545 550 555 560 Gly Gly Ala Tyr Lys Ala Ser Trp Ser Tyr Glu Cys Glu Ala Lys Lys 565 570 575 Glu Trp Ile Gly Tyr Asp Gln Tyr Ile Asn Arg Leu Phe Glu Val Ser 580 585 590 Asp Glu Met Asn Ser Phe Glu Val Lys Asp Lys Trp Leu Glu Tyr Ala 595 600 605 Asn Gly Val Gly Met Leu Ala Lys Met Glu Ser Glu Leu Lys His 610 615 620 <210> 10 <211> 1872 <212> DNA <213> Pichia stipitis <400> 10 atgaccacta ccccatttga tgctccagat aagctcttcc tcgggttcga tctttcgact 60 cagcagttga agatcatcgt caccgatgaa aacctcgctg ctctcaaaac ctacaatgtc 120 gagttcgata gcatcaacag ctctgtccag aagggtgtca ttgctatcaa cgacgaaatc 180 agcaagggtg ccattatttc ccccgtttac atgtggttgg atgcccttga ccatgttttt 240 gaagacatga agaaggacgg attccccttc aacaaggttg ttggtatttc cggttcttgt 300 caacagcacg gttcggtata ctggtctaga acggccgaga aggtcttgtc cgaattggac 360 gctgaatctt cgttatcgag ccagatgaga tctgctttca ccttcaagca cgctccaaac 420 tggcaggatc actctaccgg taaagagctt gaagagttcg aaagagtgat tggtgctgat 480 gccttggctg atatctctgg ttccagagcc cattacagat tcacagggct ccagattaga 540 aagttgtcta ccagattcaa gcccgaaaag tacaacagaa ctgctcgtat ctctttagtt 600 tcgtcatttg ttgccagtgt gttgcttggt agaatcacct ccattgaaga agccgatgct 660 tgtggaatga acttgtacga tatcgaaaag cgcgagttca acgaagagct cttggccatc 720 gctgctggtg tccaccctga gttggatggt gtagaacaag acggtgaaat ttacagagct 780 ggtatcaatg agttgaagag aaagttgggt cctgtcaaac ctataacata cgaaagcgaa 840 ggtgacattg cctcttactt tgtcaccaga tacggcttca accccgactg taaaatctac 900 tcgttcaccg gagacaattt ggccacgatt atctcgttgc ctttggctcc aaatgatgct 960 ttgatctcat tgggtacttc tactacagtt ttaattatca ccaagaacta cgctccttct 1020 tctcaatacc atttgtttaa acatccaacc atgcctgacc actacatggg catgatctgc 1080 tactgtaacg gttccttggc cagagaaaag gttagagacg aagtcaacga aaagttcaat 1140 gtagaagaca agaagtcgtg ggacaagttc aatgaaatct tggacaaatc cacagacttc 1200 aacaacaagt tgggtattta cttcccactt ggcgaaattg tccctaatgc cgctgctcag 1260 atcaagagat cggtgttgaa cagcaagaac gaaattgtag acgttgagtt gggcgacaag 1320 aactggcaac ctgaagatga tgtttcttca attgtagaat cacagacttt gtcttgtaga 1380 ttgagaactg gtccaatgtt gagcaagagt ggagattctt ctgcttccag ctctgcctca 1440 cctcaaccag aaggtgatgg tacagatttg cacaaggtct accaagactt ggttaaaaag 1500 tttggtgact tgttcactga tggaaagaag caaacctttg agtctttgac cgccagacct 1560 aaccgttgtt actacgtcgg tggtgcttcc aacaacggca gcattatccs caagatgggt 1620 tccatcttgg ctcccgtcaa cggaaactac aaggttgaca ttcctaacgc ctgtgcattg 1680 ggtggtgctt acaaggccag ttggagttac gagtgtgaag ccaagaagga atggatcgga 1740 tacgatcagt atatcaacag attgtttgaa gtaagtgacg agatgaatct gttcgaagtc 1800 aaggataaat ggctcgaata tgccaacggg gttggaatgt tggccaagat ggaaagtgaa 1860 ttgaaacact aa 1872 <210> 11 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 11 gatcggatcc atgccttcta ttaagttgaa 30 <210> 12 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 12 tcgactcgag ttagacgaag ataggaatct 30 <210> 13 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 13 gatcggatcc atggttgcta acccatcatt agtt 34 <210> 14 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 14 tcgactcgag ttataatgga ccatcaatca aaca 34 <210> 15 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 15 gatcggatcc atgactgcta acccttcctt 30 <210> 16 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 16 tcgactcgag ttactcaggg ccgtcaatga 30 <210> 17 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 17 gatcggatcc atgaccacta ccccatttga 30 <210> 18 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 18 tcgactcgag ttagtgtttc aattcacttt 30

Claims (4)

Spathaspora passalidarum represented by the amino acid sequence of SEQ ID NO: 5 Derived A gene encoding a xylitol dehydrogenase, a gene encoding a xylose reductase represented by the amino acid sequence of SEQ ID NO: 1, and a gene encoding a xylulurokinase derived from Pichia stipitis are introduced. , Recombinant yeast having the ability to produce ethanol from xylose.
The recombinant yeast according to claim 1, wherein the gene encoding the xylitol dehydrogenase has a nucleotide sequence represented by SEQ ID NO: 2.
The recombinant yeast according to claim 1, wherein the gene encoding the xylitol dehydrogenase has a nucleotide sequence of SEQ ID NO: 6.
Method for producing ethanol from xylose comprising the following steps;
(a) culturing the recombinant yeast of any one of claims 1 to 3 in a xylose-containing medium to produce ethanol; And
(b) obtaining the produced ethanol.

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