KR20140137470A - Recombinant Yeast Producing Ethanol from Xylose and Method for Producing Ethanol by Using the Recombinant Yeast - Google Patents

Abstract

The present invention relates to an yeast which is allowed to produce ethanol from xylose in a high-yield and, more specifically, to an yeast producing ethanol from xylose and a production method of ethanol using the yeast wherein the xylose is transformed by a gene which codes the xylose reduction enzyme using NADH as an extra factor, a gene which codes xylitol dehydration enzyme using NAD^+ as an extra factor and a gene which codes xylulokinase. The recombinant yeast of the present invention can produce ethanol from cellulose based biomass whose xylose is rich in a high yield while byproducts, xylitol, are hardly accumulated, so the recombinant yeast can be widely used for producing cellulosic ethanol (CE).

Description

Technical Field [0001] The present invention relates to a recombinant yeast capable of producing ethanol from xylose, and a method for producing ethanol using the recombinant yeast,

The present invention relates to a recombinant yeast having high ethanol productivity from xylose, and more particularly, to a recombinant yeast having an ability to produce ethanol from xylose, and more particularly to a gene encoding xylose reductase (XR) using NADH as a cofactor, a recombinant yeast having an ability to produce ethanol from xylose into which a gene encoding xylitol dehydrogenase (XDH) and a gene encoding xylulokinase (XK) are introduced, which is used as a cofactor, And a method for producing ethanol using yeast.

As interest in biofuels and renewable energy has increased, studies have been actively carried out to utilize cellulosic ethanol (CE) as the next generation fuel. However, the pre-treatment technology, digestion of enzymes and fermentation bacteria, Element.

In particular, unlike traditional ethanol fermentation using C ₆ glucose (glucose) as a carbon source, CE is produced by using C ₅ , Xylose as a carbon source. Therefore, ethanol is produced with high efficiency using xylose Which is capable of producing yeast strains.

A typical ethanol fermentation strain is Yeast, which is represented by Saccharomyces cerevisiae . Yeast has excellent productivity and yield, and is resistant to ethanol and the like, so that it is traditionally used in the ethanol industry and the fermentation industry Lt; / RTI > However, there is a limitation that it can not be used as carbon source. Therefore, metabolic engineering approach has been made S. cerevisiae that can develop by introducing genes involved in the metabolic pathway of xylose in S. cerevisiae using the (metabolic engineering), and the xylose as a carbon source for the fermentation of ethanol.

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

Accordingly, the present inventors have tried to overcome the problem that the ethanol productivity is lowered by the cofactor imbalance in the transformant yeast strain having ethanol production ability using xylose, and as a result, they have tried to consume glucose and xylose at the same time for that known spa dozen Fora wave sari xyl1 dealing with the known Pichia styryl blood tooth xyl2 xyl3 and the recombinant yeast, in particular S. cerevisiae strain introduction (Pichia stipitis) of (Spathaspora passalidarum), Pichia styryl blood tooth The present inventors have confirmed that they exhibit superior ethanol production yield and yield in comparison with conventional results using S. cerevisiae containing xyl1 , xyl2 and xyl3 derived from E. coli .

It is an object of the present invention to provide a recombinant yeast having the ability to efficiently convert cellulosic biomass-derived xylose into ethanol for the production of cellulose ethanol (CE).

It is another object of the present invention to provide a method for producing ethanol using recombinant yeast capable of effectively converting xylose to ethanol.

In order to achieve the above object, the present invention xylose gene (xyl2) encoding gene (xyl1), xylitol dehydrogenase that uses NAD + as cofactor encoding a reductase that uses NADH as cofactor and characters achieve Rocca or dehydratase ( Xyl < 3 > ) encoding a gene encoding xylose is introduced into a recombinant yeast having an ability to produce ethanol from xylose.

(A) culturing the yeast in a xylose-containing medium to produce ethanol; And (b) obtaining the resulting ethanol. ≪ Desc / Clms Page number 5 >

The yeast strain of the present invention can produce ethanol in a high yield without accumulating a large amount of xylitol as a by-product in the production of ethanol from xylose-rich cellulose-based biomass, and can produce biofuels using cellulose ethanol (CE) Can be very useful.

Figure 1 shows the metabolic pathway for ethanol production from xylose, showing the pathway of xylose metabolism in yeast strains into which PSxyl1 and PSxyl3 genes of S. passalidarum and P. stipitis have been introduced.
Figure 2 shows a recombinant vector containing a gene encoding xylose reductase (XR): (A) PsXR; (B) SpXR; (C) SpXR ^CO .
Figure 3 shows a recombinant vector containing a gene encoding xylitol dehydrogenase (XDH) or zyululokinase (XK): (A) PsXDH; (B) PsXK.
Figure 4 shows the enzyme activity according to cofactors (NADH and NADPH) of yeast transformed with a recombinant vector containing the gene encoding xylose reductase (XR): (A) PsXR; (B) SpXR ^CO ; (C) SpXR.

In one aspect, the present invention provides a gene encoding a xylitol dehydrogenase, wherein the gene encoding xylose reductase using NADH as a cofactor, the gene encoding xylitol dehydrogenase using NAD + as a cofactor, The present invention relates to a recombinant yeast having an ability to produce ethanol from xylose.

Xylose is a biomass in the form of a polymer, xylan, which is abundant in waste wood, and is a monosaccharide of five carbon atoms that can be metabolized into a useful product by various organisms. It is largely divided into two stages to form a pentose phosphate pathway phosphate pathway (PPP).

However, Saccharomyces cerevisiae , which is a yeast mainly used for ethanol fermentation, has a gene xyl1 encoding xylose reductase (XR) in the metabolism aspect of xylose and xylitol dehydrogenase: does not the genes coding for the present xyl2 XDH) metabolize xylose, that could be used as a 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, Is converted to xylulose-5-phosphate by xylulokinase (XK) introduced into the cellulose, and the metabolism proceeds through a pentose phosphate circuit. Zyululokanase (XK) is an enzyme present in yeast, but when XR and XDH are introduced into the strain without overexpressing it, ethanol can be produced from xylose, but yield and productivity are significantly lower.

Unless defined otherwise in the present invention, xyl1 refers to a gene encoding a xylose reductase, xyl2 refers to a gene encoding a xylitol dehydrogenase, xyl3 refers to a gene encoding a jilyl carasease , XR refers to a gene encoding xylose dehydrogenase, A reducing enzyme, XDH is xylitol dehydrogenase, and XK is xylylcarinase.

In one aspect of the invention, SpXR from NADH-dependent Spathaspora passalidarum and PsXDH from Pichia stipitis, which are not NADPH-dependent xylose reductase (XR) Overexpression of PsXK solves the cofactor imbalance problem and dramatically increases ethanol production efficiency.

The xylose metabolism pathway in the recombinant yeast strain into which Psxyl1 , Psxyl2 and Psxyl3 derived from P. stipitis has been introduced is the xylose reductase (XR) encoded by Ps xyl1 derived from P. stipitis , The xylitol reductase encoded by Ps xyl2 is an enzyme that uses NAD as a cofactor. As a result, cofactors can not be reused between the reactions of the two enzymes, resulting in cofactor imbalance.

Figure 1 illustrates the xylene loss pathways, xylose reductase, which is coded in S. SPxyl1 passalidarum derived from S. passalidarum incorporating PSxyl2 and PSxyl3 Spxyl1 of genes derived from the P. stipitis strain derived from recombinant yeast (XR ) Is an enzyme that uses NADH as a cofactor, and PSxyl2 derived from P. stipitis is an enzyme that uses NAD as a cofactor, so that cofactors are reused between the reactions of the two enzymes, and the cofactor imbalance disappears.

Therefore, in the present invention, the xylose reductase using NADH as a cofactor is derived from Spathaspora passalidarum .

Preferably, the Spathaspora passalidarum- derived xylose reductase (SpXR), which uses NADH as a cofactor, has an amino acid sequence according to SEQ ID NO: 1, and preferably the gene Spxyl1 encoding Spathaspora passalidarum- derived xylose reductase is SEQ ID NO: 2 or The nucleotide sequence of SEQ ID NO: 2 is a wild type gene sequence encoding a S. passalidarium- derived xylose reductase, and the nucleotide sequence of SEQ ID NO: 3 is a nucleotide sequence of SEQ ID NO: And the nucleotide sequence of SEQ ID NO: 2 is codon optimized so as to be optimally expressed in S. cerevisiae .

A nucleotide sequence having at least 80%, preferably at least 90%, more preferably at least 95%, and most preferably at least 97% sequence homology with the nucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 3, conservative substitutions in some amino acid residues, for a, xylose reductase mutation occurred in some amino acid sequence such that the function of the xylose reductase encoded by the Sp xyl1 of S. passalidarum derived maintain provided (conservative substitution) It is obvious to a person skilled in the art that the nucleotide sequence encoding the resulting xylose reductase is also included in the scope of the present invention. It is also known to those skilled in the art that nucleotide sequences encoding the same amino acid sequence encoded by SEQ ID NO: 2 or SEQ ID NO: 3 and having different nucleotide sequences due to codon degeneracy are also included in the scope of the present invention .

As used herein, the term "Homology" means sequence similarity or sequence identity. Said homology can be measured using standard techniques known in the art.

In the present invention, the gene ( xyl2 ) encoding xylitol dehydrogenase using NAD + as a cofactor is derived from Pichia stipitis , and the gene ( xyl3 ) encoding zyululokinase is also derived from Pichia stipitis .

In the present invention, it is preferable that the xylitol dehydrogenase using NAD < + > as a cofactor has the amino acid sequence of SEQ ID NO: 6. SEQ ID NO: 6 refers to the amino acid sequence of xylitol dehydrogenase (PsXDH) derived from Pichia stipitis . In addition, the gene ( PSxyl2 ) encoding the xylitol dehydrogenase derived from Pichia stipitis is preferably characterized by having the base sequence ( Psxyl2 ) of SEQ ID NO: 7, and the zyululokinase derived from Pichia stipitis (PsXK) It is preferable to have the amino acid sequence of SEQ ID NO: 8, and the gene ( Psxyl3 ) encoding the same preferably has the nucleotide sequence of SEQ ID NO:

In the nucleotide sequence shown in SEQ ID NO: 7 and SEQ ID NO: 9, the nucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 3 is 80% or more, preferably 90% or more, more preferably 95% , A base sequence encoding a xylitol dehydrogenase or a zyululokinase having a base sequence having 97% or more of sequence homology and a base sequence having a different base sequence due to codon degeneracy but having the same amino acid sequence, and a base sequence encoding xylitol dehydrogenase As long as the function of irulokinase is maintained, the amino acid sequence in which the mutation occurs in some amino acid sequences, particularly the nucleotide sequence encoding the xylitol dehydrogenase or zyululokinase, in which conservative substitution has occurred in some amino acid residues It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. It is

The sequence numbers of the respective enzymes and the nucleotide sequences encoding them and their definitions are as shown in Table 1.

The sequence numbers and definitions of the respective enzymes and the nucleotide sequences encoding the enzymes in the present invention order
number Display Justice One SpXR Xylose from Spathaspora passalidarum
The amino acid sequence of the reducing enzyme 2 Spxyl1 The nucleotide sequence of the natural type gene encoding SpXR 3 Spxyl1 ^CO Lt; RTI ID = 0.0 > SpXR < / RTI > and expressed in recombinant yeast
Weak codon optimized sequence 4 PsXR Of xylose reductase from Pichia stipitis
Amino acid sequence 5 Psxyl1 The nucleotide sequence of the gene encoding PsXR 6 PsXDH Of xylitol dehydrogenase derived from Pichia stipitis
Amino acid sequence 7 Psxyl2 The nucleotide sequence encoding PsXDH 8 PsXK Pichia stipitis originated from Irurokinease
Amino acid sequence 9 Psxyl3 The nucleotide sequence encoding PsXK

In the present invention, the term " vector " means a DNA product containing a DNA sequence operatively linked to a suitable host, particularly a suitable regulatory sequence capable of expressing DNA in the recombinant yeast. The vector may be a plasmid, phage particle, or simply a potential genome insert. Once transformed into the appropriate host, the vector may replicate and function independently of the host genome, or, in some cases, integrate into the genome itself. Because the plasmid is the most commonly used form of the current vector, the terms " 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.

Expression control sequence " means a DNA sequence that is essential for the expression of a coding sequence operably linked to a particular host organism. Such regulatory sequences include promoters for carrying out transcription, any operator sequences for regulating such transcription, sequences encoding suitable mRNA ribosome binding sites, and sequences controlling the 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. The most influential factor on the expression level of the gene in the plasmid is the promoter.

In order to express the DNA sequences of the present invention, any of a wide variety of expression control sequences may be used in the vector. Examples of useful expression control sequences include, for example, the lac system, the trp system, the TAC or TRC system, the T3 and T7 promoters, the major operator and promoter region of phage lambda, the regulatory region of the fd- A composition known to modulate the expression of a promoter for a lactate kinase or other glycolytic enzyme, a promoter of the phosphatase, for example, a promoter of Pho5, a yeast alpha-mating system and a prokaryotic or eukaryotic cell or a virus thereof Other sequences of induction, and various combinations thereof.

A nucleic acid is " operably linked " when placed in a functional relationship with another nucleic acid sequence. This may be the gene and regulatory sequence (s) linked in such a way that the appropriate molecule (e. G., Transcriptional activator protein) is capable of 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 whole protein participating in the secretion of the polypeptide; A promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; Or the ribosome binding site is operably linked to a coding sequence if it affects the transcription of the sequence; Or a ribosome binding site is operably linked to a coding sequence if positioned to facilitate translation. Generally, " operably linked " means that the linked DNA sequences are in contact and, in the case of a secretory leader, are in contact and present in the reading frame. However, the enhancer need not be in contact. The linkage of these sequences is carried out by ligation (linkage) at convenient restriction sites. If such a site does not exist, a synthetic oligonucleotide adapter or a linker according to a conventional method is used.

As used herein, the term " expression vector " is usually a recombinant carrier into which a fragment of different DNA is inserted, and generally means a fragment of double-stranded DNA. Herein, the heterologous DNA means a heterologous DNA that is not naturally found in the host cell. Once an expression vector is in a host cell, it can replicate independently of the host chromosomal DNA, and several copies of the vector and its inserted (heterologous) DNA can be generated.

As is well known in the art, in order to increase the expression level of a transgenic gene in a host cell, the gene must be operably linked to transcriptional and detoxification regulatory sequences that function in a selected expression host. Preferably the expression control sequence and the gene are contained within an expression vector containing a bacterial selection marker and a replication origin. If the expression host is a eukaryotic cell, the expression vector should further comprise a useful expression marker in the eukaryotic expression host.

As used herein, the term " transformation " means introducing DNA into a host and allowing the DNA to replicate as an extrachromosomal factor or by chromosomal integration. As used herein, the term " transfection " means that an expression vector, whether or not any coding sequence is actually expressed, is accepted by the host cell.

Of course, it should be understood that not all vectors and expression control sequences function equally well in expressing the DNA sequences of the present invention. Likewise, not all hosts function identically for the same expression system. However, those skilled in the art will be able to make appropriate selections among a variety of vectors, expression control sequences, and hosts without undue experimentation and without departing from the scope of the present invention. For example, in selecting a vector, the host should be considered because the vector must be replicated within 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, must also be considered. In selecting the expression control sequence, a number of factors must be considered. For example, the relative strength of the sequence, controllability and compatibility with the DNA sequences of the present invention should be considered in relation to particularly possible secondary structures. The single cell host may be selected from a selected vector, the toxicity of the product encoded by the DNA sequence of the present invention, the secretion characteristics, the ability to correctly fold the protein, the culture and fermentation requirements, the product encoded by the DNA sequence of the invention And ease of purification. Within the scope of these variables, one skilled in the art can select various vector / expression control sequences / host combinations that can express the DNA sequences of the invention in fermentation or in large animal cultures.

In another aspect, the present invention provides a method for producing ethanol, comprising: (a) culturing the recombinant yeast in a xylose-containing medium to produce ethanol; And (b) obtaining the resulting ethanol. ≪ Desc / Clms Page number 3 >

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these examples are for illustrative purposes only and that the scope of the present invention is not construed as being limited by these examples.

Example 1: xyl1 , xyl2  or xyl3  Production of a recombinant vector containing the gene

1.1 A gene encoding xylose reductase (XR) xyl1 ) ≪ / RTI >

In order to prepare a vector containing a gene encoding a xylose reductase, the expression vector pRS426 TEF vector (SEQ ID NO: 2) in Saccharomyces cerevisiae having a TEF promoter of S. cerevisiae and a CYC1 terminator (Mumberg D. et al., Gene 156: 119, 1995) was used as a backbone.

The xylose reductase (XR) gene to be introduced into the pRS426 TEF vector is derived from Psxyl1 derived from Pichia stipitis , Spxyl1 derived from Spathaspora passalidarum, and Spxyl1 ^CO ( SEQ ID NO: 1 ), which is codon optimized to optimize expression of Spxyl1 in S. cerevisiae Respectively.

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: 10)

Reverse primer PsXR: 5'-TCGACTCGAGTTAGACGAAGATAGGAATCT-3 '(SEQ ID NO: 11)

Spxyl1 of S. passalidarum is S. passalidarum (ATCC MYA-43455) was amplified by performing PCR under TaOpt (optimal annealing temperature) at 55 ° C using the following primers.

The forward primer SpXR: 5'-GATCGGATCCATGTCTTTTAAATTATCTTC-3 '(SEQ ID NO: 12)

The reverse primer SpXR: 5'-TCGACTCGAGTTAAACAAAGATTGGAATAT-3 '(SEQ ID NO: 13)

Spxyll ^CO was obtained by obtaining a nucleotide sequence (SEQ ID NO: 3) optimized in S. cerevisiae using a program co-developed by Bioneer and KAIST with Spxyll gene derived from S. passalidarum having the nucleotide sequence of SEQ ID NO: 2, Bioneer Co., Ltd., Korea).

A fragment of Spxyl1 Psxyl1 fragments, S. passalidarum of amplified P. stipitis and synthetic Spxyl1 ^CO gene was ligated to pRS426 TEF vector cut with BamHI and XhoI, respectively pRS426 TEF PsXR (xyl1), pRS426 and pRS426 SpXR TEF TEF SpXR ^CO was prepared (Fig. 2).

1.2 Xylitol dehydrogenase (XDH) gene xyl2 ) ≪ / RTI >

To construct a vector containing the xylitol dehydrogenase gene, the pRS424 TEF vector (Mumberg D. et al., Gene 156: 119, 1995), which is a S. cerevisiae expression vector having a TEF promoter of S. cerevisiae and a CYC1 terminator, Respectively.

P. stipitis derived Psxyl2 was amplified by performing PCR under the P. stipitis strain (ATCC 58785) genomic DNA as a template by, for TaOpt (optimal annealing temperature) of 60 ℃ using primers obtained from.

Forward primer PsXDH: 5'-GATCGGATCCATGACTGCTAACCCTTCCTT-3 '(SEQ ID NO: 14)

Reverse primer PsXDH: 5'-TCGACTCGAGTTACTCAGGGCCGTCAATGA-3 '(SEQ ID NO: 15)

The Psxyl2 fragment of the amplified P. stipitis was ligated to the pRS424 TEF vector digested with BamHI and XhoI to prepare pRS424 TEF PsXDH ( xyl2 ) (Fig. 3 (A)).

1.3 Xylulokinase (XK) gene xyl3 ) ≪ / RTI >

To construct a vector containing the xylulose kinase gene, the pRS425 TEF vector (Mumberg D. et al., Gene 156: 119, 1995), an expression vector for S. cerevisiae with the TEF promoter of S. cerevisiae and the CYC1 terminator, Was used as a 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: 16)

Reverse primer PsXK: 5'-TCGACTCGAGTTAGTGTTTCAATTCACTTT-3 '(SEQ ID NO: 17)

The Ps xyl 3 fragment of the amplified P. stipitis and the pRS425 TEF vector were digested with BamHI and XhoI and then ligated to produce pRS425 TEF PsXK ( xyl3 ) (Fig. 3 (B)).

Example 2: Determination of co-factor dependency of xylose reductase

Xylose reductase (XR) to confirm that the co-factor dependency, Pichia stipitis was SPxyl1 the codon optimized (codon optimization) in order to optimize the origin of Ps xyl1, Spathaspora passalidarum derived SPxyl1, expression in S. cerevisiae of Spxyl1 of ^CO , Respectively, and the degree of reduction of NADH or NADPH in the cell extract of the recombinant strain was confirmed.

First, the recombinant vectors pRS426 TEF PsXR ( xyl1 ), pRS426 TEF SpXR and pRS426 TEF SpXR ^CO containing Spxyll and Spxyll ^CO derived from Pichia stipitis- derived Psxyl1 and Spathaspora passalidarum , respectively, were transformed into Saccharomyces cerevisiae CEN-PK2-1D (euroscarf 30000D) To obtain recombinant S. cerevisiae rSC (PsXR), rSC (SpXR) and rSC (SpXR ^CO ), each of which expresses Psxyl1 , Spxyll and Spxyll ^CO alone. The obtained recombinant strains were cultured in a minimal medium (YNB w / o ura, leu, trp) at 30 ° C and 200 rpm for 24 hours, and then subjected to cell disruption using a glass bead to obtain crude cell extract ).

The activity value of xylose reductase (XR) was defined based on the degree of reduction of 1 μM of 1 μmol of NADH or NADPH for 1 minute and expressed as the value according to the total protein concentration of crude cell extract. The measurement method is as follows:

As the reaction solution, 200 μl of a solution containing 100 μl of 0.1 M potassium phosphate buffer, 20 μl of 4 mM NADH or 4 mM NADPH, 20 μl of crude cell extract and 60 μl of 0.33 M xylose solution was used, and the xylose solution was added , And the degree of reduction of coenzyme was measured at 340 nm for 3 minutes after addition of xylose solution. Protein concentrations were quantified using the Bradford assay.

Notice that as a result, as shown in FIG. 4, in the case of PsXR was used as an NADPH primarily as auxiliary factor, SpXR and SpXR ^CO For (Sp xyl1 the case of using for expression SpXR) is that the NADPH dependent decrease as auxiliary factor I could.

Example 3: Production of ethanol from xylose using NADH-dependent xylose reductase

A recombinant vector containing the xylitol dehydrogenase (XR), xylitol dehydrogenase (XDH) and xylulose kinase (XK) genes prepared in Example 1, respectively, was transformed into Saccharomyces cerevisiae CEN-PK2-1D (euroscarf 30000D) A recombinant yeast strain capable of producing ethanol from xylose was prepared and the ethanol and xylitol production of each strain was confirmed according to the type of the introduced gene.

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

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

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

Table 3 shows the types of the recombinant vectors introduced for each strain and ethanol and xylitol production of the strain into which the recombinant vector was introduced.

Metabolism of xylose and ethanol production according to introduced gene set
division Introduction gene SET Xylose consumption rate (g / L / hr) ethanol productivity (g / L / hr) ethanol titer (g / L) Yield
(g / g xylose) ethanol Xylitol NO. One Psxyl1 , Psxyl2 , Psxyl3 0.30 0.04 2.86 0.13 0.38 NO. 2 Spxyl1 , Psxyl2 , Psxyl3 0.39 0.10 7.63 0.27 0.20 NO. 3 Spxyl1 ^CO , Psxyl2 , Psxyl3 0.46 0.13 9.44 0.29 0.13

As a result, the recombinant strain expressing Sp xyl1 using NADH as a cofactor showed higher ethanol production ability and low xylitol production ability than the recombinant strain expressing Ps xyl1 using NADPH as a cofactor.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the invention is not limited to the disclosed exemplary embodiments. It will be obvious. Accordingly, the actual 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-B093 <160> 17 <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 Valle 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> 955 <212> DNA <213> Artificial Sequence <220> <223> codon optimized Xylose reductase <400> 3 atgagcttta aactatctag tggttatgaa atgcctaaaa tcggttttgg cacttggaaa 60 atggataagg ccacgattcc tcagcaaata tatgatgcta taaagggtgg tatccgatca 120 ttcgatggtg ctgaagacta tgggaatgag aaagaggttg gattaggata taagaaagct 180 attgaagacg gtttggtgaa gagggaagat ctttttatta cctccaaatt gtggaataac 240 tttcacgatc caaagaatgt ggaaaaagct ctggacagaa ctttagctga tttacaactg 300 gattatgtag acttattttt aattcatttt ccgattgcgt tcaaatttgt acctttagaa 360 gaacgttacc caccttgctt ctactgtgga gatggcgata acttccatta tgaagatgtt 420 ccattattgg agacatggaa agctttagag gcattggtta agaagggtaa aattaggtcg 480 cttggggttt caaacttcac tggtgcactg ttactcgatc tattaagagg ttctaccatt 540 aagccggcag ttttgcaagt agaacatcat ccatacctac aacaaccacg tttaattgaa 600 ttcgcgcaaa aacaaggact tgtagtcaca gcctactctt cattcggtcc tcaaagtttc 660 acagagttga accagaatag agccaacaac acccctagat tgttcgatca cgaagttatc 720 aaaaagatag ctgccagaag gggcagaact cccgctcagg ttatcttaag atgggcaacg 780 caaagaaatg tcgtgataat accaaaatcc gatactcccg aacgcctagt cgagaatttg 840 gcagtctttg actttgatct tacagaagaa gattttaaag aaattgccgc tttggacgca 900 aatttgagat ttaatgaccc atgggactgg gatcatatac caatctttgt ttaac 955 <210> 4 <211> 318 <212> PRT <213> Pchia stipitis <400> 4 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     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 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 Prop Trp Asp Trp Asp Lys Ile Pro Ile Phe Val 305 310 315 <210> 5 <211> 957 <212> DNA <213> Pchia stipitis <400> 5 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> 6 <211> 363 <212> PRT <213> Pichia stipitis <400> 6 Met Thr Ala Asn Pro Ser Leu Val Leu Asn Lys Ile Asp Asp Ile Ser   1 5 10 15 Phe Glu Thr 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 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> 7 <211> 1095 <212> DNA <213> Pichia stipitis <400> 7 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> 8 <211> 623 <212> PRT <213> Pichia stipitis <400> 8 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 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> 9 <211> 1872 <212> DNA <213> Pichia stipitis <400> 9 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> 10 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> PsXR forward primer <400> 10 gatcggatcc atgccttcta ttaagttgaa 30 <210> 11 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> PsXR reverse primer <400> 11 tcgactcgag ttagacgaag ataggaatct 30 <210> 12 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> SpXR forward primer <400> 12 gatcggatcc atgtctttta aattatcttc 30 <210> 13 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> SpXR reverse primer <400> 13 tcgactcgag ttaaacaaag attggaatat 30 <210> 14 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> PsXDH forward primer <400> 14 gatcggatcc atgactgcta acccttcctt 30 <210> 15 <211> 30 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > PsXDH reverse primer <400> 15 tcgactcgag ttactcaggg ccgtcaatga 30 <210> 16 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> PsXK forward primer <400> 16 gatcggatcc atgaccacta ccccatttga 30 <210> 17 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> PsXK reverse primer <400> 17 tcgactcgag ttagtgtttc aattcacttt 30

Claims (11)

A gene coding for a xylose reductase using NADH as a cofactor, a gene encoding a xylitol dehydrogenase using NAD + as a cofactor, and a gene coding for a xylulosease, Recombinant yeast.
2. The method according to claim 1, wherein the gene coding for the xylose reductase using NADH as a cofactor is from Spathaspora passalidarum Recombinant yeast characterized by.
2. The recombinant yeast according to claim 1, wherein the gene coding for the xylose reductase using NADH as a cofactor is a gene encoding the amino acid sequence of SEQ ID NO: 1.
4. The recombinant yeast according to claim 3, wherein the gene coding for the xylose reductase using NADH as a cofactor has the nucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 3.
2. The recombinant yeast according to claim 1, wherein the gene encoding xylitol dehydrogenase using NAD &lt; + &gt; as a cofactor is derived from Pichia stipitis .
The recombinant yeast according to claim 1, wherein the gene coding for the xylitol dehydrogenase using NAD &lt; + &gt; as a cofactor is a gene encoding the amino acid sequence of SEQ ID NO: 6.
7. The recombinant yeast according to claim 6, wherein the gene coding for the xylitol dehydrogenase using NAD &lt; + &gt; as a cofactor has the nucleotide sequence of SEQ ID NO: 7.
2. The recombinant yeast according to claim 1, wherein the gene encoding zyululokinase is derived from Pichia stipitis .
2. The recombinant yeast according to claim 1, wherein the gene encoding zyululokinase is a gene encoding the amino acid sequence of SEQ ID NO: 8.
10. The recombinant yeast according to claim 9, wherein the gene encoding zyululokinase has the nucleotide sequence of SEQ ID NO:
A method for producing ethanol from xylose comprising the steps of:
(a) culturing the recombinant yeast of any one of claims 1 to 10 in a xylose-containing medium to produce ethanol; And
(b) obtaining the resulting ethanol.

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