KR102075400B1 - 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 yeast having high ethanol production from xylose, and more particularly, a gene encoding xylose reductase using NADH as a cofactor, a gene encoding xylitol dehydrogenase using NAD + as a cofactor, and xylul. It relates to a yeast having an ethanol production capacity from xylose transformed with a gene encoding a locinase and a method for producing ethanol using the yeast.
Recombinant yeast according to the present invention can produce ethanol in high yield without producing much xylitol as a by-product in producing ethanol from cellulose-based biomass rich in xylose, thereby producing 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 ability from xylose, and more particularly, a gene encoding xylose reductase (XR) using NADH as a cofactor, NAD + as a cofactor ( Recombinant yeast having ethanol production ability from xylose having a gene encoding xylitol dehydrogenase (XDH) and a gene encoding xylulokinase (XK) used as a cofactor) It relates to a method for producing ethanol using yeast.

As interest in biofuels and renewable energy increases, researches to use cellulose ethanol (CE) as a next-generation fuel are being actively conducted, but technical obstacles such as the pretreatment technology, the discovery of saccharase and fermentation strains It is emerging as an element.

In particular, unlike traditional ethanol fermentation using C ₆ glucose (Glucose) as a carbon source, CE is produced using C ₅ Xylose (carbon sugar) as a carbon source, so ethanol with high efficiency There is an urgent need for the development of strains that can produce.

Representative ethanol fermentation strain is yeast (Yeast) represented by Saccharomyces cerevisiae , yeast has a high productivity, yield and strong resistance to ethanol, so it is used traditionally in ethanol industry and fermentation industry. It has been a strain. However, there is a limit that can not use xantose as a carbon source. Therefore, the development of S. cerevisiae capable of fermenting ethanol using xylose as a carbon source by introducing genes involved in the metabolic pathway of xylose into S. cerevisiae using metabolic engineering.

For example, xyl1 , a gene encoding xylose reductase derived 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 is known to be due to cofactor imbalance caused by NADPH-dependent xylose reductase (XR) and NAD-dependent xylose dehydrogenase (XDH).

Accordingly, the present inventors have intensively consumed glucose and xylose as a result of intensive efforts to overcome the problem of low ethanol productivity due to cofactor imbalance in a transgenic yeast strain having ethanol production ability using xylose. In the case of recombinant yeasts, especially S. cerevisiae strains, which have been introduced with xyl1 of Spathaspora passalidarum , which is known to be known, and xyl2 and xyl3 of Pichia stipitis , which have been previously known, The present invention was completed by confirming that xyl1 , xyl2 and xyl3 derived from S. cerevisiae exhibited superior ethanol production capacity and yield compared to the existing results.

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

Another object of the present invention 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 provides a gene encoding xylose reductase ( xyl1 ) using NADH as a cofactor, a gene encoding xylitol dehydrogenase using xAD2 as a cofactor ( xyl2 ), and xyrulokinase It provides a recombinant yeast having the ability to produce ethanol from xylose, in which a gene encoding xyl3 is introduced.

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.

In the yeast strain of the present invention in producing ethanol from xylose-rich cellulose-based biomass, it is possible to produce ethanol in high yield without accumulating much xylitol as a by-product, and to produce biofuel using cellulose ethanol (CE). It can be very useful.

FIG. 1 shows metabolic pathways for producing ethanol from xylose, and shows xylose metabolic pathways in yeast strains into which S. passalidarum genes SPxyl1 and P. stipitis genes PSxyl2 and PSxyl3 are introduced.
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 xylolokinase (XK): (A) PsXDH; (B) PsXK.
4 shows enzymatic titers according to cofactors (NADH and NADPH) of yeast transformed with recombinant vectors containing genes encoding xylose reductase (XR): (A) PsXR; (B) SpXR ^CO ; (C) SpXR.

In one aspect, the present invention, a gene encoding a xylose reductase using NADH as a cofactor, a gene encoding a xylitol dehydrogenase using NAD + as a cofactor, and a gene encoding a xylolokinase are introduced. It relates to a recombinant yeast having the ability to produce ethanol from xylose.

Xylose is a biomass that is abundant in waste wood in the form of a polymer called xylan, and is a five-carbon monosaccharide that can be metabolized into useful products by various organisms. It is a two-step pentose phosphate pathway. phosphate pathway (PPP).

However, Saccharomyces cerevisiae , a yeast mainly used in conventional ethanol fermentation, has a gene xyl1 and xylitol that encodes xylose reductase (XR) in terms of metabolism of xylose. The absence of the gene xyl2 , which encodes dehydrogenase (XDH), prevented the use of xylose as a metabolic, ie carbon source.

Xylose is reduced to xylitol by xylose reductase (XR) using NADH or NADPH as cofactor, xylitol is oxidized to xylulose by xylitol dehydrogenase (XDH) using NAD + as cofactor, and further xyl Xylulokinase (XK) introduced into cellulose is converted to xylulose-5-phosphate, and metabolism proceeds through the pentose phosphate cycle. Xylolokinase (XK) is an enzyme present in yeast, but if only XR and XDH are introduced into the strain without overexpressing it, ethanol can be produced from xylose, but the yield and productivity are remarkably low.

Unless defined otherwise in the present invention, xyl1 is a gene encoding xylose reductase, xyl2 is a gene encoding xylitol dehydrogenase, xyl3 is a gene encoding xylolo kinase, and XR is xylose. Reductase, XDH means xylitol dehydrogenase, XK means xylolokinase.

In one embodiment of the invention, SpXR derived from NADH dependent Spathaspora passalidarum and PsXDH derived from Pichia stipitis, and not NADPH dependent xylose reductase (XR); Overexpression of PsXK resolved cofactor imbalance and dramatically increased ethanol production efficiency.

Xylose metabolism pathways in recombinant yeast strains in which Psxyl1 , Psxyl2 and Psxyl3 derived from P. stipitis have been previously introduced are NADPH as a cofactor in the case of xylose reductase (XR) encoded by Ps xyl1 derived from P. stipitis. Xylitol reductase, which is an enzyme used and encoded by Ps xyl2, is an enzyme that uses NAD as a cofactor, so that cofactors cannot be reused between the reactions of 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, thereby eliminating cofactor imbalance.

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

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

Base sequence having at least 80%, preferably at least 90%, more preferably at least 95%, most preferably at least 97% of the base sequence of SEQ ID NO: 2 or SEQ ID NO: 3, in particular in the present invention As long as the function of the xylose reductase encoded by Sp xyl1 from the provided S. passalidarum is retained, a conservative substitution at the xylose reductase, for example at some amino acid residues, has resulted in mutations in some amino acid sequences. It will be apparent to those skilled in the art that the nucleotide sequence encoding the generated xylose reductase is also included in the scope of rights according to the present invention. In addition, those skilled in the art are those that encode the same amino acid sequence encoded by SEQ ID NO: 2 or SEQ ID NO: 3, and whose base sequences differ due to codon degeneracy, are also included in the scope of rights according to the present invention. It is self-evident to.

As used herein, the term "homology" refers to sequence similarity or sequence identity. The homology can be measured using standard techniques known in the art.

In the present invention, the gene ( xyl2 ) coding for xylitol dehydrogenase using NAD + as a cofactor is derived from Pichia stipitis , and the gene ( xyl3 ) coding for xyllurokinase is also derived from Pichia stipitis It is done.

In the present invention, the xylitol dehydrogenase using the NAD + as a cofactor preferably has an amino acid sequence of SEQ ID NO. 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 preferably is characterized by having the nucleotide sequence ( Psxyl2 ) of SEQ ID NO: 7, and the gyrullokinase ( PsXK ) derived from Pichia stipitis is a sequence It is preferable to have an amino acid sequence of No. 8, and the gene ( Psxyl3 ) encoding the same has a nucleotide sequence of SEQ ID NO: 9.

In the base sequence according to SEQ ID NO: 7 and SEQ ID NO: 9, the same as the base sequence according to SEQ ID NO: 2 or SEQ ID NO: 3 80% or more, preferably 90% or more, more preferably 95% or more, most preferably Preferably, a nucleotide sequence encoding xylitol dehydrogenase or xylolokinase having a different nucleotide sequence but having the same amino acid sequence due to codon degeneracy and a base sequence having more than 97% sequence homology, and xylitol dehydrogenase or As long as the function of the illurokinase is maintained, the amino acid sequence in which the mutation occurs in some amino acid sequences, in particular the base sequence encoding xylitol dehydrogenase or xylokinase, in which conservative substitutions occur in some amino acid residues, It is obvious to those skilled in the art that the scope of the present invention is included in the scope of the present invention. It is

The sequence numbers and definitions of the respective enzymes and base sequences encoding the same in the present invention are as described in Table 1.

Sequence number and definition of each enzyme in the present invention and the base sequence encoding the same order
number Display Justice One SpXR Xylose from Spathaspora passalidarum
Amino acid sequence of the reductase 2 Spxyl1 Nucleotide sequence of the native gene encoding SpXR 3 Spxyl1 ^CO Encodes SpXR and expresses expression in recombinant yeast
Codon-optimized nucleotide sequence 4 PsXR Of xylose reductase from Pichia stipitis
Amino acid sequence 5 Psxyl1 Nucleotide sequence of the gene encoding PsXR 6 PsXDH Of Xylitol Dehydrogenase from Pichia stipitis
Amino acid sequence 7 Psxyl2 Base sequence encoding PsXDH 8 PsXK Of xylolokinase from Pichia stipitis
Amino acid sequence 9 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 in recombinant yeast. The vector may be a plasmid, phage particles, or simply a potential genomic insert. Once transformed into the appropriate host, the vector can replicate and function independently of the host genome, or in some cases can be integrated into the genome itself. Since plasmids are the most commonly used form of the current vector, "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 effecting transcription, any operator sequence for regulating such transcription, sequences encoding suitable mRNA ribosomal binding sites, and sequences that control termination of transcription and translation. For example, suitable control sequences for prokaryotes include promoters, optionally operator sequences, and ribosomal binding sites. Eukaryotic cells include promoters, polyadenylation signals and enhancers. The factor that most influences the amount of gene expression in the plasmid is the promoter.

To express the DNA sequences of the 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 region of phage lambda, regulatory region of fd code protein, 3-phosphoglycerol A promoter known to modulate the expression of a promoter for late kinase or other glycolysis enzymes, promoters of said phosphatase, such as Pho5, a yeast alpha-crossing system and prokaryotic or eukaryotic cells or genes of their viruses; Other sequences of induction and various combinations thereof.

Nucleic acids are “operably linked” when placed in a functional relationship with other nucleic acid sequences. This may be genes and regulatory sequence (s) linked in such a way as to allow gene expression when appropriate molecules (eg, transcriptional activating proteins) bind to regulatory sequence (s). For example, DNA for a pre-sequence or secretion 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 the transcription of the sequence; Or the ribosomal binding site is operably linked to a coding sequence when it affects the transcription of the sequence; Or the ribosomal binding site is operably linked to a coding sequence when disposed to facilitate translation. In general, “operably linked” means that the linked DNA sequences are in contact, and in the case of a secretory leader, are in contact and present within the reading frame. However, enhancers do not need to touch. Linking of these sequences is performed by ligation (linking) at convenient restriction enzyme sites. If such sites do not exist, synthetic oligonucleotide adapters or linkers according to conventional methods are used.

As used herein, the term “expression vector” generally refers to a double strand of DNA as a recombinant carrier into which a fragment of heterologous DNA has been inserted. Here, heterologous DNA refers to heterologous DNA, which is DNA not naturally found in host cells. The expression vector can replicate independently of the host chromosomal DNA once it is in the host cell and several copies of the vector and its inserted (heterologous) DNA can be produced.

As is well known in the art, to raise the expression level of a transformed gene 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 gene of interest are included in one expression vector including the bacterial selection marker and the replication origin. If the expression host is a eukaryotic cell, the expression vector must further comprise 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 is replicable as an extrachromosomal factor or by chromosomal integration. The term “transfection” as used herein means that the expression vector is accepted 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 function equally in expressing the DNA sequences of the present invention. Likewise not all hosts function equally for the same expression system. However, those skilled in the art can make appropriate choices among various vectors, expression control sequences and hosts without departing from the scope of the present invention without undue experimental burden. For example, in selecting a vector, the host must be taken into account because the vector must be replicated therein. 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 expression control sequences, several 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, particularly with regard to possible secondary structures. Single cell hosts may be selected from a host for the selected vector, the toxicity of the product encoded by the DNA sequence of the invention, the secretory properties, the ability to accurately fold the protein, the culture and fermentation requirements, the product encoded by the DNA sequence of the invention from the host. It should be selected in consideration of factors such as the ease of purification. Within the scope of these variables, one skilled in the art can select a variety of vector / expression control sequence / host combinations that can express the DNA sequences of the invention in fermentation or 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. It relates to a method for preparing ethanol from xylose.

Hereinafter, the present invention will be described in more detail with reference to 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 being limited by these examples.

Example 1: xyl1 , xyl2  or xyl3  Construction of Recombinant Vectors Containing Genes

1.1 gene encoding xylose reductase (XR) xyl1 Construction of Recombinant Vectors Containing

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

The pRS426 TEF xylose reductase (XR) gene to be introduced into a vector that is codon optimized (codon optimization) such that the expression in Psxyl1, Spathaspora passalidarum of Spxyl1 and Spxyl1 derived from S. cerevisiae of Pichia stipitis-derived optimized Spxyl1 ^CO Each gene 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: 10)

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

Spxyl1 of S. passalidarum is S. passalidarum The genomic DNA template obtained from the strain (ATCC MYA-43455) was amplified by PCR under an optimal annealing temperature (TaOpt) of 55 ° C using the following primers.

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

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

Spxyl1 ^CO was obtained by optimizing the S. passalidarum- derived Spxyl1 gene having the nucleotide sequence of SEQ ID NO: 2 in S. cerevisiae (SEQ ID NO: 3) using a program co-developed by Bioneer and KAIST. It synthesize | combined and used (Bionia, 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 produced (FIG. 2).

1.2 xylitol dehydrogenase (XDH) gene ( xyl2 Construction of Recombinant Vectors Containing

In order to produce a vector containing the xylitol dehydrogenase gene and having the TEF promoter and the CYC1 terminator of S. cerevisiae S. cerevisiae expression vector pRS424 vector TEF (Mumberg D. et al, Gene 156 :. 119, 1995) the backbone Used as.

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)

Psxyl2 fragments of amplified P. stipitis were ligated to pRS424 TEF vectors digested with BamHI and XhoI to construct pRS424 TEF PsXDH ( xyl2 ), respectively (FIG. 3 (A)).

1.3 xylulokinase (XK) gene ( xyl3 Construction of Recombinant Vectors Containing

In order to construct a vector containing a xylulokinase gene, pRS425 TEF vector (Mumberg D. et al., Gene 156: 119, 1995), which is an expression vector of S. cerevisiae having a TEF promoter and a CYC1 terminator of S. cerevisiae 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: 16)

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

PRS xyl3 fragments of amplified P. stipitis and pRS425 TEF vectors were digested with BamHI and XhoI and ligated to prepare pRS425 TEF PsXK ( xyl3 ) (FIG. 3 (B)).

Example 2: Confirmation of cofactor dependency of xylose reductase

To confirm the cofactor dependence of xylose reductase (XR), Spxyl1 ^{CO with} codon optimization of SPxyl1 to optimize expression of Ps xyl1 from Pichia stipitis , SPxyl1 from Spathaspora passalidarum and S. cerevisiae To prepare a recombinant strain expressing each, and confirmed the degree of reduction of NADH or NADPH of the cell extract of the recombinant strain.

First, the recombinant Pichia stipitis containing Spxyl1 Spxyl1 and ^CO derived from the Psxyl1 and Spathaspora passalidarum derived from each vector pRS426 TEF PsXR (xyl1), pRS426 and pRS426 TEF Saccharomyces cerevisiae TEF SpXR the SpXR ^CO respectively CEN-PK2-1D (euroscarf 30000D) Was transformed to obtain recombinant S. cerevisiae rSC (PsXR), rSC (SpXR) and rSC (SpXR ^CO ), which express Psxyl1 , Spxyl1 and Spxyl1 ^CO alone, respectively. The obtained recombinant strain was incubated for 24 hours at 30 ℃ 200rpm conditions in a minimal medium (YNB w / o ura, leu, trp), crude cell extract (crude cell extract through cell disruption using glass beads) ) Was obtained.

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

As a reaction solution, 200 μl solution containing 100 μl 0.1M potassium phosphate buffer, 20 μl 4mM NADH or 4mM NADPH, 20 μl crude cell extract, 60 μl 0.33M xylose solution was added. After the mixture was allowed to stand for 3 minutes to maintain equilibrium, it was measured by observing the reduction of the coenzyme at 340 nm for 3 minutes after the addition of the xylose solution. Protein concentration was quantified using the Bradford assay.

As a result, as shown in Figure 4, in the case of PsXR, NADPH was mainly used as a cofactor, and in the case of SpXR and SpXR ^CO (when Sp xyl1 was used for the expression of SpXR), it was confirmed that the dependency of NADPH was reduced as a cofactor. Could.

Example 3: Ethanol Production from Xylose Using NADH-dependent Xylose Reductase

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

Medium 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 3 shows the types of recombinant vectors introduced for each strain and the ethanol and xylitol production of the strains into which the recombinant vectors were introduced.

Xylose Metabolism and Ethanol Production Characteristics 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 lower xylitol generating ability than the recombinant strain expressing Ps xyl1 using NADPH as cofactor.

The specific parts of the present invention have been described in detail above, and for those skilled in the art, these specific descriptions are merely examples of preferred embodiments, and the scope of the present invention is not limited thereto. Will be obvious. Thus, 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-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 Val 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 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> 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 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> 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 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> 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> <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 encoding xylose reductase using NADH represented by amino acid sequence of SEQ ID NO: 1 from Spathaspora passalidarum as a cofactor, a gene encoding xylitol dehydrogenase using NAD + from Pichia stipitis as a cofactor, and a derivative from Pichia stipitis Recombinant Saccharomyces cerevisiae with ethanol-producing ability from xylose, into which a gene encoding xylolokinase is introduced.
delete delete The recombinant Saccharomyces cerevisiae of claim 1, wherein the gene encoding the xylose reductase using NADH as a cofactor has a nucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 3.
delete The recombinant Saccharomyces cerevisiae of claim 1, wherein the gene encoding xylitol dehydrogenase using NAD + as a cofactor is a gene encoding the amino acid sequence of SEQ ID NO: 6.
The recombinant Saccharomyces cerevisiae according to claim 6, wherein the gene encoding xylitol dehydrogenase using NAD + as a cofactor has a nucleotide sequence of SEQ ID NO.
delete The recombinant Saccharomyces cerevisiae according to claim 1, wherein the gene encoding gyrullokinase is a gene encoding the amino acid sequence of SEQ ID NO: 8.
10. The recombinant Saccharomyces cerevisiae of claim 9, wherein the gene encoding the xyrulokinase has a nucleotide sequence of SEQ ID NO: 9.
A process for preparing ethanol from xylose comprising the following steps;
(a) culturing the recombinant Saccharomyces cerevisiae of claim 1 in a xylose-containing medium to produce ethanol; And
(b) obtaining the resulting ethanol.

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