WO2014185635A1 - Recombinant yeast capable of producing ethanol from xylose and method for producing ethanol using same - Google Patents

Abstract

The present invention relates to recombinant yeast having high productivity of ethanol from xylose and, more specifically, to recombinant yeast having a high productivity of ethanol from xylose in which a gene encoding xylose reductase (XR) derived from Spathaspora passalidarum, a gene encoding xylitol dehydrogenase (XDH), and a gene encoding xylulokinase (XK) are introduced, and a method for producing ethanol using the recombinant yeast. When ethanol is produced from cellulosic biomass full of xylose, yeast strains of the present invention are capable of producing a high yield of ethanol with little accumulation of xylitol as a by-product, and are very useful for producing biofuel using cellulosic ethanol (CE).

Description

Recombinant yeast capable of producing ethanol from xylose and ethanol production method using the same

The present invention relates to a recombinant yeast having high ethanol productivity from xylose, and more particularly, a gene encoding xylose reductase (XR) and xylitol dehydrogenase (XR) derived from Spathaspora passalidarum ( Spathaspora passalidarum ). Recombinant yeast having ethanol production ability from xylose into which a gene encoding XDH: xylitol dehydrogenase (XD) and a gene encoding xylulokinase (XK) is introduced, and a method for producing ethanol using the recombinant yeast will be.

As interest in biofuels and renewable energy increases, researches for using cellulose ethanol (CE) as the next generation fuel are being actively conducted. However, technical obstacles such as pretreatment technology, discovery of saccharase and fermented 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, ethanol high efficiency using xylose 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 traditionally used in ethanol industry and fermentation industry. It has been a strain. However, there is a limit that Xylose, an pentose sugar, cannot be used as a carbon source. Therefore, by introducing genes involved in the metabolic pathway of xylose into S. cerevisiae using metabolic engineering, the development of S. cerevisiae capable of fermenting ethanol with xylose as a carbon source has been made.

For example, xyl1 , which is a gene encoding xylose reductase derived from Pichia stipitis , xyl2 and xyl2 , which are genes encoding xylitol dehydrogenase, and xylolokinase ( 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 made efforts to increase ethanol productivity in a transformed yeast strain having ethanol-producing ability using xylose, surprisingly coding for xthaose reductase derived from Spathaspora passalidarum the gene is introduced to xyl1, xylitol dehydrogenase: the gene encoding a (XDH xylitol dehydrogenase) xyl2, and xylene rule Rocca or kinase: the (XK xylulokinase) a gene encoding xyl3 the introduction of recombinant yeast, in particular S. In the case of cerevisiae strains, it was confirmed that xyl1 , xyl2 and xyl3 derived from Pichia stipitis exhibited superior ethanol production capacity and yield compared to the conventional results using S. cerevisiae . .

Summary of the Invention

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.

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 .

FIG. 3 shows a recombinant vector containing a gene encoding xylitol dehydrogenase (XDH) or a gene encoding xylulose kinase (XK): (A) PsXDH; (B) SpXDH; (C) PsXK

4 shows enzymatic titers according to cofactors (NADH and NADPH) of yeast transformed with a recombinant vector containing a gene encoding xylose reductase (XR): (A) PsXR; (B) SpXR ^CO ; (C) SpXR.

Figure 5 shows the result of culturing the recombinant yeast of the present invention in anaerobic conditions, by-products reduced xylitol production and improved ethanol production capacity.

Detailed Description of the Invention and Specific Embodiments

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is well known and commonly used in the art.

In one aspect, the present invention provides a gene encoding a xylose reductase derived from Spathaspora passalidarum ; A gene encoding xylitol dehydrogenase; And it relates to a recombinant yeast having ethanol production ability from xylose, characterized in that the gene encoding xylolokinase is introduced.

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 xylose metabolism. The absence of the gene xyl2 encoding 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 xylose by xylitol dehydrogenase (XDH) using NAD + as cofactor, and further xylated Xylulokinase (XK) introduced into cellulose is converted into xylulose-5-phosphate and metabolized 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, the 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 xylulokinase , and XR is xylose reduction. The enzyme, XDH, means xylitol dehydrogenase, and XK means xylolokinase.

In the present invention, the gene encoding the xylitol dehydrogenase is preferably derived from Spathaspora passalidarum or Pichia stipitis , and the gene encoding the xylulokinase . Is derived from Pichia stipitis .

The pichia stipitis strain used in the present invention is also called Scheffersomyces stipitis .

In the present invention, the gene encoding the xylose reductase may be a gene encoding the amino acid sequence of SEQ ID NO: 1, and preferably has a nucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 3 You can do

The base sequence of the gene ( Spxyl1 ) sequence number 3 encoding the xylose reductase derived from Spathaspora passalidarum is a sequence codon optimized to be optimally expressed in S. cerevisiae .

In the present invention, the spathaspora passalidarum- derived xylitol dehydrogenase may be characterized by having an amino acid sequence of SEQ ID NO: 6, and the spadaspora passalidarum- derived xylitol dehydrogenase The gene encoding the enzyme may be characterized by having the nucleotide sequence of SEQ ID NO: 7.

In the present invention, the Pichia stipitis- derived xylitol dehydrogenase may be characterized by having an amino acid sequence of SEQ ID NO: 8, the pichia stipitis- derived xylitol dehydrogenase Gene encoding may be characterized by having the nucleotide sequence of SEQ ID NO: 9.

In the present invention, the gene encoding the gyrolokinase may be characterized in that the gene encoding the amino acid sequence of SEQ ID NO: 10, preferably characterized in that it has a nucleotide sequence of SEQ ID NO: 11. have.

In one aspect of the present invention, overexpression of SpXR derived from NADH dependent Spathaspora passalidarum and PsXDH and PsXK derived from Pichia stipitis, which is not NADPH dependent xylose reductase (XR), solves the cofactor imbalance problem, and Significantly increased production efficiency.

The xylose metabolic pathway in P. stipitis- derived Psxyl1 , Psxyl2, and Psxyl3- derived recombinant yeast strains, NADPH is used as a cofactor for P. stipitis- derived Xs. Since xylitol reductase, which is encoded by Psxyl2, is an enzyme that uses NAD as a cofactor, cofactors cannot be reused between two enzyme reactions, 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 two enzymes, thereby eliminating cofactor imbalance.

In another embodiment of the present invention, overexpression of xylitol dehydrogenase (XDH) SpXDH derived from Spathaspora passalidarum and xylose reductase (XR) SpXR derived from Spathaspora passalidarum and xylolokinase (XK) PsXK from Pichia stipitis In addition, it reduced the production of by-product xylitol and dramatically increased the efficiency of ethanol production.

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

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

Table 1 Sequence number and definition of each enzyme in the present invention and the base sequence encoding the same SEQ ID NO: Display Justice One SpXR Amino Acid Sequences of Xylose Reductase from Spathaspora passalidarum 2 Spxyl1 Nucleotide sequence of the native gene encoding SpXR 3 Spxyl1 ^CO Codon-optimized base sequence encoding SpXR and for expression in recombinant yeast 4 PsXR Amino Acid Sequences of Xylose Reductase from Pichia stipitis 5 Psxyl1 Nucleotide sequence of the gene encoding PsXR 6 SpXDH Amino Acid Sequence of Xylitol Dehydrogenase from Spathaspora passalidarum 7 Spxyl2 Base sequence encoding SpXDH 8 PsXDH Amino Acid Sequences of Xylitol Dehydrogenase from Pichia stipitis 9 Psxyl2 Base sequence encoding PsXDH 10 PsXK Amino Acid Sequences of Xylolokinase from Pichia stipitis 11 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 currently the most commonly used form of vectors, "plasmids" and "vectors" 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 performing 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. Expression control sequences for yeast are derived from genomic DNA of yeast, Saccharomyces cerevisiae. Preferably, expression control sequences for highly expressed yeast genes are used for expression of the gene of interest.

Ie promoter of TRP1 gene, ADH I or ADH II gene, acid phosphatase (PHO5) gene, isocytochrome C gene: or enolase, glycer-aldehyde-3-phosphate dehydrogenase (GAPDH), 3- Phosphoglycerate kinase (PGK), hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triose Promoters of phosphate isomerase, phosphoglucose isomerase and glucokinase genes: or promoters of yeast cross pheromone genes encoding a- or α-factors can be used.

Promoter components comprising the activation sequence (UAS) of one yeast gene in the upper part, the functional TATA box of the other yeast in the lower part, for example, the UAS (s) of the yeast PHO5 gene and the functional TATA of the yeast GAPDH gene in the lower part. Recombinant promoters containing a promoter component comprising a box can be used. Preferred vectors of the invention contain promoters with transcriptional regulation. Promoters of this type, such as promoters of the PHO5 gene and PHO5-GAPDH recombinant promoters, may or may not be activated by changing growth conditions. For example, the PHO5 promoter can be inhibited or uninhibited at will, only by increasing or decreasing the concentration of inorganic phosphate in the medium. More preferred promoters according to the invention are promoters of the GAPDH gene, in particular their functional fragments starting at nucleotides -300 to -180, in particular nucleotides -263 or -199 of the GAPDH gene and ending at nucleotide-5.

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, the DNA for a pre-sequence or secretion leader is operably linked to the DNA for the 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 positioned 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.

The process of linking different DNA sequences to obtain other DNA constructs in the present invention may be accomplished by blunt end ligation or by appropriate conventional restriction sites or synthesis in order to ensure precise linkage with careful attention to maintain the normal functioning of these DNA sequences. This can be done via linker molecules. That is, the signal sequence and the target gene can be fused by the smooth terminal linkage method. Another approach to accurately link the signal sequence with the gene of interest is, if possible, to limit the signal sequence near the 3 'end and the gene near the 5' end, so as to lack a predetermined number of base pairs, respectively.

In addition, when the synthetic oligodeoxynucleotide linker is linked via a linking oligo deoxynucleotide with a limited signal sequence and a limited target gene, the missing base pair is recovered and the target gene is present in the appropriate reading frame for the signal sequence. I can construct it.

The present invention also specifically relates to expression control sequencing, wherein the sequence containing the DNA fragment and transcription termination signal is unnecessary or less important for the action of the promoter, ie the expression of the gene of interest, but for the proliferation of cells transformed with the recombinant plasmid. It relates to a recombinant plasmid containing additional DNA sequences that are important. Additional DNA sequences may be derived from prokaryotic and / or eukaryotic cells and may include DNA sequences of chromosomes and / or chromosomes. For example, additional DNA sequences can be generated (or made) from plasmid DNA (eg bacterial or eukaryotic plasmid DNA). Preferred hybrid hybrid plasmids contain further DNA sequences derived from bacterial plasmids, in particular E. coli plasmid pBR 322 or related plasmid bacteriophage λ yeast 2μ plasmid and / or yeast chromosomal DNA.

In a preferred plasmid according to the invention further DNA sequences contain yeast replicators and selective gene markers for yeast. Plasmids containing yeast replicators, for example spontaneous replica fragments, remain extrachromosomal in yeast cells after transformation and spontaneously replicate according to mitosis Viral DNA and / or chromosomal DNA (eg bacteria Yeast or higher eukaryotic chromosomal DNA). Plasmids containing sequences homologous to yeast 2μ plasmid DNA can also be used. These plasmids can be recombinantly incorporated into 2μ plasmids already present in the cell or spontaneously replicated. Regarding the selective gene marker for yeast, any marker gene can be used that facilitates the selection of transformants due to the phenotype expression of the marker.

Suitable markers for yeast are those that express genes that complement host disorders, particularly in the case of antibiotic resistance genes or nutritional yeast mutants. Corresponding genes are for example URA-1, URA3, ARG 4, LEU 2, HIS4, HIS3, TRP5, for example to confer resistance to the antibiotic cycloheximide or for autotrophy in trophic yeast mutants. Or the TRP1 gene.

Advantageously additional DNA sequences present in the plasmids according to the invention also include replicators and preferential gene markers for bacterial hosts, in particular E. coli. It is useful when yeast recombinant plasmids are associated with the presence of E. coli replicaants and E. coli labels. Firstly, a variety of recombinant plasmid DNA can be obtained by growing and amplifying in E. coli, and secondly, construction of the recombinant plasmid is advantageously carried out using an entire repertoire of E. coli-based cloning techniques in E. coli.

E. coli plasmids such as pBR322 and the like contain both E. coli clones and E. coli gene markers that confer resistance to antibiotics such as tetracycline and ampicillin and are advantageously used as part of a yeast recombinant vector.

Additional DNA sequences containing replicants and gene markers for yeast and bacterial hosts are referred to below as "recombinant vectors" which form a recombinant plasmid according to the invention together with said DNA construct containing an expression control sequence and the gene of interest. Is mentioned.

As used herein, the term “expression vector” generally refers to a double strand of DNA as a recombinant carrier into which fragments of heterologous DNA have been inserted. Here, heterologous DNA refers to heterologous DNA, which is DNA not naturally found in host cells. Once expression vectors are within a host cell, they can replicate independently of the host chromosomal DNA and several copies of the vector and their inserted (heterologous) DNA can be produced.

Recombinant vectors according to the invention may each contain one or more DNA inserts, in particular comprising an expression manipulation sequence, a DNA sequence encoding a signal peptide and a DNA sequence encoding a target gene.

If the recombinant vectors contain multiple DNA inserts, preferably two to four DNA inserts, they may be present in series or at different positions of the recombinant vector. Preferred recombinant vectors contain one DNA insert or DNA inserts in series. DNA inserts are specifically arranged in tails in the head.

As used herein, the term “transformation” means that DNA is introduced into a host such 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 considered, since the vector must be replicated in it. The number of copies of the vector, the ability to control the number of copies, and the expression of other proteins encoded by the vector, such as antibiotic markers, must also be considered. In selecting expression control sequences, several factors must be considered. For example, the relative strength of the sequence, the controllability, and the 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 various 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) relates to a method for producing ethanol from xylose comprising the step of obtaining the produced ethanol.

Example

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, and 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: Construction of a Recombinant Vector Containing xyl1 , xyl2 or xyl3 Genes

1.1 Construction of a Recombinant Vector Containing a Gene ( xyl1 ) Encoding Xylose Reductase (XR)

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

S. passalidarum Spxyl1 was amplified by performing PCR under the (optimal annealing temperature) of 55 ℃ using the following to a genomic DNA template obtained from S. passalidarum strain (ATCC MYA-43455), the primer TaOpt.

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 S. cerevisiae nucleotide sequence (SEQ ID NO: 3) using S. passalidarum- derived Spxyl1 gene having a nucleotide sequence of SEQ ID NO: 2 jointly developed by Bioneer and KAIST. Synthesis was used. (Bionia, Korea).

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: 14)

Reverse primer PsXR:

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

Spxyl1 fragment of Psxyl1 fragments, S. passalidarum of amplified P. stipitis and synthesized Spxyl1 ^CO gene was ligated to pRS426 TEF vector cut with Bam HI and Xho I, respectively pRS426 TEF PsXR (xyl1), pRS426 and TEF SpXR pRS426 TEF SpXR ^CO was produced (FIG. 2).

1.2 Construction of Recombinant Vectors Containing Xylitol Dehydrogenase (XDH) Gene ( xyl2)

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.

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

Forward Primer SpXDH:

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

Reverse Primer SpXDH:

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

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: 18)

Reverse primer PsXDH:

 5'-TCGACTCGAGTTACTCAGGGCCGTCAATGA-3 '(SEQ ID NO: 19)

Was ligated to a fragment of amplified Spxyl2 S. passalidarum Psxyl2 the fragment of amplified and P. stipitis, respectively cut with Bam HI and Xho I vector TEF pRS424, pRS424 each TEF SpXDH (xyl2) and TEF PsXDH pRS424 (xyl2) It was produced (Fig. 3).

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

In order to construct a vector containing a xylulokinase gene, a 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: 20)

Reverse primer PsXK:

5'-TCGACTCGAGTTAGTGTTTCAATTCACTTT-3 '(SEQ ID NO: 21)

Psxyl3 fragments of amplified P. stipitis were ligated after cleaving the pRS425 TEF vector with Bam HI and Xho I to prepare pRS425 TEF PsXK ( xyl3 ) (FIG. 3).

Example 2: Confirmation of cofactor dependency of xylose reductase

Xylose reductase (XR) a cofactor, Pichia stipitis was Spxyl1 ^CO codon optimized (codon optimization) for SPxyl1 in order to optimize the resulting Psxyl1, Spathaspora passalidarum expression in Origin SPxyl1, S. cerevisiae in order to determine the dependency of the Recombinant strains each expressing the prepared, and the degree of reduction of NADH or NADPH of the cell extract of the recombinant strain was confirmed.

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) Were transformed to obtain recombinant S. cerevisiae rSC (PsXR), rSC (SpXR) and rSC (SpXR ^CO ), which express Psxyl1 , Spxyl1 and Spxyl1 ^CO , 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 as 1U based on the extent 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:

The reaction solution was a 200 μl 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. 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 mainly used NADPH as a cofactor, in the case of SpXR and SpXR ^CO (when Spxyl1 was used for the expression of SpXR) it can be confirmed that the dependence of NADPH as a cofactor there was.

Example 3: Ethanol Production from Xylose Using Recombinant Yeast

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

TABLE 2 Recombinant yeast experimental group Experimental group Introduced Gene SET Recombinant vector introduced Control PsXR / PsXDH / PsXK pRS426 TEF PsXR ( xyl1 ), pRS424 TEF PsXDH ( xyl2 ), pRS425 TEF PsXK ( xyl3 ) One PsXR / SpXDH / PsXK pRS426 TEF PsXR ( xyl1 ), pRS424 TEF SpXDH ( xyl2 ), pRS425 TEF PsXK ( xyl3 ) 2 SpXR / PsXDH / PsXK pRS426 TEF SpXR ( xyl1 ), pRS424 TEF PsXDH ( xyl2 ), pRS425 TEF PsXK ( xyl3 ) 3 SpXR ^CO / PsXDH / PsXK pRS426 TEF SpXR ^CO ( xyl1 ), pRS424 TEF PsXDH ( xyl2 ), pRS425 TEF PsXK ( xyl3 ) 4 SpXR ^CO / SpXDH / PsXK pRS426 TEF SpXR ^CO ( xyl1 ), pRS424 TEF SpXDH ( xyl2 ), pRS425 TEF PsXK ( xyl3 )

TABLE 3 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)

Types of recombinant vectors introduced for each strain and ethanol and xylitol production of the strains into which the recombinant vectors were introduced are shown in Table 4 and FIG. 5.

Table 4 Characteristics of Xylose Metabolism and Ethanol Production in Aerobic and Anaerobic Conditions According to the Introduced Gene Set division Introduction gene SET (expiratory) Xylose consumption rate (g / L / hr) ethanol productivity (g / L / hr) ethanol titer (g / L) Yield (g / g xylose) EtOH Xylitol Control Psxyl1, Psxyl2, Psxyl3 0.22 0.03 2.40 0.15 0.44 No. One Psxyl1, Spxyl2, Psxyl3 0.36 0.08 5.59 0.22 0.25 No. 2 Spxyl1, Psxyl2, Psxyl3 0.45 0.13 9.26 0.29 0.18 No. 3 Spxyl1 ^CO , Psxyl2, Psxyl3 0.50 0.17 12.25 0.34 0.18 No. 4 Spxyl1 ^CO , Spxyl2, Psxyl3 0.47 0.16 11.17 0.33 0.17 division Introduction gene SET (experimental) Xylose consumption rate (g / L / hr) ethanol productivity (g / L / hr) ethanol titer (g / L) Yield (g / g xylose) EtOH Xylitol Control Psxyl1, Psxyl2, Psxyl3 0.84 0.14 3.32 0.16 0.31 No. One Psxyl1, Spxyl2, Psxyl3 1.26 0.24 5.72 0.19 0.19 No. 2 Spxyl1, Psxyl2, Psxyl3 2.04 0.63 15.15 0.31 0.07 No. 3 Spxyl1 ^CO , Psxyl2, Psxyl3 1.63 0.49 11.76 0.30 0.14 No. 4 Spxyl1 ^CO , Spxyl2, Psxyl3 1.68 0.58 14.03 0.35 0.06

As a result, sets 2-4 containing the xyl1 gene derived from recombinant Spadaspora passalidarum had significantly higher ethanol production ability than S. cerevisiae in which xyl1, xyl2 and xyl3 derived from Pichia sphytis were introduced. Low xylitol production was shown.

Example 4: Confirmation of Integration of Recombinant Yeast

Integration of recombinant yeast transformed with SpXR ^CO / PsXDH / PsXK ( Spxyl1 ^CO , Psxyl2, Psxyl3 ) was confirmed.

As a result, ethanol was produced from xylose with high efficiency in both aerobic and anaerobic conditions, and it was confirmed that foreign genes introduced in recombinant yeast were stably integrated.

Table 5 Medium type and culture conditions Seed culture (16 hours) YNB w / o leu, trp (2% glucose) 5ml, 30 ℃, 200rpm Pre-culture (24 hours) YP (3% 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 6 Culture condition Introduction gene SET Xylose consumption rate (g / L / hr) ethanol productivity (g / L / hr) ethanol titer (g / L) Yield (g / g xylose) EtOH Xylitol Exhalation Spxyl1 ^CO , Psxyl2, Psxyl3 0.27 0.08 10.96 0.29 0.03 Narrowing Spxyl1 ^CO , Psxyl2, Psxyl3 1.09 0.42 10.07 0.38 0.14

As described above in detail a specific part of the content of the present invention, for those skilled in the art, such a specific description is only a preferred embodiment, which is not limited by the scope of the present invention Will be obvious. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

As described above, the yeast strain of the present invention can produce ethanol in high yield without accumulating much xylitol as a by-product in producing ethanol from cellulose-based biomass rich in xylose, thereby producing cellulose ethanol (CE). It can be very useful for producing biofuel.

Having described the specific part of the present invention in detail, it is obvious to those skilled in the art that such a specific description is only a preferred embodiment, thereby not limiting the scope of the present invention. something to do. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Electronic file attached.

Claims (12)

Recombinant yeast having the ability to produce ethanol from xylose, characterized by the introduction of the following genes: A gene encoding xylose reductase from Spathaspora passalidarum ; A gene encoding xylitol dehydrogenase; And Gene encoding xylolokinase. The recombinant yeast of claim 1, wherein the gene encoding xylitol dehydrogenase is derived from Spathaspora passalidarum or Pichia stipitis . The recombinant yeast according to claim 1, wherein the gene encoding xylolokinase is derived from Pichia stipitis . The recombinant yeast of claim 1, wherein the gene encoding xylose reductase is a gene encoding the amino acid sequence of SEQ ID NO: 1. The recombinant yeast of claim 1, wherein the gene encoding xylose reductase has a nucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 3. 3. The recombinant yeast of claim 2, wherein the xylitol dehydrogenase derived from Spathaspora passalidarum has the amino acid sequence of SEQ ID NO. The recombinant yeast of claim 2, wherein the gene encoding xylitol dehydrogenase derived from Spathaspora passalidarum has a nucleotide sequence of SEQ ID NO: 7. 7. The recombinant yeast of claim 2, wherein the pichia stipitis- derived xylitol dehydrogenase has an amino acid sequence represented by SEQ ID NO: 8. 8. The recombinant yeast of claim 2, wherein the gene encoding Pichia stipitis- derived xylitol dehydrogenase has a nucleotide sequence of SEQ ID NO: 9. The recombinant yeast according to claim 1, wherein the gene encoding gyrullokinase is a gene encoding the amino acid sequence of SEQ ID NO. The recombinant yeast according to claim 1, wherein the gene encoding the xylolokinase has a nucleotide sequence of SEQ ID NO. A process for preparing ethanol from xylose comprising the following steps; (a) culturing the recombinant yeast of any one of claims 1 to 11 in a xylose-containing medium to produce ethanol; And (b) obtaining the resulting ethanol.

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