KR101241346B1 - Osmosis―Resistant Yeast Strains and Uses Thereof - Google Patents

Abstract

The present invention relates to osmosis-resistant transformed yeast strains comprising the mutated SPT15 gene and uses thereof. Yeast strains of the present invention are yeast strains that can grow under high glucose, preferably 30-40% glucose or sucrose, and yeast strains that can grow in high concentration ethanol, preferably 12.5-15% ethanol. Therefore, the strain of the present invention, which exhibits high concentrations of glucose and ethanol, will be useful for more efficient ethanol production and have practical utility as a super strain of high efficiency ethanol production ability that is resistant to various stresses generated in the bioethanol production process. will be.

Description

Osmosis-Resistant Yeast Strains and Uses Thereof}

The present invention is directed to osmo-resistant yeast strains and uses thereof.

The production of bioethanol from plant or seaweed biomass is of worldwide interest in terms of long-term availability and harmful environmental aspects of fossil fuels (Jeffries, T., and P. Lindbladm, 2009; Ragauskas, AJ, et al. , 2006; Rubin, EM, 2008). However, the relatively high production costs of bioethanol have hampered investment in related industries. Thus, much effort has been made to reduce the costs of biomass acquisition, pretreatment, fermentation and product recovery (Xu, Q., A. Singh, and ME Himmel, 2009). During the bioethanol production process, ethanol-producing microorganisms face various stresses such as high concentrations of initial substrates, increased ethanol concentrations and accumulation of toxic byproducts. In addition to rapid growth and effective fermentation capacity, the ability to withstand the stresses described above is an important factor in selecting ethanol producers (Ding, J., et al. , 2009; Gibson, BR, et al. , 2007; Yoshikawa, K., et al. , 2009). One way to improve ethanol production is to develop strains with increased stress resistance.

Yeast S. cerevisiae has been used in a variety of industries, including the production of bioethanol from biomass resources. Yeast cells are always exposed to various environmental stresses such as high concentrations of ethanol that occur during industrial ethanol fermentation, which in turn results in a decrease in cell growth, cell viability and ethanol production (Casey and Ingledew, 1986). Therefore, there has been a need for the development of yeast strains that can overcome the stress caused by high concentrations of ethanol. Furthermore, the use of genome-wide assays, such as microarrays and comprehensive expression pattern analysis, has been useful in identifying novel ethanol stress related genes (Hirasawa, et al. , 2007; Teixeira, et al. , 2009; Yoshikawa, et al. , 2009). Through this approach, many genes involved in ethanol resistance have been identified as non-essential genes. In addition, results related to ethanol resistance did not match, respectively, and reported that this discrepancy was due to strain and growth conditions (Teixeira, et al. , 2009). Thus, strains constructed from the genetic information described above do not always result in changes to ethanol stress conditions (Yoshikawa, et al. , 2009). In addition, a commercially available Saccharomyces cerevisiae deletion mutant library was used for genome-wide screening of genes involved in ethanol resistance (Fujita, et al. , 2006; Teixeira, et al. , 2009; Yoshikawa, et. al. , 2009). The above studies mainly selected mutants exhibiting ethanol sensitivity to identify the corresponding genes as genes required for growth under high ethanol conditions.

In general, many genes are known to affect important intracellular phenotypes (eg, overexpression of metabolites from pathological conditions). However, most cellular and metabolic engineering approaches have been carried out through deletion or overexpression of single genes due to experimental limitations of vector construction and transformation efficiency. This ruled out the investigation through modification of several genes.

To investigate the mechanism of ethanol stress resistance, there have been many studies. In particular, unsaturated fatty acids related to membrane fluidity have been reported and considered as important determinants of ethanol resistance in yeast (Kajiwara, et al. , 2000; You, et al , 2003). In addition, intracellular accumulation of trihalose (Kim, et al. , 1996) or proline (Takagi, et al. , 2005) improves yeast ethanol resistance and ergosterol to ethanol resistance to Saccharomyces cerevisiae. Has been reported to be an important factor associated with (Inoue, et al. , 2000).

On the other hand, in order to obtain a large amount of ethanol in a short time fermentation process using the VGH fermentation (very high gravity fermentation) (very high gravity fermentation), there is an effect that can save time and budget by reducing the steps of various processes. However, in the case of general yeast, the fermentation time is long due to high glucose, which results in low ethanol production. Therefore, in the VGH fermentation technique, not only yeast resistance to ethanol but also resistance to high osmotic pressure by high glucose is essential.

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

We sought to develop osmosis-resistant yeast strains. As a result, the inventors produced a mutated SPT15 gene using PCR-mediated random mutagenesis and transformed it into yeast to isolate ethanol-resistant transformed yeast strains, which were highly concentrated glucose or sucrose. The present invention has been completed by confirming that it can grow in high concentration ethanol (eg 15% ethanol) as well as (eg 20%, 30% or 40%).

Accordingly, it is an object of the present invention to provide an osmo-resistant transformed yeast strain.

Another object of the present invention is to provide a method for producing ethanol.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to an aspect of the present invention, the present invention provides an osmosis-resistant transformed yeast strain comprising a mutated SPT15 gene.

We sought to develop osmosis-resistant yeast strains. As a result, the inventors produced a mutated SPT15 gene using PCR-mediated random mutagenesis and transformed it into yeast to isolate ethanol-resistant transformed yeast strains, which were highly concentrated glucose or sucrose. (Eg 20%, 30% or 40%) as well as high concentrations of ethanol (eg 15% ethanol).

Ethanol, a flammable, volatile colorless liquid, is the most widely used solvent. Industrially, ethanol is used as a vehicle fuel and fuel additive and also as a perfume, flavorings, colorings and medicines. Ethanol is also the main psychoactive component in alcoholic beverages and has a calming effect on the central nervous system. Ethanol can be produced petrochemically through hydration of ethylene and biologically by fermenting sugars using yeast, which is produced by petrochemical processes that depend on the price of petroleum and grain feed products. Much more economical than Therefore, the development of yeast strains for the production of biological ethanol is very important.

According to the present invention, the present invention provides a yeast strain transformed with the SPT15 gene mutated by PCR in yeast.

According to a preferred embodiment of the invention, the mutation of the invention comprises a mutated amino acid sequence within the amino acid sequence of the wild type SPT15 gene, more preferably comprises four mutated amino acid sequences, most preferably sequence listing agent It includes an amino acid sequence consisting of two sequences.

According to a preferred embodiment of the present invention, the mutated SPT15 gene of the present invention is a mutated SPT15 gene comprising an amino acid sequence of which the amino acid sequences of wild type S42, C78, S163 and I212 positions are mutated.

According to a more preferred embodiment of the present invention, the mutated SPT15 gene of the present invention is a mutant sequence wherein the amino acid sequence of positions S42, C78, S163 and I212 of the wild type SPT15 gene is mutated to S42N, C78R, S163P and I212N (SEQ ID NO: Mutated SPT15 gene).

According to the present invention, the yeast strain transformed with the above-described mutated SPT15 gene, preferably the mutated SPT15 gene consisting of SEQ ID NO: 2 sequence, comprises high concentration glucose or sucrose, more preferably 20-50% glucose or sucrose, Even more preferably 30-40% glucose or sucrose, and most preferably 40% glucose or sucrose.

According to a preferred embodiment of the invention, the yeast strain of the invention is in high concentration ethanol, more preferably 5-15% ethanol, even more preferably 10-15% ethanol, and most preferably 12.5-15% ethanol You can grow.

According to a preferred embodiment of the present invention, the mutated SPT15 gene described above may be introduced into yeast cells as a plasmid. In addition, according to a preferred embodiment of the present invention, the mutated SPT15 gene described above may be introduced into genomic DNA of yeast cells.

According to a preferred embodiment of the present invention, yeast strains that can be used for the transformation of the above-described mutated SPT15 gene are Saccharomyces spp., Schizosaccharomyces spp., Blood Pichia spp., Paffia spp., Kluyveromyces spp., Candida spp., Talaromyces spp., Bretanomyces sp . Brettanomyces spp.), Parkinson solren species (Pachysolen spp.), Deva Rio MRS species (Debaryomyces spp.) or including industrial diploid (polyploid) yeast strain, but are not limited to. More preferably, the yeast strain that can be used for the transformation of the mutated SPT15 gene described above is Saccharomyces species, even more preferably Saccharomyces cerevisiae, most preferably Saccharomyces The Seth Cervage is L3262.

According to another aspect of the present invention, the present invention provides an ethanol-resistant yeast strain comprising introducing the above-described mutated SPT15 gene copy into a yeast strain and / or mutating the endogenous SPT15 gene of the genome DNA of yeast cells. Provide a method.

According to another aspect of the present invention, the present invention provides a method for producing ethanol comprising culturing the yeast strain into which the above-described mutated SPT15 gene is inserted in a culture medium comprising at least one substrate capable of metabolizing with ethanol. do.

Since the method of the present invention includes the above-described yeast strain transformed with the mutated SPT15 gene of the present invention as an active ingredient, the overlapping content between the two is omitted in order to avoid excessive complexity of the present specification according to the overlapping description. Omit.

According to a preferred embodiment of the invention, the substrate which can be metabolized to ethanol comprises C6 sugars, and according to a more preferred embodiment of the invention, the C6 sugar comprises glucose, but is not limited thereto. no.

The present invention provides a cell transfected with a recombinant vector comprising the above mutated SPT15 gene or a transcript thereof and a transgenic cell by gene transfer. In addition, the present invention provides a recombinant vector comprising the above-mentioned mutated SPT15 gene or a transformant transformed with the aforementioned mutated SPT15 protein.

The recombinant vector of the present invention comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 2 or a complementary nucleotide sequence thereof. The vector of the present invention can typically be constructed as a vector for cloning or as a vector for expression. In addition, the vector of the present invention can be constructed by using prokaryotic cells and eukaryotic cells as hosts. For example, prokaryotic cells include bacterial cells and archaea, and eukaryotic cells include yeast cells, mammalian cells, plant cells, insect cells, stem cells and fungi, and most preferably yeast cells.

Preferably, the recombinant vector of the present invention comprises (i) a nucleotide sequence encoding the above-described expression target of the present invention; (Ii) a promoter operably linked to the nucleotide sequence of (iii) and acting on an animal cell to form an RNA molecule, and more preferably (iii) an amino acid of the second sequence of the above-listed sequence of the present invention Nucleotide sequence encoding a sequence or a nucleotide sequence thereof; (Ii) a promoter operably linked to the nucleotide sequence of (iii) and acting on animal cells to form RNA molecules; And (iii) a recombinant expression vector comprising a 3'-non-detoxification site that acts in an animal cell to cause 3'-end polyadenylation of the RNA molecule.

Preferably, the above-described expression material includes, but is not limited to, mutated SPT15 protein, more preferably mutated SPT15 protein consisting of SEQ ID NO: 2 sequence.

As used herein, the term "promoter" refers to a DNA sequence that regulates the expression of a coding sequence or functional RNA. The subject substance-coding nucleotide sequence in the combined combination expression vector of the present invention is operatively linked to the promoter. As used herein, the term “operatively linked” refers to a functional binding between a nucleic acid expression control sequence (eg, a promoter sequence, a signal sequence, or an array of transcriptional regulator binding sites) and another nucleic acid sequence. Thus, the regulatory sequence will control the transcription and / or translation of the other nucleic acid sequence.

When the vector of the present invention is a prokaryotic cell as a host, powerful promoters capable of promoting transcription (e.g., tac promoter, lac promoter, lac UV5 promoter, lpp promoter, p _L ^λ promoter, p _R ^λ promoter, rac 5 Promoters, amp promoters, recA promoters, SP6 promoters, trp promoters, T7 promoters, etc.), ribosomal binding sites for initiation of translation and transcription / detox termination sequences. Even more preferably, the host cell used in the present invention is E. coli , most preferably E. coli DH5α. In addition, when E. coli is used as the host cell, the promoter and operator site of the E. coli tryptophan biosynthetic pathway (Yanofsky, C., J. Bacteriol. , 158: 1018-1024 (1984)) and the leftward promoter of phage λ (p _L ^λ promoter, Herskowitz, I. and Hagen, D., Ann. Rev. Genet. , 14: 399-445 (1980)) can be used as regulatory sites. On the other hand, vectors that can be used in the present invention are plasmids (eg, pRS316, pSC101, ColE1, pBR322, pUC8 / 9, pHC79, pUC19, pET, etc.), phages (eg, λgt4λB, λ-Charon) which are often used in the art. , λΔz1, M13, etc.) or viruses (eg, SV40, etc.).

In addition, when the recombinant vector of the present invention is applied to eukaryotic cells, preferably yeast cells, the promoters that can be used are those capable of regulating the transcription of the expression target material of the present invention, promoters derived from yeast cells, mammals Promoters derived from animal viruses and promoters derived from genomes of mammalian cells, including, for example, the yeast ( G. cerevisiae ) GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) promoter, the yeast ( S. cerevisiae ) GAL1 to GAL10 promoters, yeast ( Pichia pastoris ) AOX1 or AOX2 promoter, CMV (cytomegalo virus) promoter, adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, tk promoter of HSV, RSV promoter, EF1 alpha promoter, metallothionine promoter, beta Actin promoter, promoter of human IL-2 gene, promoter of human IFN gene, human IL-4 gene Promoter, one containing the promoter and the promoter of the human GM-CSF gene of the human lymphotoxin gene, and the like. Most preferably, it is a yeast GAPDH promoter.

Preferably, the expression constructs used in the present invention include polyanenylation sequences (e.g., bovine growth hormone terminator and SV40-derived polyadenylation sequences).

The method of carrying the vector of the present invention into a host cell may use various methods known in the art, for example, when the host cell is a prokaryotic cell, the CaCl ₂ method (Cohen, SN et al., Proc. Natl. Acac. Sci. USA , 9: 2110-2114 (1973)), one method (Cohen, SN et al., Proc. Natl. Acac. Sci. USA , 9: 2110-2114 (1973); and Hanahan, D , J. Mol. Biol. , 166: 557-580 (1983)) and electroporation methods (Dower, WJ et al., Nucleic. Acids Res. , 16: 6127-6145 (1988)) and the like. For eukaryotic cells, electroporation, lipofection, microinjection, particle bombardment, yeast globular / cell fusion used in YAC, Agrobacterium- used in plant cells Mediated transformation can be used.

According to a preferred embodiment of the present invention, the expression-encoding nucleotide sequence used in the present invention has a structure of “promoter-expression encoding nucleotide sequence-poly adenylation sequence”.

The vector system of the present invention may be constructed through various methods known in the art, and specific methods thereof are disclosed in Sambrook et al., Molecular Cloning, A Laboratory Manual , Cold Spring Harbor Laboratory Press (2001), This document is incorporated herein by reference.

The production of transformed yeast cells using the recombinant expression vector of the present invention can be carried out by gene transfer methods commonly known in the art. For example, electroporation, lithium acetate / DMSO method (Hill, J., et al., (1991), DMSO-enhanced whole cell yeast transformation. Nucleic Acids Res. 19, 5791.), liposomes- Mediated transfer methods (Wong, et al., 1980), retrovirus-mediated transfer methods (Chen, HY, et al., (1990), J. Reprod. Fert. 41: 173-182; Kopchick, JJ et al., ( 1991) Methods for the introduction of recombinant DNA into chicken embryos.In Transgenic Animals, ed.NL First & FP Haseltine, pp. 275-293, Boston; Butterworth-Heinemann; Lee, M.-R. and Shuman, R. ( 1990) Proc. 4th World Congr. Genet. Appl. Livestock Prod. 16, 107-110).

On the other hand, in order to use the expression target protein of the present invention as an active ingredient to introduce the gene should be able to effectively penetrate into the cell. For example, when using the above-described mutated SPT15 protein as an active ingredient, it is preferable to attach a protein transduction domain (PTD) to the mutated SPT15 protein. That is, in order to introduce a mutated SPT15 protein of the present invention as an active ingredient into cells, a protein transduction domain (PTD) is fused with the protein to make a fusion protein. The protein transport domain (PTD) mainly contains basic amino acid residues such as lysine / arginine, and serves to permeate the proteins fused therewith into the cell through the cell membrane. The protein transport domain (PTD) is preferably in the HIV-1 Tat protein, the homeodomain of the drosophila antennafedia, the HSV VP22 transcriptional regulator protein, the MTS peptide derived from vFGF, the penetratin, the transpotane or the Pep-1 peptide. Including but not limited to sequences derived.

The features and advantages of the present invention are summarized as follows:

(a) The present invention relates to an osmo-resistant transgenic yeast strain comprising a mutated SPT15 gene and its use.

(b) Yeast strains of the present invention are yeast strains capable of growing under high concentrations of glucose, preferably 30-40% glucose or sucrose.

(c) Yeast strains of the present invention are yeast strains that can grow in high concentration ethanol, preferably 12.5-15% ethanol.

(d) Inventing strains that are resistant to high glucose and ethanol will be useful for the production of more efficient ethanol and also have practical utility as a super strain of high efficiency ethanol producing ability that is resistant to various stresses generated during bioethanol production process. will be.

1 is a spot assay result showing increased ethanol resistance of five ETS1-5. Cells were grown up to OD ₆₀₀ value 1 in YSCD-Ura or YPD liquid medium and then serially diluted 10-fold. Aliquots (5 μl) of cultured cells were spotted in YSCD-Ura or YPD plate medium containing appropriate concentrations of ethanol and plates were incubated at 30 ° C. for 4-6 days. Control strains were prepared by transducing parental plasmids into L3262 (C-L3262) and BY4741 (C-BY4741). FIG. 1A shows the results of a spot assay of ETS1-5 on a YSCD-Ura plate. 1B shows that the plasmid recovered from ETS1-5 was transformed back into L3262 and BY4741 to prepare rL-ETS1-5 and rBY-ETS1-5, respectively, and spot assayed on YSCD-Ura plates. 1C shows the results of spot assays of ETS2 and ETS3 on YPD plates. 1D shows the integration of parent plasmids and plasmids recovered from ETS2 and ETS3 into the genome of L3262 to prepare iL3262, iETS2 and iETS3, respectively, and spot assays on YSCD-Ura (top panel) and YPD plate (bottom panel). Was carried out.
2 shows the ethanol sensitivity of ETS2 and ETS3. After ethanol shock at the indicated time, C-L3262, ETS2 and ETS3 were grown for 2 days in YSCD-Ura plates in the presence of 12.5% (A) and 15% (B) ethanol. Relative survival was expressed as% control after counting the number of colonies. Symbol: C-L3262,; ETS2, ■; And ETS3, ▲. The experiment was conducted three times.
3 shows the results of observing the growth rate of ETS3 under different glucose concentrations. Osmotic-resistant strain, ETS3 (■) and control (Sc L3262, □) cells at 120 rpm in YPD medium containing varying concentrations of glucose (A) and sucrose (B; 20%, 30% and 40%, respectively) It was incubated at 30 ℃ shaking. After taking samples every designated time, cell density was measured to determine cell growth rate. The experiment was conducted three times.
4 is a result of observing the fermentation capacity of the osmo-resistant strain ETS3. Cell growth and ethanol production of ETS3 (•) and control (Sc L3262; ○) in YPD medium containing high concentration of glucose (50%) were shown, respectively. The cells were incubated at 30 ° C with shaking at 120 rpm. After each sample was taken at a designated time, cell growth (A, C) and ethanol production capacity (B, D) were measured, respectively. Representative results are shown.

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 embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

Materials and Experiments

Yeast Strains and Growth Conditions

S. cerevisiae L3262 ( MAT-α , ura3-52 leu2-3 ll2 his4-34 ; Biotechnology Research Institute, Daejeon) and BY4741 ( MATα his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 ) as expression recipients Was used. A non-essential haploid S. cerevisiae deletion library was kindly provided by Dr. Hur Won-ki (Seoul National University, Seoul, Korea) and used to identify the identified genes. Unless otherwise noted, yeast cells additionally added YPD medium (1% Bacteria yeast extract, 2% Bactobacillus peptone and 2w / v% glucose, and 15% Bactobacillus-agar for solid plates) for non-selective propagation. Incubated at 30 ° C. in Difco, MI or yeast synthetic complete medium for selective propagation (YSCD medium; 0.67% yeast nitrogen base without amino acids, amino acid supplement mixture and 2% dextrose, and 1.5 for solid plates And additionally containing% bacto-agar; MP, OH). For plasmid construction, the pRS316 vector (Director of the Institute; CEN -based vector, S. cerevisiae GAPDH promoter, URA3 selection marker) was used as the expression vector, E. Coli DH5α (Stratagene, Calif.) was used as a host and incubated at 37 ° C in Luria-Bertani medium (LB): Difco, MI supplemented with 100 mg / l Ampicillin (Sigma-Aldrich, Mo.).

Molecular biology method

Plasmid preparation, cloning and sequencing were performed as previously described (Sambrook, 2001). E. coli DH5α (Stratagene, USA) was used as a host for plasmid preparation.

STP15 Mutation Library Construction

Using the full open reading frame (ORF) of wild type SPT15 (SPT15wt) as genome DNA as a template, a sense primer (5'-gtag ggatcc tgagatggccgatgaggaacgtt-3 ', underlined sequence is BamH I position) and antisense primer (5'-gtag) gaTtc tcacatttttctaaattcacttag-3 ', the underlined sequence was PCR-amplified with EcoR I position) and cloned into pGEM-T easy vector to prepare pT-SPT15. The SPT15 mutation library was prepared using GeneTorph II random mutagenesis kit (Stratagene, La Jolla, CA, USA) as a template and primers described above. PCR products were digested with BamH I and EcoR I and cloned into pRS316-derived plasmid, pRS316-GCYH2gR, and cloned genes were glyceraldehyde-3-phosphate dehydrogenase promoter ( TDH3 _P ) and galactose-1-. It is located under the control of phosphate terminator (GAL7 _T ). The resulting plasmids were transformed into E. coli DH5α and incubated at 30 ° C. to prepare a primary library for SPT15 mutants with a total colony number of 4 × 10 ⁶ . Sequencing of 20 randomly selected colonies resulted in 70% molecular-based mutagenesis production. Mutations were found in at least one, mostly 3-5 positions, in the 14 colonies, with the remainder identical to the wild type. After amplification and large scale preparation, library plasmids (500 μg) were transformed into S326 cerevisiae with L3262 and incubated at 25 ° C. in solid YSCD-Ura. The total number of yeast colonies was about 5 × 10 ⁶ , indicating a transformation efficiency of about 4 × 10 ⁶ colony forming units (CFU) per 1 μg of DNA. All colonies were obtained by scraping a plate surface coated with 15 ml YSCD-Ura to prepare a yeast library for SPT15 mutants. After doubling the cell number at 25 ° C., aliquots of the cell suspension were stored at −80 ° C. in the presence of 20% glycerol until use.

Yeast transformation

All plasmids for yeast transformation were prepared manually without RNA cleavage. DNA concentration was determined by comparing the band intensities of known concentrations of control DNA. Mixtures of DNAs and RNAs were used for yeast transformation as previously described (Hirasawa, T., et al. , 2007).

Spot Assay

Aliquots (5 μl) of cells incubated with an OD ₆₀₀ value of 1 were serially diluted 10-fold and spotted on solid synthetic media or enriched media containing appropriate concentrations of ethanol. Plates were incubated at 30 ° C. for 4-6 days.

Ethanol Sensitization Assay

Cells cultured with an OD ₆₀₀ value of 1 were obtained and equally aliquoted in fresh YSCD-Ura medium containing 12.5% (v / v) and 15% (v / v) ethanol and incubated at 30 ° C. for 4-6 hours. . Aliquots were diluted at appropriate times and plated on solid YPD plates. Cell viability was measured over time and expressed in relative CFU numbers.

Genome integration (Genomic integration)

The mutated SPT15 gene was cloned into the integration vector pRS406 and linearized with the unique Apa I position in URA3 and transformed into S326 cerevisiae with L3262. In addition, a control strain, iL3262, was prepared by similarly treating the clone-free plasmid (inset-free plasmid). Genome integration was confirmed by PCR.

Fermentation

Exponentially growing cells were obtained and transferred to 100 ml of YPD30E6 [YP supplemented with 45% glucose and 6% (v / v) ethanol]. Starting cell density was adjusted to an OD ₆₀₀ value of 0.3. The cells were incubated at 30 ° C with shaking at 120 rpm. After taking samples every 12 hours, cell growth, glucose concentration and ethanol concentration were measured, respectively.

Experiment result

Identification of Ethanol-Resistant Strains

In order to identify genes that confer ethanol-resistance through high-speed screening, it is generally useful to use strains with a low concentration of ethanol resistant background. Since the laboratory strains in the various S. cerevisiae tested were most ethanol sensitive (not showing results), L3262 was selected and used to build the yeast SPT15 mutant library. For the screening of ethanol resistant strains, aliquots of 5 × 10 ⁶ CFU yeast library stocks were plated on solid YSCD-Ura medium supplemented with 12.5% or 15% ethanol. Plates were sealed and incubated at 30 ° C. to inhibit ethanol evaporation. After 10 days, 9 and 6 colonies respectively appeared in the presence of 12.5% or 15% ethanol. The ethanol resistance of 15 colonies was examined by spot assay in solid YSCD-Ura medium containing up to 15% ethanol. As a result, five ethanol resistant strains (ETS; ETS1-5) were obtained. All five strains were resistant to 15% ethanol under synthetic medium, whereas the control group was not resistant to ethanol concentrations above 10% (FIG. 1A).

To confirm whether increased ethanol resistance is conferred by mutated SPT15, plasmids were converted to ETS15 (pSPT15-M1, -M2, -M3, -M4 and -M5, respectively, for the mutated alleles of SPT15). Recovered. The plasmids described above were individually reintroduced into L3262 and By4741 to produce rL-ETS1-5 and rBY-ETS1-5, respectively. To construct the control strain, pRS316-GCYH2gR comprising SPT15wt was reintroduced into L3262 and By4741 to produce C-L3262 and C-By4741, respectively. For the spot assays carried out on the synthetic medium, rL-ETS1-5 showed ethanol resistance even in the same manner as ETS1-5 (FIG. 1B, top panel). In contrast, rBY-ETS1-5 showed resistance in high ethanol such as 17.5% ethanol (FIG. 1B, bottom panel). This is not surprising because BY4741 is basically a strain that showed higher ethanol resistance than L3262 (no results shown). Thus, increased ethanol resistance of ETS1-5 could be regarded as the effect of mutated SPT15.

Next, each plasmid was sequenced to identify mutations. Table 1 lists the mutated amino acids in each SPT15 allele: for SPT15-M1, K201N, G216S and N225Stop; L76V and L175S for SPT15-M2; For SPT15-M3, S42N, C78R, S163P and I212N; For SPT15-M4, F10S and M197K; For SPT15-M5, K15T, W26C and G192D.

Point mutations occurring in the SPT15 allele. Strain SPT15 allele Amino acid substitutions Of located point mutations
Structural domains ^a ETS1
ETS2
ETS3

ETS4
ETS5 SPT15-M1 ^b
SPT15-M2
SPT15-M3

SPT15-M4
SPT15-M5 K201N, G216S, N225Stop
L76V, L175S
S42N, C78R,
S163P, I212N
F10S, M197K
K15T, W26C, G192D S3'-S4 'Loop, S5', H2 '
S1-H1 loop, H1 '
N-terminus, S1-H1 loop,
S1 ', S5'
N-terminal, S3'-S4 'loop
N-terminal, N-terminal, S3 '

^a Chasman et al. Structural domain names as described in (1993): H, α-helix; S, β-sheet

^b 16 amino acids deleted at the C-terminus due to N225Sto.

In particular, a silent mutation in SPT15-M1 produced a truncated version with 16 residues deleted at the C-terminus. As can be seen in Table 1, point mutations spread throughout the SPT15 ORF were not located in the structural domain associated with increased ethanol resistance. Only SPT15-M2 contained a mutation (L175S) in the domain that interacts with Spt3p, which is earlier included in gene transcriptional regulation. The above data is consistent with the suggestion that mutations in various subregions of Spt15p confer ethanol resistance, which is believed to be achieved through interaction with other components of the transcriptional mechanism in addition to Spt3p.

According to the data of FIG. 1B, ETS1 showed less resistance than ETS4 and ETS5, and ETS2 and ETS3 showed slightly more (or at least the same) resistance than ETS4 and ETS5. Therefore, ETS2 and ETS3 were selected for future experiments. S. cerevisiae laboratory strains used for the expression of specific genes always have mutations that are independent of each other in many genes encoding enzymes involved in amino acid biosynthesis, so they can supplement specific amino acids for growth when grown in a limited medium. There is a need. It was controversial that increased ethanol resistance could result from low leucine supplementation rather than mutated SPT15 (Baerends, RJ, et al. , 2009). More specifically, ethanol resistance was destroyed when cells were cultured in YPD complex rich media that were not suitable for industrial applications. In addition, because leucine and histidine supplementation are required for the growth of ETS2 and ETS3, the increased ethanol resistance of these strains may not be due to SPT15 mutations. Thus, ethanol resistance of ETS2 and ETS3 was tested with a spot assay on YPD. As can be seen in FIG. 1C, ETS2 was sensitive to 15% ethanol against the data of FIG. 1A, whereas ETS3 showed ethanol resistance similar to the results seen in synthetic media. However, C-L3262 Strainin, which was very sensitive to 15% ethanol in the synthetic medium, appeared to have some ethanol resistance on YPD, which means that the base level of ethanol resistance in the rich medium is higher than the synthetic medium. The overall data were in agreement with the conclusion that ETS3 is more resistant to ethanol on YPD than ETS2.

Cell-cell heterogeneity in expression is one of the problems that may be encountered when information obtained from episomal overexpression in laboratory strains extends to industrial applications. Heterogeneity is caused by the inability to control the copy number in spite of the constant presence of selective pressures that are not arbitrarily selectable for yeast culture in industrial applications. Thus, stable expression and maintenance of genes in the absence of selection pressure (ie integration into the chromosome) is highly desirable. In the present invention, we constructed strains in which SPT15-M2 and -M3 were integrated into the genome of L3262: the corresponding constructs were named iETS2 and iETS3, respectively. Control strain iL3262 was prepared from a plasmid containing SPT15wt. 1D shows the spot assay results of the three strains in YSCD (top panel) and YPD (bottom panel). The degree of ethanol resistance of iETS2 and iETS3 in YSCD was similar as in YPD (FIG. 1C). That is, in YPD iETS2 and iETS3 had higher ethanol resistance than in YSCD, the difference between the two was not observed even at 15% ethanol concentration.

To reconfirm spot assay results, the sensitivity to 12.5% and 15% was examined. Survival of both strains was significantly increased compared to the control in both 12.5% and 15% ethanol (FIG. 2). The time to show 50% survival (T ₅₀ ) in 12.5% ethanol was 4.5 hours for ETS2 and ETS3 and 3.5 hours for the control group. In 15% ethanol, a greater difference was observed in T ₅₀ with 100 minutes for ETS2 and ETS3 and 40 minutes for control. The data described above along with the spot assay data confirmed that ETS2 and ETS3 have increased ethanol resistance by SPT15 mutations.

Five strains with increased ethanol resistance were obtained through SPT15 mutant library screening. Plasmids were recovered from these strains and transformed back into the strains used for library construction (L3262) and other strains (BY4741). In addition, all newly constructed strains also showed increased ethanol resistance in limited media. Since the increased ethanol resistance of ETS2 and ETS3 was also maintained in complex rich media, increased ethanol resistance, as previously discussed (Baerends, RJ, et al. , 2009), showed that leucine uptake by certain SPT15 mutant alleles and And / or the possibility of being imparted by activation of use. Increased ethanol resistance of ETS2 and ETS3 was confirmed by ethanol impact with ethanol sensitivity. Increased ethanol resistance was additionally observed in two integrated strains, with these SPT15 mutant alleles derived from ETS2 and ETS3 integrated on the L3262 genome.

Effect of Mutated SPT15s on Ethanol Production and Growth Rate under High Glucose

In addition to understanding the mechanisms for ethanol resistance, the purpose of producing ethanol resistant strains is to improve ethanol productivity and / or final yield. Compared to control strains, it is speculated that strains with increased ethanol resistance could better cope with the toxic effects of ethanol (Ding, J., et al. , 2009). After phenotypic characterization, the effect of increased ethanol resistance is generally determined by measuring the highest ethanol titers from batch cultures in complex rich media containing up to 30% glucose (Hong, ME, et al. , 2009; Hou, L., 2009; Hou, L., et al. , 2009; Teixeira, MC., Et al. , 2009). However, the yields in the above studies did not significantly improve and only increased by 10% compared to the control strain. This may be because most of the laboratory parent strains used are able to produce essentially maximum levels of ethanol, making it difficult to observe the effect of increased ethanol resistance. Thus, it would be possible to observe such an effect only at ethanol concentrations that rarely exceed the basic ethanol-producing capacity of the parent strains.

The ETS3 strain of the present invention is resistant to high osmotic pressure caused by high glucose or sucrose. As can be seen in Figure 3, the ETS3 strain of the present invention showed higher growth rates compared to the control at various concentrations of glucose and sucrose (eg, 20%, 30% or 40%). In particular, ETS3 showed better growth rate at 40% glucose, a higher glucose concentration than the control (Sc L3262). In addition, looking at the results of Figure 4, a fermentation experiment in 30% glucose-YPD, it is clear that the osmo-resistant strain ETS3 of the present invention is closely related to ethanol fermentation. That is, in culture under 50% glucose, the ETS3 strain of the present invention showed an increased ethanol production of 70.3% based on 72 hours compared to the control (Sc 3262) (63 g / L in ETS3; and 37 g in the control). / L). Thus, ETS3 showed about 70% higher fermentation capacity than the control under the experimental conditions used in the present invention.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

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<110> Ewha Womens University <120> Osmosis-Resistant Yeast Strains and Uses Thereof <130> PN100629 <160> 2 <170> Kopatentin 1.71 <210> 1 <211> 720 <212> DNA <213> Artificial Sequence <220> <223> SPT15_M3 <400> 1 atggccgatg aggaacgttt aaaggagttt aaagaggcaa acaagatagt gtttgatcca 60 aataccagac aagtatggga aaaccagaat cgagatggta caaaaccagc aactactttc 120 cagaatgaag aggacataaa aagagctgcc ccagaatctg aaaaagacac ctccgccaca 180 tcaggtattg ttccaacact acaaaacatt gtggcaactg tgactttggg gcgcaggtta 240 gatctgaaaa cagttgcgct acatgcccgt aatgcagaat ataaccccaa gcgttttgct 300 gctgtcatca tgcgtattag agagccaaaa actacagctt taatttttgc ctcagggaaa 360 atggttgtta ccggtgcaaa aagtgaggat gactcaaagc tggccagtag aaaatatgca 420 agaattatcc aaaaaatcgg gtttgctgct aaattcacag acttcaaaat acaaaatatt 480 gtcggtccgt gtgacgttaa attccctata cgtctagaag ggttagcatt cagtcatggt 540 actttctcct cctatgagcc agaattgttt cctggtttga tctatagaat ggtgaagccg 600 aaaattgtgt tgttaatttt tgtttcagga aagaatgttc ttactggtgc aaagcaaagg 660 gaagaaattt accaagcttt tgaagctata taccctgtgc taagtgaatt tagaaaaatg 720                                                                          720 <210> 2 <211> 240 <212> PRT <213> Artificial Sequence <220> <223> SPT15_M3 <400> 2 Met Ala Asp Glu Glu Arg Leu Lys Glu Phe Lys Glu Ala Asn Lys Ile   1 5 10 15 Val Phe Asp Pro Asn Thr Arg Gln Val Trp Glu Asn Gln Asn Arg Asp              20 25 30 Gly Thr Lys Pro Ala Thr Thr Phe Gln Asn Glu Glu Asp Ile Lys Arg          35 40 45 Ala Ala Pro Glu Ser Glu Lys Asp Thr Ser Ala Thr Ser Gly Ile Val      50 55 60 Pro Thr Leu Gln Asn Ile Val Ala Thr Val Thr Leu Gly Arg Arg Leu  65 70 75 80 Asp Leu Lys Thr Val Ala Leu His Ala Arg Asn Ala Glu Tyr Asn Pro                  85 90 95 Lys Arg Phe Ala Ala Val Ile Met Arg Ile Arg Glu Pro Lys Thr Thr             100 105 110 Ala Leu Ile Phe Ala Ser Gly Lys Met Val Val Thr Gly Ala Lys Ser         115 120 125 Glu Asp Asp Ser Lys Leu Ala Ser Arg Lys Tyr Ala Arg Ile Ile Gln     130 135 140 Lys Ile Gly Phe Ala Ala Lys Phe Thr Asp Phe Lys Ile Gln Asn Ile 145 150 155 160 Val Gly Pro Cys Asp Val Lys Phe Pro Ile Arg Leu Glu Gly Leu Ala                 165 170 175 Phe Ser His Gly Thr Phe Ser Ser Tyr Glu Pro Glu Leu Phe Pro Gly             180 185 190 Leu Ile Tyr Arg Met Val Lys Pro Lys Ile Val Leu Leu Ile Phe Val         195 200 205 Ser Gly Lys Asn Val Leu Thr Gly Ala Lys Gln Arg Glu Glu Ile Tyr     210 215 220 Gln Ala Phe Glu Ala Ile Tyr Pro Val Leu Ser Glu Phe Arg Lys Met 225 230 235 240

Claims (14)

An osmosis-resistant transformed yeast strain comprising a mutated SPT15 gene comprising a mutated amino acid sequence of the amino acid sequence at positions S42, C78, S163 and I212 of the wild type SPT15 gene.
delete delete The method of claim 1, wherein the mutated SPT15 gene is a mutated SPT15 gene comprising a mutated sequence of the amino acid sequence of the S42, C78, S163 and I212 position of the wild-type SPT15 gene to S42N, C78R, S163P and I212N Yeast strain.
The yeast strain according to claim 1, wherein the mutated SPT15 gene is introduced into yeast cells as a plasmid.
The yeast strain of claim 1, wherein the mutated SPT15 gene is introduced into genomic DNA of the yeast cell.
The yeast strain of claim 1, wherein the yeast strain is capable of growing under 30-40% glucose or sucrose culture conditions.
The yeast strain according to claim 1, wherein the yeast strain further has resistance to ethanol.
9. The yeast strain according to claim 8, wherein the yeast strain can grow under 5-15% ethanol culture conditions.
According to claim 1, wherein the yeast strain Saccharomyces spp ( Saccharomyces spp.), Schizosaccharomyces spp., Pichia spp., Paffia spp. , Cluj Vero MRS species (Kluyveromyces spp.), Candida species (Candida spp.), Tallahassee in MRS species (Talaromyces spp.), Brescia Gaetano MRS species (Brettanomyces spp.), Parkinson solren species (Pachysolen spp.), Deva Rio Yeast strain, characterized in that it is a debaryomyces spp. Or industrial polyploid yeast strain.
11. The yeast strain according to claim 10, wherein said yeast strain is Saccharomyces species.
The ethanol production method comprising the step of culturing the yeast strain into which the mutated SPT15 gene of claim 1 is inserted in a culture medium comprising at least one substrate capable of being metabolized to ethanol.
13. The method of claim 12, wherein the substrate capable of metabolizing ethanol comprises C6 sugars.
The method of claim 13, wherein the C6 sugar is glucose.

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