KR20130078049A - Furfural-tolerant yeast strains - Google Patents

Abstract

PURPOSE: A transformed yeast strain containing mutant ScSPT15 gene with furfural-resistance, which is able to grow in high concentration furfural, preferably 10-30 mM of furfural, is provided to industrially produce necessary products (biomass) with resistance to various stresses. CONSTITUTION: A furfural-resistant transformed yeast strain contains mutant ScSPT15 gene. The yeast strain is able to grow under a culture condition of 10-50 mM of furfural. The yeast strain is Saccharomyces spp., Schizosaccharomyces spp., Pichia spp., Paffia spp., Kluyveromyces spp., Candida spp., Talaromyces spp., Brettanomyces spp., Pachysolen spp., or Debaryomyces spp.

Description

Furfural-Tolerant Yeast Strains

The present invention is directed to perfural-resistant yeast strains.

Many efforts have been made until recently to produce bioethanol from low-cost renewable biomass, including lignocellulosic materials and agricultural residues. However, toxic compounds generated during biomass pretreatment by dilute acid hydrolysis inhibit microbial growth and subsequent fermentation (Klinke et al., 2004). Two major inhibitors in the compound are formed in perfural and 5-hydroxymethyl furfural (HMF) by dehydration of pentose and hexose released from hemicellulose and cellulose, respectively. The concentration of the potent inhibitors in hydrolysates varies with the type and hydrolysis of the biomass (Larsson et al., 1999; Palmqvist et al., 2000). A concentration of 10 mM perfural is sufficient to have inhibitory activity in Saccharomyces cerevisiae (Liu et al., 2004 and references therein). Perfural is more potent than HMF at the same concentration in terms of its inhibitory effect on cell growth, and the combination treatment of perfural and HMF inhibits cell growth more synergistically (Liu et al., 2004). Although additional processes are needed to decipher the inhibitors for the effective fermentation conversion of biomass to ethanol, the approaches are very problematic due to increased production costs and complexity of the process (Mussatto et al. , 2004). Thus, the acquisition of inhibitor-resistant microorganisms is another very interesting alternative for low-cost production of bioethanol from wood fiber based monthly materials.

Perfural affects several physiological properties such as cell survival, growth rate, cell budding, ethanol yield, biomass yield and enzyme activity (Palmqvist et al., 2000; Taherzadeh et al., 1999) . Furthermore, perfural induces the accumulation of reactive oxygen species (ROS) in S. cerevisiae and damages several cellular components such as mitochondria and vacuole membranes, actin cytoskeleton and nuclear chromatin. (Allen at al., 2010). Thus, ideal strains with improved resistance to perfural are strains that can reduce the levels of perfural and ROS.

Yeast cells can be detoxified by converting perfural to furfuryl alcohol under sublethal conditions. Using various strategies, various perfural-resistant strains have been previously developed, and some strains can function at concentrations above 30 mM (US Pat. No. 2009004726; Liu et al., 2005; Gorsich et al. , 2006; Heer and Sauer, 2008; Heer et al., 2009; Park et al., 2011; Lu et al. 2011). Another approach to developing ethanol resistant strains is gTME (global transcriptional machinery engineering). This approach has been used to produce strains with increased ethanol resistance by inducing mutations in the TATA-binding protein encoded by SPT15, which previously allowed to grow at lethal ethanol concentrations (Alper et al., 2006). However, other authors have reported that the increased ethanol resistance is not reproduced in rich media that is not an option in industrial applications (Baerends et al., 2009). The inventors have demonstrated the usefulness of gTME as a means for producing strains resistant to ethanol and proposed an application method for obtaining strains with increased resistance to other stresses (Yang et al., 2011). In this study, we screened the SPT15 mutant library in the presence of 20 mM perfural to obtain five perfural resistant strains.

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.

The inventors have sought to develop industrially useful yeast strains with furfural-resistance. As a result, the inventors constructed a mutated SPT15 gene using PCR-mediated random mutagenesis and transformed it into yeast to isolate perfural-resistant transformed yeast strains, which were highly concentrated perfural. The present invention was completed by confirming that it can grow at (eg, 10 mM, 15 mM, 20 mM, 30 mM).

It is therefore an object of the present invention to provide perfural-resistant yeast strains.

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 a perfural-resistant transformed strain comprising a mutated ScSPT15 gene.

We have sought to develop an industrially useful yeast strain that possesses perfural-resistance. As a result, the inventors constructed the mutated SPT15 gene using PCR-mediated random mutagenesis and transformed it into yeast to isolate perfural-resistant transformed yeast strains, which were highly concentrated perfural (eg, 10). mM, 15 mM, 20 mM, 30 mM).

Perfural is an organic compound derived from various agricultural by-products such as corn, oats, bran and sawdust and is an aromatic aldehyde having a ring structure represented by the following formula (OC ₄ H ₃ CHO):

(I)

(I)

Perfural is a colorless oily liquid with an almond flavor, but turns yellow quickly when exposed to air. Usually, furfural is used as a solvent in the petrochemical stagnation process of extracting dienes from other hydrocarbons. Perfural and its alcohol derivatives are used together with phenol, acetone or urea to produce solid resins. These resins are used in fiberglass, aircraft components, and automatic brakes. Perfural is an inhibitor that inhibits the growth of yeast by inhibiting enzymes such as ADH, PDH, ALDH, etc. of yeast and inhibits glycolysis, and develops strains resistant to ethanol production using yeast. It is very important to.

The present invention provides transformed yeast strains with increased perfural-resistance in yeast transformed with mutated ScSPT15.

The present invention comprises the steps of (a) introducing a ScSPT15 gene mutated to the yeast chromosome; And (b) culturing the mutated yeast in a medium containing high concentrations of furfural.

According to the present invention, a strain capable of growing is isolated / identified by culturing the yeast strain transformed with the mutated ScSPT15 in a high concentration of furfural. In the present invention, perpural-resistant strains were selected by performing a growth assay of yeast strain at various concentrations of perfural (10 mM, 15 mM, 20 mM, 30 mM).

According to a preferred embodiment of the present invention, the yeast strain transformed with the mutated ScSPT15 gene described above is highly concentrated perfural, more preferably 10-100 mM perfural, even more preferably 10-50 mM perfural, and Most preferably at 10-30 mM perfural.

According to a preferred embodiment of the present invention, the above-described yeast strain is selected from the group consisting of Saccharomyces species spp.), Schizosaccharomyces sp. spp.), Pichia sp. spp.), papia spp.), Kluyveromyces species spp.), Candida sp. spp.), Talaromyces sp. spp.), Brescia Gaetano MRS species (Brettanomyces spp.), Parkinson solren species (Pachysolen spp.) or Deva Rio MRS species (and Debaryomyces spp.) yeast strain, and more preferably from my process paper as Saccharomyces, and most preferably Lt; RTI ID = 0.0 > Schizosaccharomyces < / RTI > cerevisiae .

According to a preferred embodiment of the present invention, the above-mentioned mutated ScSPT15 gene can be introduced into a yeast cell as a plasmid. Further, according to a preferred embodiment of the present invention, the above-mentioned mutated ScSPT15 gene can be introduced into the genomic DNA of the yeast cell.

In addition, the present invention provides a transformed cell by transfection with a recombinant vector comprising the above-mentioned mutated ScSPT15 gene or a transcript thereof and gene introduction. In addition, the present invention provides a recombinant vector comprising the above-mentioned mutated ScSPT15 gene or a transformant transformed with the aforementioned mutated ScSPT15 protein

The vector system of the present invention can be constructed through various methods known in the art, and specific methods for this can be found in Sambrook et < RTI ID = 0.0 > al ., Molecular Cloning , A Laboratory Manual , Cold Spring Harbor Laboratory Press (2001), which is incorporated herein by reference.

Production of transformed yeast cells using the recombinant expression vector of the present invention can be carried out by a gene transfer method commonly known in the art. For example, electroporation, lithium acetate / DMSO method (Hill, et al ., (1991), DMSO-enhanced whole cell yeast transformation. Nucleic Acids Res. 19, 5791), liposome-mediated transfer methods (Wong, et al ., 1980), retroviral-mediated transfer methods (Chen, et al ., (1990), J. Reprod. Fert. 41: 173-182; Kopchick, et al ., (1991) Methods for 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), and most preferably by using a lithium acetate / DMSO method.

On the other hand, in order to utilize the expressed protein substance of the present invention as an effective ingredient for gene transfer, the protein of the expressed substance must be able to effectively penetrate into the cell. For example, when the above-mentioned mutated ScSPT15 protein is used as an active ingredient, it is preferable to attach the protein transduction domain (PTD) to the mutated ScSPT15 protein. That is, in order to introduce the mutated ScSPT15 protein of the present invention as an active ingredient into cells (permeable peptide transduction), a protein transduction domain (PTD) is fused with the protein to form 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 selected from the group consisting of the HIV-1 Tat protein, the homeodomain of Drosophila antennae, the HSV VP22 transcriptional regulatory protein, the MTS peptide derived from vFGF, the pennantatin, the transposon or the Pep- Derived sequences, but are not limited thereto.

According to a preferred embodiment of the invention, the above-mentioned transformation is Saccharomyces species spp.), Schizosaccharomyces sp. spp.), Pichia sp. spp.), papia spp.), Kluyveromyces species spp.), Candida sp. spp.), Talaromyces sp. spp.), Brescia Gaetano in MRS species (Brettanomyces spp.), Parkinson solren species (Pachysolen spp.) or Deva Rio MRS species (Debaryomyces spp.), and carried out in the yeast strain, more preferably from my process species Saccharomyces or inclusive Yves It is carried out in Mrs. species, most preferably in Saccharomyces cerevisiae .

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

(a) The present invention relates to a perfural-resistant transformed yeast strain comprising a mutated ScSPT15 gene.

(b) Yeast strains of the invention are yeast strains capable of growing at high concentrations of perfural, preferably 10-30 mM perfural.

(c) By inventing strains that are resistant to high concentrations of furfural, they can be very useful for the industrial production of desired products (biomass) that are resistant to the various stresses generated during the bioethanol production process.

1 is a result showing the identification of perfural resistant strain. FTS-1 to FTS-5 strains were serially diluted 10-fold, spotted on YPD plates in the presence or absence of perfural and incubated at 30 ° C.

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 strain and medium

S. cerevisiae BY4741 ( MAT his31 leu20 met150 ura30 ) was used as the transformation recipients. Yeast cells were incubated at 30 in YPD (1% yeast extract, 2% peptone and 2% glucose; and 1.5% agar when using solid plates).

Molecular biology method

Plasmid preparation and sequencing were performed in the same manner as previously described (Sambrook J and Russell D, 2001). Escherichia coli strain DH5 was used as a host for plasmid preparation.

SPT15  Mutant Library Screening

The S. cerevisiae SPT mutation library was constructed as described previously (Yang et al., 2011) and the library was stored at −80 ° C. in the presence of 20% glycerol until use. Aliquots of the stock (2 × 10 ⁶ CFUs) were thawed and used for screening perfural-resistant strains in YPD medium containing 20 mM perfural.

Yeast transformation

All plasmids for yeast transformation were prepared manually without RNA cleavage. The DNA concentration was approximately determined by comparing the band intensity of the control DNA of known concentration. Mixtures of DNAs and RNAs were used for yeast transformation as previously described (Gietz et al., 2002).

Spot Assay

For spot assays, aliquots (5 μl) of cells grown to an optical density of 1.0 (OD ₆₀₀ ) were serially diluted 10-fold and spotted onto solid YPD plates containing perfural up to 30 mM. The plates were incubated at 30 ° C. for an appropriate time.

Experiment result

To screen the yeast SPT15 mutant library constructed in this study to obtain perfural resistant strains, yeasts of 2 × 10 ⁶ CFUs (colony forming units) were plated on solid YPD plates added with 20 mM perfural at 30 ° C. Incubated. After a few days, 25 colonies grew. The final five strains (FTS-1 to FTS-5) were selected and examined via spot assay in solid YPD plates containing up to 30 mM perfural. The five strains showed resistance to 30 mM perfural even after 7 days, whereas the control group (BY4741) was sensitive to low concentrations of 10 mM perfural in 1 day culture (FIG. 1).

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.

references

Allen SA, Clark W, McCaffery JM, Cai Z, Lanctot A, Slininger PJ, Liu ZL, Gorsich SW. Furfural induces reactive oxygen species accumulation and cellular damage in Saccharomyces cerevisiae . Biotechnol Biofuels 3: 2-10.

Alper H, Moxley J, Nevoigt E, Fink GR, Stephanopoulos G. 2006. Engineering yeast transcription machinery for improved ethanol tolerance and production. Science 314: 1565-1568.

Baerends RJ, Qiu JL, Rasmussen S, Nielsen HB, Brandt A. 2009. Impaired uptake and / or utilization of leucine by Saccharomyces cerevisiae is suppressed by the SPT15-300 allele of the TATA-binding protein gene. Appl Environ Microbiol 75: 6055-6061.

Gietz RD, Woods RA. 2002. Transformation of yeast by lithium acetate / single-stranded carrier DNA / polyethylene glycol method. Methods Enzymol 350: 87-96.

Gorsich SW, Dien BS, Nichols NN, Slininger PJ, Liu ZL, Skory CD. 2006. Tolerance to furfural-induced stress is associated with pentose phosphate pathway genes ZWF1, GND1, RPE1, and TKL1 in Saccharomyces cerevisiae . Appl Microbiol Biotechnol 71: 339-349.

Heer D, Sauer U. 2008. Identification of furfural as a key toxin in lignocellulosic hydrolysates and evolution of a tolerant yeast strain. Microb Biotechnol 1: 497-506.

Heer D, Heine D, Sauer U. 2009. Resistance of Saccharomyces cerevisiae to high concentrations of furfural is based on NADPH-dependent reduction by at keast two oxireductases. Appl Environ Microbiol 75: 76317638.

Klinke HB, Thomsen AB, Ahring BK. 2004.Inhibition of ethanol-producing yeast and bacteria by degradation products produced during pre-treatment of biomass. Appl Microbiol Biotechnol 66: 10-26.

Larsson S, Palmqvist E, Hahn-Hgerdal B, Tengborg C, Stenberg K, Zacchi G, Nilvebrant N.O. Nilvebrant. 1999.The generation of inhibitors during dilute acid hydrolysis of softwood. Enzyme Microb Technol 24: 151-159.

Liu ZL, Slininger PJ, Gorsich SW. 2005. Enhanced biotransformation of furfural and hydroxymethylfurfural by newly developed ethanologenic yeast strains. Appl Biochem Biotechnol 121-124: 451-460.

Liu ZL, Slininger PJ, Dien BS, Berhow MA, Kurtzman CP, Gorsich SW. 2004. Adaptive response of yeasts to furfural and 5-hydroxymethylfurfural and new chemical evidence for HMF conversion to 2,5-bis-hydroxymethylfuran. J Ind Microbiol Biotechnol 31: 345352.

Lu Y, Cheng YF, He XP, Guo XN, Zhang BR. 2011.Improvement of robustness and ethanol production of ethanologenic Saccharomyces cerevisiae under co-stress of heat and inhibitors. J Ind Microbiol Biotechnol Epub ahead of print.

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Palmqvist E, Hahn-Hgerdal B. 2000. Fermentation of lignocellulosic hydrolysates II: inhibitors and mechanisms of inhibition. Bioresour Technol 74: 25-33.

Park SE, Koo HM, Park YK, Park SM, Park JC, Lee OK, Park YC, Seo JH. 2011.Expression of aldehyde dehydrogenase 6 reduces inhibitory effect of furan derivatives on cell growth and ethanol production in Saccharomyces cerevisiae . Bioresour Technol 102: 6033-6038.

Sambrook J, Russell D. 2001. Molecular cloning: a laboratory manual, 3rd ed. ed. Cold Spring Harbor Laboratory Press, N.Y.

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Claims (5)

A perfural-resistant transformed strain comprising a mutated ScSPT15 gene.
2. The yeast strain of claim 1, wherein said yeast strain is capable of growing at 10-50 mM perfural culture conditions.
3. The yeast strain of claim 2, wherein said yeast strain is capable of growing at 10-30 mM perfural culture conditions.
The yeast strain according to claim 1, wherein the yeast strain is selected from the group consisting of Saccharomyces spp., Schizosaccharomyces spp., Pichia spp., Paffia spp. , Kluyveromyces spp., Candida spp., Talaromyces spp., Brettanomyces spp., Pachysolen spp., Or Devario spp. A strain of yeast characterized by being Debaryomyces spp. Yeast strain.
5. The yeast strain of claim 4, wherein said yeast strain is Saccharomyces species.

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