KR101264294B1 - Yeast Variants Having Resistance to Low Temperatures - Google Patents

Abstract

The present invention relates to a method for increasing the growth of yeast at low temperature, and a method for producing yeast variants, comprising inactivating at least one gene selected from the group consisting of STM1 gene , SRO9 gene and YKL075C gene. In addition, the present invention relates to a yeast variant in which at least one gene selected from the group consisting of STM1 gene , SRO9 gene and YKL075C gene is inactivated to show increased growth ability at low temperature. Variants of the present invention having low temperature resistance can be stably grown less sensitively to changes in external temperature, thereby reducing production costs by using them in various applications such as food, medicine, feed, cosmetics, and the like.

Description

Yeast Variants Having Resistance to Low Temperatures

The present invention relates to yeast variants having low temperature resistance.

Low growth temperatures can reduce protein translation efficiency in most cells, reduce the ability of the protein to form tertiary structures, reduce fluidity that regulates the function of the cell membrane, and instability of the double helix structure of DNA or the secondary structure of RNA. Resulting in a number of biochemical and physiological changes, including reduced protein enzyme activity. Most organisms have developed mechanisms to adapt to these low temperature-induced changes in evolution, but they are not well understood at the genetic level.

Germinated yeast is a very good model of eukaryotic cells and is a microorganism widely used in the fields of casting, food, medicine, feed, and cosmetics.

Several genes are known for adaptation in response to low temperatures in germinating yeast S. cerevisiae , which has high industrial demand, but only recently has it been known about the overall mechanism and regulation of gene expression at the whole genome level. Full-scale research is in progress.

The results of the present studies on the expression of total genes of budding yeasts exposed to low temperatures are as follows. 1) Reduction of growth temperature induces the expression of several genes involved in transcription and translation. Some of these genes appear as cold-sensitivity phenotypes. 2) Yeast exposed to low temperatures induces the accumulation of trehalose, glycerol and thermal shock proteins to minimize the effects of freeze injury. 3) Cell membrane fluidity acts as an early signal to induce a cold shock response, which is converted and regulated by hyperosmotic glycerol mitotic-activated protein kinase pathways and traditional stress pathways and transcription factors such as Msn2 / 4p. Jaime, A et al., FEMS Microbiol Rev. 31: 327-341 (2007). In addition, as a result of confirming the ability to withstand a certain period of time in a situation where the metabolic rate was lowered due to the low temperature, when the 93 genes involved in the initial metabolic process were removed, they survived 58 months or more in low temperature conditions (Lucie, P et al. , AGING . 1: 957-960 (2009). In particular, the germinated yeast cells eliminated the enzyme Tps1 and overexpressed Hsp12 (heat shock protein 12), which is necessary for synthesizing trehalose, which plays an important role in protecting the cell membrane against external stimuli to adapt to low temperatures. It was found that growth was maintained at low temperatures. It has been shown that Hsp12 also plays an important role in refrigeration resistance (Pacheco, A et al., Microbiololy . 155: 2021-2028 (2009)). In the absence of genes that perform adaptive functions at low temperatures, cells do not adapt to changes in growth temperature, resulting in a dramatic decrease in growth and survival at low temperatures.

Known mechanisms and methods are available to accelerate the growth of sprouted yeast, which is important for many processes such as fermentation, to dramatically increase the efficiency of growth and cultivation, and to reduce production costs industrially by lowering fermentation temperature under the same growth efficiency. There are few bars.

The inventor is NST1 It has been reported that growth is promoted at low temperatures when gene activity is removed (Korean Patent No. 10-0719465). NST1 is a gene that regulates cell growth by inhibiting G1 / S metastasis of the cell cycle in which cells are replicated. Unlike the normal cells, where growth is slowed at low temperatures when the function of the gene is inactivated, NST1 is low. It did not respond to the condition and maintained growth. To further develop this invention, we searched for other genes that maintain growth at low temperatures through interaction with NST1 .

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.

Under the background of the prior art described above, the present inventors propose the existence of a specific complex that regulates growth in response to temperature by discovering a gene that does not inhibit growth at low temperatures through interaction with NST1 and to produce the yeast mutant strain described above. It was. STM1 , SRO9 , YKL075C genes of the present invention is NST1 When the STM1 , SRO9 , YKL075C is removed and interacts at the gene and gene level, the yeast grows at a normal rate even at low temperatures, and the present invention was completed.

Accordingly, it is an object of the present invention to provide a method for increasing the growth of yeast at low temperatures.

It is another object of the present invention to provide a method for preparing a yeast variant that exhibits increased growth ability at low temperatures.

It is another object of the present invention to provide yeast variants which exhibit increased growth capacity at low temperatures.

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 one aspect of the invention, the invention provides a method of increasing the growth of yeast at low temperatures comprising inactivating at least one gene selected from the group consisting of STM1 gene , SRO9 gene and YKL075C gene.

According to another aspect of the present invention, the present invention provides a method for producing a yeast variant exhibiting increased growth ability at low temperature, comprising inactivating at least one gene selected from the group consisting of STM1 gene , SRO9 gene and YKL075C gene. to provide.

According to another aspect of the invention, the invention provides yeast variants in which at least one gene selected from the group consisting of STM1 gene , SRO9 gene and YKL075C gene is inactivated and exhibits increased growth ability at low temperatures.

Under the background of the prior art described above, the present inventors propose the existence of a specific complex that regulates growth in response to temperature by discovering a gene that does not inhibit growth at low temperatures through interaction with NST1 and to produce the yeast mutant strain described above. It was. The STM1, SRO9 and YKL075C genes of the present invention interact with the NST1 gene at the gene level, and when the STM1 , SRO9 and YKL075C are removed, the yeast grows at a normal rate even at low temperatures.

The present invention is to implement a system that does not inhibit the growth even at low temperatures in yeast widely used in the fields of food, medicine, feed, cosmetics and the like. The inventors have discovered that STM1 , SRO9 , and YKL075C interact with NST1 , a gene involved in cell cycle regulation, and genetically manipulated it to implement the present invention.

NST1 is a germ-line yeast Saccharomyces cerevisiae gene that regulates cell growth by inhibiting G1 / S metastasis in the cell cycle. Responds and grows. To expand the scope of the invention, the inventors have found a variety of genes ( possibly for interaction with NST1 ) that interact with NST1 through tandem affinity purification (TAP) methods. STM1 , SRO9, and YKL075C genes were derived by selecting genes that directly interact with NST1 through computer biology.

The basic strategy of the present invention is STM1 found in various yeasts. Gene , SRO9 Genes and YKL075C Inactivating at least one gene selected from the group consisting of genes.

STM1 is known as a protein required for optimal detoxification under nutritional stress in yeast and for maintaining telomere structure (Carmody SR, et al. Mol Cell Biol. 30 (21): 5168-79 (2010); Ossareh-Nazari B , et al. EMBO Rep, 2010 Jul. PMID 20508643; Cuenca-Bono B, et al. BMC Cell Biol, 2010 Mar 15. PMID 20230609; Kaake RM, et al. J Proteome Res, 2010 Apr 5. PMID 20170199).

SRO9 is a cytoplasmic RNA-binding protein that interacts with ribosomes in the translation process and is known to be involved in the organization of actin filaments (Ossareh-Nazari B, et al. EMBO Rep, 2010 Jul. PMID 20508643; Gong Y, et. Mol Syst Biol, 2009. PMID 19536198; Wilmes GM, et al. Mol Cell, 2008 Dec 5. PMID 19061648; Hasegawa Y, et al. RNA, 2008 Nov. PMID 18805955).

YKL075C is a protein with little function so far identified in 2010 and is believed to be involved in resistance to streptozotocin and camptothecin and is considered to be a cytoplasmic protein (Yu H, et al. Science, 2008 Oct 3. PMID 18719252; Lee W, et al. PLoS Genet, 2005 Aug. PMID 16121259).

The inventors have made STM1 , SRO9 , YKL075C removed to prepare inactivated variants. Normal cells show slow growth at 18 ° C, whereas STM1 , SRO9 , YKL075C Variants without genes were found to grow normal regardless of temperature. Through this, the present inventors STM1 , SRO9 , YKL075C As a gene interacting with NST1 , it was found that when the function of the genes is inactivated, unlike normal cells which slow growth at low temperature, they do not respond to low temperature and maintain growth.

STM1 and SRO9 to be deactivated And YKL075C have been found and homologous in various yeasts. Saccharomyces in the following examples Celebrity STM1 , SRO9 And YKL075C are illustrated.

Sakaromayses Vichy serenity of her STM1 is illustrated in GenBank accession number NC_001144.4 and NM_001182037.1. In addition, various STM1 are included in the present invention. For example, GenBank Accession Numbers NC_003424.3 and NM_001020024.1 ( Schizosaccharomyces pombe ), GenBank accession number CP000500.1 ( Pichia stipitis ), GenBank accession number XM_445725.1 ( Candida The STM1 disclosed glabrata) may be inactivated in the subject invention.

Sakaromayses Vichy serenity of her SRO9 is illustrated in GenBank accession number NC_001135.4 and NM_001178682.1. In addition, various SRO9 are included in the present invention. For example, GenBank access numbers NC_006029.1 and XM_446001.1 ( Candida The SRO9 disclosed glabrata), GenBank accession number NC_005787.5 (Ashbya gossypii) may be inactivated in the subject invention.

Sakaromayses Vichy serenity of her YKL075C is illustrated in GenBank accession number NC_001143.8 and NM_001179641.1. In addition, various YKL075C are included in the present invention. For example, GenBank access numbers NC_006035.2 and XM_448994.1 ( Candida The SRO9 disclosed glabrata), GenBank accession number NC_005785.5 (Ashbya gossypii) may be inactivated in the subject invention.

STM1 , SRO9 herein And term used to refer to YKL075C , “inactivation” refers to STM1 , SRO9 , which results in transcription or translation of the gene, and impairment of the activity of the gene product. And modifications on the genome of YKL075C . Such genetic modifications include STM1 , SRO9 And inactivation of the YKL075C coding sequence as well as its promoter. The specific inactivation of only the gene of interest in the yeast genome is possible by mutation through substitution, insertion, deletion, or a combination thereof in all or at least one subdomain of the coding gene. For example, deletion of a gene and insertion of a heterogenous sequence into a gene can be accomplished by truncation of a gene, nonsense mutation, frameshift mutation, missense mutation, etc. . Specific deactivation of these genes can be accomplished through methods established in the art.

According to a preferred embodiment of the invention, STM1 , SRO9 And inactivating YKL075C is performed by deleting the nucleotide sequence encoding the STM1, SRO9 and YKL075C proteins.

STM1 , SRO9 herein And The term, referring to YKL075C, "deletion (deletion)" has a meaning that includes all the deletion mutations Kane malfunction of STM1, and SRO9 YKL075C. For example, STM1, SRO9 And YKL075C Deletion of the gene is STM1 , SRO9 And partial or complete deletion of the YKL075C coding sequence.

Deletion of such NST1 coding sequences can be accomplished through various mutagenesis methods known in the art. For example, deletion of the STM1, SRO9 and YKL075C coding sequences can be performed by PCR mutagenesis or cassette mutagenesis (Sambrook, J. et al., Molecular Cloning . A Laboratory Manual , 3rd ed. Cold Spring Harbor Press (2001).

According to a preferred embodiment of the invention, the deletion of the STM1, SRO9 and YKL075C coding sequences is carried out by homologous recombination.

According to a specific embodiment of the present invention, gene targeting by homologous recombination causes mutations in which each STM1 , SRO9 , YKL075C gene on the genome is replaced with a selection marker gene. The sequences corresponding to the upstream and downstream parts of an open reading frame (ORF) of a specific gene are identical, and in the middle part, DNA fragments carrying the selection marker gene are transferred into the cell. Induced cross-over with specific genes in the genome. The homologous recombination is inserted into the one-time occurs, DNA fragments are STM1, SRO9, YKL075C gene of the genome, when each of the front of a gene coding region and the back respectively, homologous recombination is induced on the genome STM1, SRO9, YKL075C gene DNA fragment and the nucleic acid The exchange takes place and the heterologous gene, the selection marker in the DNA fragment, is inserted instead. In this case, a selection marker commonly used is not particularly limited. Markers that confer a selectable phenotype such as drug resistance, nutritional requirements, resistance to cytotoxic agents or expression of surface proteins can be used. In the following examples, tryptophan biosynthesis gene TRP1 (N- (5'-phosphoribosyl) -anthranilate isomerase), which allows growth without tryptophan in the medium, was used as a selection marker. The method of introducing the DNA fragments into yeast includes any method of introducing nucleic acids into cells, and may be performed by selecting a suitable standard technique as known in the art. Examples include, but are not limited to, electroporation, microinjection, DEAE-dextran, cationic liposome method, and the like.

Yeast to which the present invention is applied is not particularly limited, preferably the saccharide in my process (Saccharomyces), my process (Sporobolomyces) in between the irradiation Caro My process (Schizosaccharomyces), Spokane Lobo, the mound rope sheath (Torulopsis), tree course isophorone (Trichosporon), wick keoha MIA (Wickerhamia), Ashdod Bahia (Ashbya), Blas Sat My process (Blastomyces), Candida (Candida), between the interrogating my process (Citeromyces), Crescent bromo potassium (Crebrothecium), Cryptococcus Rhodococcus ( Cryptococcus), de Bari Oh, my process (Debaryomyces), Hainaut maykop sheath (Endomycopsis), geo-tree compartment (Geotrichum), Hanse Cronulla (Hansenula), claw exciter Mosquera (Kloeckera), lipoic My process (Lipomyces), Pichia (Pichia ), also spokes lithium (Rhodosporidium) or also torulra (Rhodotorula) and yeast belonging to the genus (genus), and more preferably a yeast belonging to my process as saccharide, and most preferably Co. Romayi access It is ceremonial .

More specifically, the mutant strain of the invention deposited with No. KCCM-11108P arc deposit the Saccaromyces cerevisiae mutant YSK2835, Accession No. KCCM-11109P arc deposited Saccaromyces cerevisiae SSKcaro36 mutants and Saccaromyces deposited under accession number KCCM-11110P cerevisiae YSK2837 is a mutant strain.

The low temperature growth ability of yeast is greatly improved by the method of the present invention.

As used herein, the term “low temperature growth ability” means growth ability at temperatures of preferably 25 ° C. or lower, more preferably 18 ° C. or lower. In this case, the upper temperature associated with low-temperature growth ability is 5 ° C, more preferably 8 ° C, and more preferably 10 ° C.

As used herein referring to the yeast variants of the present invention, the expression “yeast variant showing increased growth ability at low temperature” refers to a yeast variant with increased growth ability at low temperatures compared to wild-type yeast, and such yeast variants It exhibits a growth curve that is substantially the same as the growth at the optimum temperature of wild-type yeast (eg, 30 ° C.) at low temperatures.

The mutant strains of the present invention can overcome yeast growth slowdown and low productivity at low temperatures, which are currently considered to be a barrier to fermentation engineering.

By using the yeast mutant strain of the present invention in the fields of food, medicine, feed, cosmetics and the like can reduce the production cost. Additional variations can be induced by using the mutant strain as it is or by using the mutant strain of the present invention as a parent strain depending on the purpose. For example, the metabolic mechanism of biosynthesis may be artificially altered or may be altered to overexpress pharmaceutical, industrially used proteins. Various modifications using the mutant strains that promote growth at low temperatures of the present invention can be made in a conventional manner by those skilled in the art.

As a specific example, the mutants of the present invention can be used for alcohol, such as wine and beer, liquor production and fermented food production such as soy sauce, miso, bread, cheese, yogurt, vinegar and the like. In addition, it can be used for the production of antibiotics and anticancer substances, organic acid fermentation industry, amino acid fermentation industry, protein production industry, cell production. Representative products of organic acid fermentation industry are citric acid (citric acid), lactic acid, acetic acid, citric acid is used as a raw material for the production of soft drinks, lactic acid is used in food industry, beverage, leather industry, medicine, etc., acetic acid is mainly used for food. In addition, the mutant strain of the present invention can be used for methane fermentation, waste liquid treatment, pollution prevention, desulfurization, mining and the like.

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

(a) STM1 of yeast according to the invention Gene , SRO9 Genes and YKL075C Inactivating at least one gene selected from the group consisting of genes greatly increases the yeast's ability to grow at low temperatures.

(b) Variants of the present invention having low temperature resistance can be stably grown less sensitive to changes in external temperature, thereby reducing production costs by using them in various application areas of yeast such as food, medicine, feed, cosmetics, etc. Can be.

1 is a urbanization the pFA6a- TRP1 gene deletion cassette used as a template (template) in the DNA fragment synthesized for removing STM1, SRO9, YKL075C each gene in the chromosome.
Figure 2 is a sprouted yeast wild type and STM1 Elimination Variant ( stm1 ), SRO9 Elimination variant ( sro9 ), YKL075C The same number of cells of the removal variant ( YKL075C ) were diluted 10-fold and inoculated, and then incubated at 18 ° C. (low temperature) and 30 ° C. at 3 ° C. on the plate for 3 days.
Figure 3 shows the same number of sprouted yeast wild type and STM1 The elimination variants ( stm1 ) are incubated at 18 ° C. (low temperature) for 30 hours at 30 ° C., and the number of cells is measured every 8 hours to show the result of comparing the growth.
4 shows the same number of sprouted yeast wild type and SRO9 The removal variants ( sro9 ) were incubated at 18 ° C. (low temperature) for 30 hours at 30 ° C., and the number of cells was measured every 8 hours to show the result of comparing the growth.
5 shows the same number of sprouted yeast wild type and YKL075C The removal variants ( YKL075C ) were incubated at 18 ° C. (low temperature) at 30 ° C. for 64 hours, and the number of cells was measured every 8 hours to show the result of comparing the growth.

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

Example 1: Yeast Culture Conditions and Preparation of Each Gene Inactivation Variants

(1) yeast culture conditions

In the case of the yeast wild type, it was cultured in YPAD medium or selective medium, which is the optimal condition for yeast growth. The composition of the YPAD medium consists of 1% yeast extract, 2% bacto-peptone, 100 μg / ml adenine and 2% dextrose. Synthetic Complete (SC) medium was prepared by adding 13.4 g of yeast nitrogen base, 10 g of ammonium sulfate, 4 g of 20 amino acid mixtures, and 2 ml of 200 mM inositol based on 1 L of 2 X SC medium.

SC drop-out medium was used to select and culture inactivated variants of each gene. Inactivated variants of each gene have a TRP1 (N- (5'-phosphoribosyl) -anthranilate isomerase) gene, which is required for tryptophan biosynthesis as a selection marker. -Trp). The yeast wild type cannot grow in selective medium excluding tryptophan in its amino acid mixture, and only STM1, SRO9, and YKL075C inactivated variants with selection markers are characterized by growing on this medium.

(2) STM1 inactivating variants

In order to remove and inactivate the STM1 gene in yeast, a method of replacing the STM1 gene on the genome with a selection marker gene by gene targeting by homologous recombination was used.

pFA6a- TRP1 gene deletion cassette (EUROSCARF; EUROpean Saccharomyces Cerevisiae Archive for Functional analysis, prepared and provided; Longtine, MS et al., Additional modules for versatile and economical PCR-based gene deletion and modification in Saccharomyces cerevisiae. From Yeast 14: 953-961 (1998) (see FIG. 1), the TRP1 encoding region was shown to have a primer (KS1209: see Table 1) upstream of the STM1 gene ORF and a downstream of the ORF; Amplification was carried out by polymerase chain reaction (PCR) using a homologous primer (KS1210: see Table 1). Comprises an amplifier and TRP1, the gene ORF STM1 on both sides of the upper region TRP1 (63-19 base; 45 base that) the homologous sequences and STM1 lower region of the gene ORF (93-136 base; corresponds to the base 44) Amplified DNA fragments with sequences homologous to the lithium acetate (LiAc) method in wild-type yeast (w303a; MATa ade2-1 ura3-1 trp1-1 leu2-3, 112 his3-11, 15 can1-100; ATCC) Transformation was induced. Transformation was incubated with wild type yeast (w303a) to log phase 3 X 10 ⁷ cells / ml (mid-log phase), treated with 0.1 M LiAc, and transformed mixture (50% PEG, 1 M LiAc, single Stranded DNA 2 mg / ml) and the DNA fragment to be transferred to the yeast, and then incubated for 1 hour at 30 ° C. and 30 minutes at 42 ° C. The yeast cells were plated on SC drop-out (-Trp) plates and incubated for 2-3 days at 30 ° C. Homologous recombination is induced in the upper part (63-19 bp upstream) and the lower part (93-136 bp downstream) of the STM1 gene coding region (ORF) so that the STM1 gene on the genome is directed to TRP1 , a selectable marker in the DNA fragment. Substituted STM1 inactivated variants can grow in tryptophan-free medium (SC-Trp media) because TRP1 is expressed.

The STM1 inactivated mutant prepared was named Saccaromyces cerevisiae YSK2835, and the mutant had the following genotypes: YSK2835 Saccaromyces cerevisiae (MATa ura3-1 trp1-1 ade2-3 leu2-3 112his3-11,15 Δstm1 :: TRP1 )

Saccaromyces cerevisiae YSK2835 mutant strain prepared above was deposited with Korean Culture Center of Microorganism (KCCM) on October 7, 2010, and was assigned Accession No. KCCM-11108P.

The underlined sequences are 44-45 base and homologous (44-45 mer), which correspond to the upper (63-19 bp upstream) and lower (93-136 bp downstream) portions of the STM1 gene coding region (ORF).

^* Bold sequences are homologous (20 mer) used for PCR of TRP1 in the cassette of FIG.

(3) SRO9 Inactivating variants

In order to remove and inactivate the SRO9 gene in yeast, a method of replacing the SRO9 gene on the genome with a selection marker gene by gene targeting by homologous recombination was used. pFA6a- TRP1 gene deletion cassette (EUROSCARF; EUROpean Saccharomyces Cerevisiae Archive for Functional analysis, prepared and provided; Longtine, MS et al., Additional modules for versatile and economical PCR-based gene deletion and modification in Saccharomyces cerevisiae. Yeast 14: 953-961 (1998)) (see FIG. 1), a TRP1 encoding site was identified by a primer having a homologous sequence with an upstream of the SRO9 gene ORF (KS1214; see Table 2), and a downstream of the ORF. Amplification was carried out by polymerase chain reaction (PCR) using a homologous primer (KS1215: see Table 2). Amplified Including TRP1, and, upstream region of the gene SRO9 ORF in both the TRP1; homologous sequence and the SRO9 (69-27 base 43 corresponding to the base) Amplified DNA fragments with sequences homologous to downstream regions of gene ORF (1-44 base; corresponding to 43 base) were transferred to wild-type yeast (w303a) by lithiume acetate (LiAc) to induce transformation. Transformation was incubated with wild type yeast (w303a) to the log phase 3X10 ⁷ cells / ml (mid-log phase), treated with 0.1 M LiAc, transformation mix (50% PEG, 1 M LiAc, single-strand) 2 mg / ml of DNA) and the DNA fragment to be transferred to yeast, and then used in the yeast, which is incubated for 1 hour at 30 ° C. and 30 minutes at 42 ° C. The yeast cells were plated on SC drop-out (-Trp) plates and incubated at 30 ° C for 2-3 days. Homologous recombination is induced in the upper part (69-27 bp upstream) and the lower part (1-44 bp downstream) of the SRO9 gene coding region (ORF) so that the SRO9 gene in the genome is directed to TRP1 , a selectable marker in the DNA fragment. Substituted SRO9 inactivating variants Since TRP1 is expressed, it can be grown in tryptophan-free medium (SC-Trp media).

Saccaromyces prepared SRO9 inactivated mutants cerevisiae Named YSK2836, this mutant has the following genotypes: YSK2836 Saccaromyces cerevisiae (MATa ura3 -1 trp1 -1 ade2 -3 leu2 -3 112his3-11,15 △ sro9 :: TRP1 )

Saccaromyces prepared above cerevisiae The YSK2836 mutant strain was deposited with the Korean Culture Center of Microorganism (KCCM) on October 7, 2010 and was assigned accession number KCCM-11109P.

^*

Underlined sequences are SRO9 gene coding region: 43 base homologous part (43mer) for the upper part (69-27 bp upstream) and bottom (1-44 bp downstream) of (ORF open reading frame)

^* Bold sequences are homologous (20mer) used for PCR of TRP1 in the cassette of FIG.

(4) YKL075C Inactivating variants

In order to remove and inactivate the YKL075C gene in yeast, a method of replacing the YKL075C gene on the genome with a selection marker gene by gene targeting by homologous recombination was used. pFA6a- TRP1 gene deletion cassette (EUROSCARF; EUROpean Saccharomyces Cerevisiae Archive for Functional analysis, prepared and provided; Longtine, MS et al., Additional modules for versatile and economical PCR-based gene deletion and modification in Saccharomyces cerevisiae. Yeast 14: 953-961 (1998) (see FIG. 1), a TRP1 encoding region was identified by using a primer (KS1149: SEQ ID NO: 3) having a homology with an upstream of the SRO9 gene ORF; Amplification was carried out by polymerase chain reaction (PCR) using a homologous primer (KS1150: SEQ ID NO: 4). Amplified Including TRP1, and, YKL075C upstream region of the gene in both of the TRP1 ORF; homologous sequence and the YKL075C (48-1 base on the base 48) Amplified DNA fragments with sequences homologous to the downstream region (0-48 base; 48 base) of the gene ORF were transferred to wild-type yeast (w303a) by lithiume acetate (LiAc) to induce transformation. Transformation was incubated with wild type yeast (w303a) to the log phase 3X10 ⁷ cells / ml (mid-log phase), treated with 0.1 M LiAc, transformation mix (50% PEG, 1 M LiAc, single-strand) 2 mg / ml of DNA) and the DNA fragment to be transferred to yeast, and then used in the yeast, which is incubated for 1 hour at 30 ° C. and 30 minutes at 42 ° C. The yeast cells were plated on SC drop-out (-Trp) plates and incubated at 30 ° C for 2-3 days. YKL075C Gene coding region (ORF: open reading frame) the upper part (48-1 bp upstream) and bottom (0-48 bp downstream) from each substituted with the homologous recombination is caused YKL075C gene on the genome the DNA fragment of the TRP1 selectable marker in YKL075C inactivated variants Since TRP1 is expressed, it can be grown in tryptophan-free medium (SC-Trp media).

Saccaromyces prepared YKL075C inactivated mutants cerevisiae It was named YSK2837 and this mutant has the following genotypes. YSK2837 Saccaromyces cerevisiae (MATa ura3 -1 trp1 -1 ade2 -3 leu2-3 112 his3 -11,15 △ YKL075C :: TRP1 )

Saccaromyces prepared above cerevisiae The YSK2837 mutant strain was deposited with the Korean Culture Center of Microorganism (KCCM) on October 7, 2010 and was assigned accession number KCCM-11110P.

^{* The} underlined sequence is 48 base and homologous (48mer) corresponding to the upper part (48-1 bp upstream) and the lower part (0-48 bp downstream) of the YKL075C gene coding (ORF) open reading frame.

^* Bold sequences are homologous (20mer) used for PCR of TRP1 in the cassette of FIG.

Example  2: inactivation at low temperature Mutant  Growth rate analysis

Yeast wild type and STM1 , SRO9 , YKL075C inactivated variants ( stm1, sro9, YKL075C ) were cultured in YPAD medium under the culture temperature of 30 ° C, and then the same number of cells were diluted 10-fold and containing 3% 2% glucose Dropped onto SC plate. These were incubated for 3 days by varying the culture temperature only to 18 ℃ (low temperature), 30 ℃.

As a result, STM1 , SRO9 , and YKL075C inactivated variants ( stm1, sro9, YKL075C ) significantly promoted cell growth at low temperature (18 ° C) compared to the yeast wild type (Fig. 2).

Example  3: inactivation at low temperature Mutant  Growth rate analysis

The yeast wild type and STM1 , SRO9 , YKL075C inactivating variants ( stm1, sro9, YKL075C ) were incubated for one day until saturated in 30 ° C. YPAD medium, and then the same number of cells were placed in 20 ml of YPAD medium and 30 ° C and incubated for 72 hours under the culture temperature conditions of 18 ℃. At this time, by measuring the number of cells at regular time intervals the degree of cell growth is shown as a graph.

As a result, in the case of STM1 , SRO9 , YKL075C inactivated variants ( stm1, sro9, YKL075C ), it can be seen that the growth is significantly promoted at a low temperature of 18 ° C compared to the yeast wild type (Fig. 3 and 4 and 5) .

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.

Korea Microorganism Conservation Center (overseas) KCCM11108P 20101007 Korea Microorganism Conservation Center (overseas) KCCM11109P 20101007 Korea Microorganism Conservation Center (overseas) KCCM11110P 20101007

Claims (19)

SRO9 A method of increasing the growth of yeast at low temperatures comprising inactivating a gene.
The method of claim 1, wherein the SRO9 Inactivating the gene is performed by mutating the nucleotide sequence encoding the SRO9 protein through substitution, insertion, deletion or a combination thereof.
The method of claim 2, wherein the SRO9 Inactivating the gene is performed by deleting the nucleotide sequence encoding the SRO9 protein.
4. The method of claim 3, wherein said deletion is performed by homologous recombination.

The method of claim 1 wherein the low temperature is a temperature of 18 ° C. or less.
According to claim 1, wherein the yeast Saccharomyces (Saccharomyces), Schizosaccharomyces (Schizosaccharomyces), Sprobolomyces (Sporobolomyces), Torulopsis (Trulopsis), Tricosporon (Trichosporon), Wick keoha MIA (Wickerhamia), Ashdod Bahia (Ashbya), Blas Sat My process (Blastomyces), Candida (Candida), between the interrogating my process (Citeromyces), Crescent bromo potassium (Crebrothecium), Cryptococcus Rhodococcus (Cryptococcus), de Bari Oh, my process (Debaryomyces), Hainaut maykop system (Endomycopsis), Geo tree Com (Geotrichum), Hanse Cronulla (Hansenula), Chloe exciter Mosquera (Kloeckera), lipoic My three's (Lipomyces), Pichia (Pichia), also sports a lithium ( Rhodosporidium) or also characterized in that the torulra (Rhodotorula).
7. The method of claim 6, wherein said yeast is Saccharomyces.
SRO9 A method of making a yeast variant that exhibits increased growth capacity at low temperatures comprising inactivating a gene.
10. The apparatus of claim 8, wherein the SRO9 Inactivating the gene is SRO9 And mutating the nucleotide sequence encoding the protein via substitution, insertion, deletion or combination thereof.
10. The apparatus of claim 9, wherein the SRO9 Inactivating the gene is performed by deleting the nucleotide sequence encoding the SRO9 protein.
The method of claim 10, wherein said deletion is effected by homologous recombination.
The method of claim 8, wherein the low temperature is a temperature of 18 ℃ or less.
9. The method of claim 8 wherein the yeast is Saccharomyces as MY access (Saccharomyces), between the irradiation Caro My process (Schizosaccharomyces), Spokane Robo in my process (Sporobolomyces), Toru rope sheath (Torulopsis), tree course isophorone (Trichosporon), the wick keoha MIA (Wickerhamia), Ashdod by O (Ashbya), Blas Sat My process (Blastomyces), Candida (Candida), between the interrogating my process (Citeromyces), Crescent bromo potassium (Crebrothecium), Cryptococcus Rhodococcus (Cryptococcus), de Bari Oh, my Debaryomyces, Endomycopsis, Geotrichum, Hansenula, Kloeckera, Lipomyces, Pichia, Rhodosporidium ( Rhodosporidium) or Rhodotorula .
The method of claim 13, wherein the yeast is Saccharomyces.
delete delete delete delete A yeast variant in which the SRO9 gene is inactivated and shows increased growth ability at low temperatures, wherein the yeast variant is Saccharomyces cerevisiae YSK2836 (Accession Number: KCCM-11109P).

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