JP5013448B2 - Combination of multiple gene disruptions to improve lactate tolerance or productivity of budding yeast - Google Patents

Description

  The present invention relates to a budding yeast having lactic acid tolerance or lactic acid production ability.

  A method is known in which yeast that has been modified to have lactic acid-producing ability by genetic recombination is used in the production of lactic acid (see Patent Documents 1 to 4). However, as the lactic acid fermentation progresses, the lactic acid concentration increases and the pH of the culture solution decreases, and when it reaches about pH 2 to 3, the lactic acid production efficiency of the yeast decreases. For this reason, in order to keep lactic acid production efficiency high, there existed a problem that the neutralization process of a culture solution, the desalting, and the refinement | purification process were needed and the manufacturing cost became high. Patent Document 5 discloses a technique for improving acid resistance of yeast or the like having lactic acid-producing ability, but acid resistance is not sufficient. In addition, at the 2005 Agricultural Chemistry Society conference agenda number 30F105α (Andersen Service Co., Ltd., Liquor Research Institute), there are strains in which yeast genes (SED1, EGT2, DSE2, SCW11, HOC1, SUN4, EAF3) have been destroyed individually. It has been reported to be resistant to 6.1% lactic acid (pH 2.5), 0.7% acetic acid (pH 4.1), and 0.3% hydrochloric acid (pH 2.2). However, in order to efficiently produce lactic acid using yeast, it is desired to further improve acid resistance.

  Patent Documents 6 and 7 disclose a method for producing lactic acid using a lactic acid-producing bacterium belonging to the genus Bacillus. In order to culture bacteria such as Bacillus genus, it is necessary to maintain the pH of the culture solution at 6-8, so that the production efficiency of lactic acid by the methods described in Patent Documents 6 and 7 is higher than that described in Patent Documents 1-4. Was even lower.

  Patent Document 8 discloses a method for increasing the growth and antibacterial substance productivity of lactic acid bacteria. In the method described in Patent Document 8, it is necessary to use additives such as iron porphyrin and heme protein, and there is a problem that the culture cost is increased.

JP 2006-6271 A JP 2006-20602 A Special table 2001-51658 gazette JP 2005-528106 A JP 2001-204464 A Japanese Patent No. 3682679 JP-A-9-121844 Japanese Patent No. 2991458

  As described above, in the method of producing lactic acid using yeast modified so as to have lactic acid-producing ability by genetic recombination, the lactic acid concentration increases as lactic acid fermentation proceeds, so the pH of the culture solution decreases and the yeast There was a problem that it was difficult to carry out efficient production of lactic acid because it was out of the pH range (usually about 4 to 6) suitable for culture.

  Then, this invention aims at improving the lactic acid tolerance of yeast. Another object of the present invention is to maintain or improve lactic acid productivity even after lactic acid production has progressed in yeast having lactic acid-producing ability.

The present invention includes the following inventions.
(1) A budding yeast, wherein two or more genes involved in lactic acid resistance are disrupted.
(2) The budding yeast according to (1), wherein the genes involved in lactic acid resistance are at least two genes selected from the group consisting of DSE2, SCW11, EAF3 and SED1.
(3) The budding yeast according to (1), which has lactic acid resistance.
(4) The budding yeast according to (3), which is a multigene disruption strain which is Δdse2Δeaf3, Δscw11Δeaf3, Δdse2Δeaf3Δsed1, Δscw11Δeaf3Δsed1, or Δdse2Δscw11Δeaf3Δsed1.
(5) The budding yeast according to (1), wherein a gene involved in lactic acid production is introduced and has lactic acid production ability.
(6) A gene involved in lactic acid resistance is not destroyed, and the amount of lactic acid produced is 10% or more higher than that of a budding yeast having a lactic acid producing ability into which a gene involved in lactic acid production is introduced. And the budding yeast according to (5).
(7) The budding yeast according to (6), which is a multigene-disrupted strain that is Δdse2Δeaf3, Δdse2Δscw11, Δscw11Δsed1, Δeaf3Δsed1, Δscw11Δeaf3Δsed1, or Δdse2Δscw11Δeaf3Δsed1.
(8) A method of imparting lactic acid resistance to budding yeast by destroying two or more genes involved in lactic acid resistance in budding yeast.
(9) A method of imparting lactic acid productivity to a budding yeast by destroying two or more genes involved in lactic acid resistance in the budding yeast and introducing a gene involved in lactic acid production.

  According to the present invention, the lactic acid resistance of yeast can be improved. Furthermore, according to the present invention, lactic acid productivity can be maintained or improved even after lactic acid production has progressed in yeast having lactic acid-producing ability.

(Budding yeast)
The budding yeast used in the present invention is not particularly limited as long as it is a strain classified as Saccharomyces cerevisiae.

(Genes involved in lactic acid production)
A gene involved in lactic acid production may be introduced into the budding yeast. Examples of genes involved in lactic acid production include lactate dehydrogenase (LDH) gene. The collection source of the gene involved in lactic acid production is not particularly limited. For example, the gene involved in lactic acid production is obtained from most organisms including animals such as cattle and humans, prokaryotes such as E. coli and lactic acid bacteria, and plants. Can do. Extraction of mRNA from the organism from which the gene is collected and preparation of a cDNA library can be performed by conventional methods.

  Introduction of genes involved in lactic acid production into budding yeast can be performed by conventional methods. For example, it can be introduced by transforming budding yeast with a recombinant vector incorporating the gene. The recombinant vector can be obtained by linking (inserting) a gene involved in lactic acid production into an appropriate vector. The vector for inserting a gene involved in lactic acid production is not particularly limited as long as it can be replicated in the budding yeast as a host. For example, YEp type, YRp type, and YCp type vectors can be used. Furthermore, the vector may be a vector that enables replication by integrating into a chromosome without replicating as an extranuclear gene. For efficient integration into the chromosome, a YIp type vector can be used. It can also be integrated into the chromosome by retrotransposon.

  In order to insert a gene involved in lactic acid production into a vector, first, the purified DNA is cleaved with a suitable restriction enzyme, inserted into a restriction enzyme site or a multicloning site of a suitable vector DNA, and ligated to the vector. Etc. are adopted.

  A gene involved in lactic acid production needs to be incorporated into a vector so that the function of the gene is exhibited. Therefore, the vector of the present invention includes, in addition to a promoter, a gene involved in lactic acid production, a terminator, a cis element such as an enhancer, a splicing signal, a poly A addition signal, a selection marker, a ribosome binding sequence (SD sequence) and the like. Can be linked. Examples of selectable markers include various auxotrophic markers such as LEU2, HIS3, and URA3 genes, geneticin resistance genes, methotrexate resistance genes, zeocin resistance genes, and aureobasidin.

  A specific example of a recombinant vector incorporating a gene involved in lactic acid production is the pBTrp-PDC1P-LDHKCB vector constructed in Japanese Patent Application No. 2003-334092.

  The budding yeast having the ability to produce lactic acid used in the present invention can be obtained by introducing the above recombinant vector into a host so that the target gene can be expressed.

  The transformation for obtaining the acid-resistant lactic acid-producing microorganism of the present invention is particularly a method that can introduce the recombinant vector of the present invention containing a gene involved in lactic acid production into the acid-resistant microorganism obtained by the above method. It is not limited. For example, transformation is performed by a commonly used genetic engineering technique such as a lithium acetate method, a method using calcium ions (Proc. Natl. Acad. Sci., USA, 69, 2110, 1972), an electroporation method, or the like.

(Damage of genes involved in lactate tolerance)
In the present invention, the “gene involved in lactic acid resistance” means a budding yeast gene that, when destroyed, improves the lactic acid resistance of the budding yeast. Specific examples of genes involved in lactic acid resistance include DSE2, SCW11, EAF3, and SED1.

  It has been known before the filing of the present application that the lactic acid tolerance of yeast is improved when each of these genes is destroyed alone, as shown in the background section. The present inventors have surprisingly found that when two or more of these genes are disrupted in combination, the lactic acid tolerance and / or lactic acid productivity of the budding yeast is significantly improved compared to when the genes are disrupted alone. I found out.

  A preferred form of the budding yeast according to the present invention is a budding yeast in which at least 2, preferably 3, and particularly preferably 4 genes selected from the group consisting of DSE2, SCW11, EAF3 and SED1 are disrupted.

A preferred form of the budding yeast according to the present invention is a budding yeast having significantly improved lactic acid resistance, and specifically, a budding yeast having colony forming ability in an agar medium containing 8 (w / v)% lactic acid. The specific composition of the agar medium is 8 w / v% in YPDA medium (5% YPD Broth (SIGMA; 2% glucose, 2% bactopeptone, 1% yeast extract), 0.04% adenine). As described above, L-lactic acid (manufactured by Nacalai Tesque Co., Ltd.) is added. The culture conditions for evaluating the colony forming ability are 10 ⁶ cells at a culture temperature of 30 ° C., a culture time of 264 hours, and a primary cell concentration of 5 μL. Examples of the budding yeast having such properties include a double gene disruption strain that is Δdse2Δeaf3 or Δscw11Δeaf3, a triple gene disruption strain that is Δdse2Δeaf3Δsed1 or Δscw11Δeaf3Δsed1, or a quadruple gene disruption strain that is Δdse2Δscw11Δeaf3Δsed1. Here, the notation Δdse2Δeaf3 means that the DSE2 gene and the EAF3 gene are both disrupted. The notation of other destroyed strains is interpreted in the same way.

  Another preferred form of the budding yeast according to the present invention is a budding yeast having a lactic acid-producing ability into which a gene involved in lactic acid production has been introduced, wherein the lactic acid productivity in the presence of lactic acid is significantly improved. is there. Specifically, it is compared with a budding yeast in which a gene involved in lactic acid resistance has not been destroyed and a gene involved in lactic acid production has been introduced (sometimes referred to as “parent strain” in this specification). This is a budding yeast having a lactic acid production amount of 10% or more. This comparison is performed by the following procedure. A 100 ml Erlenmeyer flask is used for culture of budding yeast having lactic acid-producing ability into which two or more genes involved in lactic acid resistance have been disrupted and a gene involved in lactic acid production is introduced. 10% YPD medium (1% Bacto Yeast extract, 2% Bacto peptone, 10% D-glucose) 40 ml of pre-cultured bacterial solution was added and left to stand at 30 ° C. for 72 hours, followed by L-lactic acid concentration in the medium (w / v%) ). And, when the value of L-lactic acid concentration in the medium of the parent strain as a control is 100, a budding yeast having a L-lactic acid concentration value of 110 or more in the medium has a lactic acid production amount higher by 10% or more. It is determined as Saccharomyces cerevisiae having such properties include a double gene disruption strain that is Δdse2Δeaf3, Δdse2Δscw11, Δscw11Δsed1, or Δeaf3Δsed1, a triple gene disruption strain that is Δscw11Δeaf3Δsed1, or a quadruple strain that is a quadruple strain that is Δdse2Δscw11Δeaf3Δsed1.

  The most preferred form of the budding yeast according to the present invention is a budding yeast in which both lactic acid resistance and lactic acid productivity when a gene involved in lactic acid production is introduced to give lactic acid production ability are improved. More specifically, it is a budding yeast having colony forming ability in an agar medium containing 8 (w / v)% lactic acid and having a lactic acid production amount of 10% or more higher than that of the parent strain. Examples of the budding yeast having such properties include a double gene disruption strain that is Δdse2Δeaf3, a triple gene disruption strain that is Δscw11Δeaf3Δsed1, or a quadruple gene disruption strain that is Δdse2Δscw11Δeaf3Δsed1.

  As a gene disruption method, in the examples of the present specification, PCR products (genes) obtained by amplifying marker genes such as the LEU2 gene (CgLEU2) and the HIS3 gene (CgHIS3) using a long primer containing a part of the gene. The DNA fragment for disruption) was subjected to gene disruption by homologous recombination.

  However, other gene disruption methods by homologous recombination can also be used. An example is shown below.

  (Alternative method 1) Recombinant vector that is capable of autonomous replication in Saccharomyces cerevisiae, incorporating a gene fragment to be disrupted in the same manner as the introduction of a gene involved in lactic acid production into Saccharomyces cerevisiae chromosome. An unretained vector such as a YIp vector or a plasmid derived from E. coli is constructed. At this time, when an incomplete recombinant vector having a gene fragment in which the N-terminus and C-terminus of the target gene are partially deleted is constructed and inserted into the target locus, two copies of the target gene are generated on the chromosome. Since it also has a deletion at the N-terminal or C-terminal coding part, it is possible to lose the function of a specific gene.

  (Alternative method 2) Similarly, a recombinant vector carrying a target gene fragment having at least one point mutation at both ends that causes loss of the function of the target gene is introduced into the target locus. Although two copies of the target gene are generated on the chromosome, each of them has a point mutation in the coding part, so that the function of the target gene can be lost.

  (Alternative method 3) In the examples in this specification, a recombinant DNA fragment having a partial sequence of 45 bp of the sequence of the gene to be disrupted was used, but the length of the partial sequence is not limited to 45 bp. That is, a recombinant DNA fragment having a partial sequence of a gene to be disrupted longer than 45 bp or a partial sequence of a gene to be disrupted shorter than 45 bp is constructed on a vector, and then excised and recovered by restriction enzyme treatment, or PCR is repeated. It is also possible to adopt a method in which a fragmented DNA fragment to which a longer or shorter partial sequence of a gene to be disrupted is added is prepared and introduced into a budding yeast to perform gene disruption.

  (Alternative method 4) Further, methods other than homologous recombination can be used if the function of the gene can be deleted. These include (1) a method of screening a point mutant using a mutation agent and (2) a method of screening from an insertion library using a transposon.

  As a gene to be inserted into a gene to be disrupted at the time of gene disruption, a gene serving as a marker for selecting a gene disruption strain is preferably used. These include antibiotic resistance genes, genes that complement auxotrophic mutations (which no longer require that nutrient source), and the like.

  The present invention will be described below based on examples, but the present invention is not limited to the following examples.

1. Methods for preparing genetically disrupted strains There are 15 combinations of strains in which 1 to 4 genes selected from the group consisting of DSE2, SCW11, EAF3 and SED1 are disrupted. Multiple disruptions of all 15 of these combinations were prepared as follows. The experiment was performed according to the method described in Method in yeast genetics 2005 (Cold spring harber press, Cold spring harbor, NY, USA).

1.1. Construction of a Δdse2Δscw11 double disruption strain from an α-type BY4742 strain-derived Δdse2 disruption strain SCW11, which is a DNA fragment partially comprising upstream and downstream sequences of a selection marker on the plasmid p3008 (pUG-CgLEU2) shown in FIG. Forward (SEQ ID NO: 3) and SCW11 reverse (SEQ ID NO: 4) were synthesized. A DNA fragment for SCW11 gene disruption was prepared by PCR amplification using p3008 as a template and SCW11 forward and SCW11 reverse as primers. The plasmid p3008 was prepared based on Biotechniques, 38 (6), 909-914, 2005.

  A Δdse2 disrupted strain was transformed with the obtained DNA fragment for disruption. Δdse2 disrupted strain, BY4742 strain (MATα leu2Δ0 his3Δ1 ura3Δ0 lys2Δ0) DSE2 gene is a geneticin resistance gene kanMX (Wach, A.etal;... New heterologous modules for classical or PCR-based gene disruptions in Saccharomyces cerevisiae Yeast 10 (13), 1793-1808 (1994)), and a strain derived from the disrupted strain set (BY4742 strain-derived non-essential gene disruption library (manufactured by Invitrogen)) was used as it was. A leucine non-requiring strain was selected to obtain a Δdse2Δscw11 double disruption strain. The selected strain was prepared by preparing genomic DNA and performing PCR using PCR with primers SCW11F1 (SEQ ID NO: 11) and SCW11R1 (SEQ ID NO: 12) having homology upstream and downstream of the SCW11 gene as shown in FIG. Confirmed gene disruption.

1.2. Construction of Δeaf3Δsed1 double disruption strain from a-type BY4741 strain EAF3 forward (SEQ ID NO: 2), which is a DNA fragment partially containing upstream and downstream sequences of the selection marker on plasmid p3009 (pUG-CgHIS3) shown in FIG. 5) and EAF3 reverse (SEQ ID NO: 6) were synthesized. A DNA fragment for disrupting the EAF3 gene was prepared by amplifying by PCR using these synthetic DNAs as primers and p3009 as a template. The plasmid p3009 was prepared based on Biotechniques, 38 (6), 909-914, 2005.

  The BY4741 strain (MATa leu2Δ0 his3Δ1 ura3Δ0 met15Δ0) (manufactured by Invitrogen) was transformed with the obtained DNA fragment for disruption, and a strain that became histidine-unrequired was selected to obtain a Δeaf3 disruption strain. The selected strain was prepared by preparing genomic DNA, and by PCR using EAF3F1 (SEQ ID NO: 13) and EAF3R1 (SEQ ID NO: 14) having homology upstream and downstream of the EAF3 gene as shown in FIG. Confirmed gene disruption.

Next, SED1 forward (SEQ ID NO: 7) and SED1 reverse (SEQ ID NO: 8), which are DNA fragments having in part the upstream and downstream sequences of the selection marker on the plasmid (pUG6-zeocin ^r ), were synthesized. A DNA fragment for SED1 gene disruption was prepared by amplifying by PCR using these synthetic DNAs as primers and pUG6-zeocin ^r as a template. The plasmid (pUG6-zeocin ^r ) was prepared by the following procedure. About 1.2 kb containing a zeocin ^r gene (zeocin resistance gene) by PCR using zeocin forward (SEQ ID NO: 17) and zeocin reverse (SEQ ID NO: 18) as primers and pPICZ-α (Invitrogen; V195-20) as a template DNA fragments were recovered. Next, the recovered fragment was digested with XhoI / BgII and inserted into the XhoI / BglII gap of pUG6 to construct pUG6-zeocin ^r .

  A Δeaf3 disruption strain constructed with the obtained SED1 gene disruption DNA fragment was transformed, and a strain resistant to zeocin was selected to obtain a Δeaf3Δsed1 double disruption strain. The selected strain was prepared by preparing genomic DNA and PCR using SED1F1 (SEQ ID NO: 15) and SED1R1 (SEQ ID NO: 16) having homology upstream and downstream of the SED1 gene as shown in FIG. Confirmed gene disruption.

1.3. Production of multiple disruption strain 1.1. And 1.2. By crossing the two haploid double disruption strains obtained in 1) on a YPDA agar medium for 24 hours, and selecting strains that are geneticin resistant, leucine non-requiring, histidine non-requiring and zeocin resistant A diploid was isolated.
The resulting diploid was grown on a YPDA agar medium for 12 hours, then applied to a spore formation medium (0.5% sodium acetate, 2% agar), and cultured at 23 ° C. for 4 days to form spores. .

Next, the following operations were performed according to the method described in “Microbial Genetics Experimental Method” (Kyoritsu Shuppan).
The cells on the plate were scraped off and treated in a 0.3 mg / ml Zymolase (manufactured by ZYMORESEARCH) solution for 5 minutes to digest the ascending wall. Next, spores were dissected using a micromanipulator (SYNGER MSM STEST SERIES 200, manufactured by SINGER Instruments), separated on a YPDA plate, and cultured at 30 ° C. for 3 days. In this way, a total of 29 ascospore 108 spores were obtained. The obtained spores were classified using markers (leucine requirement, histidine requirement, kanamycin resistance, zeocin resistance) as an index.

  As a result, as shown in Table 1, 15 types of single or multiple disrupted strains of Δdse2, Δscwl1, Δeaf3, and Δsed1 were obtained.

2. Resistance test in 6, 7, 8 w / v% lactic acid-added flat plate medium The strains shown in Table 1 were mixed with 5 ml of YPDA liquid medium (5% YPD Broth (SIGMA; containing 2% glucose, 2% bactopeptone, 1% yeast extract). ), 0.04% adenine), and cultured overnight at 30 ° C. and 170 rpm shaking. Next, the cells were inoculated again in a 5 ml YPDA liquid medium and cultured in the same manner until the OD [660 nm] reached about 1.0. Then, the cell concentration was calculated from OD according to Method in yeast genetics (Cold Spring Harbor Laboratory), and 10 dilution series of cell solutions from ¹ × 10 ⁶ cells / 5 μl to ¹ × 10 ¹ cells / 5 μl were prepared. Thereafter, 5 μl of each cell solution was added to a plate medium in which L-lactic acid (Nacalai Tesque) was added to YPDA agar medium (2% agar added to YPDA medium) to 0, 6, 7, 8 w / v%. Spot and heat retention at 30 ° C. for 24 hours (0 w / v%), 96 hours (6 w / v%), 264 hours (7 w / v%), 264 hours (8 w / v%) to confirm lactic acid resistance did.
The result is shown in FIG.

3. Growth Behavior Test on 6 w / v% Lactic Acid-Added Liquid Medium Each strain was inoculated into a 5 ml test tube YPDA liquid medium and cultured overnight at 30 ° C. and 170 rpm shaking. Next, it inoculated so that it might be set to 0.1 by OD [600nm] to the (phi) 16mm test tube containing 8 ml YPDA liquid culture medium or 6% L-lactic acid addition YPDA liquid culture medium. The culture was shaken at 30 ° C. and 170 rpm, and the OD [600 nm] of the culture solution was measured over time using a culture colorimeter CO8000 Biowave (WPA Biochrom Limited, Cambridge Science park, Cambridge, UK).
The result is shown in FIG.

  In order to verify the lactic acid-producing ability of single or multiple disruption strains of all 15 types Δdse2, Δscw11, Δeaf3, and Δsed1 shown in Table 1, the construction of recombinant vectors and the production of transformed yeast were carried out by the following methods.

  A vector for chromosomal introduction capable of expressing an L-lactate dehydrogenase gene (L-Lactate dehydrogenase gene, hereinafter referred to as L-LDH gene) in this budding yeast was constructed. The vector examined in this example was named pBHPH-LDHKCB vector. Hereinafter, although the detail of the vector construction process in a present Example is demonstrated based on FIG. 6, the procedure of vector construction is not limited to this. Details of a series of operations related to DNA subcloning such as ethanol precipitation and restriction enzyme treatment were in accordance with Molecular Cloning 'A Laboratory Manual second edition' (Maniatis et al., Cold Spring Harbor press. 1989). In addition, a series of enzymes used in the reaction were those manufactured by Takara Bio Inc. unless otherwise limited.

  A prevector pHPH-LDHKCB vector was constructed according to a general DNA subcloning method. The pBTrp-PDC1P-LDHKCB vector constructed in Japanese Patent Application No. 2003-334092 was treated with a restriction enzyme PstI, and the cut ends were blunt-ended by T4 DNA polymerase reaction. Subsequently, the sample treated with the restriction enzyme ClaI was electrophoresed (GelMate 2000, Toyobo), and the vector portion was recovered from the agarose gel by TaKaRa RECOCHIP (Takara Bio). Subsequently, the insert portion was recovered by the same operation as described below. Specifically, a pBHPH-PT vector constructed so that the hygromycin gene can be expressed in yeast is reacted in the order of the restriction enzyme SpeI, T4 DNA Polymerase, and the restriction enzyme ClaI, and this sample is subjected to electrophoresis, TaKaRa RECOCHIP, The insert part was collected.

  The vector and insert fragment collected above were ligated with T4 DNA ligase. For the DNA ligation reaction, LigaFast Rapid DNA Ligation (manufactured by Promega) was used, and the details followed the attached protocol. In addition, Escherichia coli JM109 strain (manufactured by Toyobo Co., Ltd.) was used for transformation of the Ligation reaction solution into competent cells. In either case, colonies were selected under an LB plate containing 100 μg / ml of the antibiotic ampicillin, and colony PCR reaction using each colony was performed to confirm whether the target vector was obtained. The following synthetic DNA (manufactured by Qiagen) was used as a primer for the PCR reaction, and the reaction was performed at 96 ° C for 5 minutes, followed by 25 cycles of 96 ° C for 30 seconds, 55 ° C for 30 seconds, 72 ° C for 60 seconds. The reaction was allowed to proceed at 72 ° C for 5 minutes and then at 4 ° C. After adding a dye to the reaction solution, the presence or absence of the amplified fragment was confirmed by electrophoresis.

In the PCR reaction, the following synthetic DNA was used as a primer.
TDH3P-F; 5'-AGCGTTGAATGTTAGCGCACA-3 '(22mer) (SEQ ID NO: 19)
CYC1T-R; 5′-ACATGCCGTACACCGCGTTTGTAC-3 ′ (22mer) (SEQ ID NO: 20)

  The base sequence of the prosperous vector was determined. Escherichia coli containing the plasmid was cultured in an LB medium at 37 ° C. for 18 hours, and plasmid DNA was prepared by an alkali extraction method. This was subjected to column purification using GFX DNA Purification kit (Amercham Pharmacia Biotech). Next, the DNA concentration was measured with a spectrophotometer Ultra spec 3000 (manufactured by Amercham Pharmacia Biotech), and the DNA base sequence kit BigDye Terminator Cycle Sequencing Ready Reaction Kit (manufactured by PE Applied Bioscience) was used. The reaction sample was set in the base sequence analyzer ABI PRISM 310 Genetic Analyzer (manufactured by PE Applied Biosystem), and the base sequence of the construction vector was determined. The details of how to use the equipment were in accordance with the manual attached to this device.

A map of the constructed plasmid vector pBHPH-LDHKCB is shown in FIG.
A transformant in which the LDH gene was introduced into a budding yeast that was confirmed to be involved in improving lactic acid resistance was prepared as follows. Each yeast was cultured in 5 ml of YPD culture solution at 30 ° C. until the logarithmic growth phase (OD600 nm = 0.8). A competent cell was prepared using a Frozen-EZ Yeast Transformation II kit (manufactured by ZYMO RESEARCH). According to the protocol attached to the kit, the above-described pBHPH-LDHKCB was treated with the restriction enzymes ApaI and SacI, and the gene was introduced into this competent cell. After washing these transformed samples, recovery culture was carried out overnight in 1 ml of YPD medium, and these were collected and washed, and then dissolved in 100 μl of sterilized water. This sample was smeared on a YPD medium having a hygromycin concentration of 150 μg / ml, and a transformant was selected under static culture at 30 ° C. for each.

Each of the obtained colonies was isolated again with a hygromycin selection medium at the same concentration, and a strain stably maintaining the growth ability was used as a transformation candidate strain. Next, these candidate strains were cultured overnight in 2 ml of YPD culture solution, and then genomic DNA was prepared using a genomic DNA preparation kit, GEN Torukun ^TM for Yeast (manufactured by Takara Bio Inc.). PCR analysis was performed using each of the prepared genomic DNAs as a template, and a transformant was confirmed if the presence or absence of the transgene was confirmed. The following synthetic DNA (Qiagen) was used as a primer for the PCR reaction, and the reaction conditions were 96 ° C for 5 minutes after treatment at 96 ° C for 30 seconds, 53 ° C for 30 seconds, and 72 ° C for 60 seconds. And finished at 4 ° C. after 5 minutes at 72 ° C. After adding a dye to the reaction solution, the presence or absence of the amplified fragment was confirmed by electrophoresis.

In the PCR reaction, the following synthetic DNA was used as a primer.
LDH-F; 5′-ATGGCTACTTTGAAAGATC-3 ′ (19mer) (SEQ ID NO: 21)
LDH-R; 5'-TTATTAAAAATTGCAATTCTTTTG-3 '(24mer) (SEQ ID NO: 22)

  Each of the transformed yeasts prepared as described above was inoculated into 5 ml of YPD liquid medium, and cultured overnight at 30 ° C. and 130 rpm, and those having an OD of 600 nm = 1.2 were used as the initial cells. Of these, 2 ml was inoculated into 40 ml of 10% glucose-containing YPD culture solution and fermented in a thermostatic bath (manufactured by Yamato) at 30 ° C. for 3 days under stationary conditions.

The fermentation broth after 72 hours was collected, and the L-lactic acid production (w / v%), ethanol production and glucose residual contained in this solution were measured. For the measurement, a multi-function biosensor BF-5 device (manufactured by Oji Scientific Instruments) was used, and the details of the specifications were in accordance with the attached manual.
The measurement results are shown in FIG.

It is a figure which shows the primer for producing the fragment for destroying each gene. The underlined portion indicates the annealing site with the template plasmid. It is a figure which shows the primer for amplifying each gene used in order to confirm whether each gene is destroyed. It is a figure which shows the primer for pUG-zeosin ^r construction | assembly. It is a construction figure of plasmid p3008 (pUG-CgLEU2). It is a construction figure of plasmid p3009 (pUG-CgHIS3). It is a figure which shows the relationship between the lactic acid density | concentration in a culture medium, and formation of a colony. It is a figure which shows the time-dependent change of the proliferation of each gene disruption strain. It is a figure which shows the L-lactic acid production amount of each gene disruption strain | stump | stock which has lactic acid production ability. It is a figure which shows the construction method of a plasmid vector. It is a figure which shows the map of plasmid vector pBHPH-LDHKCB.

  Sequence number 1-22: Synthetic DNA: Primer

Claims (4)

Δdse2Δeaf3, Δscw11Δeaf3, Δdse2Δeaf3Δsed1, out bud yeast, characterized in that Derutascw11derutaeaf3derutased1, or a multi-gene-disrupted strain is Derutadse2derutascw11derutaeaf3derutased1. Genes involved in lactic acid production is introduced, and Δdse2Δeaf3, Δdse2Δscw11, Δscw11Δsed1, Δeaf3Δsed1, out bud yeast, characterized in that Derutascw11derutaeaf3derutased1, or a multi-gene-disrupted strain is Derutadse2derutascw11derutaeaf3derutased1. A method of imparting lactic acid resistance to a budding yeast by disrupting a gene so that it becomes Δdse2Δeaf3, Δscw11Δeaf3, Δdse2Δeaf3Δsed1, Δscw11Δeaf3Δsed1, or Δdse2Δscw11Δeaf3Δsed1 in budding yeast. In budding yeast, a method for disrupting the gene to produce Δdse2Δeaf3, Δdse2Δscw11, Δscw11Δsed1, Δeaf3Δsed1, Δscw11Δeaf3Δsed1, or Δdse2Δscw11Δeaf3Δsed1 and introducing a gene involved in lactic acid production into lactic acid-producing yeast.

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