JP2012191885A - Method for producing ethanol - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for producing ethanol in high productivity by using yeast.SOLUTION: The method for the ethanol using the gene-disrupted strain of the yeast formed by destroying a gene encoding a stationary phase transfer promoting factor.

Description

  The present invention relates to a method for producing ethanol. More specifically, the present invention relates to a method for producing ethanol including liquor such as sake, beer and wine, bioethanol and the like using highly fermentable yeast.

  In general, ethanol contained in bioethanol for liquor and fuel is produced by fermenting sugars such as glucose with yeast belonging to the genus Saccharomyces.

  At first, yeast cells during fermentation proliferate and increase the number of cells while consuming the nutrients in the environment vigorously, but when the nutrients are depleted and the environment becomes unsuitable for growth, it is detected. Thus, the signal transduction pathway for suppressing growth works quickly, and the state shifts to a state called stationary phase where growth has stopped. A factor that plays a central role in this signal transduction pathway is a factor that promotes stationary phase transition (referred to as “stationary phase transition promoting factor”). So far, Rim15p is known as a stationary phase transition promoting factor.

  Rim15p is a protein kinase belonging to the PAS kinase family. It is activated by a signal transduction pathway via TOR kinase, protein kinase A, etc., and enters the stationary phase by phosphorylating Igo1p and Igo2p RNA binding proteins. Non-Patent Documents 1 and 2 describe that the expression of genes necessary for the control is positively controlled. Therefore, Igo1p and Igo2p are also included in the range of stationary phase transition promoting factors.

  Genes that are regulated by Rim15p are diverse, including carbon metabolism, lipid metabolism, mitochondrial proteins, cell defense reactions, and intracellular signal transduction, and Rim15p is responsible for these genes in response to the extracellular environment. It is thought that it contributes to stationary phase transition by changing expression comprehensively.

  Therefore, when the function of Rim15p becomes abnormal, a defect occurs in switching from the growth phase to the stationary phase. For this reason, there is an effect on life support after yeast has finished growing. For example, Non-Patent Document 3 reports that RIM15 gene-disrupted strains have a short lifetime over time of yeast cells.

Pedruzzi I., Dubouloz F., Cameroni E., Wanke V., Roosen J., Winderickx J., De Virgilio C., Mol. Cell, 12 (6), 1607-1613 (2003). Talarek N., Cameroni E., Jaquenoud M., Luo X., Bontron S., Lippman S., Devgan G., Snyder M., Broach JR, De Virgilio C., Mol. Cell, 38, 345-355 ( 2010). Wei M., Fabrizio P., Hu J., Ge H., Cheng C., Li L., Longo V. D., PLoS Genet., 4 (1), 139-149 (2008).

  In ethanol production using yeast, increasing the ethanol production rate is extremely important for enhancing the productivity of ethanol fermentation. However, from the viewpoint of ethanol production rate, there has never been a concept of focusing on switching from the growth phase of yeast to the stationary phase, and it was modified by introducing mutations into the RIM15 gene encoding the stationary phase transition promoting factor. However, there are no known examples of high fermentability.

  An object of the present invention is to provide a method for producing ethanol with high productivity using yeast.

  From the conventional knowledge, the present inventors continue the ethanol fermentation of yeast even in the stationary phase after the growth is stopped. Therefore, for efficient ethanol fermentation, the stationary phase transition is normally performed, and the yeast in the fermentation environment We thought it necessary to maintain a long life. In order to prove this, a fermentation test was conducted using a transformed yeast in which the RIM15 gene was disrupted, and the effect of the gene disruption on ethanol fermentation was analyzed. It was found that the converted yeast exhibits a remarkably high fermentability. This was an unexpected result from conventional knowledge. Therefore, the present inventors consider that this phenomenon can be used to improve the ethanol fermentability of yeast, and complete the present invention by conducting further detailed research on the disruption of RIM15 gene and ethanol fermentation. It came to.

That is, the gist of the present invention is as follows.
The present invention relates to a method for producing ethanol using a yeast gene disruption strain in which a gene encoding a stationary phase transition promoting factor is disrupted.

  According to the present invention, ethanol can be produced at a significantly faster production rate than before in ethanol fermentation of yeast.

FIG. 1 is a graph showing carbon dioxide generation rate (left) and total carbon dioxide generation amount (right) when sake production is performed using the parent strain laboratory yeast BY4743 strain and gene disruption strain (BY4743 Δrim15). is there. The dotted line shows the parent strain, and the solid line shows the RIM15 gene disrupted strain. FIG. 2 is a graph showing the carbon dioxide generation rate (left) and the total carbon dioxide generation amount (right) when an ethanol fermentation test was conducted using the parent strain laboratory yeast BY4743 strain and gene disruption strain (BY4743 Δrim15). It is. The dotted line shows the parent strain, and the solid line shows the RIM15 gene disrupted strain. Fig. 3 shows the carbon dioxide generation rate (left) and total carbon dioxide generation amount (right) when the ethanol fermentation test was conducted using the parent strains laboratory yeast BY4743 strain and gene disruption strains (BY4743 Δigo1 and BY4743 Δigo2). FIG. The white circle indicates the parent strain, the white square indicates the BY4743 Δigo1 strain, and the black square indicates the BY4743 Δigo2 strain. FIG. 4 shows the carbon dioxide generation rate (upper left) when ethanol was produced from a waste molasses medium using the parent strain NCYC3233-27c derived from bioethanol yeast and the gene disruption strain (NCYC3233-27c rim15Δ strain). It is a figure which shows the time passage (lower stage) of carbon dioxide total generation amount (upper stage right) and ethanol concentration. In both cases, the dotted line indicates data of the parent strain, and the solid line indicates data of the RIM15 gene disrupted strain.

  The ethanol production method of the present invention is a method for producing ethanol using yeast, and uses a gene encoding a stationary phase transition promoting factor, for example, at least one disrupted strain of RIM15 gene, IGO1 gene and IGO2 gene. Has major features.

  The RIM15 gene is described on the SGD (Saccharomyces Genome Database) website (http://genome-www.stanford.edu/Saccharomyces). Based on this sequence, PCR primers are designed to disrupt the gene. And site-specific mutations can be made. For example, RIM15 + URA3-Fw (SEQ ID NO: 4 in the sequence listing), RIM15 + URA3-Rv (SEQ ID NO: 5 in the sequence listing), and the like are exemplified as DNA primers used to destroy RIM15.

  The yeast in the present invention is a yeast that can be used for ethanol fermentation belonging to the genus Saccharomyces, and includes, for example, laboratory yeast, sake yeast, wine yeast, beer yeast, shochu yeast, baker's yeast, and bioethanol yeast. It is done. The laboratory yeast is preferably BY4743 strain belonging to Saccharomyces cerevisiae. The bioethanol yeast is preferably NCYC3233 strain belonging to Saccharomyces cerevisiae. Examples of ethanol include sake or bioethanol.

  In this specification, “disruption” refers to genetic manipulation that causes the function of the disrupted gene to be lost or inactivated, such as genetic manipulation that causes loss of all genes in the target gene region or normal operation. This refers to genetic manipulation that deletes or mutates a site necessary for function. Specifically, genetic manipulation that completely loses the RIM15 gene encoding the stationary phase transition promoting factor is exemplified. A method for performing such genetic manipulation is not particularly limited, and a known method can be used. For example, mutation by treatment with a mutation agent, gene disruption using genetic engineering, site-specific mutation using genetic engineering, etc. Can be mentioned. In the present invention, a gene disrupted strain in which disruption has occurred due to spontaneous mutation can also be used.

  When performing the above-described genetic manipulation, an auxotrophic marker such as an amino acid, a drug resistance marker, or the like may be used as a selection marker.

  The sequences of the IGO1 gene and the IGO2 gene are described on the SGD (Saccharomyces Genome Database) website (http://genome-www.stanford.edu/Saccharomyces). Therefore, a disrupted strain of such a gene can be obtained by the same method as the RIM15 gene described above.

  Representative strains of disrupted genes encoding the stationary phase transition promoting factor include BY4743 Δrim15 strain and NCYC3233-27c rim15Δ strain. The former is available as Y37281 from EUROSCARF (http://web.uni-frankfurt.de/fb15/mikro/euroscarf/index.html), and the latter is Deposit number NITE P-1017 (Deposit date: December 13, 2010). Furthermore, BY4743 Δigo1 strain is exemplified as a representative strain of the disrupted strain in which the IGO1 gene is disrupted, and BY4743 Δigo2 strain is exemplified as a representative strain of the disrupted strain in which the IGO2 gene is disrupted. These are available as EUR32055 and Y37298 respectively from EUROSCARF (http://web.uni-frankfurt.de/fb15/mikro/euroscarf/index.html). When the desired gene-disrupted strain is deposited with the depositary as described above, such a deposited strain can be used.

  The ethanol production method of the present invention is carried out using the yeast gene-disrupted strain described above, but known methods and conditions can be employed except that the yeast is a specific one.

  EXAMPLES Hereinafter, although this invention is demonstrated based on an Example, this invention is not restrict | limited at all by these Examples.

[Example 1]
Sake production test using Saccharomyces cerevisiae BY4743 strain and RIM15 gene-disrupted strain which was the parent strain was conducted. The BY4743 strain and BY4743 Δrim15 strain are both available from EUROSCARF as Y20000 and Y37281, respectively.

  Sake was produced using the parent strains BY4743 and BY4743 Δrim15 in the following manner.

One-stage charging was performed by mixing 40 g of rice, 10 g of glutinous rice, 80 mL of water, and 17.8 μL of 90% lactic acid. Alpha rice with a 70% polishing rate was used as the hanging rice, and dried rice with a 70% polishing rate was used as the polished rice. Each yeast is cultured overnight in a YPD medium (containing 1% yeast extract, 2% peptone, 2% glucose) and then washed with sterilized distilled water so that the yeast count becomes 1 × 10 ⁷ cells / mL. Was added at the time of charging. The fermentation temperature was 15 ° C. The preparation test was repeated four times for each strain. After the preparation, the amount of carbon dioxide generated during fermentation was determined using a fermentation monitor device (Farmograph II manufactured by Ato Co., Ltd.), and the average amount of carbon dioxide generated for each strain was calculated. The results are shown in FIG.

  For the peak value of the carbon dioxide generation rate and the total amount of carbon dioxide generation, a significant difference test was also performed between the parent strain and the gene-disrupted strain. Furthermore, on the 20th day after preparation, the ethanol concentration in sake recovered by centrifugation was measured, and the average ethanol concentration was calculated for each strain. In addition, the obtained average ethanol concentration was also subjected to a significant difference test between the parent strain and the gene-disrupted strain. The ethanol concentration was measured using a gas chromatograph GC-17A manufactured by Shimadzu Corporation.

  RIM15 gene-disrupted strain has a higher peak carbon dioxide generation rate during fermentation than the parent strain (parent strain: 14.7 ± 0.8mg / 30min, RIM15 gene-disrupted strain: 20.5 ± 0.7mg / 30min, risk rate less than 0.1% And significantly larger than the parent strain), and the total amount of carbon dioxide generation was also large (parent strain: 5.60 ± 0.00 g, RIM15 gene disruption strain: 10.07 ± 0.24 g, significantly higher than the parent strain with a risk rate of less than 0.1%). Furthermore, the final ethanol concentration is 11.2 ± 0.3% for the parent strain, whereas the RIM15 gene disruption strain is significantly higher at 17.0 ± 0.2% (significantly higher than the parent strain at a risk rate of less than 0.1%). When the RIM15 gene disrupted strain was used, it was found that the ethanol fermentation rate was fast and the ethanol productivity was excellent.

[Example 2]
An ethanol fermentation test was performed using the above Saccharomyces cerevisiae BY4743 strain and BY4743 Δrim15 strain.

An ethanol fermentation test using 50 mL of a high concentration glucose-containing YPD medium (yeast extract 1%, peptone 2%, glucose 20%) was carried out. Each yeast was cultured overnight in a YPD medium by shaking and then added to a high-concentration glucose-containing YPD medium so that the yeast density was OD ₆₆₀ = 0.1 / mL, followed by stationary culture for 5 days. The fermentation temperature was 30 ° C. The fermentation test was repeated four times for each strain. After the start of culture, the amount of carbon dioxide generated during fermentation was determined using a fermentation monitor device (Farmograph II manufactured by Ato Co., Ltd.), and the average amount of carbon dioxide generated for each strain was calculated. The results are shown in FIG. The peak value of carbon dioxide generation rate and the total amount of carbon dioxide generation were also tested for significant differences between the parent strain and the gene-disrupted strain.

  RIM15 gene-disrupted strain has a higher peak carbon dioxide generation rate during fermentation than the parent strain (parent strain: 11.3 ± 1.5mg / 15min, RIM15 gene-disrupted strain: 16.3 ± 2.7mg / 15min, less than 5% risk rate Because the total amount of carbon dioxide generated is significantly larger than the parent strain (parent strain: 2.74 ± 0.13g, RIM15 gene disruption strain: 3.23 ± 0.21g, significantly larger than the parent strain with a risk rate of less than 0.1%), When the RIM15 gene disrupted strain was used, it was found that the ethanol fermentation rate was fast and the ethanol productivity was excellent.

Example 3
An ethanol fermentation test was performed using Saccharomyces cerevisiae BY4743 strain, and the IGO1 gene-disrupted strain and IGO2 gene-disrupted strain, each of which was the parent strain. The BY4743 strain, BY4743 Δigo1 strain, and BY4743 Δigo2 strain are all available from EUROSCARF as Y20000, Y32055, and Y37298, respectively.

  An ethanol fermentation test was performed under the same conditions as in Example 2 and repeated three times for each strain. After the start of culture, the amount of carbon dioxide generated during fermentation was determined using a fermentation monitor device (Farmograph II manufactured by Ato Co., Ltd.), and the average amount of carbon dioxide generated for each strain was calculated. The results are shown in FIG. About the peak value of the carbon dioxide generation rate, the significant difference test of the value in a parent strain and a gene-disrupted strain was also performed.

  IGO1 gene disruption strain and IGO2 gene disruption strain have a larger peak value of carbon dioxide generation rate during fermentation than parent strain (parent strain: 301 ± 38mg / 6h, IGO1 gene disruption strain: 359 ± 23mg / 6h, IGO2 gene disruption Strains: 420 ± 71mg / 6h, significantly higher than the parent strain with a risk rate of less than 5%) Because of the large amount of carbon dioxide generation (parent strain: 3.06 ± 0.28g, IGO1 gene disruption strain: 3.41 ± 0.13g, IGO2 gene-disrupted strain: 3.55 ± 0.08 g), IGO1 gene-disrupted strain and IGO2 gene-disrupted strain were found to have a high ethanol fermentation rate and excellent ethanol productivity. The results of Examples 2 and 3 strongly suggest that the yeast gene-disrupted strain in which the gene encoding the stationary phase transition promoting factor is disrupted exhibits excellent ethanol productivity.

Example 4
A haploid uracil auxotrophic strain derived from Saccharomyces cerevisiae diploid bioethanol yeast strain NCYC3233 was used as a parent strain, and a RIM15 gene-disrupted strain was prepared, and an ethanol production test was conducted. The NCYC3233 strain is available from NCYC (National Collection of Yeast Cultures; http://www.ncyc.co.uk/).

[Create NCYC3233 haploid strain]
First, in order to facilitate genetic manipulation, a haploid strain derived from NCYC3233 strain known as diploid was prepared. After culturing NCYC3233 strain on YPD agar medium at 30 ° C overnight, transfer the cells to sporulation agar medium (containing 1% potassium acetate) and culture at 30 ° C for about 3 days to form haploid spores. Induced. After confirming spore formation with a microscope, spore separation was performed using a micromanipulator. It was confirmed that a normal haploid was obtained by PCR and flow cytometry analysis of the MAT locus. Among these, one strain having good growth was selected and named NCYC3233-27c strain. This strain was deposited with the Patent Microorganisms Depositary Center of the National Institute of Technology and Evaluation as Deposit Number NITE P-1061 (Deposit Date: January 19, 2011).

[Creation of NCYC3233 haploid uracil-requiring strain]
Next, using the obtained haploid strain as a host, uracil requirement was imparted as a selection marker. In this example, in order to confer uracil requirement, PCR for URA3 gene disruption is performed by PCR using the genomic DNA of laboratory yeast BY4743 strain having ura3Δ0 allele that has completely deleted the ORF region of URA3 gene essential for uracil synthesis as a template. A DNA fragment was created. Specifically, PCR was performed using primers URA3 (-2045) -Fw (SEQ ID NO: 1 in the sequence listing) and URA3 downstream-Rv (SEQ ID NO: 2 in the sequence listing) to amplify the ura3Δ0 allele of BY4743 strain. NCYC3233-27c strain was transformed with this PCR product, and agar medium containing 5-FOA (yeast nitrogen base without amino acids 0.67%, complete supplement mixture 0.79g / L, 5-FOA 0.1%, glucose 2%) Colonies that grow on were selected.

  Genomic DNA is extracted from each of these colonies, PCR is performed using primers URA3 (-2045) -Fw and URA3 downstream-Rv, and the size of the resulting product is measured to destroy the URA3 gene. It was confirmed. Of these strains, the URA3 gene disruption was confirmed for growth on SD-Ura agar medium (yeast nitrogen base without amino acids 0.67%, complete supplement mixture minus uracil 0.77g / L, containing 2% glucose). Confirmed to show a phenotype of requirement. This strain was named NCYC3233-27c ura3Δ0 strain, and was deposited with the Patent Microorganism Deposit Center of the National Institute of Technology and Evaluation as Deposit Number NITE P-1016 (Deposit Date: December 13, 2010).

[Create RIM15 gene disruption strain]
The RIM15 gene was disrupted by replacing the URA3 marker gene with the ORF region of the RIM15 gene. Specifically, using the Saccharomyces cerevisiae genome carrying the URA3 gene as a template, PCR was performed using primers URA3 upstream-Fw (SEQ ID NO: 3 in the sequence listing) and URA3 downstream-Rv, and the ORF region of the URA3 gene A DNA fragment containing the full length of was amplified. Next, using this PCR product as a template, PCR was performed using primers RIM15 + URA3-Fw (SEQ ID NO: 4 in the sequence listing) and RIM15 + URA3-Rv (SEQ ID NO: 5 in the sequence listing), and the marker URA3 On both sides, DNA fragments for gene disruption having upstream and downstream DNA sequences adjacent to the ORF of RIM15 were prepared. The PCR product was used to transform the NCYC3233-27c ura3Δ0 strain, and colonies that grew on the SD-Ura agar medium were selected.

  Genomic DNA was extracted from each of these colonies, PCR was performed using primers RIM15 upstream-Fw (SEQ ID NO: 6 in the sequence listing) and URA3 ORF-Rv (SEQ ID NO: 7 in the sequence listing), and the size of the resulting product Was measured to confirm that the RIM15 gene was disrupted. This strain was named NCYC3233-27c rim15Δ strain, and was deposited with the Patent Microorganism Deposit Center of the National Institute of Technology and Evaluation as Deposit Number NITE P-1017 (Deposit Date: December 13, 2010).

[Production of ethanol]
Using the parent strain NCYC3233-27c which is a haploid strain derived from bioethanol yeast and the NIMC3233-27c rim15Δ strain which is the RIM15 gene disruption strain obtained above, ethanol was produced by the method described below.

An ethanol fermentation test using 50 mL of waste molasses medium (Ken molasses stock solution adjusted to Brix 24.8% with distilled water and added with 0.025% ammonium sulfate) was performed. Each yeast was cultured overnight in a YPD medium after shaking, washed with sterilized distilled water, added to both media so that the yeast density was OD ₆₆₀ = 0.1 / mL, and cultured with shaking for 54 hours. The fermentation temperature was 35 ° C. The fermentation test was repeated 4 times for each strain. After the start of culture, the amount of carbon dioxide generated during fermentation was determined using a fermentation monitor device (Farmograph II manufactured by Ato Co., Ltd.), and the average amount of carbon dioxide generated for each strain was calculated. The time when the carbon dioxide generation rate reached 0 was regarded as the end of fermentation. The significant difference test of the value in the parent strain and the gene-disrupted strain was also performed for the carbon dioxide generation rate and the peak value at each time point. In addition, 12, 24, 30, 36, and 54 hours after the start of the culture, the ethanol concentration in the culture supernatant collected by centrifugation was measured, and the average ethanol concentration and standard deviation were calculated for each strain. In addition, the obtained average ethanol concentration was also subjected to a significant difference test between the parent strain and the gene-disrupted strain. The ethanol concentration was measured using a gas chromatograph GC-17A manufactured by Shimadzu Corporation. The results are shown in FIG.

  The RIM15 gene-disrupted strain shows the same carbon dioxide generation rate as the parent strain at the beginning of fermentation, but the peak value of the carbon dioxide generation rate is larger than that of the parent strain (parent strain: 108.3 ± 1.6 mL / h, RIM15 gene-disrupted strain: 113.9 ± 3.7mL / h, significantly higher than the parent strain at a risk rate of less than 5%, and the rapid decrease in carbon dioxide generation rate from the peak value was suppressed (carbon dioxide generation of RIM15 gene-disrupted strain in 16 to 30 hours) Speed is significantly greater than the parent strain with a risk rate of less than 5%). In addition, since the time taken to complete the fermentation is short (parent strain: 53.3 ± 0.8 hours, RIM15 gene disruption strain: 43.8 ± 0.7 hours, significantly less than the parent strain at a risk rate of less than 0.1%), the RIM15 gene disruption strain It was found that ethanol fermentation rate was high and ethanol productivity was excellent.

  As a result of testing the difference in mean value for ethanol concentration, the ethanol concentration of RIM15 gene-disrupted strain was significantly higher than the parent strain at a risk rate of less than 0.1% at 24, 30, and 36 hours after the start of culture. (Parent strain: 6.04 ± 0.06vol% (24 hours later) /7.24±0.03vol% (30 hours later) /8.23±0.06vol% (36 hours later), RIM15 gene disrupted strain: 6.80 ± 0.06vol% (24 hours later) ) /8.36±0.05 vol% (after 30 hours) /9.16±0.07 vol% (after 36 hours)), it was confirmed that the ethanol fermentation rate was fast and the ethanol productivity was excellent.

  The production method of the present invention is suitably used for producing alcoholic beverages such as sake, beer, wine, shochu, and ethanol including bioethanol.

SEQ ID NO: 1 in the Sequence Listing is a primer for URA3 disruption DNA preparation / URA3 amplification.
Sequence number 2 of a sequence table is a primer for DNA preparation and URA3 amplification for URA3 destruction.
Sequence number 3 of a sequence table is a primer for URA3 amplification.
Sequence number 4 of a sequence table is a primer for DNA preparation for RIM15 destruction.
Sequence number 5 of a sequence table is a primer for DNA preparation for RIM15 destruction.
Sequence number 6 of a sequence table is a primer for RIM15 destruction confirmation.
Sequence number 7 of a sequence table is a primer for RIM15 destruction confirmation.

Claims (6)

  A method for producing ethanol using a yeast gene disruption strain in which a gene encoding a stationary phase transition promoting factor is disrupted.   The production method according to claim 1, wherein the gene encoding the stationary phase transition promoting factor is at least one selected from the group consisting of the RIM15 gene, the IGO1 gene, and the IGO2 gene.   The production method according to claim 1 or 2, wherein the yeast is at least one selected from the group consisting of laboratory yeast, sake yeast, wine yeast, beer yeast, shochu yeast, baker's yeast, and bioethanol yeast.   The production method according to claim 3, wherein the laboratory yeast is BY4743 strain.   The manufacturing method of Claim 3 whose bioethanol yeast is NCYC3233 strain | stump | stock.   The manufacturing method of any one of Claims 1-5 whose ethanol is sake or bioethanol.

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