JP6994821B2 - Reduction of ethanol production in continuous culture of Saccharomyces cerevisiae - Google Patents

Description

The present invention mainly relates to a method for producing Saccharomyces cerevisiae cells in continuous culture.

Saccharomyces cerevisiae, a budding yeast, is an indispensable microorganism in the brewing industry such as beer and wine and the food industry such as yeast extract. However, although S. cerevisiae supplies sufficient oxygen, the addition of glucose suppresses oxygen respiration (De Deken, R. H.). This phenomenon is called the club tree effect, and it is known that in continuous culture of S. cerevisiae, when the dilution ratio is increased, ethanol fermentation is performed from the formation of biomass (Postma E et al.).

However, the details of the mechanism of this club tree effect have not yet been clarified.
Therefore, in order to produce the target substance without producing ethanol, it was necessary to limit the inflow of sugar in fed-batch culture or to culture at a low dilution rate in continuous culture, resulting in poor production efficiency. ..

Although the club tree effect has been observed in other microorganisms (Serra et al.), S. cerevisiae has been actively studied as a model organism focusing on the club tree effect in an aerobic state. For example, there is a report that overexpression of GPD1 and GPD2, which encode the isozyme of glycerol 3-phosphate dehydrogenase, increased glycerol and decreased ethanol (Cambon B et al ..). In addition, when the inflow of glucose is large, the redox balance of NADH / NAD + is disturbed to produce ethanol and glycerol, which are by-products. Therefore, NADH oxidase, which forms water, was expressed and ethanol was reduced. There are also reports (Heux S et al .., Vemuri GN et al.). However, since these methods use genetic recombination technology, they are difficult to apply to the brewing industry and the food industry.

In contrast to the approach using gene recombination technology, we are working to elucidate the club tree effect using flux analysis by taking advantage of the advantage of being able to sample in a steady state in research using a continuous culture system (Frick O et al., Kajihata S et al.). In the continuous culture, the dilution ratio (D) is the supply amount of the liquid medium / the volume of the culture solution per hour. S. cerevisiae consumes glucose completely and does not produce ethanol when the dilution ratio (D) is less than 0.3 under sufficient aerobic conditions. However, it has been reported that ethanol is produced when D ≥ 0.3 (Van Hoek P et al.). Flux analysis in a steady state has been advanced by utilizing the switching from aerobic respiration to ethanol fermentation by changing the dilution rate, but it has not been possible to reduce ethanol by using this analysis result. ..

Metabolomics is a method for comprehensively measuring metabolites that are the result of genomic information and is closely related to the macrophenotype in close proximity. Therefore, it is possible to obtain useful information that contributes to the improvement of strain performance by considering the production yield, production rate, etc. as quantitative phenotypes in the production of useful substances by microorganisms and performing metabolome analysis. (Putri SP et al.)

De Deken RH .: The Crabtree effect: a regulatory system in yeast.: J Gen Microbiol. 1966 Aug; 44 (2): 149-56. Postma E1, Verduyn C, Scheffers WA, Van Dijken JP.: Enzymic analysis of the crabtree effect in glucose-limited chemostat cultures of Saccharomyces cerevisiae.: Appl Environ Microbiol. 1989 Feb; 55 (2): 468-77. Serra A, Strehaiano P, Taillandier P .: characterization of the metabolic shift of Saccharomyces bayanus var. Uvarum by continuous aerobic culture. Appl Microbiol Biotechnol. 2003 Oct; 62 (5-6): 564-8. Cambon B1, Monteil V, Remize F, Camarasa C, Dequin S .: Effects of GPD1 overexpression in Saccharomyces cerevisiae commercial wine yeast strains lacking ALD6 genes. Appl Environ Microbiol. 2006 Jul; 72 (7): 4688-94. Heux S1, Cachon R, Dequin S .: Cofactor engineering in Saccharomyces cerevisiae: Expression of a H2O-forming NADH oxidase and impact on redox metabolism.: Metab Eng. 2006 Jul; 8 (4): 303-14. Vemuri GN, Eiteman MA, McEwen JE, Olsson L, Nielsen J.: Increasing NADH redox reduces overflow metabolism in Saccharomyces cerevisiae .: Proc Natl Acad Sci U S A. 2007 Feb 13; 104 (7): 2402-7. Frick O1, Wittmann C .: characterization of the metabolic shift between oxidative and fermentative growth in Saccharomyces cerevisiae by comparative 13C flux analysis .: Microb Cell Fact. 2005 Nov 3; 4:30. Kajihata S, Matsuda F, Yoshimi M, Hayakawa K, Furusawa C, Kanda A, Shimizu H .: 13 C-based metabolic flux analysis of Saccharomyces cerevisiae with a reduced Crabtree effect .: J Biosci Bioeng. 2015 Aug; 120 (2): 140-4. Van Hoek P1, Van Dijken JP, Pronk JT .: Effect of specific growth rate on fermentative capacity of baker's yeast.: Appl Environ Microbiol. 1998 Nov; 64 (11): 4226-33. Putri SP1, Nakayama Y, Matsuda F, Uchikata T, Kobayashi S, Matsubara A, Fukusaki E .: Current metabolomics: practical applications .: J Biosci Bioeng. 2013 Jun; 115 (6): 579-89. Yoshida R, Tamura T, Takaoka C, Harada K, Kobayashi A, Mukai Y, Fukusaki E .: Metabolomics-based systematic prediction of yeast lifespan and its application for semi-rational screening of ageing-related mutants.: Aging Cell. 2010 Aug 9 (4): 616-25. Teoh ST1, Putri S1, Mukai Y2, Bamba T1, Fukusaki E1: A metabolomics-based strategy for identification of gene targets for phenotype improvement and its application to 1-butanol tolerance in Saccharomyces cerevisiae.: Biotechnol Biofuels. 2015 Sep 15; 8: 144. 144. Ohta E1, Nakayama Y1, Mukai Y2, Bamba T1, Fukusaki E3 .: Metabolomic approach for improving ethanol stress tolerance in Saccharomyces cerevisiae.: J Biosci Bioeng. 2015 Sep 3. pii: S1389-1723 (15) 00303-5. Mitsunaga H, Meissner L, Palmen T, Bamba T, Buechs J, Fukusaki E .: Metabolome analysis reveals the effect of carbon catabolite control on the poly (γ-glutamic acid) biosynthesis of Bacillus licheniformis ATCC 9945 .: J Biosci Bioeng. 2015 Sep 23. pii: S1389-1723 (15) 00327-8. Noguchi S, Putri SP, Lan EI, Lavina WA, Dempo Y, Bamba T, Liao JC, Fukusaki E .: Quantitative target analysis and kinetic profiling of acyl-CoAs reveal the rate-limiting step in cyanobacterial 1-butanol production.: Metabolomics . 2016; 12:26.

By analyzing samples obtained from different conditions by metabolomics so far, the inventors of the present application have extended the life span of yeast (Yoshida R et al.) And improved resistance to 1-butanol and ethanol (Teoh ST et al.). , Ohta E et al.), Poly (γ-glutamic acid) and 1-butanol productivity improvement (Mitsunaga H et al., Noguchi S et al.). Using this finding, we found a way to reduce ethanol production in continuous cultures due to the club tree effect of S. cerevisiae by metabolomics, and found that reducing ethanol production in continuous cultures with high dilution in S. cerevisiae. Make it an issue. In addition, it is also an issue to efficiently produce bacterial cells and target substances.

Samples were taken from continuous cultures of S. cerevisiae at various dilutions and metabolomics were performed. That is, metabolites with large changes were identified when the dilution rate was changed from aerobic respiration to ethanol fermentation in continuous culture by metabolomics for comprehensive analysis of metabolites.

As a result, when fumaric acid or malic acid among the metabolites was added to the medium during continuous culture, ethanol could be reduced and cell production could be increased in continuous culture with a high dilution rate. The present invention has succeeded for the first time in reducing ethanol production and increasing cell production by adding metabolites identified using metabolomics in continuous culture with a high dilution rate of S. cerevisiae. Is.

That is, the present invention
(1) In culturing Saccharomyces cerevisiae yeast, a method for increasing cell production by adding a metabolite having a large change when changing from aerobic respiration to ethanol fermentation to the medium.
(2) In the continuous culture or fed-batch culture of Saccharomyces cerevisiae yeast, the above-mentioned yeast culture method in which fumaric acid or malic acid is added to a medium is provided.

According to the present invention, in the continuous culture of S. cerevisiae, by adding malic acid or fumaric acid to the medium, the ethanol concentration in the culture solution can be reduced and the cell concentration can also be increased. ..

S. cerevisiae culture results at different dilutions in continuous culture Results of principal component analysis based on metabolome data obtained from continuous cultures at different dilution rates Score plot (contribution rate: PC1 = 62.6%, PC2 = 16.2%) Results of principal component analysis based on metabolome data obtained from continuous cultures at different dilutions Loading data for PC1 Metabolite map identified by analysis (1. D = 0.05, 2. D = 0.1, 3. D = 0.2, 4. D = 0.3) Ethanol concentration in continuous culture with D = 0.3 when metabolites were added Cell concentration in continuous culture with D = 0.3 when metabolites were added Intracellular concentration of Ornithine in continuous culture with metabolite addition Intracellular concentration of Trehalose in continuous culture with metabolite addition

Hereinafter, the present invention will be specifically described.
The strain used in the present invention may be any Saccharomyces cerevisiae.

The basic medium used in the present invention is not particularly limited as long as the yeast of the present invention can grow, and for example, a normal medium used for culturing yeast can be used.
Specific examples thereof include, but are not limited to, YDP medium, SD medium, and SG medium. As the medium, for example, a medium containing a carbon source, a nitrogen source, a phosphoric acid source, a sulfur source, and other components selected from various organic components and inorganic components can be used, if necessary. The type and concentration of the medium component may be appropriately set depending on the strain to be used.

In the present invention, metabolomics analysis as shown in Examples is performed to identify metabolites that change significantly when changing from aerobic respiration to ethanol fermentation, and by adding them to the medium, ethanol production is reduced. The compounds that can be screened can be.
Specifically, malic acid and / or fumaric acid is added to the basic medium as described above so as to be 50 to 300 mg / L in the medium, and fed-batch culture or continuous culture is performed.

In the case of continuous culture, the dilution ratio D of the medium may be appropriately selected so that the target substance can be obtained most efficiently. For example, the value of D is preferably 0.1 to 0.5, more preferably 0.25 to 0.40, and even more preferably 0.30 to 0.35. The dilution ratio (D) is the supply amount of the liquid medium / the volume of the culture solution per hour.

The culture temperature may be selected from a temperature at which the target substance can be obtained most efficiently, and is, for example, 25 to 35 ° C, preferably 28 to 32 ° C. The culture time is not particularly limited as long as it is a continuous culture.

Hereinafter, the present invention will be specifically described with reference to Examples.
The present invention is not limited to these.

S. cerevisiae NBRC101557 was obtained from the Biological Resource Center, NITE (NBRC). Preculture is 100 mL YPD medium [10 g Dried yeast extract (Wako Pure Chemical Industries, Ltd., Osaka, Japan), 20 g HIPOLYPEPTON (Wako Pure Chemical Industries, Ltd., Osaka, Japan), 20 g D-Glucose (Wako) Pure Chemical Industries, Ltd., Osaka, Japan)] at 30 ° C for 18 hours. In this culture, 1 L of the culture medium was inoculated into 2 L of Jar-fermenter (Mitsuwa Frontech) so that the initial OD ₆₀₀ was 0.1, and batch culture was started. The medium composition of the culture solution is 10 g / L glucose, 1.5 g KH ₂ PO ₄ , 0.5 g / L DDL 4.7H ₂ O, 0.06 g / L CaCl ₂ , 5 g / L (NH ₄ ) ₂ SO ₄ , _0.4 . g / LK ₂ SO ₄ , 0.1 mg / L Biotin, 1.5 g / L D-pantothenic acid hemicalcium salt, 60 mg / L myo-inositol, 3 mg / L Pyridoxine Hydrochloride, 14 mg / L Thiamine Hydrochloride, 0.2 mg / L CuSO 4.5H ₂ O, ₄ mg / L ZnSO _{4.7H 2} O, 10 mg / L FeSO _{4.7H 2} O (adjusted to pH 5.0 with 4 M NaOH). In the addition test of metabolites described later, Trehalose Dihydrate, L-Ornithine Monohydrochloride, Fumaric acid, and Malic acid were added to the medium at 100 mg / L in addition to the above. All of these were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan) and Sigma (St. Louis, USA). The culture temperature was 30 ° C., the stirring rate was 700 rpm, the aeration was performed at 1 L / min, and the pH was controlled to 5.0 with 4 M NaOH. Continuous culture was started at each dilution rate in the latter half of the logarithmic growth phase of batch culture, and after confirming that the steady state was reached, a sample was obtained.

-Measurement of biomass and ethanol The dry mycelium weight (CDM) was measured after washing twice, centrifuging (6000 g, 5 min, 4 ° C) and allowing to stand overnight at 105 ° C. The ethanol concentration in the medium was measured by BF-7 (Oji Scientific Instruments, Hyogo, Japan) using the supernatant obtained by centrifuging the culture medium.

-Sample preparation for GC / MS Sample collection and metabolite extraction were performed according to the method of Hashim * et al. Samples that reached a steady state in continuous culture were set to sampling volume × OD ₆₀₀ = 80, and suction filtration was performed using a 0.45 μm pore-sized membrane filter (Millipoire, Massachusetts, USA) with a diameter of 47 mm. Washed with distilled water. After the sample was lyophilized, 5.0 mg was measured, a zirconia ball was added, a lid was placed, and the sample was immersed in liquid nitrogen and frozen. The sample was crushed with a ball mill (MM400, Verder-scientific, Germany) (20 Hz, 5 min). Add 1.0 mL of mix solvent (methanol / H ₂ O / Chroloform: 2.5 / 1/1 (v / v / v)), and add 60 μl of 0.2 mg / ml Ribitol (Wako Pure Chemical Industries, Ltd., Ltd.) as an internal standard. Osaka, Japan) was added. After mixing with vortex (VORTEX-2 GENIE, Scientific industries, inc ,, New York, USA) for 20 sec, the metabolites were extracted with a mixer mill (20 Hz, 5 min). After centrifugation (4 ° C, 5 min, 10000 rpm), 900 μl of the supernatant was transferred to a new tube, 400 μl of ultrapure water was added, and the mixture was vortexed for 10 sec. After centrifugation (10,000 rpm, 5 min, 4 ° C), 500 μl of the upper layer was transferred to a new tube. The sample was centrifuged for 2 h and then lyophilized overnight. Add 100 μl of methoxyamine hydrochloride (Sigma, St. Louis, USA) dissolved in 20 mg / ml to pyridine (Wako Pure Chemical Industries, Ltd., Osaka, Japan) in advance to the lyophilized sample, and add Thermal mixer (Thermomixer Comfort, Eppendorf). Incubated at 1200 rpm, 30 ° C., 90 min in Co., Ltd., Hamburg, Germany). Then, 50 μl of N-methyl-N- (trimethylsilyl) trifluoroacetamide (MSTFA) (GL Sciences, Kyoto, Japna) was added and incubated at 1200 rpm for 30 min with a Thermal mixer.
* Hashim Z1, Teoh ST1, Bamba T1, Fukusaki E2 .: Construction of a metabolome library for transcription factor-related single gene mutants of Saccharomyces cerevisiae .: J Chromatogr B Analyt Technol Biomed Life Sci. 2014 Sep 1; 966: 83-92 ..

・ GC / MS analysis
GC / MS analysis was performed based on the report by Tsugawa * et al. AOC-20s autosampler (Shimadzu) and AOC-20i auto injector (Shimadzu) were combined, and GCMS-QP2010 Ultra (Shimadzu Corporation, Kyoto, Japan) was used as GC and MS. Data was acquired using GCMS solution ver. 4.20β (Shimadzu) as the software.
A 30 m × 0.25 mm id DF: 0.25 μm InertCap 5MS / NP (GL science, Kyoto, Japan) was used for the column. The vaporization chamber temperature is 230 ° C., high-purity helium is used as the carrier gas, and the flow rate is 1.12 mL / min. The column temperature was maintained at 80 ° C for 2 min, then raised to 320 ° C at 15 ° C / min, and maintained at that temperature for 6 min. The interface temperature is 250 ° C, the ion source temperature is 200 ° C, the EI is 70V, and the scanning speed is 20 scan / sec. The measurement mass range is 85-500 m / z.
* Tsugawa H1, Bamba T, Shinohara M, Nishiumi S, Yoshida M, Fukusaki E .: Practical non-targeted gas chromatography / mass spectrometry-based metabolomics platform for metabolic phenotype analysis .: J Biosci Bioeng. 2011 Sep; 112 (3) : 292-8.

・ Data processing
GC / MS analysis data is exported by netCDF, peak identification and alignment are performed by Met al.ign (Ver. 041012) (Lommen. *), Compound identification and principal component analysis are performed by AI output (ver. 1.29). I went there (Tsugawa et al. *). The Pareto scaling method was used for preprocessing, and the conversion was performed by 1/4 root. The automatically identified peaks were manually confirmed by the Automated Mass Spectral Deconvolution and Identification System (AMDIS).
* Lommen A1 .: Met al.ign: interface-driven, versatile metabolomics tool for hyphenated full-scan mass spectrometry data preprocessing .: Anal Chem. 2009 Apr 15; 81 (8): 3079-86.
* Tsugawa H1, Tsujimoto Y, Arita M, Bamba T, Fukusaki E .: GC / MS based metabolomics: development of a data mining system for metabolite identification by using soft independent modeling of class analogy (SIMCA) .: BMC Bioinformatics. 2011 May 4; 12: 131.

<Result>
-Proliferation and culture results In order to search for metabolites related to the club tree effect, the dilution ratio was changed from 0.05 h ^-1 to 0.30 h ^-1 in a continuous culture in a sufficiently aerobic state. The average value of each culture result is shown in FIG. In S. cerevisae NBRC101557, the club tree effect is suppressed below D = 0.20 h ^-1 , and ethanol is not produced. On the other hand, at D = 0.30 h ^-1 , the club tree effect was induced and ethanol was produced. This dilution means that more than 25% of the consumed glucose is directly converted to ethanol. These results were previously reported in continuous cultures of S. cerevisae (Frick et al., Van Hoek P et al.). While the club tree effect was suppressed, as the dilution ratio increased, the concentration of dried cells increased, oxygen consumption increased, and DO decreased. On the other hand, at D = 0.3 h ^-1 , oxygen consumption was suppressed, DO increased, and the concentration of dried cells decreased.

-Metabolome analysis using GC / MS In this analysis, 49 metabolites including amino acids, organic acids, and sugars were identified by GC / MS. Principal component analysis (PCA) was performed on metabolome data obtained from samples with different dilutions to investigate metabolites whose intracellular content changes due to the club tree effect. According to PCA, the samples were separated along PC1 by dilution rate (Fig. 2). Therefore, the metabolites that contributed to the separation of PC1 are shown in FIG. As a result, metabolites such as Trehalose, Valine, 4-aminobenzoic acid, and Fructose 6-Phosphate were confirmed to accumulate in low dilution samples. On the other hand, Glycerol, Na-Acetyl-L-Ornithine, and Ornithine were confirmed to accumulate in the sample with high dilution rate.

The results of the metabolome analysis are shown in FIG. It can be confirmed that Glycerol is accumulated at D = 0.30. Glycerol is known as a by-product of ethanol and is consistent with previous reports (Oura *). Under aerobic conditions of D ≤ 0.20, the intracellular concentrations of Malic acid, Fumaric acid, and Succinic acid increased according to the dilution rate. It can be said that the enzyme responsible for the TCA cycle is activated according to the amount of glucose supplied. Therefore, when D ≤ 0.20, oxygen consumption increases as the dilution rate increases. On the other hand, the intracellular concentrations of Malic acid, Fumaric acid, and Succinic acid decreased at D = 0.30. This meant that the activity of the TCA cycle was suppressed, and oxygen consumption was reduced. As a result, it can be said that excess glucose was converted to ethanol. On the other hand, as the dilution rate increased, the intracellular concentration of Trehalose decreased. Trehalose is known as a metabolite that accumulates in response to various stresses such as ethanol and nutrient starvation (Muhmud et al. *, Lillie SH et al. *). In continuous culture, D = 0.30 is considered to be stressed due to exposure to ethanol, but nutritional starvation due to glucose restriction of D ≤ 0.20 may be more stressful for S. cerevisiae.
* Mahmud SA1, Hirasawa T, Shimizu H .: Differential importance of trehalose accumulation in Saccharomyces cerevisiae in response to various environmental .: J Biosci Bioeng. 2010 Mar; 109 (3): 262-6.
* Lillie SH, Pringle JR: Reserve carbohydrate metabolism in Saccharomyces cerevisiae: responses to nutrient limitation .: J Bacteriol. 1980 Sep; 143 (3): 1384-94.
* Oura E: Reaction-products of yeast fermentations.: Process biochem. 1977 12 (3): 19-21

-Selection of candidates for metabolite addition In order to reduce the ethanol concentration in the medium at D = 0.30 during ethanol fermentation, candidates for metabolites to be added were selected based on the results of metabolome analysis. Since it can be expected that the metabolites whose intracellular concentration increases and decreases as the dilution rate increases are involved in the productivity of ethanol, it is desirable to make them candidates for the metabolites to which they are added. From this, among the metabolites shown in the loading plot of FIG. 3, the metabolites located on both sides are candidates. Trehalose, Glycerol, and Ornithine are candidates for metabolites that meet these conditions. Since Glycerol is already known as a by-product of ethanol (Oura), it was excluded here and was selected as a candidate for metabolites to which Trehalose and Ornithine are added. In addition, since the phenotypes of D≤0.2 and D = 0.3 differ greatly due to the influence of the club tree effect, it is expected that the balance of metabolites will also change significantly. Therefore, at a dilution rate that does not produce ethanol (D ≤ 0.2), the intracellular concentration increases as the dilution rate increases, but at a dilution rate that produces ethanol (D = 0.3), the intracellular concentration decreases for some metabolites. It can be expected as a candidate for metabolites that reduce ethanol. From FIG. 4, the metabolites in which this tendency is observed include Malic acid, Fumaric acid, and Succinic acid. Of these, Malic acid and Fumaric acid have a larger decrease in metabolites at D = 0.3 than D = 0.2, so they were selected as candidates for the metabolites to be added this time in addition to Trehalose and Ornithine. Finally, add each of the four metabolites (Trehalose, Ornithine, Malic acid, Fumaric acid) selected from the above to the medium, and whether the ethanol concentration in the medium decreases compared to the case where no metabolite is added. investigated.

-Addition of metabolites The four metabolites (Trehalose, Ornithine, Fumaric acid, Malic acid) listed as candidates based on the above results were added to the medium so that the final concentration was 100 mg / L, and D = 0.3. And continuous culture was carried out, and the concentration of ethanol in the medium when the steady state was reached was examined (Fig. 5). Malic acid (2.81 g / L) and fumaric acid (2.74 g / L) were able to reduce the ethanol concentration compared to no additives (2.91 g / L). In addition, the dry cell concentration was 1.71 g / L without addition, whereas it was 1.81 g / L and 1.77 g / L for malic acid and fumaric acid, respectively. From this result, in the continuous culture of S. cerevisiae, we succeeded in reducing the ethanol concentration in the medium by 5.9% by adding 100 mg / ml of fumaric acid to the medium.

Moreover, when the cell concentration in the steady state in the continuous culture was measured, it was higher than the control in Trehalose, Fumaric acid, and Malic acid. (Fig. 6)

<Discussion>
When the dilution rate is increased by continuous culture in S. cerevisiae, ethanol fermentation occurs from aerobic respiration of bacterial cell growth type, the bacterial cell concentration decreases, and ethanol is produced (Fig. 1). In aerobic respiration, malic acid and fumaric acid were confirmed as metabolites in which the intracellular concentration increased when the dilution rate was increased (D ≤ 0.2), but decreased in ethanol fermentation (D = 0.3) (Fig. 4). ). On the other hand, Citrate and isocitrate and pyruvate and OAA did not decrease. According to the report of flux analysis, the flux of the TCA cycle decreases uniformly in ethanol fermentation. On the other hand, in S. cerevisiae, pyruvate is converted to Citrate and OAA during the TCA cycle by pyruvate dehydrogenase, Citrate synthase and Pyruvate carboxyrase (Nakayama. Et al. *), But these enzymatic activities are not significantly reduced (Frick et al.). al. *). Therefore, it can be considered that only Citric acid and isocitrate and Pyruvate and OAA, which are the entrances of the TCA cycle from glucose, did not decrease the intracellular metabolite content when D = 0.30 h ^-1 . This time, 100 mg / l of malic acid and fumaric acid, which decrease at D = 0.30 h ^-1 , was added to the medium and continuous culture was performed. As a result, the medium was about 3.4% for malic acid and about 5.9% for fumaric acid. A decrease in the concentration of ethanol in the medium was observed (Fig. 5). It was suggested that the addition of these metabolites led to an increase in cell concentration because energy synthesis could be performed by the TCA cycle with reduced flux. In addition, Frick et al. Show that the flux of malic enzyme increases with increasing dilution. Although malic enzyme and alcohol dehydrogenase are NADH-dependent enzymes, malic acid cell concentration decreases at D = 0.30, so when not added, alcohol is produced by alcohol dehydrogenase, as in this study. When malic acid or fumaric acid is added, NADH is consumed by the malic enzyme, so it is considered that the imbalance of NADH / NAD + is eliminated and the production of ethanol is reduced.
Varela C et al. Reported that overexpression of genes related to the TCA cycle reduced ethanol production by about 2% in malate dehydrogenase (MDH2) and fumarate reductase (FRD1) overexpressing strains. They also wondered if overexpression of multiple genes in the TCA cycle could further reduce ethanol production (Varela C et al. *). Certainly, in the overexpressing strain of one gene, the flow rate is locally increased, so that the intracellular metabolic balance is disturbed and a sufficient effect may not be obtained. On the other hand, if a metabolite is added like ours, the bottlenecked metabolite can be supplemented without disturbing the balance of the metabolite, so a sufficient effect can be expected.
* Nakayama Y, Putri SP1, Bamba T1, Fukusaki E .: Metabolic distance estimation based on principle component analysis of metabolic turnover.: J Biosci Bioeng. 2014 Sep; 118 (3): 350-5.
* Frick O, Wittmann C .: characterization of the metabolic shift between oxidative and fermentative growth in Saccharomyces cerevisiae by comparative 13C flux analysis.: Microb Cell Fact. 2005 Nov 3; 4:30.
* Varela C1, Kutyna DR, Solomon MR, Black CA, Borneman A, Henschke PA, Pretorius IS, Chambers PJ .: Evaluation of gene modification strategies for the development of low-alcohol-wine yeasts.: Appl Environ Microbiol. 2012 Sep; 78 (17): 6068-77.

On the other hand, among the four metabolites added, no decrease in ethanol concentration in the medium was observed for Ornithine and Trehalose. However, Ornithine has a report that downstream polyamines have an effect on ethanol resistance (Walters et al .. *), and Treahalose has a report that it has an effect on ethanol resistance (Mahmud et al ..). Therefore, we investigated the cell concentrations of Ornithine and Trehalose with and without the addition of their respective metabolites (Fig. 7). From FIG. 7, the intracellular concentration of ornithine increased only when ornithine was added to the medium. However, no decrease in ethanol was observed, suggesting that Ornithine does not affect ethanol production. On the other hand, in FIG. 8, it was clarified that the intracellular concentration of Trehalose was significantly increased when each metabolite was added, as compared with the control in which Trehalose was not added. In all experiments by Muhmud et al., Tolerance was investigated by adding 6% or more of ethanol (Muhmud et al.). In our experiment, the ethanol concentration was 2-3 g / L. Therefore, the effect of adding Trehalose, which is known as ethanol resistance, may have been remarkable under the culture conditions that produce a large amount of ethanol such as anaerobic culture.
* Walters D1, Cowley T .: Polyamine metabolism in Saccharomyces cerevisiae exposed to ethanol .: Microbiol Res. 1998 Aug; 153 (2): 179-84.

The present study found that ethanol was reduced by adding metabolites identified using metabolomics in continuous cultures of S. cerevisiae. The results show that by using metabolomics for different phenotypes, it is possible to narrow down the metabolites that increase or decrease the target substance. In addition, metabolomics is not limited to S. cerevisiae, which has advanced gene recombination technology as in this case, but since genomic information is not essential, it can be applied to Non conventional yeasts that do not have a gene recombination system. Therefore, this method is very effective when attempting to produce substances targeting such yeasts.

Claims (1)

A method for culturing yeast that reduces ethanol production and increases cell production by adding 50 to 300 mg / L of fumarate or malic acid to a medium in continuous culture or fed-batch culture of Saccharomyces cerevisiae.

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