JP2018019620A - Reduction of ethanol production in continuous culture of Saccharomyces cerevisiae - Google Patents

Abstract

PROBLEM TO BE SOLVED: To reduce ethanol production in continuous culture having a high dilution rate in S. cerevisiae. Moreover, it makes it a subject to efficiently produce a microbial cell and a target substance by it. SOLUTION: In the culture of Saccharomyces cerevisiae yeast, a metabolite that is largely changed when aerobic respiration is changed to ethanol fermentation by the addition of a substrate is identified, and the metabolite is added to the medium. Specifically, in continuous culture or fed-batch culture of the yeast, fumaric acid or malic acid is added to the medium in an amount of about 100 mg / L, and the yeast is cultured at a dilution rate of about 0.3. [Selection diagram] Fig. 4

Description

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

Saccharomyces cerevisiae, which is 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, even though S. cerevisiae supplies oxygen sufficiently, oxygen respiration is suppressed when glucose is added (De Deken, R. H.). This phenomenon is called Crabtree effect, and it is known that ethanol fermentation starts from biomass formation when the dilution rate is increased in continuous culture of S. cerevisiae (Postma E et al.).

However, the details of the mechanism of the Crabtree effect have not yet been clarified.
Therefore, in order to produce the target substance without producing ethanol, the inflow of sugar must be restricted in fed-batch culture or cultured at a low dilution rate in continuous culture, resulting in poor production efficiency. .

Although the Crabtree effect has been observed in other microorganisms (Serra et al.), S. cerevisiae has been actively studied as a model organism focusing on the Crabtree effect in an aerobic state. For example, there is a report of increasing glycerol and reducing ethanol by overexpressing GPD1 and GPD2 encoding isozyme of glycerol 3-phosphate dehydrogenase (Cambon B et al ..). In addition, NADH / NAD + redox balance is disrupted when glucose inflow is high, and ethanol and glycerol are produced as by-products. Therefore, NADH oxidase that forms water is expressed and ethanol is reduced. There are reports (Heux S et al., Vemuri GN et al.). However, since these techniques use genetic recombination technology, application to the brewing industry and the food industry is difficult.

Compared to approaches using genetic recombination technology, we are working on elucidating the Crabtree effect using flux analysis by taking advantage of the ability to sample in steady state in research using continuous culture systems (Frick O et al., Kajihata S et al.). In continuous culture, the dilution rate (D) is the amount of liquid medium supplied per hour / the volume of the culture solution. S. cerevisiae consumes glucose completely and does not produce ethanol when the dilution ratio (D) is less than 0.3 in a sufficiently aerobic state. However, it is reported that ethanol is produced when D ≧ 0.3 (Van Hoek P et al.). Flux analysis in steady state has been promoted by switching from aerobic respiration to ethanol fermentation by changing the dilution rate, but it has not been possible to reduce ethanol using this analysis result .

 Metabolomics is a method that comprehensively measures metabolites that are the result of genomic information, and is closely related to the close macrophenotype. Therefore, in the production of useful substances by microorganisms, it is possible to obtain useful information that contributes to the improvement of strain performance by considering the production yield, production rate, etc. as a quantitative phenotype 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 oxidation 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 .: 13C-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. 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.

The inventors of the present application have analyzed the samples obtained from different conditions by metabolomics so far, thereby extending the lifespan of yeast (Yoshida R et al.) And improving the resistance to 1-butanol and ethanol (Teoh ST et al. , Ohta E et al.), Poly (γ-glutamic acid) and 1-butanol (Mitsunaga H et al., Noguchi S et al.). Using this knowledge, we found a method that can reduce ethanol production in continuous culture due to Crabtree effect of S. cerevisiae by metabolomics, and to reduce ethanol production in serial culture with high dilution rate in S. cerevisiae. Let it be an issue. Moreover, it makes it a subject to produce a microbial cell and a target object efficiently by it.

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

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 the production of bacterial cells could be increased in continuous culture with a high dilution rate. According to the present invention, the first success in reducing ethanol production and increasing cell production by adding metabolites identified using metabolomics in continuous culture with high dilution of S. cerevisiae. It is.

That is, the present invention
(1) A method for increasing the production of bacterial cells, comprising adding a metabolite having a large change when changing from aerobic respiration to ethanol fermentation in the culture of Saccharomyces cerevisiae yeast,
(2) Provided is a method for culturing the yeast, wherein fumaric acid or malic acid is added to the medium in continuous culture or fed-batch culture of Saccharomyces cerevisiae yeast.

According to the present invention, in 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 bacterial cell concentration can also be increased. .

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

Hereinafter, the present invention will be specifically described.
The strain used in the present invention may be any strain as long as it is 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. For example, a normal medium used for yeast culture can be used.
Specific examples 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 phosphate source, a sulfur source, and other components selected from various organic components and inorganic components as necessary can be used. You may set suitably the kind and density | concentration of a culture medium component with the strain to be used.

In the present invention, metabolomic analysis as shown in the Examples is performed, metabolites having large changes are identified when changing from aerobic respiration to ethanol fermentation, and ethanol production is reduced by adding them to the medium. Possible compounds can be screened.
Specifically, malic acid and / or fumaric acid are 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 most efficiently obtained. For example, the value of D is preferably 0.1 to 0.5, more preferably 0.25 to 0.40, and still more preferably 0.30 to 0.35. The dilution rate (D) is the amount of liquid medium supplied per hour / the volume of the culture solution.

What is necessary is just to select the temperature which can obtain the target substance most efficiently, for example, 25-35 degreeC, Preferably it is 28-32 degreeC. The culture time is not particularly limited as long as it is continuous culture.

The present invention will be specifically described below with reference to examples.
The present invention is not limited to these.

S. cerevisiae NBRC101557 was obtained from Biological Resource Center, NITE (NBRC). Pre-culture was 100 mL of 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. The main culture was inoculated into 1 L of a culture solution spread on 2 L of Jar-fermenter (Mitsuwa Frontech) so that the initial OD ₆₀₀ would be 0.1, and batch culture was started. Medium composition is 10 g / L glucose in the culture _{solution, 1.5 g KH 2 PO 4,} 0.5 g / L MgSO 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 addition to the above, in addition to the above-described metabolite addition test, Trehalose Dihydrate, L-Ornithine Monohydrochloride, Fumaric acid, and Malic acid were added to the medium at 100 mg / L. 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 number of stirring was 700 rpm, aeration was performed at 1 L / min, and the pH was controlled at 5.0 with 4 M NaOH. Continuous culture was started at each dilution rate in the late logarithmic growth phase of batch culture, and after confirming that a steady state was reached, a sample was obtained.

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

-Sample preparation for GC / MS Sample collection and metabolite extraction were performed according to the method of Hashim * et al. Set the sample that reached a steady state in continuous culture to a sampling volume × OD ₆₀₀ = 80, and perform suction filtration using a 0.45 μm pore size membrane filter (Millipoire, Massachusetts, USA) with a diameter of 47 mm. Washed with distilled water. The sample was freeze-dried and weighed 5.0 mg, put a lid of zirconia balls, 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 then add 60 μl of 0.2 mg / ml Ribitol (Wako Pure Chemical Industries, Ltd. Osaka, Japan) was added. After mixing with vortex (VORTEX-2 GENIE, Scientific industries, Inc, New York, USA) for 20 sec, 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 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 freeze-dried overnight. Add 100 μl of methoxyamine hydrochloride (Sigma, St. Louis, USA) dissolved in pyridine (Wako Pure Chemical Industries, Ltd., Osaka, Japan) at 20 mg / ml in advance to the freeze-dried sample, and then add Thermal mixer (Thermomixer Comfort, Eppendorf Co., Ltd., Hamburg, Germany) at 1200 rpm, 30 ° C., 90 min. Thereafter, 50 μl of N-methyl-N- (trimethylsilyl) trifluoroacetamide (MSTFA) (GL Sciences, Kyoto, Japna) was added and incubated at 1200 rpm at 37 ° C. for 30 min in 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 of Tsugawa * et al. GCMS-QP2010 Ultra (Shimadzu Corporation, Kyoto, Japan) was used as GC and MS by combining AOC-20s autosampler (Shimadzu) and AOC-20i auto injector (Shimadzu). GCMS solution ver. 4.20β (Shimadzu) was used as software, and data was acquired.
The column used was 30 m × 0.25 mm id DF: 0.25 μm InertCap 5MS / NP (GL science, Kyoto, Japan). 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 increased to 320 ° C. at 15 ° C./min, and held 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 scan speed is 20 scan / sec. The measuring 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 is performed by Met al.ign (Ver. 041012) (Lommen. *), Compound identification and principal component analysis are AIoutput (ver. 1. 29) (Tsugawa et al. *). Pareto scaling method was used for pre-processing, and conversion was performed at 1/4 root. The automatically identified peak was manually confirmed by 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>
-Growth and culture results In order to search for metabolites related to the Crabtree effect, the dilution rate was changed from 0.05 h ^-1 to 0.30 h ^-1 in continuous aerobic culture. The average value of each culture result is shown in FIG. In S. cerevisae NBRC101557, the Crabtree effect is suppressed at D = 0.20 h ^-1 or less, and ethanol is not produced. On the other hand, at D = 0.30 h ⁻¹ , the Crabtree effect was induced and ethanol was produced. This dilution rate means that 25% or more of the consumed glucose is directly converted to ethanol. These results were as previously reported in continuous cultures of S. cerevisae (Frick et al., Van Hoek P et al.). While the Crabtree effect was suppressed, the concentration of dry cells increased, oxygen consumption increased, and DO decreased with increasing dilution rate. On the other hand, at D = 0.3 h ⁻¹ , oxygen consumption was suppressed, DO increased, and the dry cell concentration decreased.

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

The results of 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 supply amount of glucose. Therefore, when D ≦ 0.20, the oxygen consumption increases as the dilution rate increases. On the other hand, the intracellular concentrations of Malic acid, Fumaric acid, and Succinic acid were reduced at D = 0.30. This meant that the activity of the TCA cycle was suppressed, and oxygen consumption was decreasing. As a result, it can be said that excess glucose was converted to ethanol. On the other hand, as the dilution rate increased, the concentration of trehalose in the cells 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 exposed to ethanol and is considered to be stressed. However, for S. cerevisiae, nutrient starvation by D ≦ 0.20 glucose restriction may feel more stressed.
* 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 metabolite addition candidates In order to reduce the ethanol concentration in the medium at D = 0.30 where ethanol fermentation is performed, selection of metabolite candidates to be added was performed based on the results of metabolome analysis. Since it can be expected that metabolites whose intracellular concentration increases or decreases as the dilution rate increases are involved in ethanol productivity, it is desirable to make them metabolite candidates. Thus, metabolites located on both sides of the metabolites shown in the loading plot of FIG. 3 are candidates. Possible metabolites that meet these conditions include Trehalose, Glycerol, and Ornithine. Since Glycerol is already known as a byproduct of ethanol (Oura), it was excluded here and used as a metabolite candidate to which Trehalose and Ornithine were added. In addition, when D ≦ 0.2 and D = 0.3, the phenotype differs greatly due to the influence of the Crabtree effect, so the balance of metabolites is expected to change greatly. Therefore, when the dilution rate without ethanol is increased (D ≦ 0.2), the concentration in the cell increases as the dilution rate increases, but there are also metabolites whose concentration in the cell decreases at the dilution rate that makes ethanol (D = 0.3). It can be expected as a metabolite candidate for reducing ethanol. From FIG. 4, malic acid, fumaric acid, and succinic acid are listed as metabolites in which this tendency is observed. Among them, Malic acid and Fumaric acid had a large decrease in metabolites at D = 0.3 compared to D = 0.2. Therefore, in addition to Trehalose and Ornithine, they were selected as metabolites to be added this time. Finally, if the four metabolites selected above (Trehalose, Ornithine, Malic acid, and Fumaric acid) are added to the medium, respectively, will the ethanol concentration in the medium decrease compared to the case where no metabolite is added? investigated.

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

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

<Discussion>
When the dilution rate is increased in S. cerevisiae by continuous culture, ethanol fermentation starts from cell-growing aerobic respiration, and the cell concentration decreases to produce ethanol (Fig. 1). In aerobic respiration, the intracellular concentration increases when the dilution rate is increased (D ≦ 0.2), but malic acid and fumaric acid can be confirmed as metabolites that decrease during ethanol fermentation (D = 0.3) (Fig. 4). ) On the other hand, Citrate and isocitrate and pyruvate and OAA did not decrease. Flux analysis reports that the TCA cycle flux decreases uniformly during ethanol fermentation. On the other hand, pyruvate is converted to Citrate and OAA in TCA cycle by pyruvate dehydrogenase, Citrate synthase and pyruvate carboxylrase in S. cerevisiae (Nakayama. Et al. *), But these enzyme activities are not greatly reduced (Frick et al. al. *) Therefore, it can be considered that only Citrate and isocitrate and Pyruvate and OAA at the entrance of the TCA cycle from glucose did not decrease the intracellular metabolite content when D = 0.30 h- ¹ . As a result of adding 100 mg / l malic acid and fumaric acid decreasing at D = 0.30 h ^-1 to the medium and performing continuous culture, the medium was about 3.4% for malic acid and about 5.9% for fumaric acid. A decrease in the ethanol concentration was observed (Fig. 5). By adding these metabolites, it was suggested that energy synthesis could be performed by TCA cycle with decreasing flux, leading to an increase in cell concentration. In addition, according to Frick et al., An increase in malic enzyme flux is shown as the dilution rate increases. Malic enzyme and alcohol dehydrogenase are NADH-dependent enzymes, but when D = 0.30, malic acid cell concentration decreases, so ethanol is produced by alcohol dehydrogenase without addition, but as in this study When malic acid or fumaric acid is added, NADH is consumed by malic enzyme, so the NADH / NAD + imbalance is eliminated and ethanol production is considered to be reduced.
Varela C et al. Have reported that when overexpression of a gene associated with the TCA cycle is observed, ethanol production decreases by about 2% in malate dehydrogenase (MDH2) and fumarate reductase (FRD1) overexpression strains. They also considered that by overexpressing multiple genes in the TCA cycle, ethanol production could be further reduced (Varela C et al. *). Certainly, since the flow rate is locally increased in an overexpressing strain of one gene, there is a possibility that the intracellular metabolic balance is disrupted and a sufficient effect cannot be obtained. On the other hand, if a metabolite is added as in the case of us, the metabolite that is a bottleneck can be compensated without losing the balance of the metabolite, so that 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, Ornithine and Trehalose did not show a decrease in ethanol concentration in the medium. However, Ornithine reports that downstream polyamines are effective in ethanol tolerance (Walters et al. *), And Treahalose reports that it is effective in ethanol tolerance (Mahmud et al ..). Therefore, the cell concentrations of ornithine and trehalose with and without the addition of the respective metabolites were examined (FIG. 7). From FIG. 7, the intracellular concentration of Ornithine increased only when Ornithine was added to the medium. However, since there was no decrease in ethanol, it is considered that Ornithine does not affect ethanol production. On the other hand, in FIG. 8, it was revealed that the concentration of Trehalose in the cells was significantly increased when each metabolite was added, compared to the control without addition of Trehalose. In addition, in Mumumud et al.'S experiment, all of 6% or more of ethanol was added to examine tolerance (Muhmud et al.). In our experiments, the ethanol concentration was 2-3 g / L. Therefore, the effect of adding Trehalose, known as ethanol tolerance, may be noticeable under culture conditions that produce more ethanol than anaerobic culture.
* Walters D1, Cowley T .: Polyamine metabolism in Saccharomyces cerevisiae exposed to ethanol .: Microbiol Res. 1998 Aug; 153 (2): 179-84.

This study found that ethanol was reduced by adding metabolites identified using metabolomics in continuous culture of S. cerevisiae. The results show that metabolites that increase or decrease the target substance can be narrowed down by using metabolomics for different phenotypes. Metabolomics is not limited to S. cerevisiae for which genetic recombination technology has been developed as in this case, but it is applicable to non conventional yeasts that do not have a genetic recombination system because genome information is not essential. Therefore, this method is very effective when trying to produce substances targeting such yeast.

Claims (2)

A method of increasing the amount of bacterial cells produced by adding a metabolite having a large change when changing from aerobic respiration to ethanol fermentation in the culture of Saccharomyces cerevisiae. The yeast culture method according to claim 1, wherein fumaric acid or malic acid is added to the medium in continuous culture or fed-batch culture of Saccharomyces cerevisiae.