Acid-resistant torulopsis glabrata mutant strain with high yield of pyruvic acid and application thereof

Abstract

The invention discloses an acid-resistant torulopsis glabrata mutant strain with high yield of pyruvic acid and application thereof. The acid-resistant torulopsis glabrata strain is preserved in the China Center for Type Culture Collection (CCTCC), and the preservation number of the acid-resistant torulopsis glabrata strain is M 20231147. Compared with an original strain, the torulopsis glabrata strain has the advantages that the yield of pyruvic acid can be increased, the torulopsis glabrata strain can tolerate lower pH, fermentation byproducts such as heteroacid are remarkably reduced, the sugar-acid conversion rate is increased, and the production intensity is improved.

Description

Technical Field

[0001] The invention belongs to the field of microbial fermentation, and particularly relates to an acid-resistant Torulopsis glabrata mutant strain with high pyruvic acid production and application thereof. Background Art

[0002] Pyruvic acid, also known as α-ketopropionic acid, 2-oxopropionic acid and acetyl formic acid, is a colorless to pale yellow liquid. Pyruvic acid is a key intermediate product of cell metabolism and is widely used in industries such as medicine, food and agriculture, but its industrial production technology is relatively backward.

[0003] At present, the production methods of pyruvic acid mainly include chemical synthesis, enzyme conversion and microbial fermentation. The chemical method has low conversion rate, serious pollution, high raw material cost and lacks competitiveness. The enzyme conversion method has high cost, low conversion rate, difficulty in product separation and purification, and is not suitable for large-scale production. The microbial fermentation method has the advantages of being green, environmentally friendly, high yield and low cost, and is the mainstream technology for the industrial production of pyruvic acid.

[0004] The key to producing pyruvate by microbial fermentation is to obtain strains with high pyruvate production, stable performance and few fermentation byproducts. The selection of pyruvate strains mainly starts from metabolic pathway modification, mutagenesis screening and adaptive evolution. The main production strains are Escherichia coli, Saccharomyces cerevisiae and Torulopsis glabrata. The construction of pyruvate-producing Escherichia coli engineering bacteria mainly uses genetic engineering technology to achieve a large amount of pyruvate accumulation by knocking out the key enzymes that metabolize pyruvate into lactic acid and acetic acid, and weakening the TCA cycle. The pyruvate production reaches 60-90g / L (Fermentation, 2017, 3(1):8; Appl. Environ. Microbiol, 2008, 74(21), 6649-6655). Escherichia coli is easily infected with bacteriophages in industrial production, causing fermentation abnormalities and tank failure, causing serious losses to industrial production. The transformation strategy of pyruvate-producing Saccharomyces cerevisiae is focused on weakening or blocking the ethanol pathway. For example, the pyruvate decarboxylase genes pdc1, pdc5 and pdc6 are knocked out. The transformed strains cannot grow on a medium with glucose as the sole carbon source. The engineered bacteria that can accumulate pyruvate using glucose are screened by domestication. After 76 hours of fermentation, 105 g / L of pyruvate is produced and the sugar-acid conversion rate is 0.5 g / g (Li Yi, Qin Yan, Shen Naikun, et al. Construction of Saccharomyces cerevisiae pdc gene-deficient strains and their pyruvate fermentation characteristics [J]. Food and Fermentation Industries, 2020, 46(8):7). ​​Torulopsis glabrata is the most studied strain and is currently used in industrial production. The earliest strain of multivitamin-deficient smooth Torulopsis glabuata IFO 005 was screened out by Miyata and Yonehara of Japan. After mutagenesis screening, the sugar-acid conversion rate of the induced strain can reach 0.52-0.54 g / g, but the pyruvic acid production is relatively low [JP, patent 2000078996]. Liu Liming et al. used nitrosoguanidine mutagenesis technology and sodium acetate screening culture medium for screening and obtained a high-yield strain of acetic acid leakage type, with a pyruvic acid production of 63.8 g / L after fermentation for 64 hours, and a sugar-acid conversion rate of 0.588 g / g (CN1176205C). However, the poor pH tolerance (pH>4) and low acid production of Torulopsis glabuata affect its industrial application. Therefore, screening out high-acid-producing and acid-resistant engineering strains is of key significance for the industrial production of pyruvic acid. Summary of the invention

[0005] The technical problem to be solved by the present invention is to overcome the defects of the prior art of Torulopsis glabrata, such as low acid production, poor pH tolerance, high by-product content, low sugar-acid conversion rate, and low production intensity, and to provide an acid-resistant Torulopsis glabrata mutant strain with high pyruvic acid production and its application. The acid-resistant Torulopsis glabrata mutant strain of the present invention has high pyruvic acid production, high sugar-acid conversion rate, can tolerate low pH, and significantly reduces by-products α-ketoglutarate and lactic acid.

[0006] The present invention solves the above technical problems through the following technical solutions:

[0007] The first aspect of the present invention provides a Torulopsis glabrata strain, which is deposited in China Center for Type Culture Collection (CCTCC) with a deposit number of M 20231147.

[0008] The second aspect of the present invention provides a method for culturing a Torulopsis glabrata strain, wherein the culturing method comprises fermenting the Torulopsis glabrata strain as described in the first aspect of the present invention in a fermentation medium.

[0009] In some embodiments of the present invention, the fermentation medium includes 100-150 g / L of glucose, 5-10 g / L of ammonium sulfate, 5-10 g / L of sodium acetate, 1-10 g / L of potassium dihydrogen phosphate, 0.1-1 g / L of magnesium sulfate, 10-50 mg / L of ferrous sulfate, 0.1-1 mg / L of copper sulfate, 1-10 mg / L of manganese chloride, 1-10 mg / L of zinc chloride and a vitamin solution; the vitamin solution includes 0.01-0.1 mg / L of thiamine hydrochloride, 0.1-1 mg / L of pyridoxine hydrochloride, 0.01-0.1 mg / L of biotin, 1-10 mg / L of nicotinic acid and 0.1-1 mg / L of riboflavin.

[0010] In other embodiments of the present invention, the fermentation medium includes 100-150 g / L of glucose, 5-10 g / L of ammonium sulfate, 1-10 g / L of potassium dihydrogen phosphate, 0.1-1 g / L of magnesium sulfate, 1-10 mg / L of nicotinic acid, 0.01-0.1 mg / L of thiamine hydrochloride, 0.1-1 mg / L of pyridoxine hydrochloride, 0.01-0.1 mg / L of biotin, 5-20 mg / L of calcium chloride, 5-20 mg / L of ferrous sulfate, 5-50 mg / L of zinc chloride, 1-10 mg / L of manganese chloride, 1-10 mg / L of copper sulfate and 20-50 g / L of calcium carbonate.

[0011] In the present invention, the pH of the fermentation culture is preferably 4.0-5.5, for example, the pH is 4.0, 4.5 or 5.5.

[0012] In the present invention, the temperature of the fermentation culture is preferably 27-30°C.

[0013] In the present invention, the dissolved oxygen content of the fermentation culture is preferably 15-40%, more preferably 20-30%.

[0014] In the present invention, the residual sugar content of the fermentation culture is preferably 2-8 g / L, more preferably 3-5 g / L.

[0015] In some preferred embodiments of the present invention, the culture method further comprises performing expansion culture in a seed culture medium before the fermentation culture.

[0016] In the present invention, the seed culture medium preferably comprises: 20-30 g / L glucose, 0.8-1 g / L potassium dihydrogen phosphate, 0.1-1 g / L magnesium sulfate and 10-20 g / L peptone.

[0017] The third aspect of the present invention provides a method for producing pyruvate, wherein the method comprises the step of culturing the Torulopsis glabrata strain as described in the first aspect of the present invention to obtain pyruvate.

[0018] In some preferred embodiments of the present invention, the method further comprises the step of culturing the Torulopsis glabrata strain according to the culturing method described in the second aspect of the present invention.

[0019] The fourth aspect of the present invention provides a fermentation tank culture medium of a glabrata yeast strain, wherein the fermentation tank culture medium comprises 100-150 g / L of glucose, 5-10 g / L of ammonium sulfate, 5-10 g / L of sodium acetate, 1-10 g / L of potassium dihydrogen phosphate, 0.1-1 g / L of magnesium sulfate, 10-50 mg / L of ferrous sulfate, 0.1-1 mg / L of copper sulfate, 1-10 mg / L of manganese chloride, 1-10 mg / L of zinc chloride and a vitamin solution; the vitamin solution comprises 0.01-0.1 mg / L of thiamine hydrochloride, 0.1-1 mg / L of pyridoxine hydrochloride, 0.01-0.1 mg / L of biotin, 1-10 mg / L of nicotinic acid and 0.1-1 mg / L of riboflavin.

[0020] The fifth aspect of the present invention provides a primary screening fermentation medium for a glabrescent Torulopsis yeast strain, wherein the primary screening fermentation medium comprises 100-150 g / L of glucose, 5-10 g / L of ammonium sulfate, 1-10 g / L of potassium dihydrogen phosphate, 0.1-1 g / L of magnesium sulfate, 1-10 mg / L of nicotinic acid, 0.01-0.1 mg / L of thiamine hydrochloride, 0.1-1 mg / L of pyridoxine hydrochloride, 0.01-0.1 mg / L of biotin, 5-20 mg / L of calcium chloride, 5-20 mg / L of ferrous sulfate, 5-50 mg / L of zinc chloride, 1-10 mg / L of manganese chloride, 1-10 mg / L of copper sulfate and 20-50 g / L of calcium carbonate.

[0021] In some preferred embodiments of the present invention, the culture method further comprises a feeding step, wherein the feeding comprises 50% glucose solution, 40% ammonia water and feed 2 solution. The feed 2 solution comprises 2-5 g / L potassium dihydrogen phosphate, 1-4 g / L magnesium sulfate, 1-5 g / L peptone, and 1 / 2 concentration of the vitamin solution. The flow rate of the glucose solution is 2.7 g / L / h-4 g / L / h. The flow rate of the feed 2 solution is 0.5-1.5 g / L / h.

[0022] The sixth aspect of the present invention provides a use of the glabrata yeast strain as described in the first aspect of the present invention, the fermentation tank culture medium as described in the fourth aspect of the present invention, or the primary screening fermentation culture medium as described in the fifth aspect of the present invention in producing pyruvate.

[0023] On the basis of being in accordance with the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0024] The reagents and raw materials used in the present invention are commercially available.

[0025] The positive and progressive effects of the present invention are:

[0026] The Torulopsis glabrata H26-cgr02 strain of the present invention can increase the yield of pyruvate (for example, the fermentation acid yield in a 50L fermentation tank is increased by 34.5%) compared to the starting strain, tolerate a lower pH (minimum pH 4.0), significantly reduce fermentation byproducts such as α-ketoglutaric acid, lactic acid and other miscellaneous acids, improve sugar-acid conversion rate and production intensity.

[0027] Biomaterial Deposit Information

[0028] The glabrata yeast H26cgr-02 (Torulopsis glabrata H26-cgr02) of the present invention was deposited in the China Center for Type Culture Collection (CCTCC) on June 30, 2023, with the deposit address: Wuhan University, Luojia Mountain, Wuchang, Wuhan City, Hubei Province, China, postal code 430072, deposit number CCTCC NO: M 20231147, and the culture name is glabrata yeast H26-cgr02. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is the result of the initial screening of strains for pyruvate content, showing only the strains with the highest acid production in each round. DETAILED DESCRIPTION

[0030] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are selected according to conventional methods and conditions, or according to the product specifications. The commercial information of the reagents / materials, i.e., instruments used in the examples is shown in Table 1 and Table 2. All seed culture medium formulas used in the examples are the same.

[0031] Table 1 Commercial information of reagents / materials

[0032] name supplier Part Number Torulopsis glabrata hy-001 / / Peptone Angel Yeast Co., Ltd. FP101 3-Fluoropyruvic acid Aladdin F168911

[0033] Table 2 Commercial information of instruments

[0034] name supplier model ARTP Mutagenesis Apparatus Tianmu Biotechnology Co., Ltd. ARTP-M Spectrophotometer Shanghai Jinghua Technology Instrument Co., Ltd. 722S Fermentation tank (5L) Shanghai Bailun Biotechnology Co., Ltd. BLBIO-5GJ-4 Fermentation tank (50L) Shanghai Baoxing BIOTECH-50JS-900 High performance liquid chromatography Thermo Fisher Scientific Ultimate3000

[0035] In the embodiment, Torulopsis glabrata hy-001 screened from soil and stored in a strain library was used as a starting strain. The Torulopsis glabrata was a four-vitamin-deficient (VB1, VB6, VH, niacin)-deficient Torulopsis glabrata. The strain was subjected to four rounds of iterative mutagenesis using an ARTP mutagenizer, and was initially screened using a screening medium containing 3-fluoropyruvic acid to obtain a fluoropyruvic acid-sensitive strain. These strains were rescreened by shake flask fermentation, and the pyruvic acid concentration, OD and other indicators in the fermentation broth were detected. The optimal strain H26cgr-01 was screened out, and the pyruvic acid production after 48h of shake flask fermentation was 15.99 g / L, which was 269% higher than the acid production level of the starting strain (4.41 g / L).

[0036] The strain was inoculated into a seed culture medium with a pH of 4 for domestication. The obtained bacterial solution was diluted and spread on a pH 4.0 plate slope to separate single colonies. Single colonies with larger diameters and fuller colony morphology were picked and continued to the next round of domestication. After 10 consecutive rounds, the obtained strain was named H26cgr-02.

[0037] H26cgr-02 and the starting strain hy-001 were inoculated into the seed culture medium to prepare the seed solution, which was then transferred to a 5L canned fermentation medium. The pH was controlled at 5.5, 4.5, and 4.0, respectively. The fermentation was continued for 48 hours and then ended. The pyruvate production of H26-cgr02 was 72.1 g / L, 50.6 g / L, and 29.4 g / L, respectively, and the pyruvate production of the starting strain hy-001 was 57 g / L, 30 g / L, and 18 g / L, respectively.

[0038] H26cgr-02 was inoculated into seed culture medium to prepare seed liquid, which was transferred to a 15L seed tank, and the seed tank was transferred to a 50L fermentation tank for fermentation. After 48 hours of continuous fermentation, the pyruvate production reached 74 g / L, the bacterial concentration was 88 g / L, the sugar-acid conversion rate was 55%, and the by-products α-ketoglutaric acid (0.2 g / L) and lactic acid (1.1 g / L) were significantly reduced.

[0039] Example 1: Determination of the optimal mutagenesis conditions for ARTP

[0040] Take hy-001 glycerol bacteria and streak them on the seed slant medium, culture at 30℃ for 2 days, pick a single colony and inoculate it into the seed medium, and culture at 30℃ and 220rpm for 12-18h. Take 1mL of the above culture solution and centrifuge at 5000r / min at room temperature for 5min, discard the supernatant, wash twice with 1mL of sterile water, and resuspend the bacteria with an appropriate amount of 5% glycerol solution. The cell concentration of the bacterial suspension is about 1×10 7 cfu / mL. Take 10 μL of the above bacterial suspension and apply it on the surface of the stainless steel slide. Set the parameters, power is 100W, carrier gas flow rate is 10L / min, treatment time is 20s, 40s, 50s, 60s, 70s, 80s, the slide after ARTP treatment is placed in a 2mL centrifuge tube containing 1mL sterile water for 1min of oscillation elution, gradient dilution, and respectively applied to the seed culture medium plate. After culturing for 2-3 days, the viable bacteria are counted and the lethality is calculated to obtain the optimal treatment time (lethality range is 90%-95%). The relationship between the mutagenesis time and the lethality is shown in Table 3.

[0041] Lethality rate (%) = 100 × (number of live bacteria in the control group - number of live bacteria in the induced group) / number of live bacteria in the control group.

[0042] According to the experimental results, 40s-45s was selected as the optimal processing time.

[0043] Slant culture medium formula: glucose 5g / L, potassium dihydrogen phosphate 1g / L, magnesium sulfate 0.3g / L, peptone 10g / L, agar powder 20g / L.

[0044] Seed culture medium formula: glucose 20g / L, potassium dihydrogen phosphate 1g / L, magnesium sulfate 0.3g / L, peptone 10g / L.

[0045] Table 3 Relationship between mutagenesis time and lethality

[0046]

[0047]

[0048] Example 2: ARTP mutagenesis and initial screening of strains

[0049] Under the best mutagenesis conditions (treatment 45s), ARTP mutagenesis was carried out once. The steps were the same as in Example 1. The mutagenesis solution and the control group bacterial solution of equal concentration were diluted and coated on the flat plate containing the inclined surface, and cultured at 30°C for 2 days. Pick 200-300 single colonies of the mutagenesis group and transfer them to the common flat plate inclined surface and the screening plate containing 1mg / mL of 3-fluoropyruvic acid at the same time. Each colony corresponds to each other and is cultured at 30°C for 2 days. Observe the growth of bacteria, select single colonies that do not grow on the screening plate and grow well on the common inclined plate, inoculate them into 5mL seed culture medium, culture them at 30°C 220rpm overnight, and transfer them to 30mL fermentation medium with 10% transfer amount the next day for shake flask fermentation. The culture conditions are 30°C 220rpm for continuous fermentation for 48h. After the fermentation is completed, the pyruvic acid content in the fermentation supernatant and the biomass of the bacterial solution are detected. The strains with the highest acid production and biomass are selected as the starting strains for the next round of mutagenesis screening, and the mutagenesis and screening steps are the same as above. Four rounds of mutagenesis were performed continuously, and the best strain was selected and named H26-cgr01.

[0050] Screening plate formula: glucose 5g / L, peptone 10g / L, potassium dihydrogen phosphate 1g / L, magnesium sulfate 0.3g / L, 3-fluoropyruvic acid 1mg / mL, agar powder 20g / L.

[0051] Fermentation medium formula: glucose 100g / L, ammonium sulfate 8g / L, potassium dihydrogen phosphate 1g / L, magnesium sulfate 0.5g / L, niacin 8mg / L, thiamine hydrochloride 0.02mg / L, pyridoxine hydrochloride 0.5mg / L, biotin 0.04mg / L, calcium chloride 0.01g / L, ferrous sulfate 0.01g / L, zinc chloride 0.025g / L, manganese chloride 0.001g / L, copper sulfate 0.0025g / L, calcium carbonate 40g / L.

[0052] Pyruvate rapid detection method: The reaction system is ferric nitrate (0.1mol / L) indicator 80μL, the volume of pyruvate is 120μL, and the pyruvate concentration range is 0-2g / L. Detect the absorbance value at 520nm. The pyruvate concentration of the standard curve is: 0, 0.1, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6, 1.8, 2.0g / L.

[0053] The screening results (only the strains with the highest acid production in each round are shown) are as follows Figure 1 shown.

[0054] Example 3: Acclimation of acid-resistant strains

[0055] The optimal strain H26-cgr01 obtained from the initial screening was streaked on a slant plate at pH = 4.0, cultured at 30°C for 2-3 days, and single colonies with larger diameters were selected and inoculated into a seed culture medium at pH = 4.0, cultured at 30°C and 220 rpm for 16-20 h, and 1 mL of the bacterial solution was taken and diluted to 10 with sterile water. -5 , 10 -6 , 10 -7 , spread on a slant plate at pH = 4.0, and grow a single colony after culturing at 30°C for 2-3 days. Repeat the above steps, pick a single colony for inoculation, culture, dilution and coating. After 10 consecutive passages, pick 10 different single colonies with larger diameters and 1 single colony of H26-cgr01 before acclimation, inoculate them into a seed culture medium at pH = 4.0, and culture at 30°C and 220rpm for 16-20h, and detect OD 660 Value, OD of each strain 660 The values ​​are shown in Table 4. Under the same conditions, OD 660 The strain with the highest value was the most acid-resistant strain (No. 4), named H26-cgr02.

[0056] Table 4 Screening of strains with strong acid resistance

[0057] serial number <![CDATA[OD 660 ]]> serial number <![CDATA[OD 660 ]]> 1 14.32 6 12.68 2 15.60 7 14.43 3 11.38 8 11.92 4 18.29 9 12.66 5 11.79 10 12.02 H26-cgr01 10.26

[0058] Example 4: Comparison of acid production between H26-cgr02 and the starting strain in a 5L fermenter

[0059] The starting strains hy-001 and H26-cgr02 were separated by streaking on the slant plate, cultured at 30℃ for 2 days, single colonies were streaked densely on the slant plate for further activation for 16-24h, a ring of activated bacterial mud on the slant plate was streaked on the slant of the eggplant bottle for further activation and culture for 16-24h, a large amount of bacterial moss was scraped and inoculated into the first-level seed liquid culture medium, and cultured at 30℃ and 220rpm for 8-10h. 10%-20% of the seed liquid was transferred to a 5L fermenter to start fermentation, with a liquid volume of 3L, and three different pH conditions (4.0, 4.5, 5.5) were used for each strain, the dissolved oxygen was controlled between 20-30%, and the residual sugar was controlled at 3-5g / L.

[0060] Fermentation tank medium formula: glucose 120g / L, ammonium sulfate 8g / L, sodium acetate 6g / L, potassium dihydrogen phosphate 5g / L, magnesium sulfate 0.8g / L. Trace elements: ferrous sulfate 40mg / L, copper sulfate 0.5mg / L, manganese chloride 4mg / L, zinc chloride 4mg / L. Vitamins: thiamine hydrochloride 0.02mg / L, pyridoxine hydrochloride 0.4mg / L, biotin 0.04mg / L, niacin 8mg / L, riboflavin 0.1mg / L.

[0061] The feed formula includes 50% glucose solution, 40% ammonia water, feed 2 solution (4g / L potassium dihydrogen phosphate, 2g / L magnesium sulfate, 2.5g / L peptone, 1 / 2 vitamin solution). When the residual sugar is lower than 4g / L, the sugar supplement starts, and the flow rate of glucose is adjusted according to the residual sugar in the fermentation liquid, and the flow rate is 2.7g / L / h-4g / L / h (pure glucose); the concentration of vitamins in feed 2 is half of that in the fermentation medium, and the flow rate of feed 2 is 0.9g / L / h.

[0062] After the fermentation, the pyruvic acid content in the fermentation broth was detected by high performance liquid chromatography. The chromatographic column was Elite Hypersil ODS 5μm 4.6*250mm, the mobile phase was 0.05mol / L potassium dihydrogen phosphate solution, the flow rate was 1.0mL / min, the column temperature was 40℃, and the wavelength was 210nm.

[0063] Table 5 Fermentation acid production and bacterial biomass of the starting strains and screened strains under different pH conditions

[0064]

[0065] As shown in Table 5, compared with the starting strain hy-001, the pyruvic acid production of the screened strain H26-cgr02 increased by 63.3%, 68.7% and 26.5% under the conditions of pH = 4.0, pH = 4.5 and pH = 5.5, and the bacterial concentration increased by 73.1%, 38% and 19.7%, respectively.

[0066] Example 5: 50 L tank fermentation of H26-cgr02 and hy-001

[0067] H26-cgr02 and hy-001 were separated by streaking on the slant plate, cultured at 30℃ for 2 days, single colonies were streaked densely on the slant plate for further activation for 16-24h, and then a ring of bacterial mud was taken to continue activation and culture on the slant of the eggplant bottle for 16-24h, and a large amount of bacterial moss was scraped and inoculated into the primary seed culture medium, and cultured at 30℃ and 220rpm for 8-10h to obtain seed liquid. The seed liquid was transferred to a 15L seed tank containing 9L seed culture medium with an inoculation amount of 10%-20% to expand the culture, pH was controlled at 5.5, cultured at 30℃ for 12-20h, and when OD reached between 10-20, it was transferred to a 50L fermentation tank containing 30L fermentation medium with a transfer amount of 10%-20% to start fermentation, and the dissolved oxygen was controlled between 20-30%, and the residual sugar was controlled between 3-5g / L. The fermentation was stopped after 48-55h. The pyruvic acid content, bacterial concentration, by-product α-ketoglutaric acid, lactic acid, etc. were tested in the lower tank, and the sugar-acid conversion rate was calculated.

[0068] Table 6 Fermentation acid production in 50L fermenter of screened strains

[0069]

[0070] As shown in Table 6, compared with the starting strain hy-001, the screened strain H26-cgr02 was fermented in a 50L tank for 52 hours, and the pyruvate production increased by 34.5%, the sugar acid conversion rate increased by 8%, the production intensity increased by 34.0%, the production of by-products α-ketoglutarate and lactic acid decreased by 86.7% and 59.3%, respectively, and the difference in bacterial growth was not significant.

[0071] Example 6: Comparison of tyrosine production by fermentation supernatant conversion of H26-cgr02 and hy-001

[0072] The fermentation broth of hy-001 and H26-cgr02 in a 50L tank was treated with a disc centrifuge to remove the bacteria, and crude pyruvate was obtained. Phenol (the molar ratio of pyruvate to phenol was 1.08:1) was added to it. At the same time, a small amount of ammonium sulfate and PLP coenzyme (0.04‰) were added to the reaction system. Finally, tyrosine phenol lyase (enzyme number: EC:4.1.99.2, tyrosine phenol lyase: phenol = 20U: 1g) was added to carry out the catalytic reaction. The pH of the reaction process was controlled at 8.3-8.5 and the temperature was 40-45℃. The conversion was completed after 10 hours. Samples were taken to detect the pyruvate content and tyrosine production. In order to verify the effect of heteroacids on the conversion rate, a positive control was made at the same time. During the pyruvate conversion process of H26-cgr02, lactic acid and α-ketoglutaric acid were added in the same proportion as hy-001 to carry out the conversion test. The other conversion conditions remained unchanged.

[0073] Tyrosine was detected by HPLC, with a chromatographic column of Hypersil ODS 5um, 4.6*250mm, a mobile phase of 0.05mol / L potassium dihydrogen phosphate solution (pH3.4): methanol = 95:5, a flow rate of 1mL / min, a column temperature of 40°C, and a wavelength of 210nm.

[0074] Table 7 Conversion rate and tyrosine production comparison of screened strains

[0075]

[0076] As shown in Table 7, compared with the starting strain, the pyruvate fermented by the screening strain H26-cgr02 was converted by tyrosine phenol lyase, and the pyruvate conversion rate was higher, reaching 99%, and the tyrosine production was also higher, reaching 1.45 times that of the starting strain. In the positive control group, the pyruvate conversion rate was only 88%, indicating that the pyruvate in the fermentation broth had an inhibitory effect on the enzyme conversion reaction. The fermentation broth of H26-cgr02 had lower pyruvate, which had a lower impact on the enzyme activity, so the conversion rate was significantly improved.

Claims

1. A Torulopsis glabrata strain, which is deposited in China Center for Type Culture Collection (CCTCC) with the accession number of CCTCC M 20231147.

2. A method for culturing a Torulopsis glabrata strain, characterized in that: The culturing method comprises fermenting the Torulopsis glabrata strain according to claim 1 in a fermentation medium.

3. The culture method according to claim 2, characterized in that The fermentation medium comprises 100-150 g / L of glucose, 5-10 g / L of ammonium sulfate, 5-10 g / L of sodium acetate, 1-10 g / L of potassium dihydrogen phosphate, 0.1-1 g / L of magnesium sulfate, 10-50 mg / L of ferrous sulfate, 0.1-1 mg / L of copper sulfate, 1-10 mg / L of manganese chloride, 1-10 mg / L of zinc chloride and a vitamin solution; the vitamin solution comprises 0.01-0.1 mg / L of thiamine hydrochloride, 0.1-1 mg / L of pyridoxine hydrochloride, 0.01-0.1 mg / L of biotin, 1-10 mg / L of nicotinic acid and 0.1-1 mg / L of riboflavin.

4. The culture method according to claim 2, characterized in that The fermentation medium comprises 100-150 g / L of glucose, 5-10 g / L of ammonium sulfate, 1-10 g / L of potassium dihydrogen phosphate, 0.1-1 g / L of magnesium sulfate, 1-10 mg / L of nicotinic acid, 0.01-0.1 mg / L of thiamine hydrochloride, 0.1-1 mg / L of pyridoxine hydrochloride, 0.01-0.1 mg / L of biotin, 5-20 mg / L of calcium chloride, 5-20 mg / L of ferrous sulfate, 5-50 mg / L of zinc chloride, 1-10 mg / L of manganese chloride, 1-10 mg / L of copper sulfate and 20-50 g / L of calcium carbonate.

5. The culture method according to any one of claims 2 to 4, characterized in that: The pH of the fermentation culture is 4.0-5.5, for example, pH 4.0, 4.5 or 5.5; And / or, the fermentation culture temperature is 27-30°C; and / or, the dissolved oxygen content of the fermentation culture is 15-40%, preferably 20-30%; And / or, the residual sugar content of the fermentation culture is 2-8 g / L, preferably 3-5 g / L.

6. The culture method according to any one of claims 2 to 5, characterized in that: The culture method further comprises performing expansion culture in a seed culture medium before fermentation culture; Preferably, the seed culture medium comprises: 20-30 g / L glucose, 0.8-1 g / L potassium dihydrogen phosphate, 0.1-1 g / L magnesium sulfate and 10-20 g / L peptone.

7. A method for producing pyruvic acid, characterized in that: The method comprises the step of culturing the Torulopsis glabrata strain as claimed in claim 1 to obtain pyruvic acid; preferably, the method further comprises the step of culturing the Torulopsis glabrata strain according to the culturing method as claimed in any one of claims 2 to 6.

8. A fermentation tank culture medium for a Torulopsis glabrata strain, characterized in that: The fermentation tank culture medium comprises 100-150 g / L of glucose, 5-10 g / L of ammonium sulfate, 5-10 g / L of sodium acetate, 1-10 g / L of potassium dihydrogen phosphate, 0.1-1 g / L of magnesium sulfate, 10-50 mg / L of ferrous sulfate, 0.1-1 mg / L of copper sulfate, 1-10 mg / L of manganese chloride, 1-10 mg / L of zinc chloride and a vitamin solution; the vitamin solution comprises 0.01-0.1 mg / L of thiamine hydrochloride, 0.1-1 mg / L of pyridoxine hydrochloride, 0.01-0.1 mg / L of biotin, 1-10 mg / L of nicotinic acid and 0.1-1 mg / L of riboflavin.

9. A primary screening fermentation medium for a Torulopsis glabrata strain, characterized in that: The primary screening fermentation medium comprises 100-150 g / L of glucose, 5-10 g / L of ammonium sulfate, 1-10 g / L of potassium dihydrogen phosphate, 0.1-1 g / L of magnesium sulfate, 1-10 mg / L of nicotinic acid, 0.01-0.1 mg / L of thiamine hydrochloride, 0.1-1 mg / L of pyridoxine hydrochloride, 0.01-0.1 mg / L of biotin, 5-20 mg / L of calcium chloride, 5-20 mg / L of ferrous sulfate, 5-50 mg / L of zinc chloride, 1-10 mg / L of manganese chloride, 1-10 mg / L of copper sulfate and 20-50 g / L of calcium carbonate.

10. Use of the Torulopsis glabrata strain according to claim 1, the fermentation tank medium according to claim 8 or the primary screening fermentation medium according to claim 9 in producing pyruvic acid.

Images