Modified aureobasidium pullulans strain based on reduction of malic acid pathway consumption and application thereof

Abstract

The invention discloses a modified aureobasidium pullulans strain based on reduction of malic acid pathway consumption and application of the modified aureobasidium pullulans strain. Gene for coding NADPH dependent malic enzyme and phosphoenolpyruvate carboxykinase in an aureobasidium pullulans genome is directionally knocked out. The transformation blocks a key consumption branch for synthesis and accumulation of the cytoplasmic malic acid, and effectively guides a central carbon metabolic flow to be more efficiently guided to a target product. Experimental results show that the polymalic acid yield and the saccharic acid conversion rate of the double-knockout engineering strain D3N5-delta3617 / deltapepck in fermentation are remarkably improved compared with those of an original strain, and the biomass is also increased. The invention provides a high-performance strain with important application value for industrial efficient fermentation production of polymalic acid.

Description

Technical Field

[0001] This invention relates to the field of metabolic engineering, specifically to a modified budding short-stem mold strain based on reducing malic acid pathway consumption, and also to the application of this strain. Background Technology

[0002] Aureobasidum pullulan is a type of yeast fungus with various forms, such as filamentous, yeast-like, and chlamydospore-like forms. This species is widely distributed in nature and can utilize a variety of substrates. Its main metabolites include polymalic acid, pullulan, heavy oil, and melanin.

[0003] Polymalic acid (PMA), a novel, fully biodegradable polymer, has broad application potential in areas such as drug delivery systems and food packaging. This polymer is synthesized in vivo using L-malic acid as the sole monomer and exhibits excellent bioabsorbability, biocompatibility, and biodegradability.

[0004] *Brachystomum buddingum* can simultaneously produce multiple extracellular products during the synthesis of polymalic acid (PMBA). PMBA, a polypolyester compound, is synthesized in vivo using L-malic acid as the sole monomer via polymalic acid synthase and secreted extracellularly. In *Brachystomum buddingum*, malic acid is synthesized using glucose as a substrate through three pathways: the oxidative TCA pathway, the reductive TCA pathway, and the glyoxylate pathway. The reductive TCA pathway has the highest conversion rate, yielding 2 mol of malic acid from 1 mol of glucose. The glyoxylate pathway is the next most efficient, producing 1.33 mol of malic acid from 1 mol of glucose. However, the oxidative TCA pathway yields only 1 mol of malic acid from 1 mol of glucose. In natural strains, PMBA depends on the primary pathways, which contain numerous pathways that consume the substrate malic acid. The existence of these substrate-consuming pathways limits the carbon flux conversion from substrate to product, resulting in carbon and energy losses. Metabolic engineering was used to modify and optimize the polymalic acid synthesis pathway, further improving the conversion efficiency of glucose to malic acid and thus increasing the yield of polymalic acid, laying the foundation for polymalic acid industrial production strains. Summary of the Invention

[0005] In view of this, one objective of the present invention is to provide a modified budding short-stem mold strain based on reducing malic acid consumption; a second objective of the present invention is to provide a method for increasing the production of polymalic acid by budding short-stem mold; a third objective of the present invention is to provide the application of the modified budding short-stem mold strain in the fermentation production of polymalic acid; and a fourth objective of the present invention is to provide a method for producing polymalic acid and then hydrolyzing it to prepare L-malic acid.

[0006] To achieve the above objectives, the present invention provides the following technical solution: 1. Based on the modified *Brachystomia buddingensis* strain that reduces malic acid consumption, the gene encoding NADPH-dependent malate enzyme g3617 and / or the gene encoding phosphoenolpyruvate carboxykinase pepck were knocked out in the *Brachystomia buddingensis* strain. The resulting recombinant *Brachystomia buddingensis* strain is a strain with increased polymalic acid production.

[0007] Preferably, the nucleotide sequence of the NADPH-dependent malic acid oxidase encoding gene g3617 is shown in SEQ ID NO.1.

[0008] Preferably, the nucleotide sequence of the phosphoenolpyruvate carboxykinase encoding gene pepck is shown in SEQ ID NO.2.

[0009] 2. A method for increasing the production of polymalic acid by *Bacillus buddingus*, comprising knocking out the NADPH-dependent malate enzyme encoding gene g3617 and / or the phosphoenolpyruvate carboxykinase encoding gene pepck in the genome of *Bacillus buddingus* using gene editing technology.

[0010] Preferably, the gene editing technology of this invention is the CRISPR-Cas9 system.

[0011] Preferably, the nucleotide sequence of the NADPH-dependent malate enzyme encoding gene g3617 is shown in SEQ ID NO.1; and the nucleotide sequence of the phosphoenolpyruvate carboxykinase encoding gene pepck is shown in SEQ ID NO.2.

[0012] Preferably, the method for knocking out the NADPH-dependent malate enzyme encoding gene g3617 in the genome of the budding short-stalked dermal fungus is protoplast transformation; the method for knocking out the phosphoenolpyruvate carboxykinase encoding gene pepck is protoplast transformation.

[0013] 3. Application of the modified budding short-stem mold strain in the fermentation production of polymalic acid.

[0014] 4. A method for producing polymalic acid, comprising the following steps: culturing the modified budding short-stem mold strain to ferment and accumulate polymalic acid in a fermentation medium; and recovering polymalic acid from the fermentation product.

[0015] Preferably, the fermentation medium of this invention comprises: glucose 80-100 g / L, ammonium chloride 1.5-1.8 g / L, sodium nitrate 2.5-2.7 g / L, KH2PO4 0.08-0.12 g / L, ZnSO4·7H2O 0.16-0.20 g / L, MgSO4 0.08-0.12 g / L, KCl 0.9-1.1 g / L, corn steep liquor 0.9-1.1 g / L, CaCO3 28-32 g / L, and citric acid monohydrate 1.0-1.2 g / L; the fermentation conditions are: culture temperature 24-26℃, shaking speed 210-230 rpm, and culture time 110-130 hours.

[0016] In this invention, the budding short-stalked mold strain selected is CCTCCM2012223, which is disclosed in Chinese Patent Publication No. CN102827778A.

[0017] The beneficial effects of this invention are as follows: Based on an engineered budding short-stem mold strain that reduces malic acid consumption, this invention effectively blocks the non-targeted consumption of cytoplasmic malic acid and its precursor oxaloacetate at the source of the metabolic pathway by precisely knocking out two key competitive diversion genes (g3617 and pepck), forcing carbon flux to accumulate in the polymalic acid synthesis pathway, thus solving the problem of severe diversion in the natural metabolic network. The obtained engineered strains show significant improvements in polymalic acid yield and sugar-acid conversion rate. Among them, the double-knockout strain exhibits the best performance, with a 26.6% increase in yield and a simultaneous 26.6% increase in sugar-acid conversion rate compared to the original strain, demonstrating outstanding technical and economic advantages. The engineered strains constructed in this invention, especially the double-knockout strain, are characterized by high yield, high efficiency, and stable traits, providing a directly applicable core strain resource for the industrial fermentation production of polymalic acid and even its monomeric malic acid, with a clear industrialization prospect. Attached Figure Description

[0018] To make the objectives, technical solutions, and beneficial effects of this invention clearer, the following figures are provided for illustration: Figure 1 This is a schematic diagram of metabolic modification; Figure 2 Agarose gel image for verifying g3617 knockout; Figure 3 The results are from the fermentation test of D3N5-△3617. Figure 4 Agarose gel image for verification of phosphoenolpyruvate carboxykinase knockout; Figure 5 The results are from the fermentation detection of D3N5-△3617-pepck. Detailed Implementation

[0019] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0020] The process conditions in this invention are as follows: Seed culture: The strain was inoculated into YPD using an inoculation loop, incubated overnight at 25℃ and 220 rpm on a shaker until the OD600 value reached 6.0-7.0. Subsequently, it was inoculated into seed culture medium at a rate of 2%. The seed culture medium consisted of 60 g / L glucose, 2.7 g / L ammonium chloride, 4.2 g / L sodium nitrate, 0.1 g / L KH₂PO₄, 0.178 g / L ZnSO₄, 0.1 g / L MgSO₄, 0.1 g / L corn steep liquor, and 20 g / L CaCO₃. The seed culture process involved filling 250 mL shake flasks with 30 mL of the liquid, incubating at 25℃ and 220 rpm on a shaker for 60-72 hours.

[0021] Fermentation process: Shake flask fermentation was used. The fermentation formula was: glucose 90 g / L, nitrogen sources ammonium chloride and sodium nitrate 1.6875 g / L and 2.6375 g / L respectively, KH2PO4 0.1 g / L, ZnSO4·7H2O 0.178 g / L; MgSO4 0.1 g / L, KCl 1 g / L, corn steep liquor 1 g / L, CaCO3 30 g / L, and citric acid (monohydrate) 1.1 g / L. The shake flask fermentation process involved filling 250 mL shake flasks with 30 mL of liquid, incubating at 25℃, shaking at 220 rpm, and incubating for 120 hours. The seed culture inoculum ratio was 10%.

[0022] Knockout reagent preparation method: Buffer I: Accurately weigh 1.4705 g of trisodium citrate and 18.21 g of sorbitol, dissolve them in ddH2O, adjust the pH to 5.8 with 1 M citric acid, and then bring the volume to 100 mL with ddH2O.

[0023] STC Buffer: Accurately weigh 18.21 g of sorbitol, 0.27745 g of CaCl2, and 0.30275 g of Tris. Dissolve them in ddH2O, adjust the pH to 7.5 with hydrochloric acid, and then bring the volume to 100 mL with ddH2O.

[0024] PTC Solution: Accurately measure 5 mL of STC Buffer (approximately 5.27 g), and accurately weigh an equal mass of PEG-4000 into the STC Buffer. Dissolve by heating (do not boil, a warm bath is sufficient). Before use, add 40 μL / 1 mL of filtered and sterilized β-mercaptoethanol and mix well.

[0025] Driselase: Accurately weigh 50 mg of Driselase, then accurately dissolve it in 1 mL of Buffer I to prepare a 50 mg / mL stock solution. Centrifuge at 13000 rpm for 1 min, collect the supernatant, filter sterilize, and store at -20 ℃. Lysing Enzymes: Accurately weigh 20 mg of Lysing Enzymes, dissolve in 1 mL of sorbitol Buffer, mix well, filter sterilize, centrifuge at 13000 rpm for 1 min to prepare a 20 mg / mL stock solution, and store at -20 ℃. 2×HCS Stock Solution: Yeast extract 6 g / L, peptone 20 g / L, glucose 20 g / L, beef extract 2 g / L, malt extract 6 g / L, 182.1 g / L D-sorbitol, adjust pH to 5.7.

[0026] M100S solid culture medium: glucose 10 g / L, KNO3 3 g / L, M-100SS 62.5 mL / L, D-sorbitol 182.1 g / L, agar 15 g / L. Except for the enzymes, all reagents must be autoclaved at 115 °C for 30 min.

[0027] Protoplasmic transformation operation method: (1) Pick out the single colony of budding short-stalked spores D3N5 required for the experiment, culture it in HC medium at 25 ℃ and 200 rpm until the OD600 is about 1.0, and then let it stand on ice for 30 min.

[0028] (2) Collect bacterial cells by centrifuging the pre-cooled bacterial solution at 4 ℃ and 5000 rpm for 10 min.

[0029] (3) Take an equal volume of Buffer I to wash twice, centrifuge at 4 ℃ and 5000 rpm for 10 min to collect cells, and then resuspend the cells in Buffer I.

[0030] (4) Add 50 mg / mL of lysis enzyme and 20 mg / mL of lyase to the bacterial suspension and incubate at 22 ℃ and 100 rpm for about 30-60 min (the specific incubation time depends on the activity of the enzyme used; stop incubation when a large number of protoplasts appear in the bacterial cells under a microscope).

[0031] (5) After incubation, centrifuge at 3000 rpm for 10 min at 4 ℃ to collect cells. Then wash the cells twice with STC Buffer, centrifuge at 2500 rpm for 10 min at 4 ℃ to collect cells, and resuspend the cells with STC Buffer to 3.8×10 2 cells / mL. Aliquot 200 uL / tube.

[0032] (6) Add 2-3 μg of plasmid to the dispensed EP tube, then add 50 μL of PTC Solution and incubate at 22℃ for 5-10 min.

[0033] (7) Add 1 mL of PTC Solution in 3 portions: add 50 μL dropwise and mix gently for 1 min; add 200 μL dropwise and mix gently for 1 min; finally add the remaining solution and mix gently. Incubate at 22 °C and 100 rpm for 1 h.

[0034] (8) Centrifuge at 22 ℃ and 6680 rpm for 15 min to collect cells, wash cells twice with 1 mL 2×HCS, and centrifuge at 22 ℃ and 5450 rpm for 15 min to collect cells.

[0035] (9) Resuspend the cells in 0.5 mL of 2×HCS and incubate at 25 ℃ and 100 rpm for 1 h.

[0036] (10) Take an appropriate amount of the incubated bacterial solution and spread it evenly on the corresponding fungal resistance plate of M100S (M100+sorbitol, the final concentration of sorbitol is 1M), and incubate at 25 ℃ for about 3-5 days until the transformants grow.

[0037] (11) Screening and verification: Transformants grown on M100S resistance plates were transferred to M100 plates containing the corresponding fungal resistance for rescreening. After the transformants grew, an appropriate amount of bacterial cells were taken and subjected to cell disruption treatment with 10 μL of 20 Mm NaOH. The disrupted reaction solution was then used for bacterial culture PCR verification in Taq Plus DNA Polymerse. The PCR program settings are shown in Table 1.

[0038] Table 1. Verification using the PCR procedure step temperature time Cycle number 1. Prevariation 95°C 5 minutes 1 2. Cyclic amplification 30 cycles transsexual 95°C 15 seconds annealing 45 - 65°C (primer™ value) 15 seconds extend 72°C 60 seconds / kb 3. Final Extension 72°C 5 minutes 1 4. Save 4 °C unlimited 1 Polymalic acid (PMA) detection method: A certain amount of fermentation sample was centrifuged at 8000 rpm for 10 min, and the supernatant was separated from the precipitate. An equal volume of 1 mL of the supernatant was added to a 2 mol / L sulfuric acid solution, mixed thoroughly, and then placed in a 75℃ water bath for hydrolysis for 12-14 h. After appropriate dilution, the hydrolysate was filtered through a 0.22 μm aqueous microporous membrane and malic acid was detected by HPLC. The PMA data was calculated by multiplying the measured malic acid value by a proportionality coefficient of 0.86. HPLC detection conditions: Shimadzu LC-16 high-performance liquid chromatograph with a UV detector at a wavelength of 210 nm; Dima Spursil C18-EP column; mobile phase: aqueous solution containing 5 mmol / L sulfuric acid at a flow rate of 0.6 mL / min; column temperature: 40℃.

[0039] The present invention will now be described in detail with reference to embodiments. Experimental methods in preferred embodiments, where specific conditions are not specified, are generally performed under conventional conditions.

[0040] To obtain high-performance bacterial strains, the metabolic pathways of the strains were modified. The specific principles are as follows: Figure 1 As shown in SEQ ID NO.1, the nucleotide sequence of the NADPH-dependent malic acid kinase encoding gene g3617 is shown in SEQ ID NO.2, and the nucleotide sequence of the phosphoenolpyruvate carboxykinase encoding gene pepck is shown in SEQ ID NO.2.

[0041] Example 1: NADPH-dependent malicase gene knockout To construct the CRISPR-Cas9 knockout plasmid, p426-SNR52p-gRNA.CAN1.Y-SUP4t-Cas9-2μ-ori-hyg was used as the vector plasmid. A pullulan synthesis gene knockout plasmid was constructed by replacing the N20 sequence. Vector construction was performed using Phanta Max Super-Fidelity DNA Polymerase (P505) with primers g3617-N20-F and g3617-N20-R: reverse amplification to obtain a linearized vector. The amplification procedure was similar to the validation procedure, except that the amplification rate was 30 s / kb. The amplification was then performed using the ClonExpress MultiS One Step Cloning Kit (Vazyme, China). For recombinant transformation, the fragment and vector were first mixed at a 1:1 ratio. After adding the necessary reagents for the enzyme ligation system, the mixture was ligated at 50°C for 20 min. The ligation product was then added to *E. coli* DH10 competent cells, incubated on ice for 30 min, heat-shocked at 42°C for 90 s, and placed on ice for 2 min. 0.8 mL of antibiotic-free LB medium was added, and the mixture was incubated at 37°C, 100 rpm for 1 h. After centrifugation at 5000 rpm for 5 min, most of the supernatant was discarded, and the mixture was plated onto the appropriate antibiotic plates. After single colonies grew, the plasmid was obtained through verification sequencing. The donor used for knockout was amplified using 3617-up-F / R and 3617-down-F / R primers to obtain upstream and downstream homologous arms. The donor was then amplified by fusion PCR using the upstream and downstream homologous arms as templates and 3617-up-F / 3617-down-R primers. The fragment was recovered using a gel extraction kit.

[0042] Table 2. Primers used for NADPH-dependent malate gene knockout g3617-N20-F 5'-CGACACCGCATACCCCATTGGTTTTAGAGCTAGAAATAGCAAGT-3' (SEQ ID NO.3) G3617-N20-R 5'-CAATGGGGTATGCGGTGTCGGATCATTTATCTTTCACTGCGG-3' (SEQ ID NO.4) 3617-up-F 5'-GACATCGGATAAAAGCAAGGATCTGA-3' (SEQ ID NO.5) 3617-up-R 5'-TCTTTCACACTTCTTCCACCACGGCCACGACATAACCAAAA-3' (SEQ ID NO.6) 3617-down-F 5'-TTTGGTATGGTCGTGGCCGTGGTGGAAGAAGTGTGAAAGA-3' (SEQ ID NO.7) 3617-down-R 5'-CCGCCGACCGCAAAACCGTATACAGAAA-3' (SEQ ID NO.8) The knockout verification agarose gel electrophoresis size was 3000 bp. For unsuccessful knockout verification, the size should be 4846 bp. Sequencing results showed successful knockout, yielding strain D3N5-△3617. The knockout verification agarose gel image is shown below. Figure 2 As shown.

[0043] After successful knockout, strain D3N5-△3617 underwent shake-flask fermentation assays. The seed culture, fermentation culture, and detection processes were as described in the technical specifications. The fermentation test results are as follows: Figure 3 .

[0044] After knockout, the polymalic acid yield of D3N5-△3617 was 47.5 g / L, and the malic acid yield after acid hydrolysis was 55.2 g ± 2.1 g / L. The yield was 8.6% higher than that of the original strain D3N5, the sugar-acid conversion rate was 7.1% higher, and the biomass and fermentation cycle did not change significantly.

[0045] Example 2: Knockout of the phosphoenolpyruvate carboxykinase gene To construct the CRISPR-Cas9 knockout plasmid, p426-SNR52p-gRNA.CAN1.Y-SUP4t-Cas9-2μ-ori-hyg was used as the vector plasmid. A pullulan synthesis gene knockout plasmid was constructed by replacing the N20 sequence. Vector construction was performed using Phanta Max Super-Fidelity DNA Polymerase (P505) with primers pepck-n20-f and pepck-n20-r to obtain a linearized vector. The vector was then processed using the ClonExpress MultiS One Step Cloning Kit (Vazyme, China). For enzyme-linked recombination, the fragment and vector were first mixed at a 1:1 ratio. After adding the necessary reagents, the mixture was incubated at 50°C for 20 min. The enzyme-linked product was then added to *E. coli* DH10 competent cells and incubated on ice for 30 min, followed by a heat shock at 42°C for 90 s, and then placed on ice for 2 min. 0.8 mL of antibiotic-free LB medium was added, and the mixture was incubated at 37°C, 100 rpm for 1 h. After centrifugation at 5000 rpm for 5 min, most of the supernatant was discarded, and the mixture was plated onto the appropriate antibiotic plates. After single-cell colonies grew, the plasmid was obtained through verification sequencing. The donor was knocked out using pepck-up-F / R and pepck-down-F / R amplification to obtain upstream and downstream homologous arms. The donor was then amplified using fusion PCR with the upstream and downstream homologous arms as templates and pepck-up-F / pepck-down-R primers. The fragment was recovered using a gel extraction kit. Table 3. Primers used for phosphoenolpyruvate carboxykinase knockout pepck-n20-f 5'-TGAACCGGTTTCGTAGACGAGTTTTAGAGCTAGAAATAGCAAGT-3' (SEQ ID NO.9) pepck-n20-r 5'-TCGTCTACGAAACCGGTTCAGATCATTTATCTTTCACTGCGGAGAAG-3' (SEQ ID NO.10) pepck-up-F 5'-CTCGGACCTCAACTGACAGTTCC-3' (SEQ ID NO.11) pepck-up-R 5'-TTCCATTCTACATCACTCAAAGATGCTGCAACCGCTTTTTAGG-3' (SEQ ID NO. 12) pepck-down-F 5'-CTAAAAAGCGGTTGCAGCATCTTTGAGTGATGTAGAATGGAAGTTTCCCTT-3' (SEQ ID NO.13) pepck-down-R 5'-ATCTGCAGGGTGCTGGTAAGATTAA-3' (SEQ ID NO.14) The knockout validation agarose gel electrophoresis size was 3002 bp, and the validation size for unsuccessful knockout should be 5082 bp. The gel run results indicate successful knockout. Figure 4 As shown.

[0046] Fermentation results as follows Figure 5 As shown, the shake-flask fermentation results indicated that the double-knockout strain produced 55.2 g / L of polymalic acid, and after acid hydrolysis, the yield of L-malic acid was 64.2 ± 3.2 g / L, representing a 16.1% increase compared to D3N5-△3617 and a 26.6% increase compared to the original strain D3N5. The sugar-acid conversion rate was increased by 16.0% and 26.6% respectively compared to the two strains, and the biomass increased by 15.7% and 25.4% respectively.

[0047] The above-described embodiments are merely preferred embodiments provided to fully illustrate the present invention, and the scope of protection of the present invention is not limited thereto. Equivalent substitutions or modifications made by those skilled in the art based on the present invention are all within the scope of protection of the present invention. The scope of protection of the present invention is defined by the claims.

Claims

1. A modified budding short-stem fungus strain based on reducing malic acid consumption, characterized by: Knockout of the NADPH-dependent malate enzyme encoding gene g3617 and / or the phosphoenolpyruvate carboxykinase encoding gene pepck in the *Brachys buddingus* strain resulted in a recombinant *Brachys buddingus* strain with increased polymalic acid production.

2. The modified budding short-stem fungus strain according to claim 1, characterized in that: The nucleotide sequence of the NADPH-dependent malic acid oxidase encoding gene g3617 is shown in SEQ ID NO.

1.

3. The modified budding short-stem fungus strain according to claim 1, characterized in that: The nucleotide sequence of the phosphoenolpyruvate carboxykinase encoding gene pepck is shown in SEQ ID NO.

2.

4. A method for increasing the yield of polymalic acid synthesized by *Bacillus buddingus*, characterized in that: The NADPH-dependent malic acid kinase encoding gene g3617 and / or the phosphoenolpyruvate carboxykinase encoding gene pepck in the genome of the *Brucea buddingis* were knocked out using gene editing technology.

5. The method for increasing the yield of polymalic acid synthesized by *Bacillus buddingus* according to claim 4, characterized in that: The gene editing technology used is the CRISPR-Cas9 system.

6. The method for increasing the yield of polymalic acid synthesized by *Bacillus buddingus* according to claim 4, characterized in that: The nucleotide sequence of the NADPH-dependent malic acid kinase encoding gene g3617 is shown in SEQ ID NO.1; the nucleotide sequence of the phosphoenolpyruvate carboxykinase encoding gene pepck is shown in SEQ ID NO.

2.

7. The method for increasing the yield of polymalic acid synthesized by *Bacillus buddingus* according to claim 4, characterized in that: The method for knocking out the NADPH-dependent malate enzyme encoding gene g3617 in the genome of the budding short-stalked dermal fungus is the protoplast transformation method; the method for knocking out the phosphoenolpyruvate carboxykinase encoding gene pepck is the protoplast transformation method.

8. The application of the modified budding short-stem fungus strain according to any one of claims 1 to 3 in the fermentation production of polymalic acid.

9. A method for producing polymalic acid, characterized in that, The method includes the following steps: culturing the modified budding short-stem fungus strain according to any one of claims 1 to 3, causing it to ferment in a fermentation medium to produce and accumulate polymalic acid; and recovering polymalic acid from the fermentation product.

10. The method according to claim 9, characterized in that: The fermentation medium comprises: glucose 80-100 g / L, ammonium chloride 1.5-1.8 g / L, sodium nitrate 2.5-2.7 g / L, KH2PO4 0.08-0.12 g / L, ZnSO4·7H2O 0.16-0.20 g / L, MgSO4 0.08-0.12 g / L, KCl 0.9-1.1 g / L, corn steep liquor 0.9-1.1 g / L, CaCO3 28-32 g / L, and citric acid monohydrate 1.0-1.2 g / L; the fermentation conditions are: culture temperature 24-26℃, shaking speed 210-230 rpm, and culture time 110-130 hours.

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