A glutamic acid corynebacterium resistant to viscosity and its application

Abstract

The present invention relates to a glutamic acid corynebacterium resistant to viscosity and its application, belonging to the technical field of biological genetic engineering. The glutamic acid corynebacterium of the present invention is obtained by mutating glutamic acid corynebacterium ATCC 13032, and the mutation sites include: mutating cytosine at position 862902 to thymine; mutating guanine at position 862903 to adenine; mutating cytosine at position 862953 to thymine; mutating adenine at position 862961 to guanine; inserting cytosine and thymine at position 862958 and deleting and mutating guanine at position 862963. The glutamic acid corynebacterium of the present invention has significantly improved tolerance in a highly viscous environment and growth and metabolic capabilities under low dissolved oxygen conditions, thereby increasing the yield of mucopolysaccharide and avoiding the problem that the fermentation broth becomes viscous due to the produced mucopolysaccharide, resulting in insufficient dissolved oxygen and restricting the metabolism of glutamic acid corynebacterium, ultimately affecting the synthesis of mucopolysaccharide.

Description

Technical Field

[0001] The present invention relates to the technical field of biological genetic engineering, and particularly to a Corynebacterium glutamicum with viscosity resistance and its application. Background Art

[0002] Corynebacterium glutamicum is a Gram-positive bacterium. Due to its clear genetic background, stable protein secretion, low extracellular hydrolase activity, non-toxicity and other characteristics, it has now been widely used in the production processes of various value-added chemicals, amino acids and fuels. With the continuous revelation of gene regulation mechanisms, using synthetic biology technology to design and construct Corynebacterium glutamicum for biomanufacturing has become a research hotspot in this field. In addition, Corynebacterium glutamicum is a strict aerobe and requires continuous introduction of sterile air during its fermentation process. Therefore, when Corynebacterium glutamicum ferments to synthesize viscous substances, such as hyaluronic acid, chondroitin, heparin precursors, etc., or during high-density fermentation, with the accumulation of viscous substances and the increase in cell density of the bacteria, the dissolved oxygen in the fermentation broth will decrease, thus affecting its normal metabolic activities, which is also one of the difficulties in optimizing the fermentation process of Corynebacterium glutamicum. Hyaluronic acid (HA) is a type of linear acidic mucopolysaccharide with good moisturizing properties and can play roles such as inhibiting inflammation, and is widely used in the cosmetic and pharmaceutical fields. Currently, most hyaluronic acid in the industrial market is obtained by fermenting pathogenic microorganisms such as Streptococcus zooepidemicus and Escherichia coli K4. However, due to the presence of pathogenic factors such as endotoxins, its development in the pharmaceutical and other fields is severely restricted. Using microorganisms with clear genetic backgrounds and high biosafety to synthesize hyaluronic acid, such as Corynebacterium glutamicum, has become the development trend of synthesizing hyaluronic acid by the microbial fermentation method.

[0003] Although the yields of synthesizing mucopolysaccharides such as hyaluronic acid, chondroitin, heparin precursors, etc. using Corynebacterium glutamicum have been significantly improved at present, due to its strong water absorption, the fermentation broth will become viscous during the fermentation process, thereby inhibiting the normal metabolic activities of cells and restricting the further increase in the yields of viscous substances such as hyaluronic acid, chondroitin, heparin precursors, etc. Therefore, it is expected to transform Corynebacterium glutamicum through directed evolution engineering to improve its tolerance in highly viscous solutions, and then further improve the synthesis efficiency of Corynebacterium glutamicum for mucopolysaccharides. Summary of the Invention

[0004] To this end, the technical problem to be solved by the present invention is to overcome the problem in the prior art that when Corynebacterium glutamicum produces hyaluronic acid, the fermentation broth becomes viscous during the fermentation process, resulting in a decrease in the dissolved oxygen content of the fermentation broth, thereby inhibiting the metabolic activities of Corynebacterium glutamicum and ultimately affecting the yield of hyaluronic acid.

[0005] To solve the above technical problems, the present invention provides a Corynebacterium glutamicum, which is obtained by mutating Corynebacterium glutamicum ATCC 13032. The mutation sites include mutating cytosine at position 862902 to thymine; mutating guanine at position 862903 to adenine; mutating cytosine at position 862953 to thymine; mutating adenine at position 862961 to guanine; inserting cytosine and thymine at position 862958; and deleting and mutating guanine at position 862963. The Corynebacterium glutamicum of the present invention has significantly improved tolerance in a highly viscous environment and growth and metabolic capabilities under low dissolved oxygen conditions, thereby increasing the yield of mucopolysaccharide and avoiding the problem that the fermentation broth becomes viscous due to the produced mucopolysaccharide, resulting in insufficient dissolved oxygen and restricting the metabolism of Corynebacterium glutamicum, ultimately affecting the synthesis of mucopolysaccharide.

[0006] The first object of the present invention is to provide a viscous-tolerant Corynebacterium glutamicum, characterized in that the Corynebacterium glutamicum is obtained by mutating Corynebacterium glutamicum ATCC 13032, and the mutation sites include:

[0007] (1) Mutating cytosine at position 862902 to thymine;

[0008] (2) Mutating guanine at position 862903 to adenine;

[0009] (3) Mutating cytosine at position 862953 to thymine;

[0010] (4) Mutating adenine at position 862961 to guanine;

[0011] (5) Inserting cytosine and thymine at position 862958;

[0012] (6) Deleting and mutating guanine at position 862963.

[0013] Furthermore, the mutation sites include:

[0014]

[0015]

[0016]

[0017] The second object of the present invention is to provide an application of the above-mentioned Corynebacterium glutamicum in the production of mucopolysaccharide.

[0018] Furthermore, the mucopolysaccharide includes hyaluronic acid.

[0019] The third object of the present invention is to provide a recombinant Corynebacterium glutamicum, and the modification of the recombinant Corynebacterium glutamicum includes: overexpressing at least one of hyaluronic acid synthase, glutamine-fructose-6-phosphate aminotransferase, phosphoglucomutase, and uridine diphosphate-glucose dehydrogenase in the above-mentioned Corynebacterium glutamicum.

[0020] Further, the gene sequence of glutamine-fructose-6-phosphate aminotransferase (glmS) is shown in SEQ ID NO.17, the gene sequence of phosphoglucomutase (glmM) is shown in SEQ ID NO.18, and the gene sequence of uridine diphosphate-glucose dehydrogenase (ugd) is shown in SEQ ID NO.19. Hyaluronic acid is mainly synthesized through the UDP-N-acetylglucosamine pathway and the UDP-glucuronic acid pathway. The enzymes expressed by the ugd, glmS, and glmM genes are key enzymes for these two pathways, and enhancing the synthesis of these three enzymes can effectively increase the yield of hyaluronic acid.

[0021] Further, the overexpression is initiated by the Ptac promoter or the Ptrc promoter. The ugd, glmS, and glmM genes themselves also have promoters, but the promoter sequences of the ugd, glmS, and glmM genes themselves are not clear and their expression ability is weak. The Ptac promoter and the Ptrc promoter are well-known strong promoters with strong expression ability. Therefore, by introducing the Ptac promoter or the Ptrc promoter in front of the ugd, glmS, and glmM genes, the expression of ugd, glmS, and glmM is strengthened, and thus the yield of hyaluronic acid is increased.

[0022] The sixth object of the present invention is to provide a method for producing mucopolysaccharide, which is characterized by including the step of fermenting the above-mentioned recombinant Corynebacterium glutamicum.

[0023] Further, the fermentation step includes: inoculating the recombinant Corynebacterium glutamicum into a fermentation medium for cultivation, adding IPTG to induce gene expression, centrifuging the fermentation broth after fermentation, and taking the supernatant to obtain the mucopolysaccharide.

[0024] Further, the temperature of the fermentation process is 15 - 40°C. Temperature will affect the growth of Corynebacterium glutamicum. Corynebacterium glutamicum can grow at 15 - 40°C, and the optimal growth temperature of Corynebacterium glutamicum is 30°C. Preferably, the temperature of the fermentation process is 30°C.

[0025] Further, the pH of the fermentation process is 6.5 - 7. pH will also affect the growth of Corynebacterium glutamicum. Corynebacterium glutamicum can grow at a pH of 5 - 9, and the optimal pH of Corynebacterium glutamicum is 7. Preferably, the pH of the fermentation process is 6.5 - 7.

[0026] Advantages of the present invention:

[0027] The tolerance of the Corynebacterium glutamicum of the present invention in a highly viscous environment and its growth and metabolic ability in a low dissolved oxygen environment are significantly improved, thereby increasing the yield of mucopolysaccharides (such as hyaluronic acid), and avoiding the problem that the fermentation broth becomes viscous due to the produced mucopolysaccharides, resulting in insufficient dissolved oxygen and restricting the metabolism of Corynebacterium glutamicum, ultimately affecting the synthesis of mucopolysaccharides. Description of the Drawings

[0028] In order to make the content of the present invention more clearly understood, the following further describes the present invention in detail according to specific embodiments of the present invention in conjunction with the drawings, where

[0029] Figure 1 is the directed evolution screening process of viscous-tolerant Corynebacterium glutamicum;

[0030] Figure 2 is the OD 600 and glucose consumption change curves of viscous-tolerant Corynebacterium glutamicum in different concentrations of hyaluronic acid;

[0031] Figure 3 is the hyaluronic acid yield of viscous-tolerant Corynebacterium glutamicum fermented in a 5 L fermenter;

[0032] Figure 4 is the bar chart of hyaluronic acid yields obtained by expressing different genes in the hyaluronic acid synthesis pathway. Detailed Embodiments

[0033] The following further describes the present invention in conjunction with the drawings and specific embodiments, so that those skilled in the art can better understand the present invention and be able to implement it, but the examples given are not intended to limit the present invention.

[0034] Strains: Wild-type Corynebacterium glutamicum (Corynebacterium glutamicum ATCC 13032);

[0035] Plasmids: pXMJ19, pk18mobsacb;

[0036] LB medium: Yeast extract 5 g / L, peptone 10 g / L, sodium chloride 10 g / L;

[0037] BHI: Brain heart infusion 37 g / L, sorbitol 91 g / L;

[0038] Fermentation medium: Glucose 40 g / L, corn steep liquor dry powder 20 g / L, (NH 4 ) 2 SO 4 30 g / L, KH2 PO 4 1 g / L, K 2 HPO 4 1 g / L, MgSO 4 25 g / L, 3 - morpholinopropanesulfonic acid (MOPS) 42 g / L.

[0039] Preparation of competent cells of Corynebacterium glutamicum: Pick a single colony on the plate and inoculate it into a shaking tube containing 5 mL of BHI liquid medium, and culture it overnight at 30 °C. Inoculate it into a baffled Erlenmeyer flask containing 50 mL of BHI at an initial OD 600 = 0.2, and culture it at 30 °C until OD 600 = 1.4 - 1.6. Collect the seed liquid into a centrifuge tube, ice - bath for 10 min, centrifuge at 4000 rpm and 4 °C for 5 min, and collect the cells. Resuspend the cells with 20 mL of 10% glycerol solution, centrifuge at 4000 rpm and 4 °C for 5 min, and collect the cells. Repeat the above operation twice, add 2.5 mL of 10% glycerol solution to resuspend the cells, aliquot them into pre - cooled sterile EP tubes, and store them at - 80 °C for use in electrotransformation.

[0040] Purification of hyaluronic acid: Collect the fermentation broth and centrifuge it at 10000 rpm for 5 min. Take an appropriate amount of the supernatant, add 4 times the volume of absolute ethanol, place it in a 4 °C environment for overnight alcohol precipitation, centrifuge at 10000 rpm for 5 min, and discard the supernatant. After the ethanol has evaporated, add water of the original volume to resuspend it, dissolve it thoroughly, centrifuge at 10000 rpm for 10 min, collect the supernatant, and repeat the above operation. After secondary alcohol precipitation, collect the supernatant, which is the purified hyaluronic acid sample.

[0041] Determination of hyaluronic acid yield: Weigh 4.77 g of sodium tetraborate decahydrate and dissolve it in 500 mL of concentrated sulfuric acid to prepare a borax - sulfuric acid solution; dissolve 1.25 g of carbazole in 500 mL of absolute ethanol to prepare a carbazole solution; prepare a 1 g / L glucuronic acid solution.

[0042] Take the purified hyaluronic acid sample, dilute it 10 - 100 times, pipette 200 μL into a glass colorimetric tube, and simultaneously add 1 mL of borax - sulfuric acid solution. After mixing, place it in a boiling water bath for 15 min and cool it on ice. Add 50 μL of carbazole solution, mix well, and place it in a boiling water bath for 10 min. Take 200 μL of the reaction solution into a 96 - well transparent plate, and use an enzyme - linked immunosorbent assay (ELISA) reader to measure the absorbance of the sample at a wavelength of 530 nm. Gradient - dilute the 1 g / L glucuronic acid solution to 0, 10, 20, 30, 40, 50 mg / L. After the borax - sulfuric acid - carbazole color reaction, measure the absorbance at 530 nm. Take the absorbance value as the abscissa and the glucuronic acid concentration (mg / L) as the ordinate to plot the relationship curve between the absorbance value and the glucuronic acid concentration, and obtain the standard curve equation: y = 121.7x - 6.035, R2 = 0.999.

[0043] Substitute the measured absorbance value of the sample into the standard equation to calculate the content of the hyaluronic acid sample. The formula for calculating the hyaluronic acid content: hyaluronic acid content (g / L) = (concentration measured from the standard curve * dilution factor * 2.067) / 1000.

[0044] Example 1: Screening of Corynebacterium glutamicum Resistant to Viscosity

[0045] (1) Screening of Corynebacterium glutamicum CG-HAT

[0046] Pick a single colony of Corynebacterium glutamicum ATCC 13032 and inoculate it into 5 mL of BHI medium, and culture it overnight at 30 °C. Inoculate it into 25 mL of fermentation medium at an initial inoculum size of OD 600 = 0.2. When it grows to the stationary phase, inoculate it into 25 mL of fermentation medium containing 10 g / L HA at an initial inoculum size of OD 600 = 0.2. After fermentation for 20 h, add 2 M NaOH to adjust the pH of the fermentation broth to neutral. After the strain ferments to the stationary phase, inoculate it into 25 mL of fermentation medium containing 20 g / L HA at an initial inoculum size of OD 600 = 0.2. After fermentation for 20 h, add 2 M NaOH to adjust the pH of the fermentation broth to neutral. After the strain ferments to the stationary phase, inoculate it into 25 mL of fermentation medium containing 40 g / L HA at an initial inoculum size of OD 600 = 0.2. After fermentation for 20 h, add 2 M NaOH to adjust the pH of the fermentation broth to neutral. After the strain ferments to the stationary phase, inoculate it into 25 mL of fermentation medium containing 60 g / L HA at an initial inoculum size of OD 600 = 0.2. After fermentation for 20 h, add 2 M NaOH to adjust the pH of the fermentation broth to neutral. As the strain is cultured in the viscous fermentation broth, gradually increase the HA content in the medium (as Figure 1 shown).

[0047] During the subculture process, periodically streak and isolate the strains in the fermentation broth, and randomly pick single colony strains for verification of the directed evolution effect. Use the wild-type Corynebacterium glutamicum ATCC 13032 as a control, and screen for mutant strains with the highest OD 600 . Inoculate the mutant strain and the control strain into fermentation media containing 0, 10, 20, 40 g / L HA respectively, and take samples of the fermentation broth at 2 h, 4 h, 6 h, 8 h, 12 h, 16 h, 20 h, 24 h, 36 h, 48 h, 60 h after inoculation, and measure OD 600 and the remaining glucose content in the fermentation broth, and it can be significantly found that there are differences in the growth rate and glucose consumption rate between the mutant strain and the wild-type strain.

[0048] Subcultured to the 300th generation, the strains in the fermentation broth were streaked and isolated. 100 single colony strains were selected and inoculated into 5 mL of fermentation medium containing 40 g / L HA, cultured at 220 rpm and 30 °C for 20 h, and OD was measured. 600 , and the evolved strain with the highest OD 600 was the HA-tolerant Corynebacterium glutamicum CG-HAT of the present invention, and the above-mentioned directed evolution effect verification process was repeated. The verification of the directed evolution effect of the CG-HAT strain is as Figure 2 shown. With the increase in the concentration of hyaluronic acid, CG-HAT had a higher glucose consumption compared with the wild-type Corynebacterium glutamicum ATCC 13032. Therefore, it can be considered that CG-HAT has better growth and metabolic ability in high-concentration hyaluronic acid.

[0049] The strain CG-HAT was inoculated into 5 mL of BHI medium and cultured overnight, and then sent to a relevant company for whole-genome sequencing. The genome of the wild-type Corynebacterium glutamicum ATCC 13032 before evolution was used as a reference genome for comparison, and it was found that multiple gene mutations occurred in the genome of the HA-tolerant Corynebacterium glutamicum. The specific mutation information is shown in Table 1.

[0050] Table 1 Mutation information of the genome of strain CG-HAT

[0051]

[0052]

[0053]

[0054] (2) Construction of Corynebacterium glutamicum CG-HAT-M

[0055] To further identify the key mutation sites affecting the strain tolerance, we selected potential key sites 862902, 862903, 862953, 862961, 862958, and 862963 for mutation verification on the genome of wild-type Corynebacterium glutamicum ATCC 13032. Using plasmid pk18mobsacb as a template, primers PK18-F / PK18-R were designed for PCR amplification to obtain the linear vector pk18mobsacb. Corynebacterium glutamicum CG-HAT was taken out from the -80 °C refrigerator and streaked for recovery on a BHI plate. A single colony was picked and inoculated into 5 mL of LB medium, and cultured at 30 °C with 220 rpm for 24 h. Genomic DNA was extracted using a cell genomic DNA extraction kit. Using the genomic DNA of Corynebacterium glutamicum CG-HAT as a template, primers 862900-F / 862900-R were designed, and a gene fragment was amplified through a PCR amplification system and procedure. The obtained gene fragment and the linear vector pk18mobsacb were ligated. The above reaction solution was transformed into Escherichia coli Top10. Transformants were selected for plasmid sequencing, and the recombinant plasmid pk18mobsacb-862900 was successfully constructed. The above recombinant plasmid pk18mobsacb-862900 was transformed into wild-type Corynebacterium glutamicum ATCC 13032 by electroporation. Recombinants with gene replacement were obtained through plate screening to get Corynebacterium glutamicum CG-HAT-M (Corynebacterium glutamicum CG-HAT-M was obtained by mutating Corynebacterium glutamicum ATCC 13032 as follows: cytosine at position 862902 was mutated to thymine; guanine at position 862903 was mutated to adenine; cytosine at position 862953 was mutated to thymine; adenine at position 862961 was mutated to guanine; cytosine and thymine were inserted at position 862958; guanine at position 862963 was mutated with deletion). Corynebacterium glutamicum CG-HAT-M was inoculated into fermentation media containing 0, 10, 20, 40 g / L HA respectively, and samples of the fermentation broth were taken after inoculation to measure OD 600 and the remaining glucose content in the fermentation broth. The results are shown in Figure 2As shown, the growth and glucose consumption rate of strain CG-HAT-M are close to those of CG-HAT, and are also significantly faster than those of wild-type Corynebacterium glutamicum ATCC 13032. The results indicate that the mutations at positions 862902, 862903, 862953, 862961, 862958, and 862963 are the key sites affecting the viscosity resistance of Corynebacterium glutamicum. These sites have multiple mutations in the first 100 bp nucleotide sequence of pyridoxal phosphate transaminase, a gene for pyridoxal phosphate synthesis. Pyridoxal phosphate participates in nearly a hundred enzyme reactions, including transamination, decarboxylation, side-chain cleavage, dehydration, and transsulfuration. These biochemical functions are involved in multiple metabolic pathways, including protein synthesis and catabolism, gluconeogenesis, UFA metabolism, metabolism of glycogen, sphingomyelin, and steroids, synthesis of neurotransmitters (serotonin, taurine, dopamine, norepinephrine, and gamma-aminobutyric acid), metabolism of vitamin B6 and one-carbon units, vitamin B12, and folate, nucleic acid and DNA synthesis, etc. These metabolic pathways are closely related to the growth of the strain. Therefore, it is speculated that the strain may indirectly enhance the utilization of energy substances by cells and strengthen the metabolic activity of the strain under anaerobic conditions by controlling the synthesis of pyridoxal phosphate, thereby improving its anaerobic metabolic ability.

[0056] Table 2 Mutation information of the genome of strain CG-HAT-M

[0057]

[0058] Table 3 Primers required for constructing Corynebacterium glutamicum strain CG-HAT-M

[0059]

[0060] Example 2: Detection of the ability of Corynebacterium glutamicum CG-HAT and Corynebacterium glutamicum CG-HAT-M to synthesize hyaluronic acid

[0061] (1) Construction of recombinant Corynebacterium glutamicum

[0062] The hyaluronic acid synthase gene (HasA) from Streptococcus zooepidemicus was synthesized (the gene sequence of HasA is shown in SEQ ID NO. 20). After synthesis, the hyaluronic acid synthase gene was amplified by PCR using HasA-F / HasA-R as primers to obtain a 1000 bp fragment HasA; using plasmid pXMJ19 as a template, primers pXMJ-F / pXMJ-R were designed for PCR amplification reaction to obtain a 10000 bp vector pXMJ;

[0063] The fragment HasA and the vector pXMJ were subjected to restriction enzyme ligation reaction. The reaction solution was taken and transformed into Escherichia coli Top10 by heat shock method. After picking the transformants for plasmid extraction and sequencing, the pXMJ-HasA plasmid was successfully constructed.

[0064] Using an electroporator, the recombinant plasmid pXMJ-HasA was transformed into the viscous-resistant Corynebacterium glutamicum CG-HAT and Corynebacterium glutamicum CG-HAT-M obtained by screening in Example 1 using a 1 mm electroporation cuvette. The perforation voltage was 1500 V, the voltage duration was 5 ms, and the cells were electroshocked twice. Incubate at 46 °C for 6 min, then culture at 220 rpm and 30 °C for 1 h, and spread on a BHI plate containing 15 g / L chloramphenicol and culture at 30 °C for 48 h. The recombinant strains were named HVCG-HasA and HVCG-HasA-M. Prepare HVCG-HasA competent cells and HVCG-HasA-M competent cells for subsequent strain construction.

[0065] Table 4 Primers used for constructing the recombinant plasmid pXMJ-HasA

[0066]

[0067] Using the plasmid pk18mobsacb as a template, primers PK18-F / PK18-R were designed for PCR amplification to obtain the linear vector PK18; Corynebacterium glutamicum was taken out from the -80 °C refrigerator and streaked on an LB plate for resuscitation. A single colony was picked and inoculated into 5 mL of LB medium, cultured at 220 rpm and 30 °C for 24 h, and genomic DNA was extracted using a cell genomic DNA extraction kit. Using the genomic DNA of Corynebacterium glutamicum as a template, primers ugd-F / ugd-R, glmS-F / glmS-R, glmM-F / glmM-R were designed, and the Ptac promoter sequence was designed into the ugd-F, glmS-F, and glmM-F primers. Through the PCR amplification system and procedure, the ugd, glmS, and glmM genes with the Ptac promoter were amplified. The ugd, glmS, and glmM genes were ligated to the linear vector PK18 in different permutations and combinations and introduced into HVCG-HasA to obtain Corynebacterium glutamicum containing HasA-ugd, HasA-glmM, HasA-glmS, HasA-ugd-glmM, HasA-ugd-glmS, HasA-glmM-glmS, HasA-ugd-glmS-glmM respectively. Corynebacterium glutamicum containing only HasA was used as a control, and its hyaluronic acid production was measured under the same conditions. The results are as Figure 4 shown. It was found that the ugd, glmS, and glmM genes could all significantly increase the hyaluronic acid production, and Corynebacterium glutamicum containing HasA-ugd-glmS-glmM had the highest hyaluronic acid production. Therefore, Corynebacterium glutamicum containing HasA-ugd-glmS-glmM was selected to increase the hyaluronic acid production. The specific construction process is as follows:

[0068] For the fragments ugd, glmS, glmM and the linear vector PK18, restriction digestion and ligation reactions were carried out. The above reaction solution was transformed into Escherichia coli Top10. Transformants were selected for plasmid sequencing, and the recombinant plasmid PK18-Ptac-ugd-glmS-glmM was successfully constructed. The above recombinant plasmid was transformed into HVCG-HasA and HVCG-HasA-M by electroporation to construct the recombinant Corynebacterium glutamicum HVCG-HasA-Ptac-ugd-glmS-glmM and HVCG-HasA-M-Ptac-ugd-glmS-glmM.

[0069] Table 5 Primers used for the construction of the recombinant plasmid PK18-Ptac-ugd-glmS-glmM

[0070]

[0071] (2) Yield detection of hyaluronic acid produced by recombinant Corynebacterium glutamicum

[0072] The yields of recombinant Corynebacterium glutamicum were detected using the recombinant Corynebacterium glutamicum HVCG-HasA-Ptac-ugd-glmS-glmM and HVCG-HasA-M-Ptac-ugd-glmS-glmM.

[0073] 250 mL shake flask fermentation production: The recombinant Corynebacterium glutamicum HVCG-HasA-Ptac-ugd-glmS-glmM and HVCG-HasA-M-Ptac-ugd-glmS-glmM constructed in step (1) of Example 2 were respectively inoculated into shake tubes containing 5 mL of BHI and cultured overnight at 30 °C and 220 rpm. The seed solution was transferred to a baffled Erlenmeyer flask containing 25 mL of fermentation medium at an inoculation amount of initial OD 600 = 0.2 and cultured at 220 rpm and 30 °C. After 3.5 h of culture, IPTG was added at a final concentration of 0.25 mM to induce gene expression, and the fermentation cycle was 48 h. During fermentation at 20 h and 24 h, 2 M NaOH was added to adjust the pH of the fermentation broth to 6.5 - 7. The fermentation broth was collected, centrifuged at 10000 rpm for 5 min, the supernatant was taken, and after repeated alcohol precipitation twice, the hyaluronic acid content in the fermentation broth was determined by the borax sulfuric acid-carbazole method. The yields of hyaluronic acid produced by the recombinant Corynebacterium glutamicum HVCG-HasA-Ptac-ugd-glmS-glmM and HVCG-HasA-M-Ptac-ugd-glmS-glmM in shake flask fermentation were both measured to be 10 g / L.

[0074] Fermentation in a 5L fermenter: The recombinant Corynebacterium glutamicum HVCG-HasA-Ptac-ugd-glmS-glmM and HVCG-HasA-M-Ptac-ugd-glmS-glmM constructed in step (1) of Example 2 were respectively inoculated into 5 mL of BHI medium and cultured overnight at 30 °C with a rotation speed of 220 rpm. The seed solution was transferred to a baffled Erlenmeyer flask containing 25 mL of fermentation medium at an inoculation amount of OD 600 = 0.1, and cultured at 30 °C with a rotation speed of 220 rpm for 10 h, and then inoculated into a 5L fermenter at an inoculation amount of 10%. The initial temperature was set at 30 °C and the rotation speed was 3000 r / min. After 3.5 h of fermentation, IPTG with a final concentration of 0.25 mM was added to induce gene expression. During the fermentation process, the pH of the fermentation broth was controlled at about 7 with 14% ammonia water, and glucose was fed to maintain the glucose content in the fermenter at about 10 g / L. As can be seen from Figure 3 the results, the final hyaluronic acid yields of the recombinant Corynebacterium glutamicum HVCG-HasA-Ptac-ugd-glmS-glmM and HVCG-HasA-M-Ptac-ugd-glmS-glmM resistant to high-viscosity solutions were 44 g / L and 45 g / L respectively after fermentation in a 5L fermenter.

[0075] Comparative Example

[0076] The recombinant plasmid HasA-ugd-glmS-glmM constructed according to step (1) of Example 2 was transformed into the wild-type Corynebacterium glutamicum ATCC 13032, and fermentation was carried out according to the method of step (2) of Example 2. The results showed that the hyaluronic acid yield of the wild-type Corynebacterium glutamicum in the shake flask fermentation was 6.5 g / L, and the yield in the 5L fermenter was only 32 g / L. The main reason is that as the hyaluronic acid accumulates in the fermentation broth, the wild-type Corynebacterium glutamicum cannot carry out normal metabolic activities in the high-viscosity fermentation broth, thus affecting the synthesis of hyaluronic acid.

[0077] Obviously, the above examples are only for clear illustration and not a limitation of the implementation mode. For those of ordinary skill in the art, other different forms of changes or modifications can be made on the basis of the above description. It is not necessary and impossible to list all implementation modes here. And the obvious changes or modifications derived therefrom are still within the protection scope of the present invention.

Claims

1. A viscous-resistant Corynebacterium glutamicum ( Corynebacterium glutamicum ), characterized in that The Corynebacterium glutamicum is subjected to the following mutations based on Corynebacterium glutamicum ATCC 13032: mutating the cytosine at the 862902 site to thymine; mutating the guanine at the 862903 site to adenine; mutating the cytosine at the 862953 site to thymine; mutating the adenine at the 862961 site to guanine; inserting cytosine and thymine at the 862958 site; and deleting the guanine at the 862963 site.

2. The Corynebacterium glutamicum according to claim 1, characterized in that The Corynebacterium glutamicum was subjected to the following mutation based on Corynebacterium glutamicum ATCC 13032:

3. Use of Corynebacterium glutamicum according to claim 1 or 2 in producing hyaluronic acid, characterized in that: In the Corynebacterium glutamicum described in claim 1 or 2, hyaluronan synthase is overexpressed, and one or more of glutamine-fructose-6-phosphate aminotransferase, phosphoglucomutase, and UDP-glucose dehydrogenase are overexpressed.

4. A recombinant Corynebacterium glutamicum, characterized in that The transformation of the recombinant Corynebacterium glutamicum comprises: overexpressing hyaluronan synthase in the Corynebacterium glutamicum according to claim 1 or 2, and overexpressing one or more of glutamine-fructose-6-phosphate aminotransferase, phosphoglucomutase, and uridine diphosphate-glucose dehydrogenase.

5. The recombinant Corynebacterium glutamicum according to claim 4, characterized in that The overexpression is driven by the Ptac promoter or the Ptrc promoter.

6. A method for producing hyaluronic acid, characterized in that: The method comprises the step of fermenting the recombinant Corynebacterium glutamicum according to any one of claims 4 to 5.

7. The method according to claim 6, characterized in that The fermentation comprises inoculating the recombinant Corynebacterium glutamicum into a fermentation medium for cultivation, adding IPTG to induce gene expression, centrifuging the fermentation liquid after fermentation, and taking the supernatant to obtain the hyaluronic acid.

8. The method according to claim 7, characterized in that The culture temperature is 15-40°C.

9. The method according to claim 7, characterized in that: The pH of the culture is 5-9.

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