Viscosity-resistant corynebacterium glutamicum and use thereof

Abstract

Viscosity-resistant Corynebacterium glutamicum and a use thereof, relating to the technical field of biological genetic engineering. The Corynebacterium glutamicum is obtained by mutating Corynebacterium glutamicum ATCC 13032. Mutation sites include: cytosine mutation to thymine at the position 862902; guanine mutation to adenine at the position 862903; cytosine mutation to thymine at the position 862953; adenine mutation to guanine at the position 862961; cytosine and thymine insertion at the position 862958; and guanine deletion mutation at the position 862963. The tolerance of the Corynebacterium glutamicum in a highly viscous environment and the growth and metabolism ability of the Corynebacterium glutamicum in a low dissolved oxygen environment are significantly improved, thereby improving the yield of a mucopolysaccharide and avoiding the problem of the mucopolysaccharide synthesis being affected by restricted metabolism of the Corynebacterium glutamicum due to a viscous fermentation broth and insufficient dissolved oxygen resulting from the generated mucopolysaccharide.

Description

Corynebacterium glutamicum resistant to viscous and application thereof TECHNICAL FIELD

[0001] The present application relates to the field of biological genetic engineering, in particular to a corynebacterium glutamicum resistant to viscous and application thereof. BACKGROUND

[0002] Corynebacterium glutamicum is a gram-positive bacterium, which has been widely used in the production of various value-added chemicals, amino acids and fuels due to its clear genetic background, stable protein secretion, low extracellular hydrolytic enzyme activity, non-toxicity and other characteristics. With the continuous revelation of gene regulation mechanism, the design and construction of corynebacterium glutamicum for biological manufacturing using synthetic biology technology has become a research hotspot in this field. In addition, corynebacterium glutamicum is a strict aerobic bacterium, and sterile air needs to be constantly supplied during its fermentation process. Therefore, when corynebacterium glutamicum ferments to synthesize viscous substances such as hyaluronic acid, chondroitin, heparin precursor, etc., or carries out high-density fermentation process, the dissolved oxygen in the fermentation broth will decrease with the accumulation of viscous substances and the increase of cell density, thereby affecting the normal metabolic activity. This is also one of the problems in the optimization of corynebacterium glutamicum fermentation process. Hyaluronic acid (HA) is a linear acidic mucopolysaccharide with good moisturizing property and can inhibit inflammation, and is widely used in the fields of cosmetics and medicine. Most of the hyaluronic acid on the industrial market is obtained by fermentation of pathogenic microorganisms such as streptococcus zooepidemicus and escherichia coli K4, but due to the presence of endotoxin and other pathogenic factors, its development in the field of medicine is severely restricted. The use of microorganisms with clear genetic background and high biological safety, such as corynebacterium glutamicum, to synthesize hyaluronic acid has become a development trend of microbial fermentation method for synthesizing hyaluronic acid.

[0003] Although the yield of viscous polysaccharides such as hyaluronic acid, chondroitin, heparin precursor synthesized by corynebacterium glutamicum has been significantly improved, but due to its super water absorption, the fermentation broth will become viscous with the progress of the fermentation process, thereby inhibiting the normal metabolic activity of the cells and limiting the further improvement of the yield of viscous substances such as hyaluronic acid, chondroitin, heparin precursor. Therefore, it is expected to improve the tolerance of corynebacterium glutamicum to high viscous solution by directed evolution engineering, and then further improve the synthesis efficiency of corynebacterium glutamicum to viscous polysaccharides.

[0004] SUMMARY

[0005] Therefore, the technical problem to be solved by the present application is to overcome the problem that the oxygen content of the fermentation broth decreases due to the viscosity of the fermentation broth during the production of hyaluronic acid by corynebacterium glutamicum, thereby inhibiting the metabolic activity of corynebacterium glutamicum, and ultimately affecting the yield of hyaluronic acid.

[0006] To solve the above technical problems, the present application provides a corynebacterium glutamicum, which is obtained by mutating corynebacterium glutamicum ATCC 13032, and the mutation sites include: mutating cytosine at site 862902 into thymine; mutating guanine at site 862903 into adenine; mutating cytosine at site 862953 into thymine; mutating adenine at site 862961 into guanine; inserting cytosine and thymine at site 862958; and deleting guanine at site 862963. The corynebacterium glutamicum of the present application has significantly improved tolerance in a high-viscosity environment and growth and metabolism capacity in a low-dissolved oxygen environment, thereby improving the yield of mucopolysaccharide and avoiding the problem that the mucopolysaccharide produced makes the fermentation broth viscous, the dissolved oxygen is insufficient, and the metabolism of the corynebacterium glutamicum is limited, which finally affects the synthesis of mucopolysaccharide.

[0007] The first object of the present application is to provide a viscous-tolerant corynebacterium glutamicum, which is characterized in that the corynebacterium glutamicum is obtained by mutating corynebacterium glutamicum ATCC 13032, and the mutation sites include:

[0008] (1) mutating cytosine at site 862902 into thymine;

[0009] (2) mutating guanine at site 862903 into adenine;

[0010] (3) mutating cytosine at site 862953 into thymine;

[0011] (4) mutating adenine at site 862961 into guanine;

[0012] (5) inserting cytosine and thymine at site 862958;

[0013] (6) deleting guanine at site 862963.

[0014] Further, the mutation sites include:

[0015] The second object of the present application is to provide a use of the above-mentioned corynebacterium glutamicum in producing mucopolysaccharide.

[0016] Further, the mucopolysaccharide includes hyaluronic acid.

[0017] A third object of the present application is to provide a mutant of pyridoxal transaminase, characterized in that the gene sequence of the first 100 bp of the pyridoxal transaminase with NCBI No. BAB98207.1 is mutated, and the gene sequence of the first 101 bp of the mutant of pyridoxal transaminase is shown in SEQ ID NO. 5.

[0018] The gene sequence of SEQ ID NO. 5 is shown as follows:

[0019] Further, the gene sequence of the first 101 bp of the mutant of pyridoxal transaminase is obtained by mutating the gene sequence of the first 100 bp of the pyridoxal transaminase, and the mutation sites include:

[0020] (1) mutating cytosine at site 3 to thymine;

[0021] (2) mutating guanine at site 4 to adenine;

[0022] (3) mutating cytosine at site 54 to thymine;

[0023] (4) mutating adenine at site 64 to guanine;

[0024] (5) inserting cytosine and thymine at site 59;

[0025] (6) deleting guanine at site 64.

[0026] A fourth object of the present application is to provide an expression vector containing the gene encoding the mutant of pyridoxal transaminase described above.

[0027] A fifth object of the present application is to provide a recombinant C. glutamicum, characterized in that the modification of the recombinant C. glutamicum includes replacing the gene encoding the first 100 bp of wild-type pyridoxal transaminase on the genome of the C. glutamicum host strain with the gene encoding the first 101 bp of the mutant described above.

[0028] Further, the gene encoding the first 101 bp of the mutant is shown in SEQ ID NO. 5.

[0029] Further, the modification of the recombinant C. glutamicum is that the gene fragment from site 862900 to site 862999 of C. glutamicum ATCC 13032 is replaced with the gene fragment shown in SEQ ID NO. 5 to obtain the recombinant C. glutamicum.

[0030] Further, the modification of the recombinant C. glutamicum further comprises overexpression of at least one of hyaluronate synthase, glutamine-fructose-6-phosphate amidotransferase, phosphoglucomutase, and uridine diphosphate glucose dehydrogenase. The gene sequence of glutamine-fructose-6-phosphate amidotransferase (glmS) is shown as SEQ ID NO. 17, the gene sequence of phosphoglucomutase (glmM) is shown as SEQ ID NO. 18, and the gene sequence of uridine diphosphate glucose dehydrogenase (ugd) is shown as SEQ ID NO. 19. Hyaluronic acid is mainly synthesized through the UDP-N-acetylglucosamine pathway and the UDP-glucuronate pathway. The enzymes expressed by ugd, glmS, and glmM are key enzymes of the two pathways, and enhancing the synthesis of the three enzymes can effectively increase the yield of hyaluronic acid.

[0031] Further, the overexpression is initiated by a Ptac promoter or a Ptrc promoter. The ugd, glmS, and glmM genes also have promoters, but the promoter sequences of the ugd, glmS, and glmM genes are not clear, and the expression ability is weak. The Ptac promoter and the Ptrc promoter are recognized strong promoters with strong expression ability, and 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 enhanced, and the yield of hyaluronic acid is increased.

[0032] A sixth object of the present application is to provide a method for producing glycosaminoglycan, characterized in that the method comprises a fermentation step of the above-mentioned recombinant C. glutamicum.

[0033] Further, the fermentation step comprises inoculating the recombinant C. glutamicum into a fermentation medium for culture, adding IPTG to induce gene expression, and after fermentation, centrifuging the fermentation broth and taking the supernatant to obtain the glycosaminoglycan.

[0034] Further, the temperature of the fermentation process is 15-40℃. The temperature affects the growth of C. glutamicum, and C. glutamicum can grow at 15-40℃, and the optimal growth temperature of C. glutamicum is 30℃. Preferably, the temperature of the fermentation process is 30℃.

[0035] Further, the pH of the fermentation process is 6.5-7. The pH also affects the growth of C. glutamicum, and C. glutamicum can grow at pH 5-9, and the optimal pH of C. glutamicum is 7. Preferably, the pH of the fermentation process is 6.5-7.

[0036] The present application has the following beneficial effects:

[0037] The glutamic acid coryneform bacteria of the present application has significantly improved tolerance in high viscous environment and growth metabolism ability in low dissolved oxygen environment, thereby improving the yield of mucopolysaccharide (for example, hyaluronic acid), avoiding the problem that the produced mucopolysaccharide makes the fermentation broth viscous, the dissolved oxygen is insufficient, thereby limiting the metabolism of the glutamic acid coryneform bacteria, and finally affecting the synthesis of mucopolysaccharide. BRIEF DESCRIPTION OF DRAWINGS

[0038] In order to make the content of the present application more easily understood, the present application will be further described in detail below according to specific embodiments of the present application and in conjunction with the drawings, in which

[0039] Fig. 1 is a directed evolution screening process of the viscous-tolerant glutamic acid coryneform bacteria;

[0040] Fig. 2 is a change curve of OD and glucose consumption of the viscous-tolerant glutamic acid coryneform bacteria in different concentrations of hyaluronic acid; 600

[0041] Fig. 3 is the hyaluronic acid yield of the viscous-tolerant glutamic acid coryneform bacteria in a 5L fermentation tank fermentation;

[0042] Fig. 4 is a column chart of the hyaluronic acid yield obtained by respectively expressing different genes in the hyaluronic acid synthesis pathway. DETAILED DESCRIPTION

[0043] The present application will be further described below in conjunction with the drawings and specific embodiments, so that those skilled in the art can better understand the present application and implement it, but the embodiments are not as a limitation on the present application.

[0044] Strain: wild-type glutamic acid coryneform bacteria (Corynebacterium glutamicum ATCC 13032);

[0045] Plasmid: pXMJ19, pk18mobsacb;

[0046] LB medium: yeast powder 5 g / L, peptone 10 g / L, sodium chloride 10 g / L;

[0047] BHI: brain heart infusion 37 g / L, sorbitol 91 g / L;

[0048] Fermentation medium: glucose 40 g / L, corn syrup dry powder 20 g / L, (NH4)2SO4 30 g / L, KH2PO4 1 g / L, K2HPO4 1 g / L, MgSO4 25 g / L, 3-morpholinopropanesulfonic acid (MOPS) 42 g / L.

[0049] Preparation of glutamic acid coryneform bacteria competence: a single colony on the plate was inoculated into a shaking tube containing 5 mL of BHI liquid medium, and incubated at 30°C overnight, with an initial OD​600 = 0.2 was inoculated into a baffle flask containing 50 mL BHI and incubated at 30°C until OD 600 = 1.4-1.6. The seed liquid was collected into a centrifuge tube, iced for 10 min, centrifuged at 4000 rpm, 4°C for 5 min, and the bacterial cells were collected. The bacterial cells were resuspended using 20 mL of 10% glycerol solution, centrifuged at 4000 rpm, 4°C for 5 min, and the bacterial cells were collected. The above operation was repeated twice, 2.5 mL of 10% glycerol solution was added to resuspend the bacterial cells, and the bacterial cells were aliquoted into pre-cooled sterile EP tubes and stored at -80°C for use in the electroporation.

[0050] Purification of hyaluronic acid: The fermentation broth was collected and centrifuged at 10,000 rpm for 5 min. An appropriate amount of supernatant was taken, 4 volumes of absolute ethanol were added, and the mixture was placed in a 4°C environment overnight for alcohol precipitation. The mixture was centrifuged at 10,000 rpm for 5 min, and the supernatant was discarded. After the ethanol evaporated, the original volume of water was added to resuspend the mixture, which was fully dissolved and centrifuged at 10,000 rpm for 10 min. The supernatant was collected, and the above operation was repeated. After the second alcohol precipitation, the supernatant was collected, which was the purified hyaluronic acid sample.

[0051] Determination of the yield of hyaluronic acid: 4.77 g of sodium tetraborate decahydrate was dissolved in 500 mL of concentrated sulfuric acid to prepare a borax-sulfuric acid solution; 1.25 g of carbazole was dissolved in 500 mL of absolute ethanol to prepare a carbazole solution; and a 1 g / L glucuronic acid solution was prepared.

[0052] The purified hyaluronic acid sample was diluted 10-100 times, 200 μL was taken into a glass colorimetric tube, 1 mL of the borax-sulfuric acid solution was added, the mixture was mixed well, and was placed in a boiling water bath for 15 min and cooled on ice. 50 μL of the carbazole solution was added, the mixture was mixed well, and was placed in a boiling water bath for 10 min. 200 μL of the reaction solution was taken into a 96 transparent well plate, and the absorbance of the sample at 530 nm was determined using an enzyme marker. The 1 g / L glucuronic acid solution was gradient diluted to 0, 10, 20, 30, 40, and 50 mg / L. After the borax-sulfuric acid-carbazole color reaction, the absorbance at 530 nm was determined. The absorbance was taken as the abscissa, the concentration of glucuronic acid (mg / L) was taken as the ordinate, and a relationship curve between the absorbance and the concentration of glucuronic acid was drawn to obtain a standard curve equation: y = 121.7x-6.035, R 2 = 0.999.

[0053] The measured absorbance of the sample was brought into the standard equation to calculate the content of the hyaluronic acid. The formula for calculating the content of hyaluronic acid: hyaluronic acid content (g / L) = (concentration measured by the standard curve * dilution multiple * 2.067) / 1000.

[0054] Example 1: Screening of glutamic acid corynebacterium resistant to viscosity

[0055] (1) Screening of corynebacterium glutamicum CG-HAT

[0056] A single colony of corynebacterium glutamicum ATCC 13032 was picked and inoculated into 5 mL BHI medium and incubated at 30°C overnight. The culture was inoculated into 25 mL fermentation medium at an initial OD 600 = 0.2, and then inoculated into 25 mL fermentation medium containing 10 g / L HA at an initial OD 600 = 0.2, and then inoculated into 25 mL fermentation medium containing 20 g / L HA at an initial OD 600 = 0.2, and then inoculated into 25 mL fermentation medium containing 40 g / L HA at an initial OD 600 = 0.2, and then inoculated into 25 mL fermentation medium containing 60 g / L HA at an initial OD 600 = 0.2. The HA content in the medium was gradually increased as the strain was cultured in the medium (as shown in Figure 1).

[0057] During the subculture, the strain in the fermentation broth was streaked at irregular intervals, and single colony strains were randomly picked for verification of the effect of directed evolution. The wild-type corynebacterium glutamicum ATCC 13032 was used as a control, and the OD 600 The mutant strain was obtained by screening the strain with the highest OD 600 The remaining glucose content in the fermentation broth was measured, and the difference in growth rate and glucose consumption rate between the mutant strain and the wild-type strain could be significantly observed.

[0058] After subculture to the 300th generation, the strain in the fermentation broth was streaked, and 100 single colony strains were selected and inoculated into 5 mL fermentation medium containing 40 g / L HA, and incubated at 220 rpm and 30°C for 20 h. The OD 600 was measured, and the OD 600The highest evolution strain is the thick hyaluronic acid resistant C glutamicum CG-HAT of the present application, and the above-mentioned directed evolution effect verification process is repeated. The directed evolution effect verification of the CG-HAT strain is shown in Figure 2. With the increase of the concentration of hyaluronic acid, the glucose consumption of CG-HAT is higher than that of the wild type C glutamicum ATCC 13032, so it can be considered that CG-HAT has better growth and metabolism ability in high concentration of hyaluronic acid.

[0059] The strain CG-HAT is inoculated into 5 mL BHI medium for overnight culture, and the whole genome sequencing is performed by a related company. By comparing with the genome of the wild type C glutamicum ATCC 13032 before evolution as the reference genome, it is found that the genome of the thick hyaluronic acid resistant C glutamicum has multiple gene mutations, and the specific mutation information is shown in Table 1.

[0060] Table 1 Genome mutation information of strain CG-HAT

[0061] (2) Construction of C glutamicum CG-HAT-M

[0062] To further determine the key mutation sites affecting strain tolerance, we selected potential key sites 862902, 862903, 862953, 862961, 862958 and 862963 for mutation verification on the genome of wild-type C. glutamicum ATCC 13032. The linear vector pk18mobsacb was obtained by PCR amplification reaction using plasmid pk18mobsacb as a template and designing primers PK18-F / PK18-R; C. glutamicum CG-HAT was taken out from the -80°C refrigerator and inoculated on a BHI plate for resuscitation, a single colony was picked and inoculated into 5 mL LB medium, which was cultured at 220 rpm and 30°C for 24 h, and the genomic DNA was extracted using a cell genomic extraction kit. The plp gene fragment (the plp gene fragment is the gene sequence of C. glutamicum CG-HAT from site 862900 to site 863000, as shown in SEQ ID NO. 5) was obtained by PCR amplification system and program using C. glutamicum CG-HAT genomic DNA as a template and designing primers plp-F / plp-R. The fragment plp and the linear vector pk18mobsacb were ligated. The above reaction liquid was transformed into E. coli Top10. The transformants were selected for plasmid sequencing, and the recombinant plasmid pk18mobsacb-plp was successfully constructed. The above recombinant plasmid pk18mobsacb-plp was transformed into wild-type C. glutamicum ATCC 13032 by electroporation, and C. glutamicum CG-HAT-M was obtained by plate screening of gene replacement recombinants. C. glutamicum CG-HAT-M was inoculated into fermentation medium containing 0, 10, 20 and 40 g / L HA, respectively, and the fermentation broth was sampled after inoculation, and the OD 600The results are shown in Figure 2. The growth and glucose consumption rate of strain CG-HAT-M was close to that of CG-HAT, and was significantly faster than that of wild-type C. glutamicum ATCC 13032. This result indicates that the mutations at sites 862902, 862903, 862953, 862961, 862958, and 862963 are key sites affecting the viscosity tolerance of C. glutamicum. These sites are mutated in the nucleotide sequence of the phosphopantothenoyl transferase of the phosphopantothenate synthesis gene. Phosphopantothenate is involved in nearly 100 enzyme reactions, including transamination, decarboxylation, side chain cleavage, dehydration, and transsulfuration. These biochemical functions are involved in various metabolic pathways, including protein synthesis and catabolism, gluconeogenesis, UFA metabolism, glycogen, sphingomyelin, and steroid metabolism, neurotransmitter (5-hydroxytryptamine, taurine, dopamine, norepinephrine, and gamma-aminobutyric acid) synthesis, vitamin B6 and one-carbon unit, vitamin B12 and folate metabolism, nucleic acid and DNA synthesis, etc. These metabolic pathways are closely related to the growth of the strain, so it is speculated that the strain may indirectly enhance the utilization of energy substances by controlling the synthesis of phosphopantothenate, thereby enhancing the metabolic activity of the strain under anaerobic conditions, thereby improving its anaerobic metabolic capacity.

[0063] Table 2. Genome mutation information of strain CG-HAT-M

[0064] Table 3. Primers required for constructing C. glutamicum strain CG-HAT-M

[0065] Example 2: Detection of the ability of C. glutamicum CG-HAT and C. glutamicum CG-HAT-M to synthesize hyaluronic acid

[0066] (1) Construction of recombinant C. glutamicum

[0067] According to the hyaluronate synthase gene (HasA) from Streptococcus zooepidemicus, the gene sequence of HasA is shown as SEQ ID NO. 20, the synthesized hyaluronate synthase gene is amplified by PCR with HasA-F / HasA-R as primers, and a 1000 bp fragment HasA is obtained. The plasmid pXMJ19 is used as a template, and primers pXMJ-F / pXMJ-R are designed to perform PCR amplification reaction, and a 10000 bp vector pXMJ is obtained.

[0068] The enzyme digestion and ligation reaction of the fragment HasA and the vector pXMJ is performed, the reaction solution is taken, and the heat shock method is used to transform it into E. coli Top10. The transformants are picked and subjected to plasmid extraction, and then sequenced. The pXMJ-HasA plasmid is successfully constructed.

[0069] The recombinant plasmid pXMJ-HasA was transformed into the C. glutamicum CG-HAT and C. glutamicum CG-HAT-M screened in Example 1 using an electroporator with a 1 mm electrotransformation cup, a voltage of 1500 V, a voltage duration of 5 ms, and two repeated electric shocks. The cells were incubated at 46 °C for 6 min, then cultured at 220 rpm and 30 °C for 1 h, and then plated on BHI plates containing 15 g / L chloramphenicol and cultured at 30 °C for 48 h. The recombinant strains were named HVCG-HasA and HVCG-HasA-M. The HVCG-HasA and HVCG-HasA-M competent cells were prepared for subsequent strain construction.

[0070] Table 4 Primers used for construction of the recombinant plasmid pXMJ-HasA

[0071] The plasmid pk18mobsacb was used as a template to design primers PK18-F / PK18-R for PCR amplification to obtain a linear vector PK18. C. glutamicum was taken out of a -80 °C freezer, streaked on LB plates, and single colonies were inoculated into 5 mL LB medium and cultured at 220 rpm and 30 °C for 24 h. The genomic DNA was extracted using a cell genomic extraction kit. The genomic DNA of C. glutamicum was used as a template to design primers ugd-F / ugd-R, glmS-F / glmS-R, and glmM-F / glmM-R. The Ptac promoter sequence was designed into the ugd-F, glmS-F, and glmM-F primers. The ugd, glmS, and glmM genes with the Ptac promoter were amplified by a PCR amplification system and program. The ugd, glmS, and glmM genes were linked to the linear vector PK18 in different arrangements and introduced into HVCG-HasA to obtain C. glutamicum containing HasA-ugd, HasA-glmM, HasA-glmS, HasA-ugd-glmM, HasA-ugd-glmS, HasA-glmM-glmS, and HasA-ugd-glmS-glmM, respectively. The C. glutamicum containing only HasA was used as a control, and the hyaluronic acid production was determined under the same conditions. As shown in FIG. 4, the ugd, glmS, and glmM genes can significantly improve the production of hyaluronic acid, and the C. glutamicum containing HasA-ugd-glmS-glmM has the highest hyaluronic acid production. Therefore, the C. glutamicum containing HasA-ugd-glmS-glmM was selected to improve the production of hyaluronic acid, and the specific construction process is as follows:

[0072] The restriction enzyme digestion and ligation reaction was performed on fragments ugd, glmS, glmM and linear vector PK18. The above reaction solution was transformed into E. coli Top10. The transformants were selected for plasmid sequencing, and the construction of recombinant plasmid PK18-Ptac-ugd-glmS-glmM was successful. The above recombinant plasmid was transformed into HVCG-HasA and HVCG-HasA-M by electroporation method, and recombinant C. glutamicum HVCG-HasA-Ptac-ugd-glmS-glmM and HVCG-HasA-M-Ptac-ugd-glmS-glmM were obtained.

[0073] Table 5 Primers used for construction of recombinant plasmid PK18-Ptac-ugd-glmS-glmM

[0074] (2) Production detection of recombinant C. glutamicum producing hyaluronic acid

[0075] The production of recombinant C. glutamicum was detected by recombinant C. glutamicum HVCG-HasA-Ptac-ugd-glmS-glmM and HVCG-HasA-M-Ptac-ugd-glmS-glmM.

[0076] 250 mL shake flask fermentation production: the recombinant C. glutamicum HVCG-HasA-Ptac-ugd-glmS-glmM and HVCG-HasA-M-Ptac-ugd-glmS-glmM constructed in step (1) of Example 2 were inoculated into a shake flask containing 5 mL of BHI, respectively, and cultured at 30°C, 220 rpm overnight. The seed liquid was inoculated into a baffled Erlenmeyer flask containing 25 mL of fermentation medium at an initial OD 600 = 0.2, and cultured at 220 rpm, 30°C for 3.5 h, then 0.25 mM IPTG was added to induce gene expression, and the fermentation period was 48 h. During the fermentation, 2M NaOH was added to adjust the pH of the fermentation broth to 6.5-7 at 20 h and 24 h. The fermentation broth was collected and centrifuged at 10000 rpm for 5 min, and the supernatant was repeatedly precipitated twice, then the hyaluronic acid content in the fermentation broth was determined by borax sulfuric acid-carbazole method. The production of recombinant C. glutamicum HVCG-HasA-Ptac-ugd-glmS-glmM and HVCG-HasA-M-Ptac-ugd-glmS-glmM in shake flask fermentation was 10 g / L.

[0077] 5L fermenter fermentation production: the recombinant C. glutamicum HVCG-HasA-Ptac-ugd-glmS-glmM and HVCG-HasA-M-Ptac-ugd-glmS-glmM constructed in step (1) of Example 2 were inoculated into 5 mL BHI medium respectively, 220 rpm, 30°C overnight culture. Seed liquid was inoculated into a baffled triangular flask of 25 mL fermentation medium with an inoculation amount of initial OD 600 = 0.1, 220 rpm, 30°C culture for 10 h, then inoculated into a 5L fermenter with an inoculation amount of 10%. The initial temperature was set at 30°C, the rotation speed was 3000 r / min, after 3.5 h of fermentation, 0.25 mM IPTG was added to induce gene expression, the fermentation process was controlled by 14% ammonia water to keep the pH of the fermentation broth at about 7, and glucose was added to maintain the glucose content in the fermenter at about 10 g / L. As can be seen from Figure 3, the recombinant C. glutamicum HVCG-HasA-Ptac-ugd-glmS-glmM and HVCG-HasA-M-Ptac-ugd-glmS-glmM with high viscosity solution resistance produced 44 g / L and 45 g / L of hyaluronic acid respectively in a 5L fermenter after fermentation.

[0078] Comparative example

[0079] The recombinant plasmid PK18-Ptac-ugd-glmS-glmM constructed in step (1) of Example 2 was transformed into wild-type C. glutamicum ATCC 13032, and the 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 C. 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 accumulation of hyaluronic acid in the fermentation broth, the wild-type C. glutamicum cannot carry out normal metabolic activities in the high-viscosity fermentation broth, thereby affecting the synthesis of hyaluronic acid.

[0080] Obviously, the above examples are only examples for the sake of clarity, and are not limiting of the embodiments. For those skilled in the art, other different forms of changes or variations can be made on the basis of the above description. Here, it is not necessary and impossible to exhaust all the embodiments. The obvious changes or variations derived therefrom are still within the protection scope of the present invention.

Claims

1. A mass spectrometry system for IDH gene mutation marker detection, characterized by, The sampling kit comprises a sampler and a detection box; the sampler comprises a handle part and a conductive sampling sheet fixed to one end of the handle part, the conductive sampling sheet is used for collecting a sample to be detected; the detection box comprises a box body and a capillary, the box body is provided with a sample groove; the sampler is detachably clamped and fixed to the box body, and the conductive sampling sheet extends into the sample groove; the sample groove is used for containing an elution solvent, the elution solvent is used for eluting the sample to be detected collected on the conductive sampling sheet to form an analysis solution; one end of the capillary extends into the sample groove, and the other end is located outside the sample groove; and the small mass spectrometer comprises an ion source interface, an ion sampling transmission system and a vacuum system connected in sequence; the vacuum system is provided with a mass analyzer and an ion detector. The sampling kit is configured to be inserted into the ion source interface of the small mass spectrometer when the mass spectrometer is in use, and high voltage is applied to the conductive sampling sheet, so that the analysis solution in the sample groove is charged and enters the capillary, and the analysis solution is sprayed from the other end of the capillary to form a spray; the spray enters the vacuum system through the ion sampling transmission system. The conductive sampling sheet comprises a sampling part and a conductive part connected in sequence, the sampling part is used for collecting a sample to be detected, and the conductive part is used for connecting high voltage; The sampling part is provided with a notch and a circular hole, the notch communicates with the circular hole; the end of the capillary extending into the sample groove is located in the notch; The sampler further comprises a sampling fiber which is detachably fixed to the sampling part.

2. The mass spectrometry system for detecting IDH gene mutation marker according to claim 1, characterized in that, The vacuum system comprises a vacuum cavity, a molecular pump and a vortex pump, and the mass analyzer and the ion detector are located in the vacuum cavity; The ion sampling transmission system comprises a pinch valve which communicates with the vacuum cavity. When the mass spectrometer is used for detecting IDH gene mutation markers of brain tissue, the specific operation comprises the following steps:

3. The mass spectrometry system for detecting IDH gene mutation marker according to claim 1, wherein, (1) a brain tissue sample is taken and placed on a carrier, the brain tissue is wiped with the conductive sampling sheet of the sampler, and then fixed to the detection box; an elution solvent is added to the sample groove of the detection box for extracting the analyte in the brain tissue sample to form an analysis solution; the elution solvent comprises ethanol and water, and the volume percentage of ethanol in the elution solvent is 50%-90%; the analyte in the brain tissue sample includes but is not limited to 2-HG and GLU; (2) the detection box is inserted into the ion source interface of the small mass spectrometer, the test parameters of the small mass spectrometer are set, the ion source applies voltage to the conductive sampling sheet, the voltage is transmitted to the capillary tip through the analysis solution, the analysis solution forms a Taylor cone at the tip and generates an electrospray, the analyte is ionized to form ions through the electrospray, enters the vacuum system through the ion sampling transmission system of the small mass spectrometer, and the signal intensity of each ion is recorded after mass analysis.

4. The mass spectrometry system for detecting IDH gene mutation marker according to claim 1, wherein, ​ ​ ​ (3) After obtaining the mass spectrum signal, the intensity ratio of the secondary fragment ion signals of the target 2-HG and GLU is calculated according to the intensity signals of the ions with m / z 129 and m / z 128, and the IDH mutation state of the brain tissue sample is determined by comparing the calculation result with the embedded threshold.

5. A method for improving the accuracy of detection of the mass spectrometry system according to any one of claims 1 to 4, characterized by, The method comprises the following steps: S1: setting an initial injection amount as Q1, performing scanning, and obtaining an initial MS2 spectrum; determining whether the total ion number of the target in the initial MS2 spectrum meets the set limit value requirement; if the set limit value requirement is met, the optimal injection amount Q0 is determined as Q1; if the set limit value requirement is not met, an injection amount optimization adjustment scanning process is performed until the total ion number of the target in the scanning spectrum meets the set limit value requirement, and the optimal injection amount Q0 is obtained; S2: performing n times of scanning with the optimal injection amount Q0 to collect MS2 spectra; during the collection of the MS2 spectra, the following determinations are performed: first determination: determining whether the total ion number of the target in the MS2 spectrum meets the set limit value; if the set limit value requirement is met, the spectrum is a qualified spectrum, and a second determination process is entered; if the set limit value requirement is not met, the spectrum is a non-qualified spectrum, and a third determination process is entered; second determination: determining whether the number of the qualified spectra reaches a set upper limit N1; if the set upper limit N1 is reached, the scanning is ended, and a fourth determination process or step S3 is entered; if the set upper limit N1 is not reached, the third determination process is entered; third determination: determining whether the number of scanning is greater than or equal to a set upper limit n1; if the number of scanning is greater than or equal to the set upper limit n1, the scanning is ended, and the fourth determination process is entered; if the number of scanning is less than the set upper limit n1, the next scanning is performed; fourth determination: determining whether the number of the qualified spectra is greater than or equal to a set lower limit N2; if the number of the qualified spectra is greater than or equal to the set lower limit N2, step S3 is entered; if the number of the qualified spectra is less than the set lower limit N2, it is prompted to resample; S3: combining the N qualified spectra obtained in S2 into one spectrum to determine the signal intensity of the target, calculating according to a pre-designed calculation formula, comparing the calculation result with an embedded threshold, and outputting the result; wherein, N1≤n≤n1; N2≤N≤N1.

6. The method of claim 5, wherein, In S1, the injection amount optimization adjustment scanning process is specifically as follows: determining the size relationship between the total ion number of the target in the MS2 spectrum and the set limit value; if the total ion number of the target is less than the lower limit of the set limit value, the injection amount is increased based on the injection amount of the last step, and then scanning is performed; if the total ion number of the target is greater than the upper limit of the set limit value, the injection amount is reduced based on the injection amount of the last step, and then scanning is performed; the above operation is repeated until the total ion number of the target in the scanning spectrum meets the set limit value requirement, and the optimal injection amount Q0 is obtained.

7. The method of claim 6, wherein, The injection amount optimization adjustment is adjusted within a set injection amount range; and / or, the initial injection amount Q1 is 45 ms, and the set injection amount range is 20-60 ms.

8. The method of claim 5, wherein in S2, when the quantitative test is performed by the joint internal standard method, during the collection of the MS2 spectrum, an effective spectrum filtering operation is performed to determine whether the obtained MS2 spectrum is an effective spectrum; if it is an effective spectrum, the first judgment process is entered, and if it is an invalid spectrum, it is discarded. The effective spectrum filtering operation specifically includes: matching the spectral peak of the internal standard in the MS2 spectrum; if it can be matched, it is an effective spectrum; if it cannot be matched, it is an invalid spectrum.

9. The method of claim 5, wherein in S3, the original data of the target substance in N qualified spectra are respectively accumulated and summed, and an average value is calculated according to the number of qualified spectra to synthesize a spectrum. When the quantitative test is performed by the joint internal standard method, the target substance includes the test substance and the internal standard substance.

10. The method of claim 5, wherein when the mass spectrometry system is used to detect the mutation state of the IDH gene of brain tissue, the target substance is 2-HG and glutamic acid, and wherein: the peak of the target substance is located in the range of m / z 127.5-m / z 129.5; the set limit value is 100-5000; the set upper limit n1 is 10; the set upper limit N1 is 5, and the set lower limit N2 is 3.

11. The method of claim 10, wherein the calculation formula is: M1=(signal intensity of target ion m / z 129) / (0.94x signal intensity of target ion m / z 128); when the embedded threshold value is a certain range, if M1≥upper limit of the embedded threshold value, the output result is IDH mutation positive; if M1 1. [Incorporated by reference (Rule 20.6) 21.02.2025] A viscous glutamic acid corynebacterium, characterized in that the glutamic acid corynebacterium is obtained by mutation of Corynebacterium glutamicum ATCC 13032, and the mutation sites include: (1) the cytosine at position 862902 is mutated to thymine; (2) the guanine at position 862903 is mutated to adenine; (3) the cytosine at position 862953 is mutated to thymine; (4) the adenine at position 862961 is mutated to guanine; (5) a cytosine and a thymine are inserted at position 862958; and (6) the guanine at position 862963 is deleted.

3. [Incorporated by reference (Rule 20.6) 21.02.2025] Use of the glutamic acid corynebacterium of claim 1 or 2 in the production of mucopolysaccharides.

4. [Incorporated by reference (Rule 20.6) 21.02.2025] Use according to claim 3, characterized in that the mucopolysaccharides include hyaluronic acid. ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 2. [Entry into the record (Rule 20.6) 21.02.2025] Corynebacterium glutamicum as claimed in claim 1, characterized in that the mutation site comprises: ​ ​ 5. [Entry into the National Phase by Citation (Rule 20.6) 21.02.2025] A mutant of phosphopyruvate aminotransferase, characterized in that the NCBI number of the phosphopyruvate aminotransferase is BAB98207.1, the gene sequence of the first 100 bp of the phosphopyruvate aminotransferase is mutated, and the gene sequence of the first 101 bp of the mutant of the phosphopyruvate aminotransferase is shown in SEQ ID NO.

5.

6. [Entry into the National Phase by Citation (Rule 20.6) 21.02.2025] An expression vector containing the gene encoding the mutant of phosphopyruvate aminotransferase of claim 5.

7. [Entry into the National Phase by Citation (Rule 20.6) 21.02.2025] A recombinant Corynebacterium glutamicum, characterized in that the modification of the recombinant Corynebacterium glutamicum comprises: replacing the gene encoding the first 100 bp of the wild-type phosphopyruvate aminotransferase on the genome of the Corynebacterium glutamicum host strain with the gene encoding the first 101 bp of the mutant of claim 5.

8. [Reference to Entry (Rule 20.6) 21.02.2025] The recombinant C. glutamicum as claimed in claim 7, characterized in that, The modification of the recombinant Corynebacterium glutamicum further comprises: overexpressing at least one of hyaluronan synthase, glutamine-fructose-6-phosphate aminotransferase, phosphoglucomutase, and uridine diphosphate-glucose dehydrogenase.

9. [Entry into the record (Rule 20.6) 21.02.2025] Recombinant Corynebacterium glutamicum as claimed in claim 8, characterized in that, The overexpression is initiated by a Ptac promoter or a Ptrc promoter.

10. [Entry into the application (Rule 20.6) 21.02.2025] A method for producing mucopolysaccharides, characterized in that, It comprises the step of fermenting the recombinant Corynebacterium glutamicum of any one of claims 7-9.

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