Construction and application of hyaluronic acid recombinant bacteria

Abstract

The invention provides construction and application of a hyaluronic acid recombinant bacterium, and belongs to the fields of biochemical engineering, synthetic biology, genetic engineering and metabolic engineering, the recombinant corynebacterium glutamicum contains a hyaluronic acid synthase gene, a UDP-glucose dehydrogenase gene and a glutamide-6-phosphate fructose transaminase gene; the hyaluronic acid synthase gene, the UDP-glucose dehydrogenase gene and the glutamine-6-phosphate fructose transaminase gene are respectively connected with a sequence RBS1, a sequence RBS2 and a sequence RBS3; the recombinant bacterium provided by the invention also contains a dCas9 protein gene dCas9 and any one of genes sgRNA1, sgRNA2, sgRNA3, sgRNA4, sgRNA5 and sgRNA6, and the recombinant bacterium provided by the invention is high in hyaluronic acid yield and low in production cost.

Description

Technical Field

[0001] The present invention belongs to the fields of biochemistry, synthetic biology, genetic engineering, and metabolic engineering, and particularly relates to the construction and application of a recombinant bacterium for producing hyaluronic acid. Background Art

[0002] Hyaluronic acid (HA) is a linear high-molecular polysaccharide composed of monomers N-acetylglucosamine and D-glucuronic acid, and its molecular weight can reach 10 4 ~10 7 Da, and it is widely used in the fields of cosmetics, food, medicine, etc. The biological activity and use effect of HA are directly determined by its relative molecular weight (Mw). Hyaluronic acids with different molecular weights have different uses. Low-molecular-weight hyaluronic acid with a molecular weight between 10 4 ~10 6 Da is mainly added to skin care products and cosmetics, which can play a good moisturizing effect, and can also increase skin elasticity and reduce wrinkles; hyaluronic acid oligosaccharides with a molecular weight lower than 10 4 Da can be added to oral health products, which can play a role in increasing cell metabolism and preventing aging.

[0003] The biological fermentation method has become the main process for producing hyaluronic acid in China. The commonly used strains in industrial production are animal pathogenic bacteria such as Streptococcus zooepidemicus, and the yield is 6-12 g / L. Using Streptococcus zooepidemicus to produce hyaluronic acid also has certain limitations: First, Streptococcus zooepidemicus is an animal pathogenic bacterium with a pathogenic risk; second, the metabolic background of Streptococcus is not clear enough, and it is difficult to transform by molecular means; finally, the molecular weight of hyaluronic acid produced by the biological fermentation of Streptococcus zooepidemicus is relatively high and is not suitable for direct addition to cosmetics or health products. Therefore, it is of great significance to develop a more economical, safe and low-molecular-weight genetic engineering strain for producing hyaluronic acid.

[0004] To construct a genetic engineering strain for producing hyaluronic acid, it is necessary to introduce hyaluronic acid synthase. So far, hyaluronic acid has been heterologously synthesized in various microorganisms such as Escherichia coli, Lactobacillus, Bacillus subtilis, Corynebacterium glutamicum, etc. However, at present, the biological fermentation production yield of genetic engineering strains for low-molecular-weight hyaluronic acid is relatively low, and the fermentation process is relatively cumbersome. Therefore, constructing a genetic engineering strain with high yield of low-molecular-weight hyaluronic acid has become an urgent technical problem to be solved in this field. Summary of the Invention

[0005] In view of the deficiencies of the prior art, the present invention provides the construction and application of a recombinant bacterium for producing hyaluronic acid.

[0006] The recombinant Corynebacterium glutamicum constructed in the present invention constructs a synthetic pathway for expressing low-molecular-weight hyaluronic acid in the food-safety-grade microorganism Corynebacterium glutamicum. On the basis of realizing the one-step production of hyaluronic acid from glucose, the accumulation of precursor substances is regulated, the competitive pathways are weakened, and the yield of low-molecular-weight hyaluronic acid is increased.

[0007] The technical solution of the present invention is as follows:

[0008] A recombinant Corynebacterium glutamicum contains the hyaluronic acid synthase gene hasA, the UDP-glucose dehydrogenase gene udgA2, and the glutamine-fructose-6-phosphate aminotransferase gene glmS, and the host bacterium is Corynebacterium glutamicum;

[0009] The nucleotide sequence of the hyaluronic acid synthase gene hasA is as shown in SEQ ID NO.1;

[0010] The nucleotide sequence of the UDP-glucose dehydrogenase gene udgA2 is as shown in SEQ ID NO.2;

[0011] The nucleotide sequence of the glutamine-fructose-6-phosphate aminotransferase gene glmS is as shown in SEQ ID NO.3.

[0012] Preferably according to the present invention, the host bacterium is Corynebacterium glutamicum ATCC13032.

[0013] Preferably according to the present invention, the plasmid vector in the recombinant Corynebacterium glutamicum is pEC-XK99E and / or pXMJ19.

[0014] Preferably according to the present invention, in the recombinant Corynebacterium glutamicum, the hyaluronic acid synthase gene hasA, the UDP-glucose dehydrogenase gene udgA2, and the glutamine-fructose-6-phosphate aminotransferase gene glmS are ligated into the plasmid pEC-XK99E to form the recombinant plasmid pEC-XK99E-hasA-udgA2-glmS;

[0015] The hyaluronic acid synthase gene hasA, the UDP-glucose dehydrogenase gene udgA2, and the glutamine-fructose-6-phosphate aminotransferase gene glmS are respectively ligated with the sequences RBS1, RBS2, and RBS3;

[0016] The nucleotide sequence of RBS1 is as shown in SEQ ID NO.11, the nucleotide sequence of RBS2 is as shown in SEQ ID NO.12, and the nucleotide sequence of RBS3 is as shown in SEQ ID NO.13.

[0017] Preferably according to the present invention, in the recombinant Corynebacterium glutamicum, it further contains the dCas9 protein gene dCas9, and any one of the genes sgRNA1, sgRNA2, sgRNA3, sgRNA4, sgRNA5, sgRNA6;

[0018] The nucleotide sequence of the dCas9 protein gene dCas9 is shown in SEQ ID NO.4;

[0019] The nucleotide sequence of sgRNA1 is shown in SEQ ID NO.5;

[0020] The nucleotide sequence of sgRNA2 is shown in SEQ ID NO.6;

[0021] The nucleotide sequence of sgRNA3 is shown in SEQ ID NO.7;

[0022] The nucleotide sequence of sgRNA4 is shown in SEQ ID NO.8;

[0023] The nucleotide sequence of sgRNA5 is shown in SEQ ID NO.9;

[0024] The nucleotide sequence of sgRNA6 is shown in SEQ ID NO.10.

[0025] More preferably, the dCas9 protein gene dCas9 and any one of sgRNA1, sgRNA2, sgRNA3, sgRNA4, sgRNA5, sgRNA6 are ligated into the plasmid pXMJ19, which are respectively the recombinant plasmids pXMJ19-dCas9-ostAsgRNA1, pXMJ19-dCas9-ostAsgRNA2, pXMJ19-dCas9-ostAsgRNA3, pXMJ19-dCas9-treYsgRNA4, pXMJ19-dCas9-treYsgRNA5 or pXMJ19-dCas9-treYsgRNA6.

[0026] Even more preferably, in the recombinant Corynebacterium glutamicum, the dCas9 protein gene dcas9 and sgRNA1 are ligated into the plasmid pXMJ19, which is the recombinant plasmid pXMJ19-dCas9-ostAsgRNA1.

[0027] A strain of Corynebacterium glutamicum HA-Z01 was deposited at the China Center for Type Culture Collection on November 26, 2024. The deposit address is Wuhan University, Wuhan, China, and the deposit number is CCTCC NO: M20242640.

[0028] The construction method of the above-mentioned Corynebacterium glutamicum recombinant includes the following steps:

[0029] (1) Based on the codon preference of Corynebacterium glutamicum, the gene sequence hasA of hyaluronan synthase was designed, as shown in SEQ ID NO.1; the upstream primer hasA-F SEQ ID NO.14 and the downstream primer hasA-R SEQ ID NO.15 were designed, and polymerase chain reaction was carried out to amplify the RBS1-hasA gene;

[0030] (2) The plasmid pEC-XK99E was digested with EcoRⅠ and SacⅠ respectively to obtain a linearized plasmid. The linearized plasmid was ligated with the RBS1-hasA gene obtained in step (1) by seamless cloning. The ligation product was transformed into the competent cells of the host bacterium E.coli JM109, screened and the plasmid was extracted to obtain the recombinant plasmid pEC-XK99E-hasA;

[0031] (3) Using udgA2-F SEQ ID NO.16 and udgA2-R SEQ ID NO.17 as the upstream and downstream primers, and using the genomic DNA of Corynebacterium glutamicum ATCC13869 or SEQ ID NO.2 as the template, polymerase chain reaction was carried out to obtain the gene sequence RBS2-udgA2 of UDP-glucose dehydrogenase;

[0032] (4) The recombinant plasmid pEC-XK99E-hasA obtained in step (2) was digested with SacⅠ and KpnⅠ respectively to obtain a linearized plasmid. The linearized plasmid was ligated with the RBS2-udgA2 gene obtained in step (3) by seamless cloning. The ligation product was transformed into the competent cells of the host bacterium E.coli JM109, screened and the plasmid was extracted to obtain the recombinant plasmid pEC-XK99E-hasA-udgA2;

[0033] (5) Based on the codon preference of Corynebacterium glutamicum, the gene sequence glmS of glutamine-6-phosphate fructosyltransferase was designed, as shown in SEQ ID NO.3; using glmS-F SEQ ID NO.18 and glmS-R SEQ ID NO.19 as the upstream and downstream primers, polymerase chain reaction was carried out to obtain the RBS3-glmS gene;

[0034] (6) The recombinant plasmid pEC-XK99E-hasA-udgA2 obtained in step (4) was digested with KpnⅠ and BamHⅠ respectively to obtain a linearized plasmid. The linearized plasmid was ligated with the RBS3-glmS gene obtained in step (5) by seamless cloning. The ligation product was transformed into competent cells of the host bacterium E. coli JM109, screened, and the plasmid was extracted for PCR and sequencing verification to obtain the recombinant plasmid pEC-XK99E-hasA-udgA2-glmS;

[0035] (7) The recombinant plasmid pEC-XK99E-hasA-udgA2-glmS obtained in step (6) was transformed into Corynebacterium glutamicum ATCC13032, and a genetically engineered bacterium transformed with the plasmid pEC-XK99E-hasA-udgA2-glmS was screened.

[0036] Preferably according to the present invention, in the construction method, it further includes: The plasmid pXMJ19 was digested with HindⅢ and SalⅠ respectively to obtain a linearized plasmid. The linearized plasmid was ligated with the dCas9 gene as shown in SEQ ID NO.4 by seamless cloning. The ligation product was transformed into competent cells of the host bacterium E. coli JM109, screened, and the plasmid was extracted to obtain the recombinant plasmid pXMJ19-dCas9;

[0037] sgRNA sequences with different interference intensities for the ostA gene were designed, namely sgRNA1, sgRNA2, and sgRNA3 gene fragments;

[0038] The nucleotide sequence of sgRNA1 is as shown in SEQ ID NO.5;

[0039] The nucleotide sequence of sgRNA2 is as shown in SEQ ID NO.6;

[0040] The nucleotide sequence of sgRNA3 is as shown in SEQ ID NO.7;

[0041] The recombinant plasmid pXMJ19-dCas9 was digested with BamHⅠ and KpnⅠ respectively to obtain a linearized plasmid. The linearized plasmid was ligated with the sgRNA1, sgRNA2, and sgRNA3 gene fragments by seamless cloning respectively. The ligation products were transformed into competent cells of the host bacterium E. coli JM109, screened, and the plasmids were extracted to obtain the recombinant plasmids pXMJ19-dCas9-ostAsgRNA1, pXMJ19-dCas9-ostAsgRNA2, and pXMJ19-dCas9-ostAsgRNA3 respectively;

[0042] Design sgRNA sequences with different interference intensities for the treY gene, namely sgRNA4, sgRNA5, and sgRNA6 gene fragments;

[0043] The nucleotide sequence of sgRNA4 is shown in SEQ ID NO.8;

[0044] The nucleotide sequence of sgRNA5 is shown in SEQ ID NO.9;

[0045] The nucleotide sequence of sgRNA6 is shown in SEQ ID NO.10;

[0046] The recombinant plasmid pXMJ19-dCas9 was digested with BamHⅠ and KpnⅠ to obtain a linearized plasmid. The linearized plasmid was ligated with the sgRNA5, sgRNA6, and sgRNA7 gene fragments by seamless cloning. The ligation products were transformed into competent cells of the host bacterium E. coli JM109, screened, and plasmids were extracted to obtain recombinant plasmids pXMJ19-dCas9-treYsgRNA4, pXMJ19-dCas9-treYsgRNA5, and pXMJ19-dCas9-treYsgRNA6 respectively;

[0047] The recombinant plasmids pXMJ19-dCas9-ostAsgRNA1, pXMJ19-dCas9-ostAsgRNA2, pXMJ19-dCas9-ostAsgRNA3, pXMJ19-dCas9-treYsgRNA4, pXMJ19-dCas9-treYsgRNA5, and pXMJ19-dCas9-treYsgRNA6 were respectively transformed into the genetically engineered bacteria containing the plasmid pEC-XK99E-hasA-udgA2-glmS obtained in step (7), and genetically engineered bacteria CgHA-Z01, CgHA-Z02, CgHA-Z03, CgHA-Z04, CgHA-Z05, or CgHA-Z06 transformed with the plasmids pXMJ19-dCas9-ostAsgRNA1, pXMJ19-dCas9-ostAsgRNA2, pXMJ19-dCas9-ostAsgRNA3, pXMJ19-dCas9-treYsgRNA4, pXMJ19-dCas9-treYsgRNA5, or pXMJ19-dCas9-treYsgRNA6 were respectively screened.

[0048] The application of the above recombinant Corynebacterium glutamicum in the production of hyaluronic acid.

[0049] According to the preference of the present invention, the molecular weight of the hyaluronic acid is 10 4 -10 6Yes.

[0050] The method for fermenting recombinant Corynebacterium glutamicum to produce hyaluronic acid includes the following steps:

[0051] Inoculate the obtained genetically engineered bacteria into BHI liquid medium and culture at 28 - 32 °C and 150 - 250 rpm for 8 - 16 h to obtain the primary seed liquid; inoculate the primary seed liquid into the secondary seed medium with an initial OD562 between 0.05 - 0.15 and culture at 28 - 32 °C and 150 - 250 rpm for 8 - 12 h to obtain the secondary seed liquid; inoculate the secondary seed liquid into the fermentation medium with an initial OD562 of 0.05 - 0.15 and culture at 28 - 32 °C and 150 - 250 rpm for 2 - 6 h, add IPTG with a final concentration of 0.1 - 0.8 mM, and continue to culture and ferment for 24 - 84 h. Remove the bacteria to obtain the fermentation broth containing hyaluronic acid.

[0052] Preferably according to the present invention, the components of the BHI liquid medium include: yeast powder 2.5 g / L, peptone 5 g / L, sodium chloride 5 g / L, bovine brain heart infusion 18.5 g / L, kanamycin sulfate 30 mg / L, and chloramphenicol 15 mg / L.

[0053] Preferably according to the present invention, the components of the secondary seed medium include: glucose 20 g / L, ammonium sulfate 10 g / L, peptone 10 g / L, yeast powder 5 g / L, corn steep powder 10 g / L, magnesium sulfate 0.5 g / L, dipotassium hydrogen phosphate 1.5 g / L, potassium dihydrogen phosphate 1.5 g / L, pH = 7.

[0054] Preferably according to the present invention, the components of the fermentation medium include: glucose 50 g / L, ammonium sulfate 15 g / L, peptone 5 g / L, yeast powder 2.5 g / L, corn steep powder 10 g / L, magnesium sulfate 0.5 g / L, dipotassium hydrogen phosphate 1.5 g / L, potassium dihydrogen phosphate 1.5 g / L, pH = 7.

[0055] Preferably according to the present invention, the yield of hyaluronic acid in the fermentation broth reaches 50 g / L or more.

[0056] Preferably according to the present invention, the molecular weight of the hyaluronic acid produced by the method is 10 4 -10 6 Da.

[0057] Use of the hyaluronic acid produced by the above - mentioned recombinant Corynebacterium glutamicum, the hyaluronic acid produced by the recombinant Corynebacterium glutamicum constructed by the above - mentioned construction method, or the hyaluronic acid produced by the above - mentioned method for producing hyaluronic acid in the preparation of products containing hyaluronic acid in the food field, pharmaceutical field, or cosmetic field.

[0058] The beneficial effects of the present invention at least include the following:

[0059] The present invention adopts molecular biology methods and techniques, redesigned and chemically synthesized the hyaluronic acid synthase hasA gene sequence SEQ ID NO.1, UDP-glucose dehydrogenase udgA2 gene sequence SEQ ID NO.2 and glutamine-6-phosphate fructose aminotransferase glmS gene sequence SEQ ID NO.3 according to the codon preference of Corynebacterium glutamicum. Overexpressing the above three genes simultaneously can achieve high-yield production of hyaluronic acid. On this basis, the mycolic acid outer structure of Corynebacterium glutamicum is modified (trehalose forms the mycolic acid layer in the form of glycolipid), weakening the ability of mycolic acid to synthesize glucose mycolic acid under the condition of high-concentration glucose, and at the same time, it will not affect the growth of the strain, enabling more carbon sources to flow to the biosynthesis of hyaluronic acid and positively increasing the yield of hyaluronic acid. In the method of the present invention, the Corynebacterium glutamicum host is non-pathogenic to humans and animals, is a food-grade safe microorganism, the synthesized hyaluronic acid has a high yield, reaching more than 50 g / L, and the molecular weight of hyaluronic acid is low, having good industrial application prospects. Brief Description of the Drawings

[0060] Figure 1 It is a PCR verification diagram of the recombinant plasmid pEC-XK99E-hasA containing the hasA gene in the present invention;

[0061] In the figure: Lane 1 is the DNA molecular weight standard, and the 4th band from top to bottom is 1.5 kb; Lane 2 is the result of PCR amplification of the recombinant plasmid pEC-XK99E-hasA using primers hasA-F and hasA-R, approximately 1.3 kb.

[0062] Figure 2 It is a PCR verification diagram of the recombinant plasmid pEC-XK99E-hasA-udgA2 of the udgA2 gene in the present invention;

[0063] In the figure: Lane 1 is the DNA molecular weight standard, and the 4th band from top to bottom is 1.5 kb; Lane 2 is the result of PCR amplification of the recombinant plasmid pEC-XK99E-hasA-udgA2 using primers udgA2-F and udgA2-R, approximately 1.3 kb.

[0064] Figure 3 It is a PCR verification diagram of the recombinant plasmid pEC-XK99E-hasA-udgA2-glmS of the glmS gene in the present invention;

[0065] In the figure: Lane 1 is the DNA molecular weight standard, and the 4th band from top to bottom is 1.5 kb; Lane 2 is the result of PCR amplification of the recombinant plasmid pEC-XK99E-hasA-udgA2-glmS using primers glmS-F and glmS-R, approximately 1.8 kb.

[0066] Figure 4 This is the PCR verification figure of the recombinant plasmid pXMJ19-dCas9 of the dCas9 protein in the present invention;

[0067] In the figure: Lane 1 is the DNA molecular weight standard, the 1st band from top to bottom is 5 kb, and the 2nd band is 3 kb; Lane 2 is the result of PCR amplification of the recombinant plasmid pXMJ19-dCas9 using primers dCas9-F and dCas9-R, approximately 4.1 kb.

[0068] Figure 5 This is a comparison result figure of the hyaluronic acid production yields of Corynebacterium glutamicum CG containing the empty plasmid pEC-XK99E without inserted foreign genes and the genetically engineered bacteria CgHA-1, CgHA-2, and CgHA-3.

[0069] Figure 6 This is a comparison result figure of the hyaluronic acid production yields of Corynebacterium glutamicum CG containing the empty plasmid pEC-XK99E without inserted foreign genes and the genetically engineered bacteria CgHA-3, CgHA-Z01, CgHA-Z02, CgHA-Z03, CgHA-Z04, CgHA-Z05, and CgHA-Z06 in a 3L fermenter.

[0070] Figure 7 This is a comparison figure of the growth conditions of the strains during the fermentation process of Corynebacterium glutamicum CG containing the empty plasmid pEC-XK99E without inserted foreign genes and the genetically engineered bacteria CgHA-Z01, CgHA-Z02, CgHA-Z03, CgHA-Z04, CgHA-Z05, and CgHA-Z06.

[0071] Figure 8 This is the hyaluronic acid production yield figure of the genetically engineered bacterium CgHA-Z01 in a 20L fermenter. Specific implementation manners

[0072] The technical solutions of the present invention will be further described below through specific examples.

[0073] Sources of test materials

[0074] In the experiment, the strains Corynebacterium glutamicum ATCC13032, competent cell E. coli JM109, plasmid pXMJ19 and plasmid pEC-XK99E were all purchased from the market, with reliable sources and meeting the experimental requirements.

[0075] Example 1

[0076] Construction of a Corynebacterium glutamicum-Escherichia coli shuttle plasmid co-expressing the hyaluronic acid synthase hasA gene, UDP-glucose dehydrogenase udgA2 gene and glutamine-6-phosphate fructose aminotransferase glmS gene.

[0077] Construction of plasmid pEC-XK99E-hasA: Based on the codon preference of Corynebacterium glutamicum, the hyaluronic acid synthase hasA gene sequence derived from Streptococcus pyogenes was designed and chemically synthesized, and the nucleotide sequence is shown in SEQ ID NO.1.

[0078] Using hasA-F SEQ ID NO.14 and hasA-R SEQ ID NO.15 as upstream and downstream primers, and the plasmid containing the hyaluronic acid synthase hasA gene sequence as a template (synthesized by Beijing Tsingke Biotechnology Co., Ltd.), polymerase chain reaction was carried out to amplify the RBS1-hasA gene, and the primers were synthesized by Beijing Tsingke Biotechnology Co., Ltd. The polymerase used for PCR amplification was purchased from Novizan.

[0079] The PCR amplification reaction system was:

[0080]

[0081] The PCR amplification conditions were: 95°C for 30 s; 95°C for 10 s, 55°C for 30 s, 72°C for 45 s, 34 cycles; 72°C for 10 min. The amplified product was verified by sequencing to be the hyaluronic acid synthase gene RBS1-hasA containing sequence RBS1. After the amplified product was electrophoresed on a 1% agarose gel, it was purified and recovered using a product recovery kit (Novizan), and the obtained PCR product was stored at low temperature in sterile water at pH 8.0.

[0082] The Corynebacterium glutamicum-Escherichia coli shuttle plasmid pEC-XK99E was double-digested with EcoRⅠ and SacⅠ, reacted at 37 °C for 30 minutes. After the digestion product was subjected to 1% agarose gel electrophoresis, it was purified and recovered using a product recovery kit (Novizan), and the obtained linearized plasmid was stored at low temperature in sterile water with pH 8.0; then the linearized plasmid was ligated with the RBS1-hasA gene by seamless cloning, the reaction temperature was 37 °C, and the reaction time was 30 minutes to obtain a ligation product. The ligation product was transformed into competent cells of the host bacterium E. coli JM109 (TIANGEN), spread on a solid plate of LB medium (10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride) containing 30 mg / L kanamycin sulfate, resistant clones were picked for culture, and plasmids were extracted for PCR and sequencing verification (using the upstream primer hasA-F and the downstream primer hasA-R, a band of about 1.3 kb can be amplified, as Figure 1 shown), proving that the recombinant plasmid pEC-XK99E-hasA carrying the RBS1-hasA gene was successfully constructed.

[0083] Construction of plasmid pEC-XK99E-hasA-udgA2: Using udgA2-F SEQ ID NO.16 and udgA2-R SEQ ID NO.17 as upstream and downstream primers, and using the genomic DNA of Corynebacterium glutamicum ATCC13869 as a template, or using a plasmid containing the UDP-glucose dehydrogenase udgA2 gene sequence SEQ ID NO.2 (synthesized by Beijing Tsingke Biotechnology Co., Ltd.) as a template, polymerase chain reaction was carried out to obtain the UDP-glucose dehydrogenase RBS2-udgA2 gene sequence. The primers were synthesized by Beijing Tsingke Biotechnology Co., Ltd. The polymerase used for PCR amplification was purchased from Novizan.

[0084] The PCR amplification reaction system was:

[0085]

[0086] The PCR amplification conditions were: 95 °C for 30 s; 95 °C for 10 s, 55 °C for 30 s, 72 °C for 60 s, 34 cycles; 72 °C for 10 min. The amplified product was verified by sequencing to be the UDP-glucose dehydrogenase gene RBS2-udgA2 containing sequence RBS2. After the amplified product was subjected to 1% agarose gel electrophoresis, it was purified and recovered using a product recovery kit (Novizan), and the obtained PCR product was stored at low temperature in sterile water with pH 8.0.

[0087] The recombinant plasmid pEC-XK99E-hasA was digested with SacⅠ and KpnⅠ at 37°C for 30 minutes. After the digestion product was subjected to 1% agarose gel electrophoresis, it was purified and recovered using a product recovery kit (Novizan), and the obtained linearized plasmid was stored at low temperature in sterile water with a pH of 8.0. Then, the linearized plasmid was ligated with the RBS2-udgA2 gene by seamless cloning at a reaction temperature of 37°C for 30 minutes to obtain a ligation product. The ligation product was transformed into competent cells of the host bacterium E. coli JM109 (TIANGEN), spread on a solid LB medium plate (10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride) containing 30 mg / L kanamycin sulfate, and resistant clones were picked for culture. The plasmid was extracted and verified by PCR and sequencing (using the upstream primer udgA2-F and the downstream primer udgA2-R, a band of approximately 1.3 kb could be amplified, as shown in Figure 2 ), indicating that the recombinant plasmid pEC-XK99E-hasA-udgA2 carrying the RBS2-udgA2 gene was successfully constructed.

[0088] Construction of plasmid pEC-XK99E-hasA-udgA2-glmS: Based on the codon preference of Corynebacterium glutamicum, the glutamine-6-phosphofructotransferase gene glmS sequence from Sulfolobus shibatae was designed and chemically synthesized, and the nucleotide sequence is as shown in SEQ ID NO.3. Using glmS-F SEQ ID NO.18 and glmS-R SEQ ID NO.19 as the upstream and downstream primers, and the plasmid containing SEQ ID NO.3 as the template (synthesized by Beijing Tsingke Biotechnology Co., Ltd.), polymerase chain reaction was carried out to obtain the glutamine-6-phosphofructotransferase RBS3-glmS gene sequence. The primers were synthesized by Beijing Tsingke Biotechnology Co., Ltd. The polymerase used for PCR amplification was purchased from Novizan.

[0089] The reaction system for PCR amplification was:

[0090]

[0091] The PCR amplification conditions were: 95°C for 30 s; 95°C for 10 s, 55°C for 30 s, 72°C for 60 s, for 34 cycles; 72°C for 10 min. The amplified product was verified by sequencing to be the glutamine-6-phosphofructotransferase gene RBS3-glmS containing the sequence RBS3. After the amplified product was subjected to 1% agarose gel electrophoresis, it was purified and recovered using a product recovery kit (Novizan), and the obtained PCR product was stored at low temperature in sterile water with a pH of 8.0.

[0092] The recombinant plasmid pEC-XK99E-hasA-udgA2 was digested with KpnⅠ and BamHⅠ at 37°C for 30 minutes. After the digestion product was electrophoresed on a 1% agarose gel, it was purified and recovered using a product recovery kit (Novizan), and the obtained linearized plasmid was stored at low temperature in sterile water with pH 8.0. Then, the linearized plasmid was ligated with the RBS3-glmS gene by seamless cloning at 37°C for 30 minutes to obtain a ligation product. The ligation product was transformed into competent cells of the host bacterium E. coli JM109 (TIANGEN), spread on a solid LB medium plate (10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride) containing 30 mg / L kanamycin sulfate, and resistant clones were picked for culture. The plasmid was extracted and verified by PCR and sequencing (using the upstream primer glmS-F and the downstream primer glmS-R, a band of about 1.8 kb could be amplified, as Figure 3 shown), proving the successful construction of the recombinant plasmid pEC-XK99E-hasA-udgA2-glmS carrying the RBS3-glmS gene.

[0093] Example 2

[0094] Construction of a genetically engineered strain co-expressing the hyaluronic acid synthase hasA gene, UDP-glucose dehydrogenase udgA2 gene, and glutamine-6-phosphate fructose aminotransferase glmS

[0095] The recombinant plasmids pEC-XK99E-hasA, pEC-XK99E-hasA-udgA2, and pEC-XK99E-hasA-udgA2-glmS constructed in Example 1 were respectively transformed into competent cells of Corynebacterium glutamicum ATCC13032 by electroporation to obtain the genetically engineered bacteria CgHA-1, CgHA-2, and CgHA-3 for producing hyaluronic acid.

[0096] Place the prepared electrocompetent Corynebacterium glutamicum ATCC13032 on ice. After melting, add 300 - 1500 ng of the recombinant plasmid to it. After pipetting and mixing evenly, transfer the mixture into a pre-cooled electrode cup and place it on ice for about 10 - 15 min. Subsequently, dry the ice water on the outer wall and lid of the electrode cup, quickly place it in the electroporation chamber, and perform two electroporations under the condition of 1.8 kV. Immediately after electroporation, quickly add 800 - 1000 μL of BHI liquid medium (yeast powder 2.5 g / L, peptone 5 g / L, sodium chloride 5 g / L, bovine brain heart infusion 18.5 g / L) preheated to 40 - 48 °C, mix evenly, place it in a water bath at 40 - 48 °C for 5 - 10 min, recover and culture it at 25 - 35 °C for 1 - 2 h, spread it on a solid BHI medium plate (containing 30 mg / L kanamycin sulfate), pick resistant clones for culture, extract plasmids, and use hasA-F / hasA-R, udgA2-F / udgA2-R, and glmS-F / glmS-R for PCR and sequencing verification respectively to prove the successful construction of the genetically engineered bacteria CgHA-1, CgHA-2, and CgHA-3.

[0097] CgHA-1 is Corynebacterium glutamicum ATCC13032 / pEC-XK99E-hasA;

[0098] CgHA-2 is Corynebacterium glutamicum ATCC13032 / pEC-XK99E-hasA-udgA2;

[0099] CgHA-3 is Corynebacterium glutamicum ATCC13032 / pEC-XK99E-hasA-udgA2-glmS.

[0100] Example 3

[0101] Construct CRISPR interference plasmids with different strengths of weakening the expression of otsA and treY genes in the trehalose synthesis pathway

[0102] First, site-directed mutagenesis was performed on the Cas9 protein to construct the dCas9 protein. Using dCas9-F1 (including the mutant base D10A) SEQ ID NO.22 and dCas9-R1 SEQ ID NO.23 as primers, and the pCas plasmid as a template, a 1.2 kb dCas91 gene fragment was amplified; using dCas9-F2 SEQ ID NO.24 and dCas9-R2 (including the mutant base H840A) SEQ ID NO.25 as primers, a 1.3 kb dCas92 gene fragment was amplified; using dCas9-F3 SEQ ID NO.26 and dCas9-R3 SEQ ID NO.27 as primers, a 1.6 kb dCas93 gene fragment was amplified. Subsequently, using dCas9-F SEQ ID NO.20 and dCas9-R SEQ ID NO.21 as primers, and the three fragments of dCas91, dCas92, and dCas93 as templates, an overlapping amplification was performed to obtain a 4.2 kb dCas9 gene fragment with mutated Cas9 protein, as shown in SEQ ID NO.4. The primers were synthesized by Beijing Tsingke Biotechnology Co., Ltd. The polymerase used for PCR amplification was purchased from Novizan.

[0103] The reaction system for PCR amplification was:

[0104]

[0105] The PCR amplification conditions were: 95°C for 30 s; 95°C for 10 s, 55°C for 30 s, 72°C for 60 s, for 34 cycles; 72°C for 10 min.

[0106] The reaction system for overlapping PCR amplification was:

[0107]

[0108] The PCR amplification conditions were as follows: 95°C for 30 s; 95°C for 10 s, 55°C for 30 s, 72°C for 130 s, for 34 cycles; 72°C for 10 min. The amplified product was verified by sequencing to be the dCas9 protein gene sequence (the sequence is shown in SEQ ID NO.4). The Corynebacterium glutamicum-Escherichia coli shuttle plasmid pXMJ19 was double digested with HindⅢ and SalⅠ, reacted at 37°C for 30 minutes. After the digested product was subjected to 1% agarose gel electrophoresis, it was purified and recovered using a product recovery kit (Novizan Co., Ltd.), and the obtained linearized plasmid was stored at low temperature in sterile water with pH 8.0; then the linearized plasmid and the dCas9 gene were ligated by seamless cloning, the reaction temperature was 37°C, and the reaction time was 30 minutes to obtain a ligation product. The ligation product was transformed into competent cells of the host bacterium E. coli JM109, spread on a solid plate of LB medium (10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride) (containing 15 mg / L chloramphenicol), resistant clones were picked for culture, and plasmids were extracted for PCR and sequencing verification to obtain the recombinant plasmid pXMJ19-dCas9 carrying the dCas9 gene. The nucleic acid gel diagram for PCR verification is shown in Figure 4 .

[0109] Subsequently, the CHOPCHOP software was used to design sgRNA sequences with different interference intensities for the ostA gene and the treY gene. A total of 10 sgRNA sequences with different interference intensities were designed according to the different binding positions to the target genes, and all were transformed into the host bacterium. After screening and verification by shake flask fermentation experiments, 6 sgRNA sequences were obtained that promoted the production of hyaluronic acid and had little effect on the growth of the host bacteria. Using the pTargetF plasmid as a template and gRNA-F1 SEQ ID NO.28 and gRNA-R SEQ ID NO.29 as primers, the pJ23119-sgRNA1 scaffold structural gene fragment was amplified, as shown in SEQ ID NO.5. The primers were synthesized by Beijing Tsingke Biotechnology Co., Ltd. The polymerase used for PCR amplification was purchased from Novizan Co., Ltd.

[0110] The PCR amplification reaction system was:

[0111]

[0112] The PCR amplification conditions were as follows: 95°C for 30 s; 95°C for 10 s, 55°C for 30 s, 72°C for 15 s, for 34 cycles; 72°C for 10 min. The amplification methods for sgRNA2, sgRNA3, sgRNA4, sgRNA5, and sgRNA6 were the same as above. Using gRNA-F2 SEQ ID NO.30 and gRNA-R as primers, the pJ23119-sgRNA2 scaffold structural gene fragment, as shown in SEQ ID NO.6, was amplified. Using gRNA-F3 SEQ ID NO.31 and gRNA-R as primers, the pJ23119-sgRNA3 scaffold structural gene fragment, as shown in SEQ ID NO.7, was amplified. Using gRNA-F4 SEQ ID NO.32 and gRNA-R as primers, the pJ23119-sgRNA4 scaffold structural gene fragment, as shown in SEQ ID NO.8, was amplified. Using gRNA-F5 SEQ ID NO.33 and gRNA-R as primers, the pJ23119-sgRNA5 scaffold structural gene fragment, as shown in SEQ ID NO.9, was amplified. Using gRNA-F6 SEQ ID NO.34 and gRNA-R as primers, the pJ23119-sgRNA6 scaffold structural gene fragment, as shown in SEQ ID NO.10, was amplified.

[0113] The recombinant plasmid pXMJ19-dCas9 was double digested with BamHⅠ and KpnⅠ at 37°C for 30 minutes. After the digestion product was subjected to 1% agarose gel electrophoresis, it was purified and recovered using a product recovery kit (Novizan). The obtained linearized plasmid was stored at low temperature in sterile water at pH 8.0. Then, the linearized plasmid was ligated with the sgRNA1, sgRNA2, sgRNA3, sgRNA4, sgRNA5, and sgRNA6 gene fragments by seamless cloning at 37°C for 30 minutes to obtain the ligation product. The ligation product was transformed into the competent cells of the host bacterium E. coli JM109 and spread on a solid LB medium plate (10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride) containing 15 mg / L chloramphenicol. Resistant clones were picked for culture, and the plasmids were extracted for PCR and sequencing verification. The recombinant plasmids pXMJ19-dCas9-ostAsgRNA1, pXMJ19-dCas9-ostAsgRNA2, pXMJ19-dCas9-ostAsgRNA3, pXMJ19-dCas9-treYsgRNA4, pXMJ19-dCas9-treYsgRNA5, and pXMJ19-dCas9-treYsgRNA6 were successfully constructed.

[0114] Example 4

[0115] Construction of genetically engineered bacteria with different intensities of weakened expression of otsA and treY genes in the trehalose synthesis pathway

[0116] The recombinant plasmids pXMJ19-dCas9-ostAsgRNA1, pXMJ19-dCas9-ostAsgRNA2, pXMJ19-dCas9-ostAsgRNA3, pXMJ19-dCas9-treYsgRNA4, pXMJ19-dCas9-treYsgRNA5 and pXMJ19-dCas9-treYsgRNA6 in Example 3 were respectively transformed into the competent cells of the genetically engineered bacterium CgHA-3 by electroporation, and the genetically engineered bacteria CgHA-Z01, CgHA-Z02, CgHA-Z03, CgHA-Z04, CgHA-Z05 and CgHA-Z06 for producing hyaluronic acid could be obtained.

[0117] Place the prepared electrocompetent cells of the genetically engineered bacterium CgHA-3 on ice. After melting, add 300 - 1500 ng of the recombinant plasmid to it. After pipetting and mixing evenly, transfer the mixture into a pre-cooled electrode cup and place it on ice for about 10 - 15 min. Subsequently, dry the ice water on the outer wall and the lid of the electrode cup, and quickly put it into the electroporation chamber, and perform two electroporations under the condition of 1.8 kV. After electroporation, quickly add 800 - 1000 μL of BHI liquid medium (yeast powder 2.5 g / L, peptone 5 g / L, sodium chloride 5 g / L, bovine brain heart infusion 18.5 g / L) preheated at 40 - 48 °C, mix evenly and place it in a water bath at 40 - 48 °C for 5 - 10 min, and recover and culture it at 25 - 35 °C for 1 - 2 h. Spread it on a solid BHI medium plate (containing 30 mg / L kanamycin sulfate and 15 mg / L chloramphenicol), pick resistant clones for culture, extract plasmids, and verify them by sequencing respectively, proving that the genetically engineered bacteria CgHA-Z01, CgHA-Z02, CgHA-Z03, CgHA-Z04, CgHA-Z05 and CgHA-Z06 were successfully constructed.

[0118] CgHA-Z01 is Corynebacterium glutamicum ATCC13032 / pEC-XK99E-hasA-udgA2-glmS / pXMJ19-dCas9-ostAsgRNA1;

[0119] CgHA-Z02 is Corynebacterium glutamicum ATCC13032 / pEC-XK99E-hasA-udgA2-glmS / pXMJ19-dCas9-ostAsgRNA2;

[0120] CgHA-Z03 is Corynebacterium glutamicum ATCC13032 / pEC-XK99E-hasA-udgA2-glmS / pXMJ19-dCas9-ostAsgRNA3;

[0121] CgHA-Z04 is Corynebacterium glutamicum ATCC13032 / pEC-XK99E-hasA-udgA2-glmS / pXMJ19-dCas9-treYsgRNA4;

[0122] CgHA-Z05 is Corynebacterium glutamicum ATCC13032 / pEC-XK99E-hasA-udgA2-glmS / pXMJ19-dCas9-treYsgRNA5;

[0123] CgHA-Z06 is Corynebacterium glutamicum ATCC13032 / pEC-XK99E-hasA-udgA2-glmS / pXMJ19-dCas9-treYsgRNA6.

[0124] Example 5

[0125] Using genetically engineered bacteria CgHA-1, CgHA-2, CgHA-3, CgHA-Z01, CgHA-Z02, CgHA-Z03, CgHA-Z04, CgHA-Z05 and CgHA-Z06 to produce hyaluronic acid in a 3L fermenter

[0126] Inoculate Corynebacterium glutamicum (ATCC13032) CG transfected with empty plasmids pEC-XK99E and pXMJ19, genetically engineered bacteria CgHA-1, CgHA-2 and CgHA-3 obtained in Example 2, and genetically engineered bacteria CgHA-Z01, CgHA-Z02, CgHA-Z03, CgHA-Z04, CgHA-Z05 and CgHA-Z06 obtained in Example 4 into BHI liquid medium (yeast powder 2.5 g / L, peptone 5 g / L, sodium chloride 5 g / L, bovine brain heart infusion 18.5 g / L, containing 30 mg / L kanamycin sulfate and 15 mg / L chloramphenicol), culture at 30 °C and 220 rpm for 16 h, and inoculate into the fermentation medium (glucose 50 g / L, ammonium sulfate 15 g / L, peptone 5 g / L, yeast powder 2.5 g / L, corn steep powder 10 g / L, magnesium sulfate 0.5 g / L, dipotassium hydrogen phosphate 1.5 g / L, potassium dihydrogen phosphate 1.5 g / L, pH = 7) at an initial OD562 of 0.1, culture at 30 °C and 220 rpm for 3 h, add IPTG (final concentration 0.3 mM), and continue to culture and ferment for 48 h. Centrifuge at room temperature at 12,000 rpm to remove the cells to obtain the fermentation broth containing hyaluronic acid.

[0127] Take 1 mL of the obtained fermentation broth, add 1 mL of 0.1% w / v SDS solution thereto for mixing, and incubate for 30 minutes at room temperature after mixing evenly; transfer the supernatant to a new EP tube after centrifuging at 12,000 rpm for 10 min, add 2 - 3 volumes of ethanol at 4°C thereto, and place at 4°C for 2 h; remove the supernatant after centrifuging again at 12,000 rpm for 10 min, and after completely volatilizing the ethanol at room temperature, resuspend the precipitate with deionized water. Take 300 μL of the resuspended liquid, add 700 μL of acetate buffer (0.2 mol / L sodium acetate, 0.15 mol / L sodium chloride, adjusted to pH 6 with acetic acid) and 2 mL of 2.5 g / L CATB solution thereto, and measure OD400 after reacting for 5 min.

[0128] The measurement results are shown in Figure 5 、 Figure 6 and Figure 7 , hyaluronic acid was detected in all of the genetically engineered bacteria CgHA-1, CgHA-2 and CgHA-3, indicating that the heterologous synthesis pathway of hyaluronic acid was successfully constructed in the host bacteria. The yield of hyaluronic acid in the genetically engineered bacteria CgHA-1 was 18.95 g / L, the yield of hyaluronic acid in the genetically engineered bacteria CgHA-2 was 23.67 g / L, and the yield of hyaluronic acid in the genetically engineered bacteria CgHA-3 reached 31.88 g / L, as shown in Figure 5 , while the hyaluronic acid yields of CgHA-Z01, CgHA-Z02, CgHA-Z03, CgHA-Z04, CgHA-Z05 and CgHA-Z06 reached more than 35 g / L, which were 38.55 g / L, 36.34 g / L, 36.99 g / L, 37.01 g / L, 35.69 g / L and 37.21 g / L respectively, as shown in Figure 6 , higher than the existing industrial production level of Streptococcus zooepidemicus. In addition, during the fermentation of the engineered bacteria, the growth of the bacterial cells was detected in real time. Compared with the original strain CG, the growth of the engineered bacteria was good and there was little difference from the original strain, as shown in Figure 7 .

[0129] The molecular weight of the product was analyzed by high performance liquid chromatography. The chromatographic column was Shodex SB-806M OHpak (8 * 300 mm), the mobile phase was 0.2 M sodium chloride, and the flow rate was 0.5 mL / min. The molecular weight standard was purchased from Lifecore Biomedical. The results showed that the molecular weight of hyaluronic acid in the genetically engineered bacteria CgHA-Z01, CgHA-Z02, CgHA-Z03, CgHA-Z04, CgHA-Z05 and CgHA-Z06 could reach between 10 4 -10 6 Da.

[0130] Example 6

[0131] Production of hyaluronic acid using genetically engineered bacterium CgHA-Z01 in a 20 L fermenter

[0132] The genetically engineered bacterium CgHA-Z01 obtained in Example 4 was inoculated into BHI liquid medium (2.5 g / L yeast extract, 5 g / L peptone, 5 g / L sodium chloride, 18.5 g / L bovine brain heart infusion, containing 30 mg / L kanamycin sulfate and 15 mg / L chloramphenicol), and cultured at 30 °C and 220 rpm for 16 h to obtain a primary seed solution; the primary seed solution was inoculated into a secondary seed medium (20 g / L glucose, 10 g / L ammonium sulfate, 10 g / L peptone, 5 g / L yeast extract, 10 g / L corn steep liquor powder, 0.5 g / L magnesium sulfate, 1.5 g / L dipotassium hydrogen phosphate, 1.5 g / L potassium dihydrogen phosphate, pH = 7) with an initial OD562 of 0.1, and cultured at 30 °C and 220 rpm for 10 h to obtain a secondary seed solution; the secondary seed solution was inoculated into a fermentation medium (50 g / L glucose, 15 g / L ammonium sulfate, 5 g / L peptone, 2.5 g / L yeast extract, 10 g / L corn steep liquor powder, 0.5 g / L magnesium sulfate, 1.5 g / L dipotassium hydrogen phosphate, 1.5 g / L potassium dihydrogen phosphate, pH = 7) with an initial OD562 of 0.1, cultured at 30 °C and 220 rpm for 3 h, IPTG (final concentration 0.3 mM) was added, and fermentation was continued for 72 h. The cells were removed by centrifugation at 12,000 rpm at room temperature to obtain a fermentation broth containing hyaluronic acid.

[0133] Take 1 mL of the obtained fermentation broth, add 1 mL of 0.1% w / v SDS solution and mix. After mixing, incubate at room temperature for 30 minutes; after centrifugation at 12,000 rpm for 10 min, transfer the supernatant to a new EP tube, add 2 - 3 volumes of 4 °C ethanol, and place at 4 °C for 2 h; after centrifugation at 12,000 rpm for 10 min again to remove the supernatant, completely volatilize the ethanol at room temperature, and resuspend the precipitate with deionized water. Take 300 μL of the resuspended liquid, add 700 μL of acetate buffer (0.2 mol / L sodium acetate, 0.15 mol / L sodium chloride, pH adjusted to 6 with acetic acid) and 2 mL of 2.5 g / L CATB solution, and measure OD400 after reacting for 5 min.

[0134] The measurement results are shown in Figure 8, when fermented for 66 h, the yield of hyaluronic acid in the genetically engineered bacterium CgHA-Z01 reached 52.29 g / L. In the hyaluronic acid fermentation experiments of the genetically engineered bacteria CgHA-1, CgHA-2, CgHA-3, CgHA-Z01, CgHA-Z02, CgHA-Z03, CgHA-Z04, CgHA-Z05 and CgHA-Z06, three parallel samples were set up for each strain, and each group of experiments was repeated three times or more. The genetically engineered bacterium CgHA-Z01 was fermented in a 5 L fermenter for 144 hours, and the hyaluronic acid was still increasing, proving that the above engineered bacteria had good stability.

[0135] Among the above genetically engineered bacteria, the highest yield of hyaluronic acid in CgHA-Z01 reached 52.29 g / L. CgHA-Z01 has been preserved, and the preservation information is as follows:

[0136] A strain of Corynebacterium glutamicum HA-Z01, deposited at the China Center for Type Culture Collection on November 26, 2024, with the deposit address being Wuhan University, Wuhan, China, and the deposit number being CCTCC NO: M20242640.

Claims

1. A recombinant Corynebacterium glutamicum, characterized in that It contains the hyaluronan synthase gene hasA, the UDP-glucose dehydrogenase gene udgA2, and the glutamine-6-phosphofructotransaminase gene glmS, and the host bacteria is Corynebacterium glutamicum; The nucleotide sequence of the hyaluronan synthase gene hasA is shown in SEQ ID NO.1; The nucleotide sequence of the UDP-glucose dehydrogenase gene udgA2 is shown in SEQ ID NO.2; The nucleotide sequence of the glutamine-6-phosphofructotransaminase gene glmS is shown in SEQ ID NO.

3.

2. The recombinant Corynebacterium glutamicum according to claim 1, characterized in that The host bacteria is Corynebacterium glutamicum ATCC13032.

3. The recombinant Corynebacterium glutamicum according to claim 1, characterized in that The plasmid vector in the recombinant Corynebacterium glutamicum is pEC-XK99E and / or pXMJ19.

4. The recombinant Corynebacterium glutamicum according to claim 1, characterized in that In the recombinant Corynebacterium glutamicum, the hyaluronan synthase gene hasA, the UDP-glucose dehydrogenase gene udgA2, and the glutamine-6-phosphofructotransaminase gene glmS are connected to the plasmid pEC-XK99E to obtain the recombinant plasmid pEC-XK99E-hasA-udgA2-glmS; The hyaluronan synthase gene hasA, the UDP-glucose dehydrogenase gene udgA2, and the glutamine-6-phosphofructotransaminase gene glmS are connected to sequences RBS1, RBS2, and RBS3, respectively; The nucleotide sequence of RBS1 is shown in SEQ ID NO.11, the nucleotide sequence of RBS2 is shown in SEQ ID NO.12, and the nucleotide sequence of RBS3 is shown in SEQ ID NO.

13.

5. The recombinant Corynebacterium glutamicum according to claim 1, characterized in that The recombinant Corynebacterium glutamicum also contains a dCas9 protein gene dCas9, and any one of the genes sgRNA1, sgRNA2, sgRNA3, sgRNA4, sgRNA5, and sgRNA6; The nucleotide sequence of the dCas9 protein gene dCas9 is shown in SEQ ID NO.4; The nucleotide sequence of sgRNA1 is shown in SEQ ID NO.5; The nucleotide sequence of sgRNA2 is shown in SEQ ID NO.6; The nucleotide sequence of sgRNA3 is shown in SEQ ID NO.7; The nucleotide sequence of sgRNA4 is shown in SEQ ID NO.8; The nucleotide sequence of sgRNA5 is shown in SEQ ID NO.9; The nucleotide sequence of sgRNA6 is shown in SEQ ID NO.10; Preferably, the dCas9 protein gene dCas9 and any one of sgRNA1, sgRNA2, sgRNA3, sgRNA4, sgRNA5, and sgRNA6 are connected to plasmid pXMJ19, respectively, to form recombinant plasmids pXMJ19-dCas9-ostAsgRNA1, pXMJ19-dCas9-ostAsgRNA2, pXMJ19-dCas9-ostAsgRNA3, pXMJ19-dCas9-treYsgRNA4, pXMJ19-dCas9-treYsgRNA5, or pXMJ19-dCas9-treYsgRNA6; Preferably, in the recombinant Corynebacterium glutamicum, the dCas9 protein gene dcas9 and sgRNA1 are connected to the plasmid pXMJ19 to form the recombinant plasmid pXMJ19-dCas9-ostAsgRNA1.

6. A strain of Corynebacterium glutamicum HA-Z01, deposited in the China Center for Type Culture Collection on November 26, 2024, with the deposit address at Wuhan University, Wuhan, China, and the deposit number is CCTCC NO: M20242640.

7. The method for constructing a recombinant Corynebacterium glutamicum according to any one of claims 1 to 5, characterized in that: The steps include: (1) Based on the codon preference of Corynebacterium glutamicum, the gene sequence of hyaluronan synthase, hasA, was designed, as shown in SEQ ID NO.1; the upstream primer hasA-F SEQ ID NO.14 and the downstream primer hasA-R SEQ ID NO.15 were designed, and polymerase chain reaction was performed to amplify the RBS1-hasA gene; (2) The plasmid pEC-XK99E was double-digested with EcoRⅠ and SacⅠ to obtain a linearized plasmid, and the linearized plasmid was connected with the RBS1-hasA gene obtained in step (1) by seamless cloning, and the connection product was transformed into the host bacteria E. coli JM109 competent cells, and the plasmid was screened and extracted to obtain the recombinant plasmid pEC-XK99E-hasA; (3) Using udgA2-F SEQ ID NO.16 and udgA2-R SEQ ID NO.17 as upstream and downstream primers, and using genomic DNA of Corynebacterium glutamicum ATCC13869 as a template or SEQ ID NO.2 as a template, polymerase chain reaction was performed to obtain the UDP-glucose dehydrogenase RBS2-udgA2 gene sequence; (4) The recombinant plasmid pEC-XK99E-hasA obtained in step (2) was double-digested with SacⅠ and KpnⅠ to obtain a linearized plasmid, and the linearized plasmid was connected with the RBS2-udgA2 gene obtained in step (3) by seamless cloning, and the connection product was transformed into the host bacteria E. coli JM109 competent cells, and the plasmid was screened and extracted to obtain the recombinant plasmid pEC-XK99E-hasA-udgA2; (5) Based on the codon preference of Corynebacterium glutamicum, the gene sequence glmS of glutamine-6-phosphofructotransaminase was designed, as shown in SEQ ID NO.3; Using glmS-F SEQ ID NO.18 and glmS-R SEQ ID NO.19 as upstream and downstream primers, polymerase chain reaction was performed to obtain the RBS3-glmS gene; (6) The recombinant plasmid pEC-XK99E-hasA-udgA2 obtained in step (4) was subjected to double enzyme digestion with KpnⅠ and BamHI to obtain a linearized plasmid, and the linearized plasmid was connected with the RBS3-glmS gene obtained in step (5) by seamless cloning. The connection product was transformed into E. coli host bacteria JM109 competent cells, and the plasmid was screened and extracted for PCR and sequencing verification to obtain the recombinant plasmid pEC-XK99E-hasA-udgA2-glmS; (7) transforming the recombinant plasmid pEC-XK99E-hasA-udgA2-glmS obtained in step (6) into Corynebacterium glutamicum ATCC13032, and screening to obtain genetically engineered bacteria transformed with the pEC-XK99E-hasA-udgA2-glmS plasmid; Preferably, the construction method further comprises: performing double restriction enzyme digestion of plasmid pXMJ19 with HindⅢ and SalⅠ, respectively, to obtain a linearized plasmid, connecting the linearized plasmid and the dCas9 gene as shown in SEQ ID NO.4 by seamless cloning, transforming the connection product into host bacteria E.coli JM109 competent cells, screening and extracting the plasmid, and obtaining the recombinant plasmid pXMJ19-dCas9; Design sgRNA sequences with different interference strengths for the ostA gene, which are sgRNA1, sgRNA2, and sgRNA3 gene fragments; The nucleotide sequence of sgRNA1 is shown in SEQ ID NO.5; The nucleotide sequence of sgRNA2 is shown in SEQ ID NO.6; The nucleotide sequence of sgRNA3 is shown in SEQ ID NO.7; The recombinant plasmid pXMJ19-dCas9 was double-digested with BamHⅠ and KpnⅠ to obtain a linearized plasmid, which was connected to the sgRNA1, sgRNA2 and sgRNA3 gene fragments by seamless cloning, and the ligation products were transformed into host bacteria E. coli JM109 competent cells, screened and plasmids were extracted to obtain recombinant plasmids pXMJ19-dCas9-ostAsgRNA1, pXMJ19-dCas9-ostAsgRNA2 and pXMJ19-dCas9-ostAsgRNA3, respectively; Design sgRNA sequences with different interference strengths for the treY gene, which are sgRNA4, sgRNA5, and sgRNA6 gene fragments; The nucleotide sequence of sgRNA4 is shown in SEQ ID NO.8; The nucleotide sequence of sgRNA5 is shown in SEQ ID NO.9; The nucleotide sequence of sgRNA6 is shown in SEQ ID NO.10; The recombinant plasmid pXMJ19-dCas9 was double-digested with BamHⅠ and KpnⅠ to prepare a linearized plasmid, which was connected with the sgRNA5, sgRNA6 and sgRNA7 gene fragments by seamless cloning, and the ligation products were transformed into host bacteria E. coli JM109 competent cells, screened and plasmids were extracted to obtain recombinant plasmids pXMJ19-dCas9-treYsgRNA4, pXMJ19-dCas9-treYsgRNA5 and pXMJ19-dCas9-treYsgRNA6, respectively; The recombinant plasmids pXMJ19-dCas9-ostAsgRNA1, pXMJ19-dCas9-ostAsgRNA2, pXMJ19-dCas9-ostAsgRNA3, pXMJ19-dCas9-treYsgRNA4, pXMJ19-dCas9-treYsgRNA5 and pXMJ19-dCas9-treYsgRNA6 were transformed into the genetically engineered bacteria containing the pEC-XK99E-hasA-udgA2-glmS plasmid obtained in step (7), and the transformed bacteria were screened to obtain The genetically engineered bacteria CgHA-Z01, CgHA-Z02, CgHA-Z03, CgHA-Z04, CgHA-Z05 or CgHA-Z06 containing pXMJ19-dCas9-ostAsgRNA1, pXMJ19-dCas9-ostAsgRNA2, pXMJ19-dCas9-ostAsgRNA3, pXMJ19-dCas9-treYsgRNA4, pXMJ19-dCas9-treYsgRNA5 or pXMJ19-dCas9-treYsgRNA6 plasmid.

8. Use of the recombinant Corynebacterium glutamicum according to any one of claims 1 to 5, the Corynebacterium glutamicum HA-Z01 according to claim 6, or the recombinant Corynebacterium glutamicum constructed by the method according to claim 7 in the production of hyaluronic acid; Preferably, the hyaluronic acid has a molecular weight of 10 4 -10 6 Da.

9. A method for producing hyaluronic acid by fermentation using the recombinant Corynebacterium glutamicum according to any one of claims 1 to 5, the Corynebacterium glutamicum HA-Z01 according to claim 6, or the recombinant Corynebacterium glutamicum constructed by the method according to claim 7, characterized in that: The steps include: The obtained genetically engineered bacteria were inoculated into a BHI liquid culture medium, and cultured at 28-32° C. and 150-250 rpm for 8-16 hours to obtain a primary seed solution; the primary seed solution was inoculated into a secondary seed culture medium with an initial OD562 between 0.05 and 0.15, and cultured at 28-32° C. and 150-250 rpm for 8-12 hours to obtain a secondary seed solution; The secondary seed liquid is inoculated into the fermentation medium at an initial OD562 of 0.05-0.15, cultured at 28-32°C and 150-250rpm for 2-6h, IPTG is added to a final concentration of 0.1-0.8mM, and the culture and fermentation are continued for 24-84h, and the bacterial cells are removed to obtain a fermentation liquid containing hyaluronic acid; Preferably, the components of the BHI liquid medium include: 2.5 g / L yeast powder, 5 g / L peptone, 5 g / L sodium chloride, 18.5 g / L bovine brain heart infusion, 30 mg / L kanamycin sulfate and 15 mg / L chloramphenicol; Preferably, the components of the secondary seed culture medium include: 20 g / L glucose, 10 g / L ammonium sulfate, 10 g / L peptone, 5 g / L yeast powder, 10 g / L corn syrup powder, 0.5 g / L magnesium sulfate, 1.5 g / L dipotassium hydrogen phosphate, 1.5 g / L potassium dihydrogen phosphate, pH=7; Preferably, the components of the fermentation medium include: 50 g / L glucose, 15 g / L ammonium sulfate, 5 g / L peptone, 2.5 g / L yeast powder, 10 g / L corn syrup powder, 0.5 g / L magnesium sulfate, 1.5 g / L dipotassium hydrogen phosphate, 1.5 g / L potassium dihydrogen phosphate, pH=7; Preferably, the yield of hyaluronic acid in the fermentation broth is above 50 g / L; Preferably, the hyaluronic acid produced by the method has a molecular weight of 10 4 -10 6 Da.

10. Use of the hyaluronic acid produced by the recombinant Corynebacterium glutamicum according to any one of claims 1 to 5, the hyaluronic acid produced by Corynebacterium glutamicum HA-Z01 according to claim 6, the hyaluronic acid produced by the recombinant Corynebacterium glutamicum constructed by the method according to claim 7, or the hyaluronic acid produced by the method according to claim 9 in the preparation of products containing hyaluronic acid in the food, pharmaceutical or cosmetic fields.

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