Genetically engineered bacteria for producing hyaluronic acid and application thereof

Abstract

The application provides a genetically engineered bacterium for producing hyaluronic acid and application thereof, and belongs to the technical field of bioengineering. The genetically engineered bacterium is obtained by introducing hyaluronic acid synthase StHAS from Streptococcus thermophilus into Bacillus amyloliquefaciens NF as a host, and the genetically engineered bacterium can synthesize hyaluronic acid from scratch. Subsequently, by using system metabolic engineering, the fermentation yield of hyaluronic acid is further improved by optimizing the StHAS synthase regulation mode, up-regulating the precursor UDP-glucuronide metabolic pathway and removing the polysaccharide byproduct pathway by using a self-designed CRISPR-Cas9n system. Finally, by using the recombinant strain, the fermentation yield of hyaluronic acid reaches 5.59+ / -0.06 g / L, and the molecular weight is 1.75 MDa. The genetically engineered bacterium constructed in the application is safe and stable, has a short production cycle, and has an industrial application prospect.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of bioengineering and relates to a genetically engineered bacterium for producing hyaluronic acid and application thereof. BACKGROUND

[0002] Hyaluronic acid (HA), also known as hyaluronan, is a linear, unbranched glycosaminoglycan (GAG) composed of alternating β-1, 3 and β-1, 4 glycosidic bonds between D-glucuronic acid (GlcA) and N-acetylglucosamine (GlcNAc). Figure 1 Due to the unique viscoelasticity, hygroscopicity, non-immunogenicity and biocompatibility of hyaluronic acid, it has been widely used in the fields of medicine, food and cosmetics.

[0003] For a long time, the industrial production of hyaluronic acid mainly relied on extraction from animal tissues (such as chicken combs). Due to the limitation of raw material quality and quantity, the production cost of hyaluronic acid has been high. In addition, the frequent occurrence of animal epidemic cross-infection events leads to increasing health and safety concerns, which limits the application of hyaluronic acid in biomedicine and clinical practice. At present, the acquisition of HA has been changed from traditional animal tissue extraction to microbial fermentation. The most widely used microbial host is the pathogenic weak hemolytic Streptococcus species C group, and the maximum yield of fermented hyaluronic acid can reach 6-7 g / L. However, due to the potential pathogenicity of Streptococcus and the difficulty of genetic manipulation, the host system modification and biosynthesis of hyaluronic acid are greatly limited. Thanks to the rapid development of synthetic biology technology, the use of safe level microorganism fermentation to synthesize hyaluronic acid is a new type of polysaccharide synthesis technology in recent years. Although some safe level microbial hosts such as Bacillus subtilis and Corynebacterium glutamicum have been successfully developed for the synthesis of hyaluronic acid, the recombinant HA synthase used is mainly derived from the potential risk of Streptococcus class I enzyme and pathogenic bacteria Pasteurella multocida class II enzyme. Therefore, it is particularly important to find and develop a new type of HA synthase with high efficiency and food safety properties from natural sources, and to develop a corresponding production process to solve the green biological manufacturing of hyaluronic acid.

[0004] Streptococcus thermophilus is a traditional probiotic commonly used for the preparation of dairy products, which is generally considered as a food safety level microbial strain and has the ability to produce hyaluronic acid. The current reported production process mainly uses Streptococcus thermophilus for fermentation, and the maximum yield of hyaluronic acid is 260 mg / L. The low yield of hyaluronic acid synthesized by it cannot meet the large-scale cultivation and industrial production (202110884758.6). SUMMARY

[0005] The technical problem solved by the present application is to provide a novel HA synthetase with food safety properties and a genetically engineered bacterium for producing hyaluronic acid to overcome the deficiencies of the prior art.

[0006] The present application also aims to provide a method for constructing the genetically engineered bacterium for producing hyaluronic acid.

[0007] The present application also aims to provide a method for constructing the genetically engineered bacterium for producing hyaluronic acid.

[0008] Invention idea: To solve the problem of green biological manufacturing of hyaluronic acid, a method for establishing an industrial production process of hyaluronic acid by using a novel HA synthetase with high efficiency, natural source and food safety is explored. Therefore, based on synthetic biology technology, the present application genetically modifies a safe microorganism Bacillus amyloliquefaciens to construct a high-yield strain and use it for the production of hyaluronic acid.

[0009] To solve the above technical problem, the technical solution of the present application is as follows:

[0010] The present application provides a genetically engineered bacterium for producing hyaluronic acid, which uses Bacillus amyloliquefaciens NF as a host and expresses hyaluronic acid synthetase coding gene sthasA by using a constitutive strong promoter pHpaII; simultaneously expresses UDP-glucose dehydrogenase coding gene cghasB heterologously; and knocks out polysaccharide synthesis operon gene epsA-O and Levan synthetase coding gene sacB.

[0011] The Bacillus amyloliquefaciens NF host is a modified strain of Bacillus amyloliquefaciens NX-2S (the detailed construction process of which is disclosed in patent CN106047780B), i.e., the gamma-polyglutamic acid synthetase coding gene PgsBCA is knocked out based on the original strain.

[0012] The expression vector of the genetically engineered bacterium is pMA5 (purchased from BioVector NTCC plasmid vector strain cell protein antibody gene preservation center).

[0013] The hyaluronic acid synthetase coding gene sthasA is derived from Streptococcus thermophiles SMQ-301, and the optimized coding sequence is shown in SEQ ID NO. 1.

[0014] The UDP-glucose dehydrogenase encoding gene cghasB is derived from Corynebacterium glutamacium with strain number ATCC 13032, and the coding sequence is shown as SEQ ID NO. 2.

[0015] The application further provides a construction method of the genetically engineered bacterium for producing hyaluronic acid, comprising the following steps:

[0016] (a) Construction of Bacillus amyloliquefaciens CF strain: the epsA-O and sacB genes of Bacillus amyloliquefaciens NF are knocked out to obtain the Bacillus amyloliquefaciens CF strain;

[0017] (b) Construction of recombinant Bacillus amyloliquefaciens: the sthasA gene sequence and the codon-optimized sequence of cghasB gene are cloned into an expression vector pMA5, and verification is performed to obtain a recombinant plasmid pMA5-sthasA-cghasB; the recombinant plasmid pMA5-sthasA-cghasB is transformed into competent cells prepared by using the Bacillus amyloliquefaciens CF obtained in step (a) to construct the recombinant Bacillus amyloliquefaciens.

[0018] In step (a), the Bacillus amyloliquefaciens CF strain is constructed according to the following specific operation steps:

[0019] 1) Construction of knockout plasmid

[0020] The gene knockout of Bacillus amyloliquefaciens used the CRISPR-Cas9n system developed by the research group, pNX was used to express Cas9n protein, and PDR temperature-sensitive plasmid was used for sgRNA transcription and to provide donor DNA for homologous repair template. The Cas9n protein used in this experiment was synthesized by Shanghai Bioengineering Co., Ltd. using Cas9n-F / Cas9n-R primers to amplify the cas9n gene with the Clostridium CRISPR working plasmid pNICKclos 2.0 as the template. The size of the gene is about 4107 bp. The Pgrac strong promoter was amplified from the Bacillus subtilis expression vector pHT01 (purchased from BioVector NTCC Plasmid Vector Strain Cell Protein Antibody Gene Preservation Center) using Pgrac-F / Pgrac-R primers, in addition, the amylase Tamy from Bacillus amyloliquefaciens was selected and amplified using Tamy-F / Tamy-R primers as the transcription terminator of Cas9n gene. After recovering the strong promoter Pgrac, Cas9n and Tamy fragments by gel recovery kit, the Cas9n protein expression frame was obtained by overlapping PCR using Pgrac-F / Tamy-R primers. The fused fragment Pgrac-Cas9n-Tamy was cloned by Sma I and Xba I enzyme digestion and connected to the expression vector pNX01 (the detailed construction process of the expression vector has been disclosed in the patent CN108624546A), to obtain the recombinant plasmid pNX-Cas9n.

[0021] The expression of sgRNA uses PDR (purchased from BioVector NTCC Plasmid Vector Strain Cell Protein Antibody Gene Preservation Center) as the carrier, and the primers SgsacB-F / SgsacB-R and SgespA-O-F / SgespA-O-R are used to obtain the sgRNA of sacB and espA-O genes according to the target gene design, and the upstream and downstream of each 800 bp of the knockout gene is used as the homologous arm repair, wherein the primer SacBL-F / SacBL-R is used to amplify the sacB gene upstream arm, and the primer SacBR-F / SacBR-R is used to amplify the sacB gene downstream arm, and the three fragments are overlapped to obtain sacB sgRNA-SacBL-SacBR. The espsgRNA-EspL-EspR is obtained in the same way. The amplified sacB sgRNA-SacBL-SacBR and espsgRNA-EspL-EspR are inserted into the Sal I and Xho I sites of the PDR plasmid, respectively. The pDR-sacBsgupp and pDR-espA-Osgupp plasmids are obtained, respectively.

[0022] 2) CRISPR-Cas9n knockout of byproduct pathway

[0023] To knock out epsA-O and sacB genes of B. amyloliquefaciens NF. Using Cas9n expression and sgRNA double-plasmid system based on CRISPR method, transform cells were spread on LB agar plates containing 1 mM IPTG, spectinomycin and chloramphenicol double-antibiotic, incubated at 30°C for 12 h, and transformants were picked and then verified by primers SacBOut-F / SacBOut-R and EspA-OOut-F / EspA-OOut-R. To remove the expression plasmids of Cas9n and sgRNA in edited cells, single colonies of edited cells were inoculated in 5 mL LB medium and cultured at 42°C without antibiotics for 12 h, and after 2 times of continuous passage, dilution spread was performed on LB solid medium. When no growth was observed on spectinomycin and chloramphenicol, the strain was preserved. The strain with epsA-O and sacB genes knocked out was named CF.

[0024] wherein, in step (b), the construction of the recombinant B. amyloliquefaciens is specifically performed as follows:

[0025] 1) Codon optimization of hyaluronan synthase StHAS

[0026] A hyaluronan synthase StHAS is derived from Streptococcus thermophilus, and its nucleotide sequence is shown in SEQ ID NO. 1. The N-acetylglucosamine transferase is optimized according to the codon bias of Bacillus amyloliquefaciens CF, and then the whole gene is synthesized, and its nucleotide sequence is shown in SEQ ID NO. 2. The codon optimization and whole gene synthesis of the target gene are completed by General Biosystems (Anhui) Co., Ltd.

[0027] 2) Construction of recombinant B. amyloliquefaciens

[0028] The nucleotide sequences shown in SEQ ID NO. 1 and SEQ ID NO. 2 are amplified separately, and after overlapping PCR, they are cloned into the expression vector pMA5 to obtain the recombinant plasmid pMA5-sthasA-cghasB. The recombinant plasmid pMA5-sthasA-cghasB after ligation is transformed into the E. coli GM2163 strain by a chemical transformation method, and the positive transformants verified correctly are subjected to plasmid extraction. The recombinant plasmid pMA5-sthasA-cghasB verified successfully is transformed into the prepared Bacillus amyloliquefaciens CF competent cells by an electroporation method, and the recovered transformation liquid is coated on an LB agar plate containing kanamycin sulfate (25 μg / mL), and incubated at 37°C for 12 h. The single colonies grown on the plate are observed for the colony morphology of the recombinant strain and the colony PCR verification of the positive strain, and the genetically engineered bacteria are obtained.

[0029] The genetically engineered bacteria for producing hyaluronic acid are also within the scope of the present application.

[0030] The application of the genetically engineered bacteria for producing hyaluronic acid in the preparation of hyaluronic acid by fermentation includes the following steps:

[0031] (1) The genetically engineered bacteria are activated at 28-37°C, inoculated into a seed culture medium, and cultured to obtain a seed liquid containing the genetically engineered bacteria.

[0032] (2) The seed liquid obtained in step (1) is inoculated into a fermentation medium at an inoculation amount of 1%-10% for fermentation in a fermenter.

[0033] In step (1), the culture conditions are as follows: a shaking speed of 220 rpm, 37°C, and a culture time of 10-16 h to an OD 660 greater than 5.0 and a pH of 6.0-8.0. Preferably, the culture conditions are as follows: a shaking speed of 220 rpm, 37°C, and a culture time of 10 h to an OD 660 greater than 5.0 and a pH of 6.0-8.0.

[0034] In step (1), the seed culture medium is as follows: yeast powder 5 g / L, peptone 10 g / L, NaCl 10 g / L, and the rest is water, with a pH of 6.0-8.0.

[0035] In step (2), the inoculation amount of the seed liquid is 1%-10%, and the preferred inoculation amount is 4%.

[0036] The fermentation in step (2) is carried out under the following conditions: 28-37 DEG C, pH 5.0-8.0, 220 rpm of shaking speed for 48 h.

[0037] The fermentation medium in step (2) is composed of a carbon source, a nitrogen source, inorganic salt, an osmotic regulator and water; the carbon source is any one or combination of inulin, inulin crude extract, inulin, glucose, fructose, sucrose, maltose, xylose, arabinose, molasses and glycerol, with a concentration of 20-60 g / L; the nitrogen source is any one or combination of yeast powder, peptone, soybean meal powder, beef extract, fish meal peptone, ammonium sulfate and urea, with a concentration of 5-20 g / L; the inorganic salt is any one or combination of MgSO4, MnSO4, CoCl2, CaCl2 and FeSO4, with a concentration of 5-10 g / L; and the osmotic regulator is betaine and sorbitol, with a concentration of 1-10 g / L; preferably, the carbon source is inulin, with a concentration of 40 g / L; the nitrogen source is yeast powder, with a concentration of 10-15 g / L; the inorganic salt is MgSO4, with a concentration of 6 g / L; and the osmotic regulator is betaine, with a concentration of 2 g / L.

[0038] Compared with the prior art, the present application has the following advantages:

[0039] (1) In order to exploit the food safety of HA synthetase for the production of hyaluronic acid, the present application restructures the hyaluronic acid synthetase StHAS from Streptococcus thermophilus in Bacillus amyloliquefaciens through a synthetic pathway, thereby providing a new method for the microbial fermentation production of hyaluronic acid.

[0040] (2) The Bacillus amyloliquefaciens used in the present application is a food safety level new host, and meanwhile, it exhibits a high hyaluronic acid synthesis capacity.

[0041] (3) The genetically engineered bacterium for stably and efficiently producing hyaluronic acid according to the present application can produce 5.59 g / L of hyaluronic acid after being cultured in a 7.5 L fermenter for 48 h. The fermentation process for producing hyaluronic acid has a high level and has great application value and industrialization potential. BRIEF DESCRIPTION OF DRAWINGS

[0042] Figure 1 Fig. 1 is a structural schematic diagram of hyaluronic acid;

[0043] Figure 2 Fig. 2 is a schematic diagram of the synthesis pathway of hyaluronic acid in Bacillus amyloliquefaciens;

[0044] Figure 3 The schematic diagram of PCR electrophoresis detection of CRISPR-Cas9n double-plasmid tool (A) and CRISPR-Cas9n knockout of exopolysaccharide synthesis operon gene epsA-O and Levan synthesis enzyme coding gene sacB (B), note: lane 1: DL 15000 Marker; lane 2: PCR result of control strain before gene knockout; lane 3: PCR result after gene knockout;

[0045] Figure 4 The plasmid construction map of recombinant plasmid pMA5-sthasA-cghasB (the meaning of pMA5-hasA-cghasB and pMA5-sthasA-cghasB in the figure is the same);

[0046] Figure 5 The schematic diagram of electrophoresis detection of recombinant plasmid pMA5-sthasA-cghasB, note: lane 1: DL 15000 Marker; lane 2: Nde I and BamH I double enzyme digestion of pMA5-sthasA-cghasB (the target band is 1208 and 1320 bp, and Nde I enzyme digestion site exists in front of cghasB);

[0047] Figure 6 The results of optimization of HA fermentation culture conditions and culture components. (A) Effect of temperature on HA synthesis; (B) Effect of pH on HA synthesis; (C) Effect of inoculum size on HA synthesis; (D) Effect of inoculum age on HA synthesis; (E) Effect of inulin concentration on HA synthesis; (F) Effect of nitrogen source type on HA synthesis; (G) Effect of nitrogen source concentration on HA synthesis; (H) Effect of different inorganic salts on fermentation synthesis of HA;

[0048] Figure 7 The relationship diagram of fermentation time and hyaluronic acid production and biomass (dry weight DCW);

[0049] Figure 8 The liquid chromatogram result diagram of fermentation broth for detecting hyaluronic acid. DETAILED DESCRIPTION

[0050] The experimental methods in the following examples are conventional methods unless otherwise specified; the reagents and materials can be obtained from commercial channels unless otherwise specified.

[0051] The application will be further described below in combination with specific examples.

[0052] The experimental materials and kits used in the following examples

[0053] Table 1 main experimental materials

[0054] Table 1 main experimental materials

[0055]

[0056] In the following examples, the method for detecting the production of hyaluronic acid (HA) in the fermentation broth

[0057] After the fermentation broth was diluted appropriately, the bacterial cells were removed by centrifugation, and the supernatant of the fermentation broth was filtered through a 0.22 μm filter membrane, and the filtrate was collected for gel permeation chromatography (GPC) to determine the HA production. The chromatographic column was Shodex Ohpak SB-806M HQ, the mobile phase was 0.1 M Na2SO4 solution, and the flow rate was 1 mL / min. The yield of HA was monitored by the peak area of GPC, and the HA used to make the standard curve of HA concentration was purchased from Bloomage BioCo., Ltd.

[0058] Example 1: Knockout of polysaccharide by-product pathway by CRISPR-Cas9n system

[0059] (1) Construction of knockout plasmid

[0060] The gene knockout of B. amyloliquefaciens used the CRISPR-Cas9n system independently developed by the research group. pNX was used to express Cas9n protein, and PDR temperature-sensitive plasmid was used for sgRNA transcription and to provide donor DNA for homologous repair template. The Cas9n protein used in this experiment was synthesized by Shanghai Bioengineering Co., Ltd. using Cas9n-F / Cas9n-R primers for amplification of the cas9n gene with the Clostridial CRISPR working plasmid pNICKclos 2.0 as the template. The size of this gene is about 4107 bp. The Pgrac strong promoter was amplified from the B. subtilis expression plasmid pHT01 (purchased from BioVector NTCC Plasmid Vector Strain Cell Protein Antibody Gene Preservation Center) using Pgrac-F / Pgrac-R primers, and in addition, the amylase Tamy from B. amyloliquefaciens was amplified using Tamy-F / Tamy-R primers as the transcription terminator of the Cas9n gene. After recovering the strong promoter Pgrac, Cas9n and Tamy fragments by gel recovery kit, the Cas9n protein expression frame was obtained by overlapping PCR using Pgrac-F / Tamy-R primers. The fused fragment Pgrac-Cas9n-Tamy was cloned by Sma I and Xba I enzyme digestion and ligated into the expression vector pNX01 (the detailed construction process of this expression vector has been disclosed in the patent CN108624546B), to obtain the recombinant plasmid pNX-Cas9n.

[0061] The expression of sgRNA was carried out by using PDR (purchased from BioVector NTCC plasmid vector strain cell protein antibody gene preservation center) as a carrier, and the sgRNA of sacB and espA-O genes was obtained by using the primers SgsacB-F / SgsacB-R and SgespA-O-F / SgespA-O-R designed according to the target gene, respectively, so as to knock out the upper and lower 800 bp of the gene as a homologous arm repair, wherein the arm of the upper sacB gene was obtained by using the primers SacBL-F / SacBL-R for amplification, the arm of the lower sacB gene was obtained by using the primers SacBR-F / SacBR-R for amplification, and the sacB sgRNA-SacBL-SacBR was obtained by using the three fragments for overlapping. The esp sgRNA-EspL-EspR was obtained by using the same method. The sacB sgRNA-SacBL-SacBR and esp sgRNA-EspL-EspR obtained by amplification were respectively inserted into the Sal I and Xho I sites of the PDR plasmid. The pDR-sacBsgupp and pDR-espA-Osgupp plasmids were obtained, respectively.

[0062] The primer sequences used in the construction of the knockout plasmid are shown in Table 2.

[0063]

[0064] The PCR reaction system was as follows: 2 μL of template DNA, 2 μL of upstream primer, 2 μL of downstream primer, 12.5 μL of 2x mix DNA polymerase, and 6.5 μL of ddH2O.

[0065] The PCR reaction parameters were as follows: 94 ℃ pre-denaturation for 5 min; 94 ℃ denaturation for 30 s, 55 ℃ annealing for 30 s, 72 ℃ extension for 1 min, 30 cycles; 72 ℃ final extension for 10 min, and 4 ℃ termination.

[0066] The overlapping PCR reaction system was as follows: 2 μL of template DNA1, 2 μL of template DNA2, 2 μL of upstream primer, 2 μL of downstream primer, 25 μL of 2x mix DNA polymerase, and 17 μL of ddH2O.

[0067] The overlapping PCR reaction parameters were as follows: 94 ℃ pre-denaturation for 5 min; 94 ℃ denaturation for 30 s, 55 ℃ annealing for 1 min, 72 ℃ extension for 2 min, 30 cycles; 72 ℃ final extension for 10 min, and 4 ℃ termination; after the reaction, the product was detected by agarose gel electrophoresis, and the target DNA fragment was recovered by using a kit.

[0068] (2) CRISPR-Cas9n knockout of byproduct pathway Figure 3 )

[0069] To knock out epsA-O and sacB genes of B. amyloliquefaciens NF, the transformed cells were spread on LB agar plates containing 1 mM IPTG, spectinomycin and chloramphenicol double-antibiotic plates, incubated at 30°C for 12 h, and then the transformants were picked and verified by primers SacBOut-F / SacBOut-R and EspA-OOut-F / EspA-OOut-R. To remove the expression plasmids of Cas9n and sgRNA in the edited cells, the single colony of the edited cells was inoculated in 5 mL LB medium and cultured at 42°C without antibiotics for 12 h, and after 2 times of continuous passage, dilution and spread on LB solid medium. When no growth was observed on spectinomycin and chloramphenicol, the strain was preserved. The strain with epsA-O and sacB genes knocked out was named CF.

[0070] Table 3 Primer sequences used for CRISPR-Cas9n knockout of byproduct pathways

[0071]

[0072] Example 2: Construction of recombinant B. amyloliquefaciens

[0073] (1) Codon optimization of hyaluronan synthase StHAS

[0074] A hyaluronan synthase StHAS derived from Streptococcus thermophilus has a nucleotide sequence as shown in SEQ ID NO. 1. The N-acetylglucosamine transferase was optimized according to the codon bias of Bacillus amyloliquefaciens CF, and the optimized full gene was synthesized, and the nucleotide sequence is shown in SEQ ID NO. 2. The codon optimization and full gene synthesis of the target gene were completed by General Biosystems (Anhui) Co., Ltd.

[0075] (2) Construction of recombinant B. amyloliquefaciens

[0076] The nucleotide sequences shown in SEQ ID NO. 1 and SEQ ID NO. 2 were amplified separately, and after overlap PCR, they were cloned into the expression plasmid pMA5 (purchased from BioVector NTCC Plasmid Vector Strain Cell Protein Antibody Gene Preservation Center) to obtain the recombinant plasmid pMA5-sthasA-cghasB. Then the recombinant plasmid pMA5-sthasA-cghasB was transformed into B. amyloliquefaciens by electroporation to obtain an engineering strain capable of fermenting and synthesizing hyaluronic acid, and the specific process is as follows:

[0077] 1) The nucleotide sequence of the hyaluronic acid synthase-encoding gene SthasA shown in SEQ ID NO. 1 was amplified using primers Fl and primer Rl, and the nucleotide sequence of the UDP-glucose dehydrogenase-encoding gene CgHasB shown in SEQ ID NO. 2 was amplified using primers F2 and primer R2.

[0078] Table 4 Primer sequences used for amplifying the encoding genes SthasA and CgHasB

[0079]

[0080] The PCR amplification system was as follows: template plasmid pUC57-sthasA (sthasA was whole gene synthesized by Anhui General Biotechnology Co., Ltd.): DNA 2 μL, primer Fl and primer Rl: 2 μL each, PrimeSTAR high-fidelity enzyme: 12.5 μL, ddH2O: 6.5 μL; template genome Corynebacterium glutamacium ATCC 13032: DNA 2 μL, primer F2 and primer R2: 2 μL each, PrimeSTAR high-fidelity enzyme: 12.5 μL, ddH2O: 6.5 μL.

[0081] The PCR reaction program was as follows: 94°C pre-denaturation for 4 min, 94°C denaturation for 2 min; then 55°C annealing for 30 s, 72°C extension for 1 min, for 30 cycles;

[0082] The PCR amplification system was as follows: template 1 sthasA cloning product and template 2 cghasB cloning product: 2 μL each, primer Fl and primer R2: 2 μL each, PrimeSTAR high-fidelity enzyme: 12.5 μL, ddH2O: 4.5 μL;

[0083] The overlapping PCR reaction program was as follows: 94°C pre-denaturation for 4 min, 94°C denaturation for 2 min; then 55°C annealing for 1 min, 72°C extension for 2 min, for 30 cycles;

[0084] The overlapping PCR amplification product was recovered, and was ligated with the plasmid pMA5 which was digested with restriction endonucleases Nde I and BamH I using one-step cloning method under the action of Exnase II, to obtain the recombinant plasmid pMA5-sthasA-cghasB Figure 4

[0085] ​2) Transform the recombinant plasmid pMA5-sthasA-cghasB into E. coli GM2163, and coat on LB solid medium containing 100 μg / mL ampicillin, and incubate at 37°C for 10-12 h to obtain primary positive clones; pick the primary positive clones into 5 mL LB liquid medium containing 100 μg / mL ampicillin, and incubate at 37°C, 200 rpm overnight, extract the plasmid, and double-enzyme cut the plasmid with restriction endonuclease Nde I and BamH I; according to the electrophoresis result, the plasmid with the DNA fragment of SEQ ID NO: 1 and SEQ ID NO: 2 is the recombinant plasmid pMA5-sthasA-cghasB, and the colony with the plasmid is the positive clone strain Figure 5 ).

[0086] 3) Preparation of competent cells

[0087] B. amyloliquefaciens stored in a glycerol tube is streaked on an LB plate and incubated at 37°C overnight. A single colony is picked into 5 mL of competent preparation medium as a seed liquid and incubated at 37°C for 12 h. 2 mL of the seed liquid is inoculated into a 500 mL flask containing 100 mL of competent medium, and incubated at 37°C with shaking until the OD 600 of the culture liquid is 0.5-0.7. The competent culture liquid is placed in an ice bath for 0.5 h, and then centrifuged at 8000 x g at 4°C for 10 min. The supernatant is discarded, and the competent cells are collected. The collected competent cells are resuspended with an electroporation buffer washing solution, centrifuged at 8000 x g at 4°C for 10 min, the supernatant is discarded, and the competent cells are collected. The above operation is repeated for 2-3 times. An appropriate amount of suspension is added to the bacteria obtained by centrifugation, mixed, and then the competent cells are aliquoted (100 μL per tube) and stored at -80°C.

[0088] The 2 mm electroporation cup is washed with 75% alcohol, dried in a sterile environment, and pre-cooled on ice before electroporation. 100 ng of the recombinant plasmid is added to 100 μL of the competent cells, mixed, and then transferred to the pre-cooled electroporation cup for electroporation. The voltage is set to 2.5 kV, the resistance is set to 200 Ω, and the shock time is set to 4 ms for electroporation.

[0089] 3) The recombinant plasmid pMA5-sthasA-cghasB is transformed into Bacillus amyloliquefaciens CF strain by electroporation, and the recovered transformation liquid is coated on an LB agar plate containing kanamycin sulfate (25 μg / mL) and incubated at 37°C for 12 h. Single colonies growing on the plate are picked to observe the colony morphology of the recombinant strain and the positive strain verified by colony PCR, which is the genetically engineered bacteria.

[0090] Example 3: Fermentation production of hyaluronic acid (HA) by genetically engineered strain

[0091] (1) The genetically engineered strain obtained in Example 2 was activated at a culture temperature of 28-37°C, and then inoculated into a seed culture medium. The shaking speed was 220 rpm, and the culture was carried out at 37°C for 10-16 h until the OD 660 was greater than 5.0, as a seed liquid.

[0092] (2) The obtained seed liquid was inoculated into a fermentation medium at an inoculation amount of 1%-10%, and the fermentation tank fermentation was carried out at 28-37°C, pH 5.0-8.0, and a shaking speed of 220 rpm for 48 h.

[0093] The seed culture medium was composed of 5 g / L of yeast powder, 10 g / L of peptone, 10 g / L of NaCl, and the rest was water, with a pH of 6.0-8.0.

[0094] The initial fermentation medium was composed of 30 g / L of carbon source (inulin), 10 g / L of nitrogen source, 7 g / L of K2HPO4·3H2O, 3 g / L of KH2PO4, and 3 g / L of MgSO4·7H2O, and the rest was water.

[0095] The fermentation process for the fermentation production of hyaluronic acid was optimized by investigating the effects of fermentation temperature, pH, seed liquid inoculation amount and inoculation age, carbon source concentration in the fermentation medium, type and concentration of nitrogen source, and different metal ions in inorganic salts on the HA yield.

[0096] 1. Effect of temperature on HA yield

[0097] 28°C, 30°C, 32°C, and 37°C were selected as different fermentation temperatures for shake flask fermentation. The HA product accumulation amount at different temperatures was measured after fermentation at pH 7.0 and 200 rpm for 48 h. The fermentation temperature had a significant effect on the biomass and HA synthesis of the strain. As shown in FIG. A, the HA yield of the recombinant strain increased synchronously with the increase of the culture temperature. When the culture temperature increased from 28°C to 32°C, the HA yield increased significantly. The highest HA yield was 3.2 g / L ± 0.05 g / L under the fermentation condition of 32°C, and therefore, 32°C was selected as the fermentation temperature for the fermentation preparation of HA by the recombinant strain. Figure 6

[0098] 2. Effect of pH on HA yield

[0099] The pH of the fermentation medium was adjusted to 5.0, 6.0, 6.5, 7.0, 7.5, and 8.0 before inoculation. The medium composition was the same as above, the fermentation temperature was 32°C, and the rotation speed was 200 rpm. The effects of different pH values on the HA synthesis of the strain were investigated. The effects of different initial pH of the medium on the HA synthesis were investigated at the optimal temperature, as shown in FIG.​Figure 6 B. At pH 7.0, the cell growth was good and the yield of target product was the highest, reaching 3.37±0.02 g / L, while the synthesis of HA was significantly inhibited in alkaline environment, thus it can be seen that the fermentation pH is one of the important factors affecting cell growth and product synthesis.

[0100] 3. The effect of inoculum size and inoculum age on HA yield

[0101] Under the condition of constant medium composition, at pH 7.0, 32℃, 200 rpm, the seed liquid of different growth periods was inoculated into the fermentation medium with inoculum size of 1, 2, 4, 6, 8, 10% respectively to investigate the effect of different inoculum size on HA synthesis. As can be seen from Figure 6 C, when the inoculum size is higher than 4%, the cell growth and reproduction are rapid, resulting in excessive production of inhibitory by-products in the early stage of fermentation, affecting normal fermentation and being not conducive to the accumulation of HA product. When the inoculum size is 4%, the cell growth is good and the yield of hyaluronic acid is the highest. On the other hand, the seed culture period should be selected as the logarithmic phase of seed growth, and the seed is too tender or too old, which will not only prolong the fermentation period but also reduce the yield. Figure 6 D data show that the different lengths of seed liquid culture have a significant effect on the subsequent fermentation production of HA. When the seed liquid is cultured for 10h, inoculation into the fermentation medium can maximize the synthesis of HA.

[0102] 4. The effect of carbon source concentration on HA yield

[0103] Different carbon source concentrations (g / L) were selected: 20, 30, 40, 50, 60, and the other medium component concentrations and culture conditions were the same as above. The fermentation was carried out at 32℃, 200 rpm for 48h, and the effect of different carbon source concentrations on HA yield was compared after 48h of fermentation. As shown in Figure 6 E, there was a significant difference in the yield of hyaluronic acid with different concentrations of carbon source. When the carbon source concentration was 40 g / L, the synthesis of HA was the largest, reaching 4.12±0.04 g / L. Therefore, the selected concentration of inulin for the growth of microorganisms and the accumulation of target product has important influence.

[0104] 5. The effect of nitrogen source type and concentration on HA yield

[0105] Yeast powder, peptone, soybean meal, beef extract, fish meal peptone, ammonium sulfate, urea were selected as the nitrogen source of fermentation medium, with a concentration of 10 g / L, and the rest of the medium concentration was the same as the initial fermentation medium. The fermentation was carried out at 32℃, 200 rpm for 48h, and the effect of different nitrogen sources on HA yield was determined. As shown in Figure 6F. Compared with other nitrogen sources, yeast powder and peptone have significant advantages in the production of HA. The recombinant strain has high selectivity for organic nitrogen source. When yeast powder is used as the nitrogen source, the yield of HA synthesis is high and the cell growth is good. After selecting the nitrogen source, different nitrogen source concentrations (g / L) are selected, i.e. 5, 10, 15, and 20. The other medium component concentrations and culture conditions are the same as above. After 48 h of fermentation, the effect of nitrogen source concentration on the yield of HA is compared. Figure 6 G. Based on the fermentation of HA with different concentrations of yeast powder, the results show that the yeast powder concentration is preferably 10-15 g / L, at which the highest accumulation of HA is 4.4 g / L. The subsequent optimization is based on 10 g / L of yeast powder.

[0106] 6. Effect of inorganic salts of different metal ions on the yield of HA

[0107] MgSO4 in the initial fermentation medium is replaced by other inorganic salts. The effects of MgSO4, MnSO4, CoCl2, CaCl2, and FeSO4 on the yield of HA are investigated. The above-mentioned carbon source and nitrogen source and the corresponding concentrations are selected. The other medium components are the same as the initial medium. Figure 7 H. The effects of different metal ions on the synthesis of HA are shown. It is found that Fe 2+ and Co 2+ almost completely inhibit the generation of HA, while Mg 2+ can significantly improve the yield of HA, which is consistent with the results of alcohol precipitation of the fermentation broth. Metal ions mainly affect the activity of protease, thereby affecting the accumulation of target products in the metabolic process of the cell. HA synthase is activated by Mg 2+ , so Mg 2+ is an indispensable component in the synthesis of HA. According to the fermentation results of different concentrations of Mg 2+ , the most suitable concentration of MgSO4 is 6 g / L.

[0108] (3) Finally, batch fermentation is carried out in a 7.5 L fermenter to investigate the potential of the recombinant strain for industrial production of HA. According to the experimental results in steps (1) and (2), after 10 h of seed culture, the seed liquid is inoculated into a 7.5 L fermenter with a liquid volume of 3 L at an inoculation amount of 4%. The initial rotation speed is set to 400 r / min and the aeration amount is 2vvm. At the same time of inoculation, kanamycin sulfate and carbon source inulin are added to make the concentration of kanamycin sulfate 25 mg / L and the final concentration of inulin 45 g / L. During the fermentation process, the dissolved oxygen and pH in the fermentation broth are detected in real time by the dissolved oxygen and pH electrodes. Ammonia is used for pH control during the fermentation process. The dissolved oxygen is controlled at about 30% by coordinated change of aeration amount and rotation speed. The carbon source content, OD, and HA yield in the fermentation broth are detected every 6 h during the fermentation process. The experimental results are as follows:Figure 8 As shown, the content of HA in the fermentation broth finally stabilized at 48h 5.59±0.07g / L.

[0109] (4) In order to detect the molecular weight level of the hyaluronic acid synthesized by the genetically engineered bacteria, the fermentation broth after filtration in step (2) was used for gel permeation chromatography (GPC) determination.

[0110] The method for detecting hyaluronic acid in the fermentation broth: After the fermentation broth was appropriately diluted, the bacterial bodies were removed by centrifugation, and the fermentation broth supernatant was filtered by a 0.22μm filter membrane, and the filtrate was collected for gel permeation chromatography (GPC) determination of HA yield. The chromatographic column was Shodex Ohpak SB-806M HQ, the mobile phase was 0.1M Na2SO4 solution, and the flow rate was 0.8mL / min. The sample molecular weight was calculated by the peak retention time of GPC, and the different molecular weight dextran standard for making the molecular weight standard curve was purchased from sigma company. The results are as follows ​ , which shows that the molecular weight of the hyaluronic acid synthesized by the genetically engineered bacteria constructed in the present application is 1.75MDa.

[0111] The chromatographic column was Shodex Ohpak SB-806M HQ, the mobile phase was 0.1M Na2SO4 solution, and the flow rate was 1mL / min. The peak area of GPC was used to monitor the yield of HA, and the HA for making the HA concentration standard curve was purchased from Shandong Bloomage Biotechnology Co., Ltd. (Bloomage BioCo., Ltd.).

[0112] The present application provides a genetically engineered bacteria for producing hyaluronic acid and its application ideas and methods, and there are many methods and ways to realize the technical scheme. The above description is only the preferred embodiment of the present application, and it should be pointed out that for ordinary skilled persons in the technical field, some improvements and refinements can be made without departing from the principles of the present application, and these improvements and refinements should also be regarded as the protection scope of the present application. The components not explicitly described in the embodiments can be realized by existing technology.

Claims

1. A genetically engineered bacterium for producing hyaluronic acid, characterized by comprising a nucleic acid encoding a hyaluronan synthase and a nucleic acid encoding a hyaluronidase. Using the strong constitutive promoter P43 as a host, the hyaluronic acid synthase gene was expressed in Bacillus amyloliquefaciens NF pHpaII The hyaluronic acid synthase gene was expressed in Bacillus amyloliquefaciens NF sthasA The UDP-glucose dehydrogenase gene was expressed in Bacillus amyloliquefaciens NF cghasB The polysaccharide synthesis operon gene was knocked out in Bacillus amyloliquefaciens NF epsA-O The fructosyltransferase gene was expressed in Bacillus amyloliquefaciens NF sacB The fructosyltransferase gene was expressed in Bacillus amyloliquefaciens NF The hyaluronic acid synthase encoding gene sthasA derived from Streptococcus thermophilus Streptococcus thermophilus SMQ-301; The fermentation conditions of the genetically engineered bacteria in the preparation of hyaluronic acid are as follows: 32 DEG C, pH 7.0, 220 rpm of shaking speed, 48 h of fermentation time, 40 g / L of inulin as the carbon source, 10-15 g / L of yeast powder as the nitrogen source, 6 g / L of MgSO4 as the inorganic salt, and 2 g / L of betaine as the osmotic regulator. The molecular weight of the hyaluronic acid prepared by the genetically engineered bacteria is 1.75 Mda. The hyaluronic acid synthase encoding gene sthasA The coding sequence after codon optimization is shown in SEQ ID NO. 1; the UDP-glucose dehydrogenase encoding gene cghasB derived from Corynebacterium glutamicum Corynebacterium glutamacium strain number ATCC 13032, and the coding sequence is shown in SEQ ID NO. 2; The oligofructose synthetase gene has a coding sequence as shown in SEQ ID NO. 3; and the polysaccharide synthesis operon gene has a coding sequence as shown in SEQ ID NO.

4. sacB The oligofructose synthetase gene has a coding sequence as shown in SEQ ID NO. 3; and the polysaccharide synthesis operon gene has a coding sequence as shown in SEQ ID NO.

4. epsA-O The oligofructose synthetase gene has a coding sequence as shown in SEQ ID NO. 3; and the polysaccharide synthesis operon gene has a coding sequence as 2. The genetically engineered bacteria according to claim 1, characterized in that, The said B. amyloliquefaciens NF host is a modified strain of B. amyloliquefaciens NX-2S, i.e. the γ-polyglutamic acid synthetase encoding gene is knocked out on the basis of the original strain PgsBCA . 3.The genetically engineered bacteria according to claim 1, characterized in that, The expression vector of the genetically engineered bacteria is pMA5.

4. The method for constructing a genetically engineered bacterium for producing hyaluronic acid according to claim 1, characterized by, The method comprises the following steps: (a) Construction of Bacillus amyloliquefaciens CF strain: Bacillus amyloliquefaciens CF strain was obtained by knocking out the epsA-O and sacB gene of Bacillus amyloliquefaciens NF. (b) Construction of recombinant Bacillus amyloliquefaciens: sthasA Gene sequence and cghasB The codon-optimized gene sequence was cloned into the expression vector pMA5 and verified to obtain the recombinant plasmid pMA5-sthasA-cghasB. The recombinant plasmid pMA5-sthasA-cghasB was transformed into competent cells prepared using Bacillus amyloliquefaciens CF obtained in step (a) to construct recombinant Bacillus amyloliquefaciens.

5. The genetically engineered bacteria for producing hyaluronic acid according to any one of claims 1-3 are applied to the fermentation preparation of hyaluronic acid.

6. Use according to claim 5, characterized in that, The method comprises the following steps: (1) The genetically engineered bacteria according to any one of claims 1-3 are activated at 28-37 DEG C, inoculated into seed culture medium, and cultured to obtain seed liquid containing the genetically engineered bacteria; (2) The seed liquid obtained in step (1) is inoculated into fermentation culture medium at an inoculation amount of 1%-10% for fermentation tank fermentation.

7. Use according to claim 6, characterized in that, In step (1), the culture is under the conditions of a shaking speed of 220 rpm and a culture at 37 °C for 10-16 h to an OD 660 greater than 5.0, and a pH of 6.0-8.0.

Images