CN101935679A - Heterologous Synthesis of Hyaluronic Acid Based on Gram-Positive Safe Microorganisms - Google Patents

Abstract

A method for heterologous synthesis of hyaluronic acid based on Gram-positive safe microorganisms in the field of genetic engineering technology, by cloning genes related to the synthesis of hyaluronic acid precursors from Gram-positive safe microorganism hosts (in the sequence listing SEQ ID No.31 to SEQ ID No.43); According to the gene codon preference of the Gram-positive safe microbial host, redesign and synthesize the hyaluronan synthase gene (SEQ ID No.1 to SEQ ID in the sequence listing No.27), and the genes related to the synthesis of hyaluronic acid precursors and the optimized hyaluronic acid synthase gene are combined into gene expression cassettes, and finally the gene expression cassettes are transformed into Gram-positive microbial host bacteria to obtain hyaluronic acid secretion Gram-positive genetically engineered safe host bacteria for acid, and hyaluronic acid based on Gram-positive safe microorganisms is obtained through fermentation and cultivation. The invention greatly improves the ability of host cells to synthesize hyaluronic acid.

Description

Heterologous Synthesis of Hyaluronic Acid Based on Gram-Positive Safe Microorganisms

technical field

The invention relates to a method in the technical field of genetic engineering, in particular to a heterologous synthesis method of hyaluronic acid based on Gram-positive safe microorganisms.

Background technique

Hyaluronic acid (Hyaluronic Acid, hereinafter referred to as HA) is a linear unbranched polymer polysaccharide, with two precursors: UDP-glucuronic acid (UDP-GlcUA) and UDP-N-acetylglucosamine (UDP-N-GlcNAc). The body is synthesized catalyzed by hyaluronan synthases (HA synthases, HAS), and its average molecular weight can reach several million Daltons. As a multifunctional matrix, it is widely distributed in human tissues and organs. With its unique molecular structure and physical and chemical properties, it plays a very important role in the normal physiological activities of the body, such as lubricating joints, maintaining good elasticity of the skin, and regulating blood vessel walls. It is closely related to the physiology and pathology of the body. HA has been widely used in the preparation of drugs for the treatment of joint diseases (Mazieres, et al 2007), drug delivery vehicles (Fuente, et al 2008; Bechara, et al 2008), dermal fillers (Romagnoli, et al 2008; Tezel and Fredrickson, 2008) and drug cross-linking agents (Leonelli, et al 2008) and other aspects of medicine.

At present, there are two production methods of HA, one is to extract from animal tissues such as cockscombs, and the other is to use some attenuated strains of Streptococcus group C (Streptococcus zooepidemicus or Streptococcus equine, etc.) to ferment, from Extracted from the fermentation broth. Although the HA extracted from the cockscomb has been approved for the above-mentioned medical field, yet, the HA extracted therefrom is combined with a large amount of glycoproteins and glycolipids, making its separation and purification very difficult, and the yield is very low (only one hectogram of the cockscomb can be extracted 0.4 g of pure product). Moreover, hyaluronic acid derived from rooster combs is susceptible to contamination by interspecies viral agents and other pathogenic agents. Due to the above shortcomings of extracting HA from animal tissues, people gradually turn their attention to microbial fermentation to prepare HA. At present, the most studied is to use Streptococcus zooepidemicus or Streptococcus equine to ferment and produce HA. However, since the above-mentioned streptococcus is an opportunistic pathogen, there is potential harm to the health of production personnel and the environment. More and more studies have shown that this type of streptococcus can also produce a certain amount of endotoxin and other toxic factors ( Virulence factors) (Leonard, et al 1998; Steiner and Malke, 2002; Hashikawa, et al 2004; Lindsay, et al 2009), making the HA extracted from the fermentation broth potentially contaminated by virulence factors including endotoxin . At present, HA used in the field of medicine is still extracted from tissues such as cockscombs. The complex extraction process and very limited sources of raw materials make the product very expensive. Therefore, it is very necessary to study a novel preparation method of hyaluronic acid with simple extraction method, easy-to-obtain raw materials, safe production and no toxin factors.

After searching the prior art, it was found that Chinese Patent Document No. CN1636052A, published date 2005-7-6, records a "method for producing hyaluronic acid in recombinant host cells", Chinese Patent Document No. CN101426925A, published date 2009 -5-6, which records a "method for producing hyaluronic acid in Bacillus cells", and US Patent Document No. US2002/0160489, published on 2002-10-31, which records a "Streptococcus equisimilis hyaluronan synthase gene and expression thereof in Bacillus subtilis (the streptococcus equisimilis hyaluronic acid synthase gene and its expression of Bacillus subtilis), this technology uses Bacillus subtilis as the host, and integrates the hyaluronic acid synthase gene and its regulatory sequence from Streptococcus equisimilis into the subtilis host genome and enables hyaluronic acid synthesis.

The commonality of the above-mentioned prior art is that it describes the use of Bacillus subtilis as a host, and the gene related to the synthesis of hyaluronic acid and the promoter of the constitutive amylase gene constitute an artificial operon, and all genes utilize the constitutive amylase gene The promoter to initiate transcription, the synthesis of hyaluronic acid is synchronized with the proliferation of host cells. Although the above technology can realize the heterologous synthesis of hyaluronic acid in Bacillus subtilis, there is an obvious problem: with the synthesis of hyaluronic acid, the viscosity of the fermentation medium also gradually increases, and the dissolved oxygen also decreases rapidly. The ability to obtain oxygen also decreases rapidly, and the biomass of cells also increases slowly or stops increasing, which leads to a rapid decline in the ability of cells to synthesize hyaluronic acid.

Contents of the invention

Aiming at the above-mentioned deficiencies in the prior art, the present invention provides a method for heterologous synthesis of hyaluronic acid based on Gram-positive safe microorganisms. By separating the growth of host cells from the synthesis of hyaluronic acid, the host cell growth rate is greatly improved. The ability to synthesize hyaluronic acid not only realizes the synthesis of hyaluronic acid in safe Gram-positive microorganisms, but also its synthesis can be either constitutive or induced.

The present invention is achieved through the following technical solutions, and the present invention comprises the following steps:

The first step, isolating genes related to the synthesis of hyaluronic acid precursors (i.e. UDP-glucuronic acid and UDP-N-acetyl-glucosamine) from Gram-positive safe microbial hosts, these genes include: UDP-glucose Dehydrogenase gene (UDP-glucose dehydrogenase gene), namely SEQ ID No.31, SEQ ID No.32, SEQ ID No.33, SEQ ID No.34; UTP-glucose-1-phosphotransferase gene (UTP-glucose -1-phosphate Uridyltransferasegene), namely SEQ ID No.35, SEQ ID No.36, SEQ ID No.37; glucose-6-phosphate mutase gene (Phosphoglucomutase gene), namely SEQ ID No.38, SEQ ID No. .39; Glucose-6-phosphate isomerase gene (Phosphoglucoisomerase gene), namely SEQ ID No.40; Aminotransferase (Aminotransferase), namely SEQ ID No.41, N-acetyl-1-phosphate-glucosamine - uracil base transferase gene (N-Acetyl-glucosamine-1-phosphate uridyltransferase gene), namely SEQ ID No.42, SEQ ID No.43.

Described Gram-positive safe microorganism host refers to any one in the following bacterium: Clostridium butyricum (clostridium butyricum or butyricum), Bacillus licheniformis (Bacillus licheniformis), Bacillus amyloliquefaciens (Bacillus amyloliquefaciens), Bacillus cereus (beneficial or nontoxic Bacillus cereus), Bacillus brevis (Bacillus brevis), Bacillus pumilus (Bacillus pumilus), Brevibacillus brevis (Bacillus brevis), Bacillus stearothermophilus (Bacillus stearothermophilus), Bacillus megaterium ( Bacillus megaterium), Bacillus natto, Bacillus subtilis, Geobacillus stearothermophilus, Bacillus coagulans, Bacillus lentus, Bactroides amylophilus (Bacteroides amylovora).

The second step, according to the codon preference of the Gram-positive safe microbial host described in step 1, to SEQ ID No.44, SEQ ID No.45, SEQ ID No.46, SEQ ID No.47, SEQ ID No .48 and SEQ ID No.49 were subjected to base substitution and codon optimization, and a total of 27 different sequences from SEQ ID No.1 to SEQ ID No.27 were obtained respectively. Better expression in the host;

The microorganisms containing the genes of SEQ ID No.44, SEQ ID No.45, SEQ ID No.46, SEQ ID No.47 and SEQ ID No.48 are respectively: Streptococcus zooepidemicus, equine Streptococcus equisimilis, Paramecium bursaria Chlorella virus 1, Streptococcus pygenes, and Streptococcus uberis.

The paramecium chlorella is a pair of symbiotic systems, the chlorella grows and reproduces in the paramecium, and at the same time can provide the nutrition required by the paramecium. In chlorella there is a virus whose genome contains a hyaluronic acid synthase gene whose expression can synthesize hyaluronic acid in chlorella.

The base replacement refers to codon optimization according to the codon preference of the host genome, and the genes related to the synthesis of hyaluronic acid from other non-host bacteria are translated into proteins according to the genes of the host bacteria. The codon bias of the base is replaced without changing the type of amino acid encoded by the base, so that the genes related to hyaluronic acid synthesis from other non-host bacteria can be efficiently expressed in the host bacteria.

The third step, combining any gene from SEQ ID No.1 to SEQ ID No.27 and more than one gene from SEQ ID No.31 to SEQID No.43 gene with a constitutive promoter or an inducible promoter Subunits constitute a gene expression cassette.

The constitutive promoter refers to a promoter that the host bacteria has transcriptional activity at any stage of culture.

The inducible promoter refers to a promoter that has transcriptional activity only in a specific period of host bacterial culture, specifically including chemically induced promoters and physically induced promoters.

Described chemical induction promoter comprises: the promoter (Pbm-sacB) of the promotor (Pbm-sacB) of the levansucrase (levansucrase) gene of Bacillus megaterium (giant bacillus), the initiation of the xylose isomerase gene of Bacillus megaterium (giant bacillus) (Pbm-xylA), the promoter (Pbs-sacB) of the levansucrase (levansucrase) gene of Bacillus subtilis (Bacillus subtilis), the promoter of the xylose isomerase gene of Bacillus subtilis (Bacillus subtilis) ( Pbs-xylA), promoter (Para) of the arabinose utilization operon of Eschelichia coli (Escherichia coli), promoter (Plac) of the lactose utilization operon of Eschelichia coli (Escherichia coli), Eschelichia coli (Escherichia coli), preferably Bacillus The promoter (Pbm-sacB) of the levansucrase gene of megaterium (Bacillus megaterium), the promoter (Pbs-sacB) of the levansucrase gene of Bacillus subtilis (Bacillus subtilis) and Bacillus Promoter (Pbm-xylA) of the xylose isomerase gene of megaterium (Bacillus megaterium). Most preferred is the promoter of the levansucrase gene (Pbm-sacB) of Bacillus megaterium.

The transcription of the chemically induced promoter is achieved by:

The promotor of the described levansucrase (levansucrase) gene may initiate the transcription of its downstream gene only after adding sucrose, and the mass percentage concentration of sucrose is 0.5% to 10%, preferably 2% to 6% (mass volume percentage ).

The promoter of the described xylose isomerase gene may initiate the transcription of its downstream gene only after adding xylose, and the mass percent concentration of xylose is 0.5% to 8%, preferably 2%-6% (mass volume percentage).

The promoter of the arabinose utilization operon may initiate the transcription of its downstream genes only after adding arabinose, and the mass percent concentration of arabinose is 0.5% to 8%, preferably 2%-6% (mass volume percentage) .

The promoter of the lactose utilization operon can initiate the transcription of its downstream genes only after adding lactose, and the mass percentage concentration of lactose is 0.5% to 8%, preferably 2%-6% (mass volume percentage).

The promoters of described physical induction include: the heat-inducible promoter derived from the groESL gene of Bacillus subtilis (Bacillus subtilis), the heat-induced promoter derived from the dnaK gene of Bacillus subtilis (Bacillus subtilis), the heat-induced promoter derived from Bacillus licheniformis (Bacillus licheniformis) heat-inducible promoter of the groE gene.

The fourth step is to transform the gene expression cassette into Gram-positive microbial host bacteria by means of electroporation, preparation of protoplasts or preparation of competent cells, and screening with selection markers to obtain Gram-positive microorganisms capable of secreting hyaluronic acid. Positive genetically engineered safe host bacteria;

The selectable marker refers to: the gene used to effectively screen host cells containing nucleic acid constructs related to hyaluronic acid synthesis, such as: erythromycin resistance gene, chloramphenicol resistance gene, D-arabitol Dehydrogenase gene, ribitol dehydrogenase gene, spectinomycin resistance gene.

The fifth step is to ferment and cultivate Gram-positive genetically engineered safe host bacteria, and add an inducer to induce hyaluronic acid synthesis during the cultivation stage to improve the synthetic level of hyaluronic acid. After the fermentation is completed, it is separated and purified from the medium to obtain Hyaluronic Acid Based on Gram-Positive Safe Microorganisms.

The inducer for inducing the synthesis of hyaluronic acid refers to: sucrose, xylose, arabinose or lactose.

Described fermentation culture refers to: prepare glucose 5-20 grams/liter, yeast extract powder 0.5-5 grams/liter, magnesium sulfate heptahydrate 0.3-3 grams/liter, potassium dihydrogen phosphate 3-8 grams/liter, Disodium hydrogen phosphate 2-8 g/L, ammonium sulfate 2-8 g/L, sodium citrate 2-5 g/L, ferrous sulfate heptahydrate 1-20 mg/L, manganese sulfate monohydrate 1-20 mg /L, anhydrous copper sulfate 0.2-10 mg/L, zinc chloride 0.2-10 mg/L, and adjust the pH to 6.5-7.5 with citric acid or sodium hydroxide. The fermentation temperature is 28-37 degrees, preferably 30-37 degrees, most preferably 33-35 degrees, and the stirring speed is 300-600 rpm.

The hyaluronic acid is a straight-chain macromolecular acidic mucopolysaccharide formed by alternately linking D-glucuronic acid (GlcUA) and N-acetylglucosamine (GlcNAc) with disaccharide units, and its molecular weight is 50,000 to 50,000. million daltons.

In order to enhance the synthetic ability of hyaluronic acid in Gram-positive host bacterial cells, the present invention combines genes (SEQ ID NO.1 to 27; SEQ ID No.31 to 43) related to hyaluronic acid synthesis to form a The different gene expression cassettes are then transformed into the chromosome of the host cell. In addition to containing one or more genes in SEQ ID No.31 to 43, the gene expression cassette must contain a codon-optimized hyaluronan synthase gene, which is activated in a constitutive promoter or an inducible promoter It is transcribed and expressed and translated into active enzyme proteins under the control of the protease, and these enzyme proteins can enhance the ability of Gram-positive host cells to synthesize hyaluronic acid precursors and hyaluronic acid. For example, UDP-glucose dehydrogenase gene (UDP-glucose dehydrogenase gene), UTP-glucose-1-phosphate transferase gene (UTP-glucose-1-phosphate Uridyltransferase gene) and hyaluronic acid synthase gene can be combined Together, the genes join ribosome binding sites, with constitutive or inducible promoters controlling the expression of all three genes. UDP-glucose dehydrogenase gene (UDP-glucose dehydrogenase gene), UTP-glucose-1-phosphate transferase gene (UTP-glucose-1-phosphate uridyltransferase gene), glucose-6-phosphate mutase gene ( Phosphoglucomutase gene), N-acetyl-1-phosphate-glucosamine-uracil-based transferase gene (N-Acetyl-glucosamine-1-phosphate uridyltransferase gene) and hyaluronic acid synthase gene are combined together, Genes are added to add ribosome binding sites, and constitutive or inducible promoters are used to initiate the transcription of these five genes to form an artificial operon. It is also possible to combine all the above-mentioned genes related to hyaluronic acid synthesis to form a gene cluster.

In a preferred version, the present invention will UDP-glucose dehydrogenase gene (UDP-glucose dehydrogenase gene), i.e. SEQ ID No.31-34, UTP-glucose-1-phosphotransferase gene (UTP-glucose-1-phosphateUridyltransferase gene), namely SEQ ID No.35-37, N-acetyl-1-phosphate-glucosamine-uracil transferase gene (N-Acetyl-glucosamine-1-phosphate uridyltransferase gene), namely SEQ ID No. 43, and the optimized hyaluronan synthase gene (any one of SEQ ID No.1-27) is combined to form a gene expression cassette, and the promoter of the sucrose-inducible fructanase gene is used to start these four gene transcription.

The hyaluronan synthase gene used above is optimized according to the codon preference of Gram-positive host bacteria.

In order to further enhance the transcriptional activity of the inducible promoter, the present invention also introduces an enhancer (enhancer) upstream or downstream of the inducible promoter. The presence of an enhancer can further enhance the transcriptional activity of an inducible promoter. The enhancer in the present invention refers to any nucleic acid sequence capable of enhancing the transcriptional activity of the promoter connected thereto.

In order to obtain hyaluronic acid-producing Gram-positive genetically engineered bacteria more effectively, when constructing an expression vector containing and synthesizing hyaluronic acid precursor and hyaluronan synthase gene, the following selection markers can be introduced into the expression vector: D-arabitol dehydrogenase gene (D-arabitol dehydrogenase gene), ribitol dehydrogenase gene (ribitol dehydrogenase gene), erythromycin resistance gene (erm), chloramphenicol resistance gene (cat ), kanamycin resistance gene (km). Since the D-arabitol dehydrogenase gene is a safe selection marker, it is preferred to use this gene as a selection marker in the present invention. However, it does not mean that other selection markers are not used in the present invention. Any source of the D-arabitol dehydrogenase gene encoding the product D-arabitol dehydrogenase that converts D-arabitol to D-xylulose in the presence of NAD(H) or D-ribulose. A ribitol dehydrogenase gene from any source that encodes a ribitol dehydrogenase product that converts ribitol to D-ribulose in the presence of NAD(H) can also be used.

Bacillus subtilis, Bacillus licheniformis, Bacillus megaterium, Bacillus amyloliquefaciens, beneficial or nontoxic Bacillus cereus, Bacillus brevis, Bacillus brevis, Bacillus stearothermophilus, Bacillus amyloliquefaciens described in the present invention , Geobacillus stearothermophilus, Bacillus coagulans, Bacillus lenticularis, Bacteroides amylovora, and Bacillus pumilus are obtained from the China Ordinary Microorganisms Collection and Management Center. The preservation information of coccus and streptococcus uberis is obtained from the German DSMZ preservation center.

In order to obtain a relatively stable genetically engineered Gram-positive host bacterium capable of synthesizing hyaluronic acid, the present invention connects the gene of a hyaluronic acid-synthesizing enzyme to an integrated expression plasmid vector or an episomal expression plasmid vector.

The hyaluronic acid produced by the fermentation of a genetically engineered safe microbial host can be used in the fields of food, cosmetics and pharmaceuticals because of its very good biological safety and the absence of any endotoxin and any therapeutic factor. In the field of food, it can be made into oral liquids, capsules, etc.; in the field of cosmetics, it can be made into various water-retaining skin care products (emollient cream, body lotion, etc.); in the field of medicine, it can be made into joint lubricating injections to promote wound healing ; It can also be used for modification and derivatization to make hyaluronic acid derivatives.

Description of drawings

Figure 1 is a Bacillus integrated expression vector containing hyaluronan synthase gene and xylose-induced promoter from Streptococcus zooepidemicus and optimized according to the codon preference of Bacillus subtilis;

In the picture:

lacA' and 'lacA represent the 5' and 3' homologous integration arms of the beta-galactosidase gene respectively; erm represents the erythromycin resistance gene;

szhas represents the hyaluronan synthase gene from Streptococcus zooepidemicus and optimized according to the codon preference of Bacillus subtilis;

PxylA represents the xylose-inducible promoter from Bacillus megaterium;

bla represents the ampicillin resistance gene;

ori represents the replication initiation DNA sequence in E. coli.

Figure 2 is a Bacillus integrated expression vector containing hyaluronan synthase gene and sucrose-induced promoter from Streptococcus zooepidemicus and optimized according to the codon preference of Bacillus subtilis;

In the picture:

PsacB represents the inducible promoter of the fruccanase gene from Bacillus subtilis.

Figure 3 is a bacillus episomal expression vector containing hyaluronic acid synthase gene and lactose-induced promoter from Streptococcus zooepidemicus and optimized according to the codon preference of Bacillus subtilis;

In the picture:

szhasA: a hyaluronan synthase gene from Streptococcus zooepidemicus optimized according to the codon preference of Bacillus subtilis;

Amp: Ampicillin resistance gene;

ori-Bs: the autonomous replication origin sequence of the plasmid of Bacillus;

ori-Ec: the autonomous replication origin sequence of the plasmid of Escherichia coli;

cat: chloramphenicol resistance gene.

Figure 4 is an episomal Bacillus expression vector containing the hyaluronan synthase gene from Streptococcus zooepidemicus and optimized according to the codon preference of Bacillus subtilis, the sucrose-inducible promoter of Bacillus subtilis;

In the picture:

PsacB: promoter sequence of fructanase gene from Bacillus subtilis; other symbols in the figure are the same as those shown in FIG. 3 .

Figure 5 is an integrated Bacillus expression vector containing the hyaluronan synthase gene from Streptococcus zooepidemicus and optimized according to the codon preference of Bacillus subtilis, the sucrose-induced promoter of Bacillus megaterium;

In the picture:

lacA' and 'lacA represent the 5' and 3' homologous integration arms of the beta-galactosidase gene, respectively;

erm means erythromycin resistance gene;

has represents the hyaluronan synthase gene from Streptococcus zooepidemicus and optimized according to the codon preference of Bacillus subtilis;

PbmsacB represents the inducible promoter of the fruccanase gene from Bacillus megaterium;

bla represents the ampicillin resistance gene;

ori represents the replication initiation DNA sequence in E. coli.

Figure 6 is an integrated Bacillus expression vector containing the hyaluronan synthase gene, Bacillus megaterium sucrose-induced promoter derived from PBCV and optimized according to the codon preference of Bacillus subtilis;

In the picture:

cvhas denotes the hyaluronan synthase gene from PBCV optimized according to Bacillus subtilis codon bias.

Figure 7 is an integrated Bacillus expression vector containing the hyaluronan synthase gene, Bacillus subtilis sucrose-induced promoter from PBCV and optimized according to Bacillus subtilis codon preference;

In the picture:

rep: a fragment of the origin of replication that can replicate autonomously in Bacillus;

ORF2: open reading frame of unknown function;

bla: ampicillin resistance gene;

cat: chloramphenicol resistance gene;

cvhas: hyaluronic acid synthase gene from PBCV, codon-optimized according to Bacillus subtilis;

PsacB: the promoter of the fructanase gene from Bacillus subtilis;

ORF3: open reading frame of unknown function.

Figure 8 is an integrated spore containing hyaluronic acid synthase gene, UDP-glucose dehydrogenase gene of Bacillus megaterium, sucrose-inducible promoter of Bacillus megaterium derived from PBCV and optimized according to the codon preference of Bacillus subtilis Bacillus expression vector;

In the picture:

cvhas represents the hyaluronan synthase gene derived from PBCV and optimized according to the codon preference of Bacillus subtilis;

PbmsacB represents the inducible promoter of the fruccanase gene from Bacillus megaterium;

Pamy represents the α-amylase gene promoter from Bacillus subtilis;

tuaD denotes the UDP-glucose dehydrogenase gene from Bacillus megaterium.

Figure 9 is a hyaluronic acid synthase gene, UDP-glucose dehydrogenase gene of Bacillus megaterium, N-acetyl-1-phosphate- Glucosamine-uracil transferase gene, Bacillus megaterium sucrose-inducible promoter integrated Bacillus expression vector;

In the picture:

gcaD denotes the N-acetyl-1-phosphate-glucosamine-uracilyltransferase gene from Bacillus megaterium.

Figure 10 is a hyaluronic acid synthase gene, UDP-glucose dehydrogenase gene of Bacillus megaterium, N-acetyl-1-phosphate- Glucosamine-uracil-based transferase gene, UTP-glucose-1-phosphouracil-based transferase gene, integrated Bacillus expression vector of the sucrose-induced promoter of Bacillus megaterium;

In the picture:

gtaB represents UTP-glucose-1-phosphate uracil transferase gene; other symbols in the figure are the same as those shown in FIG. 9 .

Figure 11 is a hyaluronic acid synthase gene, UDP-glucose dehydrogenase gene of Bacillus megaterium, N-acetyl-1-phosphate- Integrative Bacillus expression vectors for the glucosamine-uracil-based transferase gene, the sucrose-inducible promoter of Bacillus subtilis and its regulatory elements;

In the picture:

P43 represents the P43 gene promoter of Bacillus subtilis;

cvhas represents the hyaluronan synthase gene from PBCV and optimized according to the codon preference of Bacillus subtilis; degQ and degU represent the regulatory protein genes from Bacillus subtilis;

PbssacB represents the inducible promoter of the fruccanase gene from Bacillus subtilis;

Pamy represents the α-amylase gene promoter from Bacillus subtilis;

tuaD represents the UDP-glucose dehydrogenase gene from Bacillus megaterium;

gtaB means UTP-glucose-1-phosphate uracil transferase gene from Bacillus megaterium;

gcaD denotes the N-acetyl-1-phosphate-glucosamine-uracilyltransferase gene from Bacillus megaterium.

Figure 12 is the hyaluronic acid synthase gene, the UDP-glucose dehydrogenase gene of Bacillus megaterium, the N-acetyl-1- Integrated Bacillus expression vectors for phosphate-glucosamine-uracilyltransferase gene, sucrose-inducible promoter of Bacillus subtilis and its regulatory elements;

In the picture:

pyhas denotes the hyaluronan synthase gene from Streptococcus pyogenes optimized according to the codon bias of B. subtilis.

Fig. 13 is the hyaluronic acid synthase gene, the UDP-glucose dehydrogenase gene of Bacillus megaterium, the N-acetyl-1- Integrated Bacillus expression vectors for phosphate-glucosamine-uracilyltransferase gene, sucrose-inducible promoter of Bacillus subtilis and its regulatory elements;

In the picture:

puhas indicates the hyaluronan synthase gene from Streptococcus pyogenes optimized according to the codon bias of B. subtilis.

Figure 14 is a hyaluronic acid synthase gene from Streptococcus zooepidemicus and optimized according to the codon preference of Bacillus subtilis, UDP-glucose dehydrogenase gene of Bacillus licheniformis, UTP-glucose-1-phosphate uracil base transfer Enzyme gene, N-acetyl-1-phosphate-glucosamine-uracil transferase gene, Bacillus subtilis sucrose-inducible promoter and its regulatory elements integrated Bacillus expression vector;

In the picture:

szhas represents the hyaluronan synthase gene from Streptococcus zooepidemicus and optimized according to the codon preference of Bacillus subtilis; degQ and degU represent the regulatory protein genes from Bacillus subtilis;

PbssacB represents the inducible promoter of the fruccanase gene from Bacillus subtilis;

tuaD represents the UDP-glucose dehydrogenase gene from Bacillus licheniformis;

gtaB represents UTP-glucose-1-phosphate uracil transferase gene from Bacillus licheniformis;

gcaD represents the N-acetyl-1-phosphate-glucosamine-uracilyltransferase gene from Bacillus licheniformis.

Fig. 15 is the hyaluronic acid synthase gene, the UDP-glucose dehydrogenase gene of Bacillus megaterium, the alpha-amylase gene promoter of Bacillus subtilis, weak An integrated Bacillus expression vector of the D-arabitol dehydrogenase gene of Acetobacter oxidans and the sucrose-inducible promoter of Bacillus subtilis;

lacA' and 'lacA represent the 5' and 3' homologous integration arms of the beta-galactosidase gene, respectively;

aArDH; represents the D-arabinitol dehydrogenase gene from Acetobacter suboxidans;

cvhas represents the hyaluronan synthase gene derived from PBCV and optimized according to the codon preference of Bacillus subtilis;

PbmsacB represents the inducible promoter of the fruccanase gene from Bacillus megaterium;

bla represents the ampicillin resistance gene;

ori represents the replication initiation DNA sequence in Escherichia coli;

Pamy represents the α-amylase gene promoter from Bacillus subtilis;

tuaD denotes the UDP-glucose dehydrogenase gene from Bacillus megaterium.

Figure 16 is a hyaluronic acid synthase gene from Streptococcus zooepidemicus and optimized according to the codon preference of Bacillus subtilis, UDP-glucose dehydrogenase gene of Bacillus licheniformis, UTP-glucose-1-phosphate uracil base transfer Enzyme gene, N-acetyl-1-phosphate-glucosamine-uracil transferase gene, D-arabinitol dehydrogenase gene of Klebsiella pneumoniae and sucrose-inducible promoter of Bacillus subtilis and its Integrative Bacillus expression vectors for regulatory elements;

In the picture:

lacA' and 'lacA represent the 5' and 3' homologous integration arms of the beta-galactosidase gene, respectively;

DalD represents the D-arabinitol dehydrogenase gene from Klebsiella pneumoniae; P43 represents the P43 gene promoter of Bacillus subtilis;

szhas represents the hyaluronan synthase gene from Streptococcus zooepidemicus and optimized according to the codon preference of Bacillus subtilis; degQ and degU represent the regulatory protein genes from Bacillus subtilis;

PbssacB represents the inducible promoter of the fruccanase gene from Bacillus subtilis;

bla represents the ampicillin resistance gene; ori represents the replication initiation DNA sequence in Escherichia coli;

Pamy represents the α-amylase gene promoter from Bacillus megaterium;

tuaD represents the UDP-glucose dehydrogenase gene from Bacillus licheniformis;

gtaB represents UTP-glucose-1-phosphate uracil transferase gene from Bacillus licheniformis;

gcaD represents the N-acetyl-1-phosphate-glucosamine-uracilyltransferase gene from Bacillus licheniformis.

Fig. 17 is a schematic diagram of PCR verification results in step (1) of Example 3.

Fig. 18 is a schematic diagram of PCR verification results in step (2) of Example 3.

Fig. 19 is a schematic diagram of PCR verification results in step (7) of Example 3.

Figure 20 is a schematic diagram of Embodiment 7.

Figure 21 is a schematic diagram of Embodiment 8.

Detailed ways

The embodiments of the present invention are described in detail below. This embodiment is implemented on the premise of the technical solution of the present invention, and detailed implementation methods and specific operating procedures are provided, but the protection scope of the present invention is not limited to the following implementation example.

Example 1: Cloning or synthesis of genes and promoter sequences related to the synthesis of hyaluronic acid precursors

(1) Cloning of hyaluronan synthase gene from Streptococcus zooepidemicus

The total DNA of Streptococcus zooepidemicus was extracted using the method for extracting total bacterial DNA described in the literature (Cheng H and Jiang N, 2006, 28: 55-59, Biotechnol. Letters). Design a pair of primers, and use the extracted total DNA of Streptococcus zooepidemicus as a template to carry out PCR reaction. The specific reaction conditions are 94 degrees pre-denaturation for 5 minutes, followed by 30 cycles at 94 degrees for 30 seconds, 56 degrees for 40 seconds, and 72 degrees for 60 seconds. , with a final extension of 10 min for the procedure. The sequence of a pair of primers designed is as follows:

5'-CTTCTAGAATGAGAACATTAAAAAACCTCATA-3' (with XbaI restriction site at the 5' end, TCTAGA)

5'-ATCCGCGGTTATAATAATTTTTTACGTGTTCC-3'(with SacII restriction site at the 5' end, CCGCGG)

The size of the hyaluronan synthase gene of Streptococcus zooepidemicus amplified according to the above method is 1254bp, namely SEQ ID No.44.

(2) Cloning of Streptococcus pyogenes hyaluronan synthase gene

The total DNA of Streptococcus pyogenes was extracted using the method for extracting total bacterial DNA described in the literature (Cheng H and Jiang N, 2006, 28: 55-59, Biotechnol. Letters). Design a pair of primers, and use the extracted total DNA of Streptococcus pyogenes as a template for PCR reaction. The specific reaction conditions are 94 degrees pre-denaturation for 5 minutes, followed by 32 cycles of 94 degrees for 30 seconds, 55 degrees for 40 seconds, and 72 degrees for 70 seconds. , with a final extension of 10 min for the procedure. The sequence of a pair of primers designed is as follows:

5'-CTTCTAGAATGCTTATTTTTAAAAAAACTTTTA-3' (with XbaI restriction site at the 5' end, TCTAGA)

5'-ATCCGCGGTTATTTAAAAATAGTGACCTTTTTAC-3' (with SacII restriction site at the 5' end, CCGCGG)

The hyaluronan synthase gene size of Streptococcus pyogenes amplified according to the above method is 1260bp, namely SEQ ID No.47.

(3) Cloning of Streptococcus uberis hyaluronan synthase gene

The total DNA of Streptococcus uberis was extracted using the method for extracting total bacterial DNA described in the literature (Cheng H and Jiang N, 2006, 28: 55-59, Biotechnol. Letters). Design a pair of primers, and use the extracted total DNA of Streptococcus uberis as a template to carry out PCR reaction. The specific reaction conditions are 94 degrees pre-denaturation for 5 minutes, followed by 32 cycles of 94 degrees for 30 seconds, 55 degrees for 40 seconds, and 72 degrees for 70 seconds. , with a final extension of 10 min for the procedure. The sequence of a pair of primers designed is as follows:

5'-CTTCTAGAATGGAAAAACTAAAAAATCTCATTAC-3' (with XbaI restriction site at the 5' end, TCTAGA)

5'-ATCCGCGGTTATTTACTTGTCTTTTTACGAGTTCC-3' (with a SacII restriction site at the 5' end, CCGCGG)

The hyaluronan synthase gene size of Streptococcus uberis amplified according to the above method is 1254bp, namely SEQ ID No.48.

(4) Optimization and synthesis of paramecium chlorella virus hyaluronan synthase gene

According to the gene sequence (SEQ ID No.46) of Paramecium bursaria Chlorella virus 1 (PBCV1) publicly reported by GenBank, the size of the gene coding frame is 1707 bases (starting from the start codon ATG to stop codon TAA end). Optimize according to the codon preference of the Gram-positive host bacterium genome that the present invention adopts, the method for optimizing is to adopt online molecular biology software OptimumGene Codon Optimization Analysis, Gene Designer, Codon Optimizer or Optimizer, after analysis, different software is given The results were manually corrected, so that the CAI value reached above 0.80. Then entrust a specialized gene synthesis department to carry out the whole gene synthesis to obtain the codon-optimized hyaluronic acid synthase gene of paramecium chlorella virus. The sequence of the optimized paramecium chlorella virus hyaluronan synthase gene is SEQ ID No.15-21.

(5) Cloning of UDP-glucose dehydrogenase gene and UTP-glucose-1-phosphotransferase gene of Bacillus megaterium.

The method for extracting total bacterial DNA described in the literature (Cheng H and Jiang N, 2006, 28: 55-59, Biotechnol. Letters) was used to extract the total DNA of Bacillus megaterium. Design a pair of primers, and use the extracted total DNA as a template to carry out PCR reaction. The specific reaction conditions are 94 degrees pre-denaturation for 5 minutes, followed by 35 cycles of 94 degrees for 30 seconds, 58 degrees for 40 seconds, and 72 degrees for 100 seconds, and finally extended A 10-minute program is performed. The sequence of a pair of primers designed is as follows:

5'-ATGGATCCGGTACCAGGAGGAAAAGTAAATGAAAATTC-3' (with BamHI site GGATCC and KpnI site GGTACC at the 5' end)

5'-ATCTCGAGTTATAAAGTAGAAACTTTAGAATCACC-3' (with XhoI restriction site CTCGAG at the 5' end)

The size of the amplified DNA containing two genes of UDP-glucose dehydrogenase gene and UTP-glucose-1-phosphotransferase gene is 2.4kb. Its DNA sequence is SEQ ID No: 28.

(6) Cloning of Bacillus megaterium N-acetyl-1-phosphate-glucosamine-uracil transferase gene.

The method for extracting total bacterial DNA described in the literature (Cheng H and Jiang N, 2006, 28: 55-59, Biotechnol. Letters) was used to extract the total DNA of Bacillus megaterium. Design a pair of primers, and use the extracted total DNA as a template to carry out PCR reaction. The specific reaction conditions are 94 degrees pre-denaturation for 5 minutes, followed by 35 cycles of 94 degrees for 30 seconds, 58 degrees for 40 seconds, and 72 degrees for 100 seconds, and finally extended A 10-minute program is performed. The sequence of a pair of primers designed is as follows:

5'-ATCTCGAGTAGGAGGCCTGTTATGTCAAAAAGATATGCAGTC-3' (with XhoI restriction site CTCGAG and ribosome binding site AGGAGG at the 5' end)

5'-ATGAGCTCTTAGGATTTTTTATTAATATCAAGC-3' (with SacI restriction site GAGCTC at the 5' end)

The size of the amplified N-acetyl-1-phosphate-glucosamine-uracil transferase gene is 1380bp. Its DNA sequence is SEQ ID No: 29.

(7) Cloning of the promoter sequence of the Bacillus megaterium alpha-amylase gene.

Using the total DNA of Bacillus megaterium as a template, design a pair of primers for PCR amplification. The PCR reaction conditions are: pre-denaturation at 94 degrees for 5 minutes, followed by 35 cycles at 94 degrees for 30 seconds, 58 degrees for 40 seconds, and 72 degrees for 30 seconds. , and finally extended for 10 minutes. The alpha-amylase gene promoter sequence with a size of about 390bp was obtained. The designed primer sequences are:

5'-CTTCTAGACATCGTTTCATCCCCTTTTTTTTGCAA-3' (with XbaI restriction site TCTAGA at the 5' end)

5'-CTGGATCCTTGCAGATAGTAAATAAAATTCAA-3' (with a BamHI restriction site GGATCC at the 5' end)

The obtained Bacillus megaterium alpha-amylase gene promoter sequence is SEQ ID No: 30.

(8) Cloning of the promoter sequence of the Bacillus subtilis fructanase gene.

Design the following pair of primers:

5'-AAGCGAAAACATACCACCTATCAGATCCTTTTTAACCCATCACATATACCTG-3'

5'-CTTCTAGACATATAAAACACCTCCTTTTTTATGTACTGTGTTAGCGG-3' (with XbaI restriction site TCTAGA at the 5' end)

Using the above-mentioned pair of primers and using the total DNA of Bacillus subtilis as a template to perform PCR, a DNA fragment of about 450 bp of the fruccanase gene promoter was obtained. The conditions of the PCR reaction were: pre-denaturation at 94 degrees for 5 minutes, followed by 35 cycles at 94 degrees for 30 seconds, 56 degrees for 30 seconds, and 72 degrees for 30 seconds, and finally extended for 10 minutes.

(9) Cloning of the promoter sequence of Bacillus megaterium fructanase gene.

Design the following pair of primers:

5'-AAGCGAAAACATACCACCTATTCACTGATTCCAGCCGTGAAGGAAAAG-3'

5'-CTTCTAGA ATGTTTTTCTCTTTTGTGTTAGTAAAG-3' (with XbaI restriction site TCTAGA at the 5' end)

Using the above-mentioned pair of primers and using the total DNA of Bacillus subtilis as a template to perform PCR, a DNA fragment of about 630 bp of the fruccanase gene promoter was obtained. The conditions of the PCR reaction were: pre-denaturation at 94 degrees for 5 minutes, followed by 35 cycles at 94 degrees for 30 seconds, 56 degrees for 30 seconds, and 72 degrees for 30 seconds, and finally extended for 10 minutes.

(10) Cloning of the promoter of the Bacillus subtilis P43 gene.

Design the following pair of primers:

5'-CAATTGTTTTACTTCTTCAAGTTTCTTTTCCATGTGTACATTCCTCTCTTACCTATAATG-3'

5'-CAGGTATATGTGATGGGTTAAAAAGGATCTGATAGGTGGTATGTTTTCGCTT-3'

Using the above-mentioned pair of primers and using the total DNA of Bacillus subtilis as a template to carry out PCR, a DNA fragment of about 300 bp of the fruccanase gene promoter is obtained. The conditions of the PCR reaction were: pre-denaturation at 94 degrees for 5 minutes, followed by 35 cycles at 94 degrees for 30 seconds, 56 degrees for 30 seconds, and 72 degrees for 30 seconds, and finally extended for 10 minutes.

(11) Cloning of the Bacillus subtilis degQ sequence.

Design the following pair of primers:

TCACGCAATTTTCATTGCATAATTGTATTTATCG 3’5'AT GGATCC

TCACGCAATTTTCATTGCATAATTGTATTTATCG 3'

The underline at the 5' end indicates the BamHI recognition sequence (GGATCC), and the framed base indicates the complementary chain sequence of the Escherichia coli trpA terminator (AAAAAAGCCCGCTCATTAGGCGGGCTGC).

5'CATTATAGGTAAGAGAGGAATGTACACATGGAAAAGAAACTTGAAG AAGTAAAACAATTG-3'

Using the above pair of primers, PCR was carried out using the total DNA of Bacillus subtilis as a template to obtain a degQ DNA fragment of about 200 bp. The conditions of the PCR reaction were: pre-denaturation at 94°C for 5 minutes, followed by 35 cycles at 94°C for 30 seconds, 56°C for 30 seconds, and 72°C for 20 seconds, and finally extended for 10 minutes.

Example 2: Construction of expression vectors containing genes related to synthetic hyaluronic acid and promoter sequences

(1) Construction of an integrated expression vector containing hyaluronan synthase gene from Streptococcus zooepidemicus codon-optimized according to Bacillus subtilis.

The hyaluronan synthase gene from Streptococcus zooepidemicus was optimized according to the codon preference of Bacillus subtilis, and after double digestion with BamHI and SacII, the double-digested hyaluronan synthase gene was recovered and connected to the In the integrated expression plasmid vector pAX01 double digested with SacII, a new integrated expression vector pAX-szhas1 containing the Streptococcus zooepidemicus hyaluronan synthase gene optimized according to the codon preference of Bacillus subtilis was constructed. The map of the carrier is shown in Figure 1.

(2) Construction of an integrated expression vector containing the hyaluronic acid synthase gene from Streptococcus zooepidemicus and optimized according to the codons of Bacillus subtilis and the promoter of the Bacillus subtilis fructanase gene.

The hyaluronan synthase gene from Streptococcus zooepidemicus was optimized according to the codon preference of Bacillus subtilis, and after double digestion with BamHI and SacII, the double-digested hyaluronan synthase gene was recovered and connected to the In the integrated expression plasmid vector pAX01 double-digested with SacII, a new integrated expression vector pAX-szhas1 containing the Streptococcus zooepidemicus hyaluronan synthase gene optimized according to the codon preference of Bacillus subtilis was formed (the map is shown in Figure 1 ), and then double digested pAX-szhas1 with SpeI and XhoI, and recovered the vector fragment DNA by gel recovery. The fruccanase gene promoter from Bacillus subtilis was digested with SpeI and XhoI, and connected with the vector pAX-szhas1, which was double digested with SpeI and XhoI, to obtain a new integrated expression vector pAX-szhas2, which contained optimized transparent The promoters of the uronic acid synthase gene and the inducible sucrase gene. The map of the vector pAX-szhas2 is shown in Figure 2.

(3) Construction of an episomal expression vector containing the hyaluronic acid synthase gene from Streptococcus zooepidemicus and optimized according to the codons of Bacillus subtilis and the promoter of the Bacillus subtilis fruccanase gene.

The hyaluronan synthase gene from Streptococcus zooepidemicus was optimized according to the codon preference of Bacillus subtilis, and after double digestion with KpnI and SacI, the double-digested hyaluronan synthase gene gel was recovered and connected to the In the integrated expression plasmid vector pNW33N double-digested with SacI, a new episomal expression vector pNW-szhas1 containing the Streptococcus zooepidemicus hyaluronan synthase gene optimized according to the codon preference of Bacillus subtilis was formed (the map is shown in Figure 3 ), then use BamHI and KpnI to double-enzyme digest the vector, and simultaneously use BamHI and KpnI to double-enzyme-cut the promotor of the Bacillus subtilis fruccanase gene. And a new episomal expression vector pNW-szhas2 of the Streptococcus zooepidemicus hyaluronan synthase gene optimized according to the codon preference of Bacillus subtilis (the map is shown in Figure 4).

(4) Construction of an integrated expression vector containing hyaluronic acid synthase gene from Streptococcus zooepidemicus and codon-optimized according to Bacillus subtilis and Bacillus megaterium frucanase gene promoter.

After optimizing the hyaluronan synthase gene from Streptococcus zooepidemicus according to the codon preference of Bacillus subtilis, after double-enzyme digestion with SpeI and BamHI, the double-enzyme-digested hyaluronan synthase gene gel was recovered and connected to the In the integrated expression plasmid vector pAX01 cut with SpeI and BamHI, the vector pAX-has1 was constructed, and then pAX-has1 was cut with BamHI and SacII, and the Bacillus megaterium fruccanase gene was cut with BamHI and SacII The promoter DNA was ligated to form the integrated expression vector pAX-has2 (the map is shown in Figure 5). The integrated expression vector contains the promotor of the Bacillus megaterium fructanase gene and the hyaluronic acid synthase gene from Streptococcus zooepidemicus and optimized according to the codons of Bacillus subtilis.

(5) Construction of an integrated expression vector containing the hyaluronan synthase gene derived from PBCV and codon-optimized according to Bacillus subtilis.

After optimizing the hyaluronan synthase gene from PBCV according to the codon preference of Bacillus subtilis, after double-digestion with SpeI and SacII, the double-enzyme-digested hyaluronan synthase gene was recovered and connected to the In the integrated expression plasmid vector pAX01 cut with SacII, a new integrated expression vector pAX-cvhas1 containing the Streptococcus zooepidemicus hyaluronan synthase gene optimized according to the codon preference of Bacillus subtilis was constructed, and then SpeI and XhoI were used to construct the integrated expression vector pAX-cvhas1. Digest pAX-cvhas1 with double enzymes, and recover the vector fragment DNA from the gel. The promotor of the sucrase gene from Bacillus megaterium was double-digested with SpeI and XhoI, and connected to the vector pAX-cvhas1, which was double-digested with SpeI and XhoI, to obtain a new integrated expression vector pAX-cvhas2, which contained the optimized expression vector from The promoters of the hyaluronan synthase gene of PBCV and the inducible sucrase gene of Bacillus megaterium. The map of the vector pAX-cvhas2 is shown in Figure 6.

(6) Construction of an episomal expression vector containing the hyaluronic acid synthase gene and the Bacillus subtilis frucanase gene promoter derived from PBCV and optimized according to the codons of Bacillus subtilis.

The hyaluronic acid synthase gene derived from PBCV and optimized according to the codons of Bacillus subtilis was double-digested with SpeI and SacI, and the 1.7kb DNA fragment was digested by gel recovery. The vector pHCMC04 was double-digested with SpeI and SacI, and the two were connected to obtain a new vector containing the hyaluronan synthase gene, and then this vector was double-digested with BamHI and SpeI, and the B. The fructanase gene promoter was connected to form a new episomal expression plasmid vector pHC-cvhas2 (the map is shown in Figure 7).

(7) An integrated type containing the hyaluronic acid synthase gene optimized according to the codons of Bacillus subtilis from PBCV and the 6-phosphate glucose dehydrogenase gene from Bacillus megaterium and the promotor of the fructanase gene of Bacillus megaterium Construction of expression vectors.

The constructed pAX-cvhas2 expression vector (as shown in Figure 6) was digested with XhoI and SacI to obtain a large linear vector fragment, which was recovered by gel. The α-amylase gene promoter sequence from Bacillus subtilis was directional cloned into the XhoI and BamHI sites of the cloning plasmid vector pBlueScriptSK(-) to form the vector pBS-Amy, and then the vector was double-digested with BamHI and SacI, and the vector from The 6-phosphate glucose dehydrogenase gene of Bacillus megaterium was directional cloned into this vector with BamHI and SacI double digestion to form a new vector pBS-AG. Then pBS-AG was digested with XhoI and SacI, and a 1.9 kb DNA fragment was recovered, which was ligated with the pAX-cvhas2 expression vector digested with XhoI and SacI to form a new Bacillus integrated expression vector pAX-AGH2. The glucose-6-phosphate dehydrogenase gene in the vector is constitutively transcribed under the control of the α-amylase gene promoter, while the hyaluronan synthase gene is transcribed under the control of the inducible promoter PsacB. Induced synthesis of hyaluronic acid can be achieved. The map of this carrier is shown in Figure 8.

(8) Containing hyaluronic acid synthase gene optimized according to the codon of Bacillus subtilis from PBCV and 6-phosphate glucose dehydrogenase gene from Bacillus megaterium and N-acetyl-1-phosphate-glucose from Bacillus megaterium Construction of integrated expression vectors of glycosamino-uracil-based transferase gene and Bacillus megaterium sucrase gene promoter.

The α-amylase gene promoter sequence from Bacillus subtilis was directional cloned into the XhoI and HindIII sites of the cloning vector pBlueScriptSK(-) to form the vector pBS-Amy2, and then the vector was double-digested with HindIII and BamHI, and the The 6-phosphate glucose dehydrogenase gene of Bacillus was directional cloned into this vector with HindIII and BamHI double enzyme digestion to form a new vector pBS-AU2, and then BamHI and SacI double enzyme digestion pBS-AU2, the Bacillus megaterium The gene of N-acetyl-1-phosphate-glucosamine-uracil transferase was directional cloned into this vector, and then the vector was digested with XhoI and SacI to recover a 3.3kb DNA fragment, which was connected to XhoI and SacI A new Bacillus integrated expression vector pAX-cvhas3 was constructed from the pAX-cvhas2 vector cut with double restriction enzymes. The hyaluronan synthase gene of this vector is transcribed with the promoter of the fructanase gene, while the 6-phosphate glucose dehydrogenase gene and the N-acetyl-1-phosphate-glucosamine-uracil transferase gene Transcription is controlled by a constitutive promoter, so hyaluronic acid synthesis is induced by sucrose. The map of the vector pAX-cvhas3 is shown in Fig. 9 .

(9) Contain hyaluronic acid synthase gene, glucose-6-phosphate dehydrogenase gene, UTP-glucose-1-phosphouracil transferase gene, N-acetyl- Construction of integrated expression vectors of 1-phosphate-glucosamine-uracil-transferase gene and Bacillus megaterium fructanase gene promoter.

The α-amylase gene promoter sequence from Bacillus subtilis was directional cloned into the XhoI and HindIII sites of the cloning vector pBlueScriptSK(-) to form the vector pBS-Amy2, and the vector was double-digested with HindIII and BamHI, and the Bacillus subtilis The 6-phosphate glucose dehydrogenase gene was directional cloned into this vector to construct pBS-AU3, pBS-AU3 was digested with BamHI and SpeI, and the UTP-glucose-1-phosphotransferase gene was directional cloned into this vector, Construct the pBS-AUT1 vector, and then use SpeI and SacI to double-enzyme digest the vector, and clone the N-acetyl-1-phosphate-glucosamine-uracil transferase gene into this vector to form the pBS-AUTG vector. The vector was then digested with XhoI and SacI, and a 4.3 kb DNA fragment was recovered, which was ligated with the pAX-cvhas2 vector digested with XhoI and SacI to form a new integrated expression vector pAX-cvhas4. The hyaluronic acid synthetase gene of the vector is transcribed under the control of the sucrase gene promoter of the bacillus megaterium, and induced to express. The 6-phosphate glucose dehydrogenase gene, UTP-glucose-1-phosphotransferase gene, and N-acetyl-1-phosphate-glucosamine-uracil-based transferase gene are found in the α-amylase gene of Bacillus subtilis. Constitutive transcription takes place under the control of a gene promoter. Therefore the synthesis of hyaluronic acid is induced by sucrose. The map of the integrated expression vector pAX-cvhas4 is shown in Fig. 10 .

(10) Containing hyaluronic acid synthase gene optimized according to the codon of Bacillus subtilis from PBCV and 6-phosphate glucose dehydrogenase gene from Bacillus megaterium and N-acetyl-1-phosphate-glucose from Bacillus megaterium Construction of integrated expression vectors of glucosamine-uracil-based transferase gene and Bacillus subtilis fructanase gene promoter and its regulatory sequence.

The promoter of the sucrase gene in the vector pAX-cvhas3 was replaced with the promoter of the sucrase gene containing the enhancing element. The vector pAX-cvhas3 was double-digested with SpeI and XhoI, and the large fragment DNA was recovered from the gel. The P43 gene promoter, degQ gene and degU gene cloned in Example 1 are combined together according to SOE (spicing overlap extension PCR) technology (with reference to Heckman and Pease, 2007, Vol 2 (4), Nature Protocols), wherein degQ and degU Transcription is strongly initiated under the control of the p43 gene promoter. The expression cassette of P43-degQ-degU was obtained, and the expression cassette was directionally cloned into the pAX-cvhas3 vector digested with SpeI and XhoI to form a new Bacillus expression vector pAX-cvhas5. The fructanase gene promoter of this vector is enhanced by degQ and degU, so the downstream hyaluronan synthase gene transcription is stronger. The map of the vector pAX-cvhas5 is shown in Fig. 11 .

(11) Contains hyaluronic acid synthase gene from Streptococcus pyogenes codon-optimized according to Bacillus megaterium and glucose-6-phosphate dehydrogenase gene from Bacillus megaterium and N-acetyl-1-phosphate from Bacillus megaterium - Construction of integrated expression vectors for glucosamine-uracil-based transferase gene and Bacillus megaterium fructanase gene promoter and its regulatory sequence.

Replace the hyaluronic acid synthase gene from PBCV and optimized according to the codon of Bacillus subtilis in the vector pAX-cvhas5 with the hyaluronan synthase gene from Streptococcus pyogenes and optimized according to the codon of Bacillus megaterium to form a new Integrate the expression vector pAX-sphas1. The spectrum is shown in Figure 12.

(12) Contains hyaluronic acid synthase gene from Streptococcus uberis codon-optimized according to Bacillus subtilis and glucose-6-phosphate dehydrogenase gene from Bacillus megaterium and N-acetyl-1-phosphate from Bacillus megaterium - Construction of integrated expression vectors for glucosamine-uracil-based transferase gene and Bacillus megaterium fructanase gene promoter and its regulatory sequence.

The hyaluronan synthase gene from Streptococcus pyogenes and optimized according to the codons of Bacillus megaterium in the integrated expression vector pAX-sphas1 was replaced with the hyaluronan synthase gene from Streptococcus uberis and optimized according to the codons of Bacillus subtilis, A new integrated expression vector pAX-suhas1 was constructed. The spectrum is shown in Figure 13.

(13) Contains the hyaluronan synthase gene from Streptococcus zooepidemicus codon-optimized according to Bacillus licheniformis, and the 6-phosphate glucose dehydrogenase gene, UTP-glucose-1-phosphate uracil-transferred gene from Bacillus licheniformis Construction of integrated expression vectors of enzyme gene, N-acetyl-1-phosphate-glucosamine-uracil transferase gene, promotor of Bacillus subtilis fruccanase gene and its regulatory elements.

The α-amylase gene promoter sequence from Bacillus megaterium was directional cloned into the XhoI and HindIII sites of the cloning vector pBlueScriptSK(-) to form the vector pBS-Amy3, and the vector was double-digested with HindIII and BamHI, and the Bacillus licheniformis The 6-phosphate glucose dehydrogenase gene was directional cloned into this vector to form pBS-AU4, pBS-AU4 was digested with BamHI and SpeI, and the UTP-glucose-1-phosphouracil transferase gene was directional cloned here In the vector, construct the pBS-AUT2 vector, and then use SpeI and SacI to double-enzyme digest this vector, and clone the N-acetyl-1-phosphate-glucosamine-uracil transferase gene into this vector to construct pBS-AUT2 The AUTG2 vector was double-digested with XhoI and SacI to recover a 4.3 kb DNA fragment, which was ligated with the pAX-szhas2 vector digested with XhoI and SacI to form a new integrated expression vector pAX-szhas3. Then double digest pAX-szhas3 with BamHI and SacII, and replace the hyaluronic acid of Streptococcus zooepidemicus optimized according to the codon preference of Bacillus subtilis with the hyaluronic acid synthase gene of Streptococcus zooepidemicus optimized according to the codon preference of Bacillus licheniformis The synthetase gene constitutes the new vector pAX-szhas5. The hyaluronic acid synthetase gene of the carrier is transcribed under the control of the fruccanase gene promoter of the bacillus subtilis, and induced and expressed. The 6-phosphate glucose dehydrogenase gene, UTP-glucose-1-phosphotransferase gene, and N-acetyl 1-phosphate-glucosamine-uracil-based transferase gene are found in the α-amylase gene of Bacillus megaterium. Constitutive transcription takes place under the control of the promoter. Therefore the synthesis of hyaluronic acid is induced by sucrose. The map of the integrated expression vector pAX-szhas5 is shown in Figure 14.

(14) Contains the tandem hyaluronan synthase gene from Streptococcus zooepidemicus codon-optimized according to Bacillus subtilis, and the 6-phosphate glucose dehydrogenase gene, UTP-glucose-1-phosphotransferase gene from Bacillus licheniformis Gene, N-acetyl-1-phosphate-glucosamine-uracil transferase gene and the construction of integrated expression vectors for the promotor of Bacillus megaterium fructanase gene.

The fruccanase gene promoter of Bacillus megaterium was directional cloned into the BamHI and XbaI sites of pBlueScript SK(-), and then the hyaluronan synthase gene from Streptococcus zooepidemicus codon-optimized according to Bacillus subtilis was directional cloned Cloning into the XbaI and NotI sites, and then directionally cloning the same copy of the hyaluronan synthase gene from Streptococcus zooepidemicus and optimized according to the codons of Bacillus subtilis into the NotI and SacII sites. Two copies of the gene were added with the ribosome binding site sequence AGGAGGTG. In this way, two copies of the same hyaluronan synthase gene are transcribed under the control of the same promoter. Use BamHI and SacII to excise and recover the PsacB-szhas-szhas expression cassette, connect it to the pAX-szhas5 vector with BamHI and SacII double digestion, and replace the PsacB-szhas expression cassette in pAX-szhas5 to form a new vector pAX-szhas6 . Compared with pAX-szhas5, pAX-szhas6 added a hyaluronan synthase gene.

(15) Containing hyaluronic acid synthase gene from PBCV and codon-optimized according to Bacillus subtilis, D-arabitol dehydrogenase gene from Acetobacter suboxidans, and glucose-6-phosphate dehydrogenase from Bacillus megaterium Construction of the integrated expression vector of the gene and the promotor of the sucrase gene of Bacillus megaterium.

On the basis of the expression vector pAX-AGH2, the erythromycin resistance gene was replaced with the D-arabinitol dehydrogenase gene from Acetobacter suboxidans. SnaBI and SacII were used to double-digest pAX-AGH2, and the D-arabitol dehydrogenase gene was directional cloned into this double-digested vector to obtain vector pAX-AGH3, the map of which is shown in Figure 15 .

(16) Containing the hyaluronic acid synthase gene from Streptococcus zooepidemicus and optimized according to the codon of Bacillus subtilis, and the 6-phosphate glucose dehydrogenase gene and UTP-glucose-1-phosphotransferase gene from Bacillus licheniformis , N-acetyl-1-phosphate-glucosamine-uracil-based transferase gene and Bacillus subtilis fruccanase gene promoter and its regulatory sequence and integrated expression vector of D-arabitol dehydrogenase gene build.

On the basis of the expression vector pAX-szhas5, the hyaluronan synthase gene from Streptococcus zooepidemicus and optimized according to the codons of Bacillus subtilis was used to replace the hyaluronan synthase gene optimized according to the codons of Bacillus licheniformis in pAX-szhas5 Gene, and then double-digested with SacII and SnaBI, the D-arabinitol dehydrogenase gene from Klebsiella pneumoniae was directional cloned into the SacII and SnaBI sites. A new vector pAX-szhas6 was constructed. After the host cell is transformed with this vector, the erythromycin resistance selection marker is not required, but a safe D-arabitol dehydrogenase metabolic marker is used. The map of pAX-szhas6 is shown in Figure 16.

Embodiment 3: Transform the expression vector constructed in embodiment 2 into different Gram-positive cell hosts

This example describes several methods for transforming the expression vector constructed in Example 2 into a Gram-positive cell host to obtain host cells capable of synthesizing hyaluronic acid. In this embodiment, the hosts of Bacillus subtilis cells, Bacillus licheniformis cells, Bacillus megaterium cells and Bacillus brevis cells are listed as synthetic hyaluronic acid, but it does not mean that the present invention does not use other unlisted genetic materials. Lambert-positive host cells.

(1) Transform the constructed integrated expression vector pAX-cvhas2 into Bacillus subtilis cells.

Prepare the competent state of Bacillus subtilis according to the following method: Streak Bacillus subtilis on the LB plate to obtain a single colony, then inoculate the single colony into 3 ml of liquid LB medium, shake it at 37 degrees for 5 hours, and take out 500 microliters Inoculate into 10 ml of SPI liquid medium, shake and culture at 37 degrees for 5 hours to logarithmic growth phase. Then suck out 200 microliters from the SPI medium containing the thalli to 2 milliliters of SPII liquid medium and cultivate it at 37 degrees at 150 rpm for 90 minutes, then add 20 microliters of 10 mmol/L EGTA, and incubate at 37 degrees at 150 rpm. Incubate for 10 minutes, then divide into 200 microliter tubes, add 1 microgram of plasmid vector pAX-cvhas2, shake and cultivate at 37 degrees for 90 minutes, and spread all the bacterial solution on LB containing 4 micrograms/ml erythromycin On the plate, cultured at 37 degrees for 48 hours, 65 single colonies were obtained. Pick 5 strains to extract the total DNA, and use the following pair of primers to amplify the cvhasA gene. A DNA band with a size of 1.7 kb was obtained, which was consistent with the actual size of the hyaluronan synthase gene from PBCV. The PCR verification result is shown in FIG. 17 .

Swimming lanes 1, 2, 3, 5 and 6 in Figure 17 are 1.7 kb DNA obtained by PCR with the total DNA of a single colony resistant to erythromycin.

Swimming lane 4 is the situation after PCR of the total DNA of the control bacteria (single colony not resistant to erythromycin), and no DNA band appears.

M is the DNA molecular weight control of 500bp interval size. From bottom to top: 500bp, 1000bp, 1500bp, 2000bp (the brightest band), 2500bp, 3000bp, etc.

The primer pair used was:

5’-CT TCT AGA ATG GGA AAA AAC ATC ATC ATC ATG G-3’

5’-CT CCGCGG TTA TAC AGA CTG GGC GTT CGT TGT AGC-3’

The amplification conditions are:

Denaturation at 94°C for 40 seconds, annealing at 58°C for 40 seconds, extension at 72°C for 90 seconds, a total of 35 cycles.

The preparation method of the solution is:

SPI solution: Add 1% volume of 50% glucose and 100 times CAFE solution of 1% volume in SP salt solution; wherein the SP salt solution composition is: 0.2% ammonium sulfate, 1.4% dipotassium hydrogen phosphate (with 3 crystal water ), 0.6% potassium dihydrogen phosphate, 0.02% magnesium sulfate heptahydrate, 0.1% sodium citrate. The ingredients of 100 times CAFE solution are: 2% hydrolyzed tyrosine, 10% yeast extract powder.

SPII solution: 1% volume of 50mmol/L calcium chloride solution and 1% volume of 250mmol/L magnesium chloride solution were added to the SPI solution.

(2) Transform the vector pHC-cvhas2 into Bacillus brevis.

The episomal plasmid pHC-cvhas2 was transformed into Bacillus brevis according to the method described in the published literature (Zhou Haiyan et al., Journal of Zhejiang University (Agriculture and Life Sciences), 34(4):389-394, 2008). Then spread on the nutrient broth agar screening plate containing 10 micrograms/ml chloramphenicol and 50 micrograms/ml ampicillin, and cultivate at 37 degrees for 24-36 hours. Extract the plasmid from the grown resistant colony as a template, use the pair of primers described in (1) above to carry out PCR amplification under the same conditions, and obtain a specific DNA band with a size of 1.7kb, which is synthesized with hyaluronic acid from PBCV The actual size of the enzyme gene is the same, and the PCR verification result is shown in Figure 18.

Swimming lanes 2, 3, 4, 5 and 6 in Fig. 18 are 1.7 kb DNA obtained after PCR with the total DNA of a single colony resistant to chloramphenicol and ampicillin.

Swimming lane 1 is the situation after PCR of the total DNA of the control bacteria (a single colony not resistant to chloramphenicol and ampicillin), and no DNA band appears.

M is the DNA molecular weight control of 500bp interval size. From bottom to top: 500bp, 1000bp, 1500bp, 2000bp (the brightest band), 2500bp, 3000bp, etc.

It indicated that the vector pHC-cvhas2 was transformed into Bacillus brevis.

(3) Transform the vector pAX-cvhas5 into Bacillus licheniformis cells.

The integrated plasmid vector pAX-cvhas5 was transformed into Bacillus licheniformis cells according to the method described in the published literature (Mo Jingyan et al., Biotechnology, 19(5); 75-77, 2009). Then spread on the LB screening plate containing 4 μg/ml erythromycin and 50 μg/ml ampicillin, and culture at 37 degrees for 24-36 hours. Extract the total DNA from the grown resistant colony as a template, use the pair of primers described in (1) above to carry out PCR amplification under the same conditions, and obtain a specific DNA band with a size of 1.7kb, which is compatible with hyaluronic acid from PBCV The actual size of the synthetase gene is the same. It indicated that the vector pAX-cvhas5 was transformed into Bacillus licheniformis cells.

(4) Transform the episomal expression vector pNW-szhas2 into Bacillus coagulans.

Cultivate Bacillus coagulans according to the method described in the published literature (Moro A et al, Biotechnology Techniques, 1995, Vol9 (8), pp.589-590), then add the plasmid pNW-szhas2 to the culture medium containing the bacteria, at 1500V Under the conditions of voltage, 25 μF capacitance and 200 ohm resistance, the electroporation transformation was carried out (the instrument used was Bio-Rad Gene Pulser). Aspirate the bacterial solution and add it to the rich medium to mix evenly, spread it in the rich medium containing 15 μg/ml chloramphenicol and culture it at 37°C for 2 days. Extract the plasmid from the grown colony, transform it into Escherichia coli DH5α, culture the extracted plasmid in a rich medium containing 50 μg/ml, and use the following pair of primers for PCR to obtain 1.2kb DNA.

5'-CTTTCTAGAATGCGTACACTGAAAAAATCTTATTACGG-3'

5'-ATCCGCGG TTACAATAATTTTTTGCGCGTTCCCC-3'

It indicated that the vector pNW-szhas2 was transformed into the genome of Bacillus coagulans.

(5) The episomal expression vector pNW-szhas1 was transformed into Bacillus natto.

Cultivate Bacillus natto according to the method described in the published literature (Moro A et al, Biotechnology Techniques, 1995, Vol9 (8), pp.589-590), then add the plasmid pNW-szhas1 in the culture medium containing the bacteria, in Under the conditions of 1200V voltage, 25μF capacitance and 200 ohm resistance, the electroporation transformation was carried out (the instrument used was Bio-Rad Gene Pulser). Aspirate the bacterial solution and add it to the rich medium to mix evenly, spread it in the rich medium containing 15 μg/ml chloramphenicol and culture it at 37°C for 2 days. Extract the plasmid from the grown colony, transform it into Escherichia coli DH5α, culture the extracted plasmid in a rich medium containing 50 μg/ml, and use the following pair of primers for PCR to obtain 1.2kb DNA.

5'-CTTTCTAGAATGCGTACACTGAAAAAATCTTATTACGG-3'

5'-ATCCGCGG TTACAATAATTTTTTGCGCGTTCCCC-3'

The results proved that the expression vector pNW-szhas1 was transformed into the genome of Bacillus natto.

(6) Transform the integrated expression vector pAX-szhas6 into Bacillus stearothermophilus.

Cultivate Bacillus stearothermophilus according to the method described in the published literature (Moro A et al, Biotechnology Techniques, 1995, Vol9 (8), pp.589-590), then add the plasmid pAX-szhas6 in the medium containing the bacteria, Electroporation transformation was carried out under the conditions of 1500V voltage, 25 μF capacitance and 200 ohm resistance (the instrument used was Bio-Rad GenePulser). Aspirate the bacterial solution and add to the rich medium to mix well, spread in the rich medium containing 4 μg/ml erythromycin and culture at 37°C for 2 days. The total DNA was extracted from the grown colonies, and PCR was performed with the following pair of primers to obtain 1.2kb DNA.

5'-CTTTCTAGAATGCGTACACTGAAAAAATCTTATTACGG-3'

5'-ATCCGCGG TTACAATAATTTTTTGCGCGTTCCCC-3'

The results proved that the expression vector pAX-szhas6 had been transformed into the genome of Bacillus stearothermophilus.

(7) Transform the vector pAX-szhas5 into Bacillus megaterium.

The integrated plasmid vector pAX-szhas5 was transformed into Bacillus megaterium cells according to the method described in the published literature (Zhang Xinjian et al., Yunnan Plant Research, 29(6):666-670, 2007). Then spread on the LB screening plate containing 4 μg/ml erythromycin and 50 μg/ml ampicillin, and culture at 37 degrees for 24-36 hours. Extract the total DNA from the grown resistant colony as a template, and use the following pair of primers for PCR amplification to obtain a specific DNA band with a size of 1.2kb, which is consistent with the actual size of the hyaluronic acid synthase gene from Streptococcus zooepidemicus. The PCR verification results are shown in Figure 19.

Swimming lanes 1, 2, 3, 4, 5 and 6 in Fig. 19 are 1.2 kb DNA obtained after PCR with the total DNA of a single colony resistant to erythromycin.

M is the DNA molecular weight control of 500bp interval size. From bottom to top: 500bp, 1000bp, 1500bp, 2000bp (the brightest band), 2500bp, 3000bp, etc.

The primer pair used was:

5'-CTTTCTAGAATGCGTACACTGAAAAAATCTTATTACGG-3'

5'-ATCCGCGG TTACAATAATTTTTTGCGCGTTCCCC-3'

The conditions for PCR amplification are:

Denaturation at 94°C for 40 seconds, annealing at 58°C for 40 seconds, extension at 72°C for 60 seconds, a total of 35 cycles.

Prove that the vector pAX-szhas5 has been transformed into the Bacillus megaterium genome.

Embodiment 4: Lactose-induced fermentation test of genetically engineered gram-positive bacteria producing hyaluronic acid

After transforming Bacillus natto cells with the plasmid vector pNW-szhas1, a single colony resistant to chloramphenicol was obtained. After verification, the resistant colony contained the hyaluronic acid synthase gene, and one strain was selected for a shake-flask fermentation test. First inoculate the bacterial strain in 2 milliliters of LB medium (adding 15 micrograms/microliter of chloramphenicol) and cultivate overnight, then inoculate 1 milliliter of bacterial liquid into a 500 milliliter shaking flask containing 100 milliliters of basic medium (the composition is: KH2PO4 7.0g/L, Na2HPO4 5.0g/L, (NH4)2SO4 5.0g/L, trisodium citrate 2.5g/L, MgSO4.7H2O 2.5g/L, CaCl2.2H2O 0.25g/L, glucose 10g/L , 5 milliliters trace element mother liquor; trace element mother liquor composition is: citric acid 50g/L, FeSO4.7H2O 10g/L, MnSO4.H2O 5g/L, CuSO4.5H2O 1g/L, ZnCl2 2g/L). Under the conditions of 37 degrees, pH value 7.0, and 200 rpm, cultivate for a period of time until the glucose is basically completely consumed, and then add lactose with a final concentration of 1% as an inducer to induce for 36 hours. After the fermentation is over, centrifuge the supernatant and add 3 times Volume of absolute ethanol produces a white flocculent precipitate.

Explain that lactose can induce the synthesis of hyaluronic acid.

Example 5: Xylose-induced fermentation test of hyaluronic acid produced by genetically engineered gram-positive bacteria

After transforming Bacillus amyloliquefaciens cells with the plasmid vector pAX-szhas1, a single colony resistant to erythromycin was obtained. After verification, the resistant colony contained the hyaluronic acid synthase gene, and one strain was selected for a shake-flask fermentation test. First inoculate the bacterial strain in 2 milliliters of LB medium (add 4 micrograms/microliter of erythromycin) and cultivate overnight, then inoculate 1 milliliter of bacterial liquid into a 500 milliliter shake flask containing 100 milliliters of basic medium (the composition is: KH2PO4 7.0g/L, Na2HPO4 5.0g/L, (NH4)2SO4 5.0g/L, trisodium citrate 2.5g/L, MgSO4.7H2O 2.5g/L, CaCl2.2H2O 0.25g/L, glucose 10g/L , 5 milliliters trace element mother liquor; trace element mother liquor composition is: citric acid 50g/L, FeSO4.7H2O 10g/L, MnSO4.H2O 5g/L, CuSO4.5H2O 1g/L, ZnCl2 2g/L). Under the conditions of 37 degrees, pH value 7.0, and 200 rpm, cultivate for a period of time until the glucose is basically consumed completely, and then add xylose with a final concentration of 0.5% as an inducer for 48 hours. After the fermentation, centrifuge to take the supernatant and add 3 Doubling the volume of absolute ethanol produces a white flocculent precipitate.

It shows that xylose can induce the synthesis of hyaluronic acid.

Embodiment 6: Sucrose-induced fermentation test of hyaluronic acid produced by genetically engineered Gram-positive bacteria

After transforming Bacillus licheniformis cells with plasmid vector pAX-cvhas5, a single colony resistant to erythromycin was obtained. After verification, the resistant colony contained hyaluronic acid synthase gene, and one strain was selected for shake-flask fermentation test. First inoculate the bacterial strain in 2 milliliters of LB medium (add 4 micrograms/microliter of erythromycin) and cultivate overnight, then inoculate 1 milliliter of bacterial liquid into a 500 milliliter shake flask containing 100 milliliters of basic medium (the composition is: KH2PO4 7.0g/L, Na2HPO4 5.0g/L, (NH4)2SO4 5.0g/L, trisodium citrate 2.5g/L, MgSO4.7H2O 2.5g/L, CaCl2.2H2O 0.25g/L, glucose 5g/L , 5 milliliters trace element mother liquor; trace element mother liquor composition is: citric acid 50g/L, FeSO4.7H2O 10g/L, MnSO4.H2O 5g/L, CuSO4.5H2O 1g/L, ZnCl2 2g/L). Under the conditions of 37 degrees, pH value 7.0, and 200 rpm, cultivate for a period of time until the glucose is basically completely consumed, then add 2% sucrose as an inducer to induce the synthesis of hyaluronic acid, and continue to cultivate until the end of fermentation. After the fermentation was finished, the fermented liquid became more viscous, and the supernatant was collected by centrifugation and added with 3 times the volume of absolute ethanol to produce white flocculent precipitates.

It shows that sucrose can induce the synthesis of hyaluronic acid.

Embodiment 7: Fermentation tank fermentation test of genetically engineered gram-positive bacteria producing hyaluronic acid

After transforming Bacillus subtilis cells with the integrated expression vector pAX-szhas6, erythromycin-resistant colonies were obtained. After verification, the resistant colonies contained the hyaluronic acid synthase gene, and one strain was selected for fermenter fermentation test. First inoculate the strain in 4 ml of LB medium (add 4 μg/μl of erythromycin) and cultivate overnight to obtain the first-level test tube seeds, and then inoculate 1 ml of the bacterial liquid into a 500 ml shake flask containing 100 ml of basic medium Secondary shake flask seeds were obtained by medium culture, and 400 milliliters of secondary seeds were co-cultivated. Put all the 400 ml shake flask seeds into a 3.0 liter fermenter, cultivate for a period of time under the conditions of 37 degrees, pH 7.0, and 600 rpm until the glucose is basically completely consumed, then reduce the stirring speed to 400 rpm , then add sucrose to make the final concentration reach 2.5%, and at the same time add yeast extract powder as a nitrogen source, and continue to cultivate until the end of fermentation. After the fermentation is over, the fermented liquid becomes more viscous. Take 10 milliliters of the fermented liquid, centrifuge the supernatant and add 3 times the volume of absolute ethanol to produce a white flocculent precipitate. The white flocculent precipitate produced is shown in Figure 20. The white flocculent precipitate was preserved in absolute ethanol.

The basic medium components are: KH ₂ PO ₄ 7.0g/L, Na ₂ HPO ₄ 5.0g/L, (NH4) ₂ SO ₄ 5.0g/L, trisodium citrate 2.5g/L, MgSO ₄ .7H ₂ O 2.5g/L, CaCl ₂ .2H ₂ O 0.25g/L, glucose 5g/L, 5ml trace element mother liquor; the composition of trace element mother liquor is: citric acid 50g/L, FeSO ₄ .7H ₂ O 10g/L, MnSO _4. H ₂ O 5g/L, CuSO ₄ .5H ₂ O 1g/L, ZnCl ₂ 2g/L. The concentration of the sucrose rehydration bottle is 50% (mass volume percentage), and the concentration of the yeast extract powder rehydration bottle is 20% (mass volume percentage).

The refining of hyaluronic acid in the fermented liquid of embodiment 8

Get embodiment 7 to obtain fermented liquid 800mL, add chlorobenzene while stirring, add altogether 200 milliliters, with 5000rpm centrifugal 10min, remove thalline precipitation, add people's 10g sodium chloride in the supernatant and fully dissolve, then add 3 Double the volume of 95% ethanol, mix and stir, let stand for 30 minutes, and centrifuge at 3000 rpm for 10 minutes to obtain a white precipitate that is the HA extract.

Concentration is the NaHCO of 4.2g/L Aqueous solution 450mL and concentration are the Na of 5.3g/L CO Aqueous solution 50mL is added in the above-mentioned extract, then add the trypsin of 20mg (per mg protein adds the enzyme amount of 100 activity units), in Stir and hydrolyze at 35°C and 120rpm for 2h. Then add sodium chloride to the above solution until the concentration reaches 10g/L and stir to dissolve it completely, then add 3 times the volume of 95% ethanol, let stand for 1h to coagulate and precipitate, and centrifuge at 3000rpm to obtain the precipitate of hyaluronic acid.

The precipitation obtained is added to the 500mL aqueous solution containing NaCl concentration of 10g/L, Na2HPO4 concentration of 0.2g/L, NaH2PO4 concentration of 0.06g/L, stirring and dissolving, adding 3 times of volume of 95% ethanol wherein, Mix and stir, centrifuge at 3000rpm to recover hyaluronic acid precipitate, wash with 30mL 80% ethanol, and dry to obtain solid hyaluronic acid. Dissolve the solid HA obtained above in a 400mL aqueous solution with a Na2HPO4 concentration of 0.1g/L, a NaH2PO4 concentration of 0.05g/L, and a NaCl concentration of 10g/L, stir and dissolve, and then add 5g of acid-pretreated HA to the solution. Activated carbon was stirred at 80 rpm at 50° C. for 1 h, and then filtered through a 0.22 m filter membrane to obtain a clear filtrate. The filtrate was dialyzed through a 35000 Da dialysis bag against 1000 mL deionized water overnight at 4°C. Add 3 times the volume of 95% ethanol into the dialysis bag, mix and stir to obtain flocculent hyaluronic acid, centrifuge to obtain a precipitate, and wash the precipitate with 20 mL of acetone. The solid HA is frozen, vacuum-dried and ground to obtain a white powder hyaluronic acid refined product.

The medicines used are all medical-grade medicines, and are operated in a clean environment with an air purification level of 10,000.

By adopting the method, the hyaluronic acid pure product conforming to the cosmetic level and external medicine can be prepared from the fermented liquid.

Example 9: Infrared detection and identification of hyaluronic acid prepared by fermentation of genetically engineered gram-positive bacteria

The white hyaluronic acid obtained in Example 8 was pressed into potassium bromide tablets, and then detected and analyzed by infrared spectroscopy to obtain a characteristic infrared absorption spectrum, which was compared with the characteristic infrared absorption spectrum of standard hyaluronic acid. The results can be seen It is found that the white flocculent precipitate produced by genetic engineering is hyaluronic acid. The infrared absorption spectrum of the white flocculent precipitate produced by the secretion of genetically engineered Gram-positive bacteria is shown in Figure 21.

It can be seen from Figure 21 that there is a strong OH stretching vibration around 3400cm ^-1 , indicating that the substance has a polyhydroxyl structure, and hyaluronic acid belongs to mucopolysaccharides containing polyhydroxyl groups. There are strong absorptions at 1620, 1043 and 1542cm ^-1 , indicating that it contains C=O, CN stretching vibration and NH bending vibration, indicating that the substance contains acetamido structure, and hyaluronic acid contains acetamido. There is an O=CO stretching vibration absorption band at 1427cm ^-1 , indicating the presence of dissociated carboxyl and hydroxyl structures on glucuronic acid. There is no absorption band at 1240, 800-850 cm ^-1 , indicating that the substance does not contain sulfated groups. The infrared absorption peak of the substance coincides completely with the standard characteristic absorption peak of hyaluronic acid, indicating that the white flocculent precipitate produced by genetically engineered Gram-positive bacteria is hyaluronic acid mucopolysaccharide.

The method can prove that the white flocculent precipitate produced by the genetically engineered Gram-positive bacteria is hyaluronic acid.

Embodiment 10: Preparation of body lotion containing hyaluronic acid produced by genetically engineered Gram-positive safe microorganisms Prepare a body lotion containing hyaluronic acid according to the following formula: (units are mass volume percentages)

Element content Fat-soluble licorice extract 0.1% Methylparaben 0.05% Linoleic acid 0.5% citric acid 0.05% Linolenic acid 0.5% Sodium citrate 0.05%

Glycerin 6.0% Fragrance 0.1% ethanol 5.0% Water Soluble Placenta Extract 1.0% Hydrogenated castor oil polyoxyethylene ether 0.8% Coenzyme Q10 0.3% The hyaluronic acid prepared by embodiment 8 1.0% Deionized water 85.0%

The body lotion prepared by this method can effectively prevent the dryness of the skin and keep the skin lubricated.

Embodiment 11: Preparation of moisturizing face cream containing hyaluronic acid produced by genetically engineered Gram-positive safe microorganisms According to the formula in the following table, a moisturizing face cream containing hyaluronic acid was prepared: (units are mass volume percentages)

Element content (%) Group A: Lanolin 10.00 mineral spirits 13.00 fatty acid 3.00 Glyceryl monostearate 3.00 cetyl alcohol 4.00 Tween 85 1.00 Propylparaben 0.10 Methylparaben 0.15 Group B: polyacrylic acid resin (Carbopol 940, 3% solution) 5.00 Deionized water 52.25 Propylene Glycol 5.00

Group C: Triethanolamine (85-87%) 1.00 The hyaluronic acid prepared by embodiment 8 1.5

Add polyacrylic acid resin to water, mix thoroughly, and then add propylene glycol; mix the ingredients in group A according to the content relationship and heat to 80°C, and mix the ingredients in group B according to the content relationship and heat to 80°C. Then add group A to group B and mix well. Add the ingredients of Group C to the mixture of Groups A and B and stir to cool.

The emollient face cream prepared by the method can effectively prevent dryness of the skin and keep the skin smooth and clean.

Example 12: Preparation of Eye Moisturizing Solution Containing Hyaluronic Acid Produced by Genetically Engineered Gram-positive Safe Microorganisms According to the formula in the following table, an eye moisturizing solution containing hyaluronic acid was prepared: (units are mass volume percentages)

Element content Centella Asiatica Extract 0.25ml Pearl Extract 0.25ml Cassia Seed Extract 0.025g Vitamin _B6 0.005g Taurine 0.01g aspartic acid 0.005g The hyaluronic acid that embodiment 8 makes 0.010g Menthol 0.005g Sterile deionized water 14.5ml

The eye drops prepared in Example 12 can effectively relieve dry eyes.

Claims (10)

1. a heterologous synthesis method of hyaluronic acid based on Gram-positive safe microorganisms, characterized in that: comprise the following steps: The first step is to isolate genes related to the synthesis of hyaluronic acid precursors, UDP-glucuronic acid and UDP-N-acetyl-glucosamine, including: UDP-glucose dehydrogenase from Gram-positive safe microbial hosts Genes, namely SEQ ID No.31, SEQ ID No.32, SEQ ID No.33, SEQ ID No.34; UTP-glucose-1-phosphotransferase genes, namely SEQ ID No.35, SEQ ID No.36 , SEQ ID No.37; glucose-6-phosphate mutase gene, i.e. SEQ ID No.38, SEQ ID No.39; glucose-6-phosphate isomerase gene, i.e. SEQ ID No.40; aminotransferase , namely SEQ ID No.41 and N-acetyl-1-phosphate-glucosamine-uracil transferase gene, namely SEQ ID No.42 and SEQ ID No.43; The second step, according to the codon preference of the Gram-positive safe microbial host described in step 1, to SEQ ID No.44, SEQ ID No.45, SEQ ID No.46, SEQ ID No.47, SEQ ID No .48 base substitutions were performed to obtain a total of 27 different sequences from SEQ ID No.1 to SEQ ID No.27; In the third step, any gene from SEQ ID No.1 to SEQ ID No.27 and more than one gene from SEQ ID No.31 to SEQ ID No.43 gene and a constitutive promoter or an inducible promoter Substitutes form a gene expression cassette; The fourth step is to transform the gene expression cassette into Gram-positive microbial host bacteria by means of electroporation, preparation of protoplasts or preparation of competent cells, and screening with selection markers to obtain Gram-positive microorganisms capable of secreting hyaluronic acid. Positive genetically engineered safe host bacteria; The fifth step is to ferment and cultivate Gram-positive genetically engineered safe host bacteria, and add an inducer to induce hyaluronic acid synthesis during the cultivation stage to improve the synthetic level of hyaluronic acid. After the fermentation is completed, it is separated and purified from the medium to obtain Hyaluronic acid based on Gram-positive safe microorganisms; The hyaluronic acid is a straight-chain macromolecular acidic mucopolysaccharide formed by alternately linking D-glucuronic acid and N-acetylglucosamine with disaccharide units, and its molecular weight is 50,000 to 5 million Daltons. 2. the heterologous synthesis method of the hyaluronic acid based on Gram-positive safe microorganism according to claim 1, it is characterized in that, described Gram-positive safe microorganism host refers to any one in the following bacteria: Clostridium butyricum or butyricum, Bacillus licheniformis, Bacillus amyloliquefaciens, beneficial or nontoxic Bacillus cereus, Bacillus brevius, Bacillus pumilus, Bacillus brevius, Bacillus stearothermophilus, Bacillus megaterium, Bacillus natto, Bacillus subtilis, Geobacillus stearothermophilus, Bacillus coagulans, Bacillus lentus or Bacteroides amylovora. 3. the heterologous synthesis method based on the hyaluronic acid of Gram-positive safe microorganism according to claim 1, it is characterized in that, described containing SEQ ID No.44, SEQ ID No.45, SEQ ID No. 46. The microorganisms with genes of SEQ ID No.47 and SEQ ID No.48 are: Streptococcus zooepidemicus, Streptococcus equiepidemicus, Paramecium chlorella virus 1, Streptococcus pyogenes and Streptococcus uberis respectively. 4. The method for heterologous synthesis of hyaluronic acid based on Gram-positive safe microorganisms according to claim 1, wherein said base substitution refers to: performing code according to the codon preference of the host genome Sub-optimization, the genes related to hyaluronic acid synthesis from other non-host bacteria are base-substituted according to the codon preference adopted by the genes of the host bacteria when they are translated into proteins. 5. The method for heterologous synthesis of hyaluronic acid based on Gram-positive safe microorganisms according to claim 1, wherein said gene expression cassette comprises a constitutive promoter or a chemically induced promoter, wherein: A constitutive promoter refers to a promoter that is transcriptionally active at any stage of the culture of the host bacterium; the inducible promoter refers to a promoter that has transcription activity only during a specific period of the culture of the host bacterium, specifically including: Chemically inducible promoters and physically inducible promoters. 6. the heterologous synthesis method of hyaluronic acid based on Gram-positive safe microorganism according to claim 5, is characterized in that, described chemical induction promoter comprises: the initiation of the fruccanase gene of bacillus megaterium promoter of the xylose isomerase gene of Bacillus megaterium, the promoter of the fructanase gene of Bacillus subtilis, the promoter of the xylose isomerase gene of Bacillus subtilis, the arabinose utilization operator of Escherichia coli The promoter of the lactose utilization operon of Escherichia coli, the promoter of the lactose utilization operon of Escherichia coli. 7. the heterologous synthesis method based on the hyaluronic acid of Gram-positive safe microorganism according to claim 6, it is characterized in that, the transcription of described chemical induction promoter refers to: The promoter of the fruccanase gene may initiate the transcription of its downstream genes only after adding sucrose, and the concentration of sucrose is 0.5% to 10%; The promoter of the xylose isomerase gene may initiate the transcription of its downstream genes only after xylose is added, and the mass percentage concentration of xylose is 0.5% to 8%. The promoter of the arabinose utilization operon may initiate the transcription of its downstream genes only after adding arabinose, and the mass percent concentration of arabinose is 0.5% to 8%; The promoter of the lactose utilization operon may initiate the transcription of its downstream genes only after adding lactose, and the mass percent concentration of lactose is 0.5% to 8%. 8. The method for heterologous synthesis of hyaluronic acid based on Gram-positive safe microorganisms according to claim 5, characterized in that, the physically induced promoter comprises: a thermal gene derived from the groESL gene of Bacillus subtilis Inducible promoters, heat-inducible promoters from the dnaK gene of Bacillus subtilis, and heat-inducible promoters from the groE gene of Bacillus licheniformis. 9. The method for heterologous synthesis of hyaluronic acid based on Gram-positive safe microorganisms according to claim 1, characterized in that, the selection marker refers to: be used to effectively screen for genes containing hyaluronic acid synthesis-related Genes of Gram-positive safe microbial hosts. 10. The method for heterologous synthesis of hyaluronic acid based on Gram-positive safe microorganisms according to claim 1, wherein said fermentation culture refers to: 5-20 g/L of glucose, yeast infusion Powder 0.5-5 g/L, magnesium sulfate heptahydrate 0.3-3 g/L, potassium dihydrogen phosphate 3-8 g/L, disodium hydrogen phosphate 2-8 g/L, ammonium sulfate 2-8 g/L, Sodium citrate 2-5 g/L, ferrous sulfate heptahydrate 1-20 mg/L, manganese sulfate monohydrate 1-20 mg/L, anhydrous copper sulfate 0.2-10 mg/L, zinc chloride 0.2-10 mg/L, adjust the pH to 6.5-7.5 with citric acid or sodium hydroxide; the fermentation temperature is 28-37 degrees, and the stirring speed is 300-600 rpm.

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