阅读说明：本技术 一种提高透明质酸产量的方法及菌株 (Method and strain for improving hyaluronic acid yield ) 是由 刘浩 高伟霞 张晓亮 张广通 于 2020-11-23 设计创作，主要内容包括：本发明涉及一种提高透明质酸产量的方法与菌株,将glnA基因插入pSET4S::repA::Ppgk(pLH275)载体的多克隆位点处,构建成功pSET4S::repA::Ppgk::GlnA(pLH860)；转入兽疫链球菌ATCC 39920,获得的菌株glnA/OP的HA产量明显高于初始菌株。以菌株glnA/OP进行透明质酸的发酵罐发酵生产,10h时其透明质酸的产量比野生型提高了70％,发酵进行到16h时比野生型提高了21％,最终发酵24h时,工程菌glnA/OP HA产量为3.55g/L,较野生型提高了7.2％。为提升透明质酸产量提供了新方法,并获得一株高产透明质酸的菌株。(The invention relates to a method and a strain for improving the yield of hyaluronic acid, wherein a glnA gene is inserted into a polyclonal locus of a pSET4S vector, repA vector, Ppgk vector (pLH275), and pSET4S vector, repA vector, Ppgk vector, GlnA vector (pLH860) are successfully constructed; the strain obtained by transferring the streptococcus zooepidemicus ATCC39920 HAs obviously higher HA yield than the original strain. When the strain glnA/OP is used for fermentation production of the hyaluronic acid in a fermentation tank, the yield of the hyaluronic acid is improved by 70% compared with that of a wild type after 10 hours, the yield of the hyaluronic acid is improved by 21% compared with that of the wild type after 16 hours of fermentation, and the yield of the engineering strain glnA/OP HA is 3.55g/L and is improved by 7.2% compared with that of the wild type after 24 hours of final fermentation. Provides a new method for improving the yield of hyaluronic acid and obtains a strain with high hyaluronic acid yield.)

1. A genetically recombinant plasmid for increasing production of hyaluronic acid, comprising: the genetic recombinant plasmid is characterized in that a glnA gene is inserted into a gene expression vector pSET4S: repA: Ppgk is a polyclonal site of pLH275, and pSET4S: repA: Ppgk: GlnA is pLH860 is successfully constructed;

wherein the nucleotide sequence of the gene expression vector pSET4S repA Ppgk is SEQ ID NO. 2.

2. The genetic recombinant plasmid for increasing hyaluronic acid production according to claim 1, wherein: the length of the glnA gene is 1382bp, the nucleotide sequence is SEQ ID NO.1, the glnA gene starts from 15bp at the upstream of an initiation codon ATG and ends at 20bp at the downstream of a termination codon TAA.

3. The genetic recombinant plasmid for increasing hyaluronic acid production according to claim 1 or 2, wherein: the glnA gene is derived from the genome of Streptococcus zooepidemicus (Streptococcus zoepidemicus) ATCC 39920.

4. An engineered bacterium comprising the genetic recombinant plasmid for enhancing production of hyaluronic acid according to any one of claims 1 to 3.

5. The engineered bacterium of claim 4, wherein: the construction method of the engineering bacteria comprises the following steps:

transferring the genetic recombinant plasmid for improving the yield of the hyaluronic acid into host bacteria to construct engineering bacteria; wherein the host bacterium is Streptococcus zooepidemicus (Streptococcus zoepidemicus) ATCC 39920.

6. The engineered bacterium of claim 5, wherein: the transfer is electrotransfer.

7. Use of the engineered bacterium of any one of claims 4 to 6 for increasing the production of hyaluronic acid.

8. Use according to claim 7, characterized in that: the application is the application in the aspect of fermentation production of hyaluronic acid by taking sucrose as a substrate.

9. A method for highly producing hyaluronic acid by using the engineered bacterium of any one of claims 4 to 6, wherein: obtained by fermenting the engineering bacteria.

10. The method for highly producing hyaluronic acid according to claim 9, wherein the method comprises: the method comprises the following steps: inoculating engineering bacteria into a fermentation culture medium of engineering bacteria according to the inoculation amount of 2-8%, fermenting at 37 ℃ and pH7.0 at the rotation speed of 150-;

wherein the fermentation medium of the engineering bacteria is as follows: sucrose: 30-50g/L, yeast extract: 2-3.5g/L, casein peptone: 8-10g/L, NaCl: 1-1.5g/L, K₂HPO₄：2-7g/L，MgSO₄·7H₂O：0.4-0.6g/L。

Technical Field

The invention belongs to the technical field of genetic engineering, and particularly relates to a method and a strain for improving the yield of hyaluronic acid.

Background

Hyaluronic Acid (HA) HAs great commercial value in the aspects of cosmetics, clinical medicine, health products and the like due to its moisture retention, biocompatibility and non-immunity. In the cosmetic industry, HA is increasingly regarded by the cosmetic industry at home and abroad as a cosmetic additive with excellent properties, and HAs a very considerable application prospect in cosmetics. In clinical medicine, HA is widely distributed in the outer matrix of soft connective tissue cells, and HAs been widely used in clinical medicine due to its good biodegradability and biocompatibility. In health food, the health food containing HA can increase the precursor of HA synthesis in human body, increase HA content in human body, protect joint, increase skin elasticity, and resist aging.

Currently, microbial fermentation is one of the major methods for HA production. However, the production of HA synthesized by microbial fermentation still HAs many problems at present, for example, the rate-limiting step of HA synthesis is not clear, and the metabolic pathway modification target for improving HA yield is less; after the HA yield reaches 4-5g/L, the viscosity of the culture medium is as high as 400-500mPas, which can seriously affect dissolved oxygen and mass transfer, thereby limiting the HA yield; HA synthesis competes with bacterial growth for precursor substances, UDP-N-acetylglucosamine and UDP-glucuronic acid.

Through searching, no patent publication related to the present patent application has been found.

Disclosure of Invention

The invention aims to overcome the problems that in the prior art, the market HAs great demand on HA, the existing strain improvement method for improving the HA yield is less, and the yield improvement difficulty is higher, and provides a method and a strain for improving the HA yield, so that the method for improving the HA yield is enriched.

The technical scheme adopted by the invention for solving the technical problems is as follows:

a genetic recombinant plasmid for improving the yield of hyaluronic acid is characterized in that a glnA gene is inserted into a gene expression vector pSET4S: repA: Ppgk is a polyclonal site of pLH275, and pSET4S: repA: Ppgk: GlnA is pLH860 is successfully constructed;

wherein the nucleotide sequence of the gene expression vector pSET4S repA Ppgk is SEQ ID NO. 2.

Moreover, the length of the glnA gene is 1382bp, the nucleotide sequence is SEQ ID NO.1, the glnA gene starts from 15bp at the upstream of an initiation codon ATG and ends at 20bp at the downstream of a termination codon TAA.

Moreover, the glnA gene is derived from the genome of Streptococcus zooepidemicus (Streptococcus zoepidemicus) ATCC 39920.

An engineering bacterium containing the genetic recombinant plasmid for improving the yield of the hyaluronic acid.

Moreover, the construction method of the engineering bacteria comprises the following steps:

transferring the genetic recombinant plasmid for improving the yield of the hyaluronic acid into host bacteria to construct engineering bacteria; wherein the host bacterium is Streptococcus zooepidemicus (Streptococcus zoepidemicus) ATCC 39920.

Moreover, the transfer is an electrotransfer.

The application of the engineering bacteria in improving the yield of the hyaluronic acid.

Moreover, the application is the application in the aspect of fermentation production of hyaluronic acid by taking sucrose as a substrate.

A method for highly producing hyaluronic acid by using the strain, which is obtained by fermentation by using the engineering bacteria.

Moreover, the steps are as follows: taking a fermentation culture medium of the strain, inoculating engineering bacteria into the fermentation culture medium according to the inoculation amount of 2-8%, fermenting at 37 ℃, pH7.0, the rotation speed of 150-;

wherein the fermentation medium of the engineering bacteria is as follows: sucrose: 30-50g/L, yeast extract: 2-3.5g/L, casein peptone: 8-10g/L, NaCl: 1-1.5g/L, K₂HPO₄：2-7g/L，MgSO₄·7H₂O：0.4-0.6g/L。

The invention has the advantages and positive effects that:

1. the HA yield of the strain (glnA/OP) is obviously higher than that of the original strain, the strain glnA/OP is used for performing fermentation production of HA in a 1.2L fermentation tank, the HA yield of the original strain is 1.3g/L when the fermentation is performed for 10 hours, and the glnA/OP HA yield of the strain is 2.2g/L, which is improved by 70 percent compared with the original strain; when the fermentation is carried out for 12 hours, the yield of the spawn-producing HA is 1.76g/L, the yield of the strain glnA/OP HA is 1.97g/L, and is improved by 10 percent compared with the spawn-producing HA; when fermentation is carried out for 16 hours, the yield of the grown strain HA is 2.26g/L, the yield of the strain glnA/OP HA is 2.75g/L, and the yield is improved by 21 percent; when the fermentation is carried out for 16 hours, the sucrose serving as a carbon source is basically consumed, but the thalli still have activity, so that 30g of sucrose is supplemented, the yield of the starting strain and the yield of the strain glnA/OP HA are improved when the fermentation is carried out for 20 hours, the yield of the starting strain HA is 2.71g/L, the yield of the strain glnA/OP HA is 3.64g/L, and the yield is improved by 34%; the yield of the grown strain HA is 3.31g/L and the yield of the strain glnA/OP HA is 3.55g/L after fermentation is carried out for 24 hours, which is improved by 7.2 percent. Provides a new effective target for improving the metabolic modification of hyaluronic acid yield.

2. The expression vector pSET4S, repA, Ppgk, GlnA (pLH860) constructed by the invention can stably replicate in streptococcus zooepidemicus, and antibiotics do not need to be added in the fermentation culture process.

3. The present invention has been accomplished on the basis of the intensive studies of the glnA glutamine synthetase gene by the present applicant. Glutamine is the major nitrogen source donor in bacterial cells. Intracellular glutamine is synthesized by glutamic acid and amino under the catalysis of glutamine synthetase GlnA, transaminase GlmS transfers the amino of glutamine to fructose-6-phosphate to generate glucosamine-6-phosphate, glucosamine-1-phosphate is generated under the action of mutase GlmM, and then another precursor UDP-N-acetylglucosamine (UDP-GlcNAc) required by hyaluronic acid synthesis is generated under the action of acetyltransferase and pyrophosphorylase GlmU, so that the yield of hyaluronic acid is improved. Based on this finding, the present invention HAs constructed a novel HA high-producing strain.

Drawings

FIG. 1 is a glnA gene expression vector in the present invention;

FIG. 2 is a PCR verification chart of the colonies of the glnA gene expression vector of the present invention; wherein M is a DNA Marker, N is a negative control, P is a positive control, a genome is used as a template, 1-10 are PCR amplified glnA genes, and the size is 2318 bp;

FIG. 3 is a restriction enzyme digestion verification diagram of the glnA gene expression vector of the present invention; wherein M is a DNA Marker; 1.2, 3 and 4 are EcoR I and BamH I double enzyme digestion verification plasmids;

FIG. 4 is a graph showing the growth of strain glnA/OP constructed in the present invention and WT shake flask fermentation;

FIG. 5 is a graph of HA yield from shake flask fermentation of strain glnA/OP and WT constructed in the present invention;

FIG. 6 is a graph showing the production of fermentation in a fermentor for strain glnA/OP and WT constructed in the present invention;

FIG. 7 is a graph showing HA productivity in fermentation in a fermentor of strain glnA/OP and WT constructed in the present invention;

FIG. 8 is a graph showing the residual amount of sucrose fermented in a fermentor of strain glnA/OP and WT constructed in the present invention;

Detailed Description

The following detailed description of the embodiments of the present invention is provided for the purpose of illustration and not limitation, and should not be construed as limiting the scope of the invention.

The raw materials used in the invention are conventional commercial products unless otherwise specified; the methods used in the present invention are conventional in the art unless otherwise specified.

A genetic recombinant plasmid for improving the yield of hyaluronic acid is characterized in that a glnA gene is inserted into a gene expression vector pSET4S: repA: Ppgk is a polyclonal site of pLH275, and pSET4S: repA: Ppgk: GlnA is pLH860 is successfully constructed;

wherein the nucleotide sequence of the gene expression vector pSET4S repA Ppgk is SEQ ID NO. 2.

Preferably, the length of the glnA gene is 1382bp, the nucleotide sequence is SEQ ID NO.1, the glnA gene starts from 15bp upstream of an initiation codon ATG and ends at 20bp downstream of a termination codon TAA.

Preferably, the glnA gene is derived from the genome of Streptococcus zooepidemicus (Streptococcus zoepidemicus) ATCC 39920.

An engineering bacterium containing the genetic recombinant plasmid for improving the yield of the hyaluronic acid.

Preferably, the construction method of the engineering bacteria comprises the following steps:

transferring the genetic recombinant plasmid for improving the yield of the hyaluronic acid into host bacteria to construct engineering bacteria; wherein the host bacterium is Streptococcus zooepidemicus (Streptococcus zoepidemicus) ATCC 39920.

Preferably, the transfer is an electrotransfer.

The application of the engineering bacteria in improving the yield of the hyaluronic acid.

Preferably, the application is the application in the aspect of fermentation production of hyaluronic acid by using sucrose as a substrate.

A method for highly producing hyaluronic acid by using the engineering bacteria, which is obtained by fermenting the engineering bacteria.

Preferably, the steps are as follows: inoculating engineering bacteria into a fermentation culture medium of engineering bacteria according to the inoculation amount of 2-8%, fermenting at 37 ℃, pH7.0, rotation speed of 150-;

wherein the fermentation medium of the engineering bacteria is as follows: sucrose: 30-50g/L, yeast extract: 2-3.5g/L, casein peptone: 8-10g/L, NaCl: 1-1.5g/L, K₂HPO₄：2-7g/L，MgSO₄·7H₂O：0.4-0.6g/L。

More specifically, the steps are as follows:

and (3) shaking flask fermentation:

taking out strains from a refrigerator at the temperature of minus 80 ℃, scribing on a THY solid culture medium, culturing for 12h at 37 ℃ at the rotating speed of 200r/min, selecting a single grown colony, inoculating the single colony into a test tube filled with 4mL of THY liquid culture medium, culturing for 12h at 37 ℃ at the rotating speed of 200r/min, inoculating the bacterial liquid into a 250mL conical flask filled with 50mL of THY liquid culture medium by 1 percent of inoculation amount, culturing for 12h at 37 ℃ at the rotating speed of 200r/min, inoculating the bacterial liquid into a 500mL conical flask filled with 100mL of FSB fermentation culture medium by 1 to 4 percent of inoculation amount, fermenting for 24h at 37 ℃, pH7.0 and the rotating speed of 150-;

wherein the FSB fermentation medium is: sucrose: 30-50g/L of yeast extractFetching: 2-3.5g/L, casein peptone: 8-10g/L, NaCl: 1-1.5g/L, K₂HPO₄：2-7g/L，MgSO₄·7H₂O：0.4-0.6g/L。

Fermentation in a fermentation tank:

taking out strains from a refrigerator at minus 80 ℃, streaking on a THY solid culture medium, culturing for 12h at 37 ℃ at the rotating speed of 200r/min, selecting a single colony which grows out, inoculating the single colony into a test tube containing 4mL of THY liquid culture medium, culturing for 12h at 37 ℃ at the rotating speed of 200r/min, inoculating the bacterial liquid into a 500mL conical flask containing 100mL of TSB seed culture medium at the inoculation amount of 4%, culturing for 12h at 37 ℃ at the rotating speed of 200r/min, inoculating the seed liquid into a 2L fermentation tank containing 1.2LFSB fermentation culture medium at the inoculation amount of 2-8%, fermenting for 16-24h at 37 ℃, pH7.0, rotating speed of 150-.

Wherein the THY culture medium is as follows: soaking beef in powder: 10.0g/L, tryptone: 20.0g/L, glucose: 2.0g/L, yeast extract: 2.0g/L of NaHCO₃：2.0g/L，NaCl：2.0g/L，Na₂HPO₄：0.4g/L。

The TSB seed culture medium is: soybean peptone: 3.0g/L, tryptone: 17.0g/L, anhydrous glucose: 2.5g/L, NaCl: 5.0g/L, K₂HPO₄：2.5g/L。

The fermentation medium is as follows: sucrose: 30-50g/L, yeast extract: 2-3.5g/L, casein peptone: 8-10g/L, NaCl: 1-1.5g/L, K₂HPO₄：2-7g/L，MgSO₄·7H₂O：0.4-0.6g/L。

Specifically, the preparation and detection are as follows:

the gene glnA is cloned to a vector pSET4S:: repA:: Ppgk (pLH275) by taking a Streptococcus zooepidemicus ATCC39920 genome as a template, and after colony PCR (figure 2) EcoRI/BamHI double enzyme digestion (figure 3) and sequencing verification, a glnA overexpression vector pSET4S:: repA:: Ppgk:: GlnA (figure 1) is successfully constructed. And (3) electrically transferring the expression vector to a host bacterium streptococcus zooepidemicus ATCC39920 starting strain to successfully obtain an overexpression strain glnA/OP. The strain is subjected to shake flask fermentation and tank fermentation analysis.

The method for producing HA by shake flask fermentation is as follows: respectively inoculating the WT and glnA/OP strains into an FSB liquid culture medium, performing fermentation culture, sampling every 2h for the first 16h, sampling every 24h every other day, and finishing fermentation. The HA production was obtained by sample treatment and analysis. The fermentation culture conditions are that the temperature is 37 ℃, the culture time is 24h, the pH value is 7.0 and the rotating speed is 200 r/min.

In the process of shake flask fermentation, the growth conditions of the strain glnA/OP and the starting strain WT are not obviously different, the strain glnA/OP and the strain enter a logarithmic growth phase within about 4 hours, the logarithmic growth phase is finished within about 6 hours, the strain reaches a stationary phase within 8 hours, and the growth of the strain is slowed (fig. 4). When the strain is fermented in a shake flask for 12 hours, the HA yield of the strain glnA/OP is 0.44g/L, which is 12.82% higher than that of the strain WT; after the strain is fermented for 24 hours in a shake flask, the HA yield of the strain glnA/OP is 0.48g/L, which is improved by 9.09 percent compared with the original strain (figure 5).

The method for producing HA by tank fermentation comprises the following steps: respectively inoculating WT and glnA/OP strains into an FSB liquid culture medium, performing fermentation culture, collecting fermentation liquor every 2 hours within 12 hours after the start of fermentation, collecting fermentation liquor every 4 hours after 12 hours, supplementing 30g of sucrose when the fermentation is performed for 16 hours, and performing sample treatment and analysis to obtain the HA yield and the sucrose residual quantity of the strain, wherein the fermentation culture conditions are that the temperature is 37 ℃, the culture time is 24 hours, the pH value is 7.0 and the rotation speed is 200 r/min.

As shown in figure 6, in the process of fermentation in a tank, the growth conditions of the strain glnA/OP and the strain are not obviously different, the strain glnA/OP and the strain are slowly grown for 4 hours before starting, enter the logarithmic phase for about 4 hours, finish the logarithmic phase for about 8 hours, reach the stationary phase for about 10 hours, slowly grow thalli, enter the recession phase after 16 hours, reduce the biomass of the thalli, and finish the fermentation for 24 hours.

As shown in FIG. 7, the HA yield of the strain glnA/OP is obviously higher than that of the starting strain, the HA yield of the starting strain is 1.3g/L after fermentation is carried out for 10 hours, and the HA yield of the strain glnA/OP is 2.2g/L which is 70% higher than that of the starting strain; when the time is 12 hours, the HA yield of the starting strain is 1.76g/L, and the HA yield of the strain glnA/OP is 1.97g/L, which is 10 percent higher than that of the starting strain; when the fermentation is carried out for 16 hours, the HA yield of the starting strain is 2.26g/L, is 2.75g/L compared with the HA yield of the starting strain, and is 21% higher than the HA yield of the starting strain; when the fermentation is carried out for 20 hours, due to the addition of 30g of sucrose, the HA yield of the starting strain and the strain glnA/OP is improved, the HA yield of the starting strain is 2.71g/L, the HA yield of the strain glnA/OP is 3.64g/L and is 34% higher than that of the starting strain, and when the fermentation is carried out for 24 hours, the HA yield of the starting strain is 3.31g/L, the glnA/OP HA yield of the strain is 3.55g/L and is 7.2% higher than that of the starting strain.

As shown in FIG. 8, the initial sucrose concentration in the fermentation medium was 50g/L, the sucrose consumption rate of the strain glnA/OP was faster than that of the original strain in the same period of time, and the sucrose consumption was increased after 4 hours, 8.58g/L of sucrose remained in the fermentation medium after 16 hours, and the sucrose remaining in the strain glnA/OP was only 4.71g/L, at this time, 30g of sucrose was added to the fermentation medium, the strain glnA/OP was more than that of the original strain between 16 hours and 20 hours, and after 20 hours, the sucrose consumption by the original strain was slowed down, while the sucrose was hardly consumed by the strain glnA/OP, and the fermentation was completed.

More specific implementation details are as follows:

construction of glnA expression vector

An upstream primer glnA-F is designed at 15bp upstream of an ATG (initiation codon) of a glnA gene by taking a streptococcus zooepidemicus ATCC39920 genome as a template, and a downstream primer glnA-R is designed at 20bp downstream of a TAA (termination codon TAA) to amplify the glnA gene.

The PCR reaction system is as follows: 5 XPS Buffer 10. mu.L, dNTP (2mM) 4. mu.L, forward and reverse primers (10. mu.M) each 2. mu.L, template 0.5. mu.L, PrimeSTAR DNA polymerase (Takaru, R010A) 0.5. mu.L, sterile water to a final volume of 50. mu.L.

The PCR reaction conditions are as follows: pre-denaturation at 98 deg.C for 2min, denaturation at 98 deg.C for 15s, annealing at 55 deg.C for 15s, extension at 72 deg.C for 2min, reaction for 30 cycles, and extension at 70 deg.C for 10 min.

The complete glnA gene nucleotide sequence is obtained by the PCR system amplification, and the nucleotide sequence is shown in a sequence table SEQ ID NO. 1.

TABLE 1 primer sequences used

The amplified glnA gene fragment was recovered and ligated with EcoRI/BamHI-endonuclease-treated plasmid fragment pSET4S:: repA:: Ppgk (pLH275), and the nucleotide sequence of pSET4S:: repA:: Ppgk (pLH275) was the sequence shown in SEQ ID No.2 of the sequence Listing.

The ligation products were transformed into competent cells of Escherichia coli JM109, and uniformly spread on LB plates with spectinomycin resistance (50. mu.g/ml), cultured overnight at 37 ℃, and single clones were picked for colony PCR verification and enzyme digestion verification, as shown in FIGS. 2 and 3. And the sequencing comparison is correct, and the gene expression vector pSET4S, repA, Ppgk, GlnA (pLH860) is obtained as shown in figure 1.

Wherein, the LB culture medium is: and (3) aging of trypsin: 10.0g/L, yeast extract: 5.0g/L, NaCl: 10.0g/L, dissolving in deionized water, fixing the volume to 1.0L, adjusting the pH to 7.0-7.2, and adding 1.5% agar powder into a solid culture medium. Sterilizing at 121 deg.C for 20 min.

Second, construction of recombinant strain glnA/OP

2.1 preparation of the zooepidemicus streptococci competence.

(1) Taking out the preserved strain glycerin tube from-80 deg.C, performing three-zone streaking on THY solid plate, and culturing in 37 deg.C incubator for 12-24 hr until clear single colony appears.

(2) A single colony is picked and inoculated into a 250mL triangular flask filled with 50mL THY (containing 0.5mol/Lsucrose, 2% glycine) liquid culture medium, and cultured for 12-14 h at 37 ℃ and 200 r/min.

(3) Inoculating 2% of the culture medium into fresh l00mL THY liquid culture medium, and culturing at 37 deg.C and 200r/min to OD₅₃₀About 0.38, hyaluronidase (12.5kU/l00mL) (Shanghai Biotech engineering Co., Ltd., 37326-33-3) was added and the culture was continued for 30 min. Ice-bath for 5 min.

(4) And subpackaging the bacterial solution in the ice-bath triangular flask into a sterilized 50mL centrifuge tube on a sterile operating table, and centrifuging for 10min at 8000r/min and 4 ℃.

(5) The supernatant was discarded, and 20mL of an ice-bath 0.5M sucrose solution was added to resuspend the cells, and the cells were centrifuged at 8000r/min at 4 ℃ for 10 min.

(6) The supernatant was discarded, and 500. mu.L of a 0.5M sucrose solution containing 15% by mass of glycerol was added to the supernatant, and the cells were resuspended, aliquoted into sterile 1.5mL EP tubes (50. mu.L per tube), and stored in a freezer at-80 ℃.

2.2. Electrotransformation of streptococcus zooepidemicus and screening of gene expression strains.

(1) The electric revolving cup is slightly flushed by clean water.

(2) Adding 75% alcohol into the electric rotary cup, and soaking for 2 h.

(3) Discarding alcohol, washing with distilled water for 2-3 times, and repeatedly blowing the electric rotary cup for more than 10 times by sucking ultrapure water with a gun of lmL.

(4) Adding 2mL of absolute ethyl alcohol into the electric revolving cup, and soaking for 30 min.

(5) The absolute ethanol is discarded and the ethanol is blown dry in a ventilated kitchen.

(6) And putting the cleaned electric revolving cup into a refrigerator at the temperature of-20 ℃ for later use.

(7) The electric rotor was taken out at-20 ℃ and placed on ice.

(8) The cells were taken out of the freezer at-80 ℃ and placed on ice. After the plasmid is dissolved, 5 mu L of plasmid is added, the gun head is blown and uniformly mixed, then the plasmid is added into the electric rotating cup along the wall of the electric rotating cup, and the mixture is placed back on ice again.

(9) After the electric transfer at 2500V (2mm electric rotating cup), 950 mu L of resuscitation solution is added into the electric rotating cup, evenly blown, sucked out and placed into a centrifuge tube.

(10) Recovering at 37 deg.C for 2h at 200 r/min.

(11) Coating a plate: after centrifugation at 8000r/min for 3min, 900. mu.L of the mixture was aspirated, and the remaining 100. mu.L was applied to a spectinomycin-resistant THY plate.

(12) After culturing at 37 ℃ for 48h, transformants were found to grow. The transformants were subjected to resistance spot plate validation on a THY plate with resistance to spectinomycin (100. mu.g/mL) to obtain a gene-overexpressed strain glnA/OP.

Wherein, the THY culture medium is: 10.0g/L of beef extract powder, tryptone: 20.0g/L, glucose: 2.0g/L, yeast extract: 2.0g/L of NaHCO₃:2.0g/L，NaCl:2.0g/L，Na₂HP0₄0.4g/L, dissolving in deionized water, diluting to 1.0L, adjusting pH to 68, adding 1.5% agar powder into the solid culture medium, and sterilizing at 121 ℃ for 20 min.

Analysis of metabolites of glnA-expressing strains

3.1 fermentation of glnA expressing strains

(1) Shake flask fermentation of glnA expression strain

The WT and glnA strains were cultured in 500mL shake flask liquid l00mL for shake flask fermentation. The fermentation medium used was sucrose: 30-50g/L, yeast extract: 2-3.5g/L, casein peptone: 8-10g/L, NaCl: 1-1.5g/L, K₂HPO₄：2-7g/L，MgSO₄·7H₂O: 0.4-0.6 g/L. Fermentation conditions are as follows: 500mL conical flask, liquid loading amount of 100mL, inoculum size of 1%, 37 ℃, pH7.0, rotation speed of 200r/min, fermentation for 24h, sampling every 2h for the first 16h, sampling every 24h every other day, and ending fermentation.

(2) Fermentation tank fermentation of glnA expression strain

WT and glnA were each subjected to fermentation culture in a 2L fermentor containing 1.2L of solution. The fermentation medium used: sucrose: 30-50g/L, yeast extract: 2-3.5g/L, casein peptone: 8-10g/L, NaCl: 1-1.5g/L, K₂HPO₄：2-7g/L，MgSO₄·7H₂O: 0.4-0.6 g/L. Fermentation conditions are as follows: 2L fermentation tank, liquid loading capacity of 1.2L, inoculum size of 2-8%, 37 ℃, Ph7.0, rotation speed of 150-.

3.2 the detection of the HA yield is carried out,

the method comprises the following specific steps:

(1) and (4) pretreating fermentation liquor. Accurately measuring 30mL of fermentation liquid, adding 0.1% SDS with the same volume, fully and uniformly mixing, and standing at room temperature for l0 min. The sample was centrifuged at 12,000r/min at 4 ℃ for 15min, 50mL of the supernatant was transferred to a clean centrifuge tube, the cells were removed, 3 volumes of absolute ethanol were added and mixed well, and lh was precipitated at 4 ℃. Centrifuging at 4 ℃ for 15min at 12,000r/min, and placing the supernatant in another centrifuge tube to store the residual sucrose to be measured. Washing the precipitate with anhydrous ethanol, centrifuging at 4 deg.C for 15min at 12,000r/min, removing supernatant, and air drying the precipitate at room temperature. Accurately measure 50mL ddH₂0, adding the precipitate into a test tube to fully dissolve the precipitate, and performing gradient dilution on a dissolved sample until the concentration is below 0.lg/L for detecting the HA content.

(2) And (4) preparing a hyaluronic acid standard product. Accurately weighing 10mg glucuronic acid with electronic balance, and using ddH₂And (4) fixing the volume of the O to 10ml, adding water to dissolve the O, and fixing the volume to the scale. Accurately measuring standard solutions 0.5 mL, 1.0 mL, 1.5mL, 2.0 mL and 2.5mL by using lmL pipette, adding into 10mL volumetric flasks respectively, diluting with water, diluting to constant volume to scale, preparing into reference solutions with concentrations of 10, 20, 30, 40 and 50 μ g/mL, and storing at 4 ℃ for later use.

(3) And (4) detecting the sample. Cetyl trimethylammonium bromide (CTAB)2.5g was dissolved beforehand in 100mL of 2% NaOH for use. 50. mu.L of the hyaluronic acid standard solution and 50. mu.L of the sample solution were accurately pipetted into 96-well plates containing 50. mu. L0.1M of phosphate buffer (pH 7), set at 37 ℃ in a microplate reader and incubated for 15min, and after the end of the incubation, 100. mu.L of CTAB solution dissolved in 2% sodium hydroxide was added to each sample and incubated again at 37 ℃ for 10 min. At the beginning and end of the incubation, the plates were shaken for 10s and the values read at 600 nm. And (4) making a standard curve of the hyaluronic acid standard solution, calculating a numerical value corresponding to the data of the sample solution and the standard curve, and performing plotting analysis.

3.3 detection of residual sucrose content was as follows:

(1) and (3) diluting the supernatant sample preserved in the HA extraction process to a sucrose concentration of less than 250mg/L in a gradient manner.

(2) 0.9mL (the content of sucrose is 40-250mg/L) of the sample solution to be tested and 0.lmL2mol/L of sodium hydroxide are sucked, and then the sample solution is heated in a boiling water bath at 100 ℃ for 10min and immediately cooled in running water. Adding resorcinol solution 1mL and 10mol/L hydrochloric acid 3mL, shaking, heating in 80 deg.C water bath for 8min, cooling, and adding into OD₅₀₀At the wavelength, blank zero setting is carried out, the absorbance of the sample is measured and compared with a standard sample, and the content of the sucrose in the sample is calculated.

In conclusion, the HA high-yield strain glnAO is obtained by cloning a glnA gene segment in the genome of Streptococcus zooepidemicus ATCC39920, constructing an expression vector pSET4S:: repA:: Ppgk:: GlnA (pLH860) stably existing in the Streptococcus zooepidemicus ATCC39920 and electrically transferring the successfully constructed expression vector into the Streptococcus zooepidemicus ATCC39920 strain.

The constructed strain HAs the HA yield obviously higher than that of the original strain in the processes of shake flask fermentation and tank fermentation.

Although the embodiments of the present invention have been disclosed for illustrative purposes, those skilled in the art will appreciate that: various substitutions, changes and modifications are possible without departing from the spirit and scope of the invention and the appended claims, and therefore the scope of the invention is not limited to the disclosure of the embodiments and the accompanying drawings.

Sequence listing

The gene of SEQ ID No. 1glnA:

SEQ ID NO.2pSET4S: repA: Ppgk vector:

sequence listing

<110> Tianjin science and technology university

<120> method and strain for improving hyaluronic acid yield

<160> 4

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1382

<212> DNA/RNA

<213> glnA Gene (Unknown)

<400> 1

gctgagtgag ttttgttttt ctaatagtta tgtaagtaat tatcaatctc ccattgcgaa 60

acaaaggtcg cataggagga ccattcaatc cgtttggcct ctaaaaagtt ggtgtaaata 120

tggtcaccta aagccttttg aataacctcg tccttttgaa gagcctttag agcattatgg 180

agtgttgatg gcaaatcaac aataccagca gcattgcgct cctctgctgt catcatgtaa 240

atgttggcct caatcggtgc tggtgcctca atgcgattaa tgattccatc taagcctgcc 300

tctagtaaaa ctgcaagtgc taaataagga ttggctgttg gatcaaccga gcgcaattct 360

aaacgagtcc ccacaccacg agaagcagga acacgaataa gaggtgaacg attgcttcct 420

gcccaggcaa cataaacagg agcctcatag ccaggaacca agcgcttgta agaattaact 480

gttggatttg taatagcggt gtaattgtaa gcatgcttca tcaacccacc taagaaataa 540

taggcatcat ctgatagctt catgccacgc ttatcatttt catcgtaaaa ggcattgttg 600

ccctccttgt caaacaagga catgttacag tgcatacctg agccggcaat accaaatttc 660

ggctttgcca taaaggtggc gtagagcccg tgctcacgag caattgtctt gacgacaagc 720

ttaaagatct gaatattatc acaggccttc aaggcatctg catatttaaa gtcaatttca 780

tgctgaccga cagctacctc atggtggctg gcttcgacct caaagcccat ttttgtcaag 840

acattaacaa tttcacgacg ggtattgtct gccagatcaa tcggagcgag gtcaaaatag 900

cctcccttgt cattgacctc aagcgttggc tctccagctt catctatctt aaacaagaaa 960

aattctggct ctggaccaag gttaaaggac tgatagccaa cctcatccat atgcttcaag 1020

gctcttttta ggttgccacg tgggtcacca gcaaagggct tgccttcagc agtgtaaata 1080

tcacaaatca agccaccaac agctccattt tcatctcccc aaggaaagac gatccaagtg 1140

tctaaatcag gatagagata catatctgat tcattgatac gcacaaagcc ttcaatcgag 1200

gagccatcaa acatgacctt attagataag actttatcta attgctcttc agttgcaggt 1260

atctcaacat ttttcataac acccataatg tccgtaaaca tcaaccgaag aaaggtcaca 1320

tgcttttctt tgacctcgcg acgaacatca gctgctgtga ttgccattaa tcaatctcct 1380

tt 1382

<210> 2

<211> 4721

<212> DNA/RNA

<213> Gene expression vector pSET4S: repA: Ppgk (Unknown)

<400> 2

actagttatc ggcataatcg ttaaaacagg cgttatcgta gcgtaaaagc ccttgagcgt 60

agcgtggctt tgcagcgaag atgttgtctg ttagattatg aaagccgatg actgaatgaa 120

ataataagcg cagcgccctt ctatttcggt tggaggaggc tcaagggagt atgagggaat 180

gaaattccct catgggtttg attttaaaaa ttgcttgcaa ttttgccgag cggtagcgct 240

ggaaaatttt tgaaaaaaat ttggaatttg gaaaaaaatg gggggaaagg aagcgaattt 300

tgcttccgta ctacgacccc ccattaagtg ccgagtgcca atttttgtgc caaaaacgct 360

ctatcccaac tggctcaagg gtttaagggg tttttcaatc gccaacgaat cgccaacgtt 420

ttcgccaacg ttttttataa atctatattt aagtagcttt attgttgttt ttatgattac 480

aaagtgatac actaacttta taaaattatt tgattggagt tttttaaatg gtgatttcag 540

aatcgaaaaa aagagttatg atttctctga caaaagagca agataaaaaa ttaacagata 600

tggcgaaaca aaaaggtttt tcaaaatctg cggttgcggc gttagctata gaagaatatg 660

caagaaagga atcagaacaa aaaaaataag cgaaagctcg cgtttttaga aggatacgag 720

ttttcgctac ttgtttttga taaggtaatt atatcatggc tattaaaaat actaaagcta 780

gaaattttgg atttttatta tatcctgact caattcctaa tgattggaaa gaaaaattag 840

agagtttggg cgtatctatg gctgtcagtc ctttacacga tatggacgaa aaaaaagata 900

aagatacatg gaatagtagt gatgttatac gaaatggaaa gcactataaa aaaccacact 960

atcacgttat atatattgca cgaaatcctg taacaataga aagcgttagg aacaagatta 1020

agcgaaaatt ggggaatagt tcagttgctc atgttgagat acttgattat atcaaaggtt 1080

catatgaata tttgactcat gaatcaaagg acgctattgc taagaataaa catatatacg 1140

acaaaaaaga tattttgaac attaatgatt ttgatattga ccgctatata acacttgatg 1200

aaagccaaaa aagagaattg aagaatttac ttttagatat agtggatgac tataatttgg 1260

taaatacaaa agatttaatg gcttttattc gccttagggg agcggagttt ggaattttaa 1320

atacgaatga tgtaaaagat attgtttcaa caaactctag cgcctttaga ttatggtttg 1380

agggcaatta tcagtgtgga tatagagcaa gttatgcaaa ggttcttgat gctgaaacgg 1440

gggaaataaa atgacaaaca aagaaaaaga gttatttgct gaaaatgagg aattaaaaaa 1500

agaaattaag gacttaaaag agcgtattga aagatacaga gaaatggaag ttgaattaag 1560

tacaacaata gatttattga gaggagggat tattgaataa ataaaagccc cctgacgaaa 1620

gtcgaagggg gtttttattt tggtttgatg ttgcgattaa tagatctcgg tgatgacggt 1680

gaaaacctct gacacatgca gctcccggag acggtcacag cttgtctgta agcggatgcc 1740

gggagcagac aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg gggctggctt 1800

aactatgcgg catcagagca gattgtactg agagtgcacc atatgcggtg tgaaataccg 1860

cacagatgcg taaggagaaa ataccgcatc aggcgccatt cgccattcag gctgcgcaac 1920

tgttgggaag ggcgatcggt gcgggcctct tcgctattac gccagctggc gaaaggggga 1980

tgtgctgcaa ggcgattaag ttgggtaacg ccagggtttt cccagtcacg acgttgtaaa 2040

acgacggcca gtgaattcga gctcggtacc cggggatcct ctagagtcga cctcgagagt 2100

caatttagcc atttcataga ctccttaata ttttttagta cactctatta taacacaatt 2160

cttttgataa ggataaaatc tgaaaacaat aaattaacta tttcacaatt tttcaaagcg 2220

cttgaaaaat cccttgaata accccctttt cagcagatca tgatcaccta agtaggcagg 2280

aagccacaac atagtcatga catcacgcaa atgcatgcaa gcttggcgta atcatggtca 2340

tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga 2400

agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg 2460

cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc 2520

caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac 2580

tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata 2640

cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa 2700

aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct 2760

gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa 2820

agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg 2880

cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcaatgctca 2940

cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa 3000

ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg 3060

gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg 3120

tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaagg 3180

acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc 3240

tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag 3300

attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac 3360

gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc 3420

ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag 3480

taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt 3540

ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactag tgttcgtgaa 3600

tacatgttat aataactata actaataacg taacgtgact ggcaagagat atttttaaaa 3660

caatgaatag gtttacactt actttagttt tatggaaatg aaagatcata tcatatataa 3720

tctagaataa aattaactaa aataattatt atctagataa aaaatttaga agccaatgaa 3780

atctataaat aaactaaatt aagtttattt aattaacaac tatggatata aaataggtac 3840

taatcaaaat agtgaggagg atatatttga atacatacga acaaattaat aaagtgaaaa 3900

aaatacttcg gaaacattta aaaaataacc ttattggtac ttacatgttt ggatcaggag 3960

ttgagagtgg actaaaacca aatagtgatc ttgacttttt agtcgtcgta tctgaaccat 4020

tgacagatca aagtaaagaa atacttatac aaaaaattag acctatttca aaaaaaatag 4080

gagataaaag caacttacga tatattgaat taacaattat tattcagcaa gaaatggtac 4140

cgtggaatca tcctcccaaa caagaattta tttatggaga atggttacaa gagctttatg 4200

aacaaggata cattcctcag aaggaattaa attcagattt aaccataatg ctttaccaag 4260

caaaacgaaa aaataaaaga atatacggaa attatgactt agaggaatta ctacctgata 4320

ttccattttc tgatgtgaga agagccatta tggattcgtc agaggaatta atagataatt 4380

atcaggatga tgaaaccaac tctatattaa ctttatgccg tatgatttta actatggaca 4440

cgggtaaaat cataccaaaa gatattgcgg gaaatgcagt ggctgaatct tctccattag 4500

aacataggga gagaattttg ttagcagttc gtagttatct tggagagaat attgaatgga 4560

ctaatgaaaa tgtaaattta actataaact atttaaataa cagattaaaa aaattataaa 4620

aaaattgaaa aaatggtgga aacacttttt tcaatttttt tgttttatta tttaatattt 4680

gggaaatatt cattctaatt ggtaatcaga ttttagaaaa c 4721

<210> 3

<211> 38

<212> DNA/RNA

<213> primer glnA-F (Unknown)

<400> 3

gtcgactcta gaggatccaa aggagattga ttaatggc 38

<210> 4

<211> 39

<212> DNA/RNA

<213> primer glnA-R (Unknown)

<400> 4

acgacggcca gtgaattcgc tgagtgagtt ttgtttttc 39