CN106190939B - Recombinant corynebacterium glutamicum capable of producing hyaluronic acid at high yield, and preparation method and application thereof - Google Patents
Recombinant corynebacterium glutamicum capable of producing hyaluronic acid at high yield, and preparation method and application thereof Download PDFInfo
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Abstract
The invention provides a recombinant corynebacterium glutamicum capable of producing hyaluronic acid with high yield and a preparation method thereof. The invention also provides application of the recombinant corynebacterium glutamicum capable of producing hyaluronic acid with high yield in fermentation production of hyaluronic acid. The invention utilizes the food safety level microorganism corynebacterium glutamicum which can not secrete endotoxin, simplifies the post-extraction process, constructs the corynebacterium glutamicum recombinant strain with high hyaluronic acid yield through the genetic engineering technology, can produce hyaluronic acid (3000 Da-200 ten thousand Da) with different molecular weights, can reach 7g/L-20g/L according to the output of the hyaluronic acid with different molecular weights, and can meet the requirements of food, cosmetics and pharmaceuticals without complex separation.
Description
Technical Field
The invention relates to the field of genetic engineering and microbial fermentation, in particular to recombinant corynebacterium glutamicum for high-yield hyaluronic acid and a preparation method and application thereof.
Background
Hyaluronic acid is a high molecular polysaccharide substance formed by connecting D-glucuronic acid and N-acetylglucosamine through β -1,3 and β -1,4 glycosidic bonds, and is also called hyaluronic acid, which widely exists in various parts of a human body and has extremely important physiological effects, such as assisting diffusion and transportation of water electrolytes, lubricating joints, regulating permeability of blood vessel walls, promoting wound healing and the like.
The production method of hyaluronic acid mainly comprises two methods of animal tissue extraction and microbial fermentation. The former has high production cost and is easy to generate immune reaction due to factors such as limited source, low yield and the like. Therefore, hyaluronic acid is currently produced mainly by fermentation. At present, the industrial hyaluronic acid production strains mainly comprise conditionally pathogenic strains such as streptococcus zooepidemicus, streptococcus equi and streptococcus equisimilis. Since such strains secrete endotoxin, the isolation process is extremely demanding and product applications are limited.
Disclosure of Invention
The invention aims to provide a recombinant corynebacterium glutamicum capable of producing hyaluronic acid with high yield and a preparation method thereof.
Another object of the present invention is to provide the use of the recombinant Corynebacterium glutamicum for producing hyaluronic acid in a fermentation manner.
In order to achieve the object of the present invention, the present invention firstly provides a recombinant Corynebacterium glutamicum Δ zwf, wherein a glucose 6-phosphate dehydrogenase gene zwf in Corynebacterium glutamicum (Corynebacterium glutamicum) is inactivated by genetic engineering means, so as to construct the recombinant Corynebacterium glutamicum Δ zwf. The preferred starting strain is Corynebacterium glutamicum MB 001.
For example, the same objective can be achieved by knocking out the zwf gene in C.glutamicum, knocking out the whole gene or a part of the gene, or by inactivating the function of glucose 6-phosphate dehydrogenase by mutation at a key site.
The invention also provides a construction method of the recombinant corynebacterium glutamicum delta zwf, which comprises the following steps:
s11, carrying out PCR by taking the genome DNA of Corynebacterium glutamicum MB001 as a template and zwf-up-F and zwf-up-R as primers to obtain a homologous fragment zwf-up of about 1000bp at the upstream of zwf of a glucose 6-phosphate dehydrogenase gene and purifying a PCR product;
s12, carrying out PCR by taking the genome DNA of Corynebacterium glutamicum MB001 as a template and zwf-down-F and zwf-down-R as primers to obtain a homologous fragment zwf-down of about 1000bp at the downstream of zwf, and purifying a PCR product;
s13, carrying out double digestion on a Corynebacterium glutamicum suicide plasmid pK18mobsacB (Journal of Biotechnology 104(2003)287-299) by EcoRI/XbaI, connecting zwf-up and zwf-down fragments to the pK18mobsacB after digestion by a Gibson Assembly kit (NEB) in one step, and obtaining a recombinant plasmid named pK 18-zwf;
s14, converting pK18-zwf into Corynebacterium glutamicum MB001, and screening positive clones to obtain the corynebacterium glutamicum MB 001;
wherein, the sequence of the glucose 6-phosphate dehydrogenase gene zwf is shown as SEQ ID NO. 1. The primer sequences used were as follows:
zwf-up-F：5’-acagctatgacatgattacgggcgattcctacgacgctca-3’
zwf-up-R：5’-taaattatggcacgatggtagtgtcacgatcc-3’
zwf-down-F：5’-taccatcgtgccataatttaggggcaaaaaatgatctttgaact-3’
zwf-down-R：5’-tgcatgcctgcaggtcgactgtgcgcgtactacatcaaccatag-3’。
pK18-zwf is preferably transferred into C.glutamicum MB001 by electroporation using an electroporator (Burley) under conditions of a voltage of 2.5KV, a resistance of 200. omega. and a capacitance of 25. mu.F (cuvette width of 2 mm). A first selection of recombinant bacteria was performed on 25mg/L kanamycin LB plates. Positive recombinants were picked and further cultured overnight in liquid LB medium, and then subjected to secondary screening on a sucrose LB plate containing 100 g/L. Colony PCR is carried out by using zwf-up-F and zwf-down-R as primers, a recombinant of the zwf gene is knocked out, a fragment with the size of 2Kb can be amplified, and the recombinant bacterium is named as C.
To verify the effect of the knock-out of the glucose 6-phosphate dehydrogenase gene zwf on hyaluronic acid production, the following experiment was performed:
based on the amino acid sequence of hyaluronic acid synthetase of streptococcus zooepidemicus, a codon-optimized hyaluronic acid synthetase gene hasA (SEQ ID NO:7) is designed, and the Wuxi Qinglan biological technology limited company is entrusted to carry out gene synthesis; PCR was carried out using the artificially synthesized hasA gene fragment as a template and hasA-F (5'-acagctatgacatgattacgcgaaccacgcaatgcgtctc-3') and hasA-R (5'-gcctgcaggtcgactcgtctcggttggcagtgac-3') as primers to obtain a hasA fragment of about 1400bp, and purification of the PCR product was carried out. Plasmid pXYJ-12 (purchased from Addge) carrying the lacY gene (SEQ ID NO:8) was double digested with EcoRI/XbaI, and the hasA fragment was ligated to pXYJ-12 using Gibson Assembly kit (NEB), and the resulting recombinant plasmid was named pXYJ-hasA. The plasmid pXYJ-hasA is transformed into wild Corynebacterium glutamicum MB001 and a recombinant bacterium C. glutamicum delta zwf by an electrotransfer method (the conditions are the same as above), and the obtained recombinant bacterium is named Cg/pXYJ-hasA and Cg delta zwf/pXYJ-hasA respectively.
Cg/pXYJ-hasA and Cg Δ zwf/pXYJ-hasA were fermented in a shake flask, and the hyaluronic acid production was examined. The fermentation medium is as follows: glucose 40g/L, (NH)4)2SO430g/L of corn steep liquor and 20g/L, KH of corn steep liquor2PO41g/L、K2HPO40.5g/L、MgSO45g/L、FeSO4·7H2O0.01g/L、MnSO4·H20.01g/L of O, 1.5g/L of glutamine and 0.5g/L of arginine. The temperature of the fermentation process is controlled at 32 ℃, and the rotating speed is 200 r/min. And when the fermentation is carried out for 3 hours, adding 10.0mM lactose to induce the expression of the protein and the production of hyaluronic acid, and detecting the content of the hyaluronic acid in the fermentation liquor after the fermentation is carried out for 72 hours.
The detection method of the hyaluronic acid comprises the following steps: taking 1mL of fermentation liquor, adding equivalent 0.1% sodium dodecyl sulfate solution (SDS), standing at room temperature for 30min, centrifuging the fermentation liquor at 12000rpm for 10min, collecting supernatant, adding 2 times volume of anhydrous ethanol, standing at 4 deg.C for 1h, and fully precipitating. Centrifuging at 4 deg.C and 12000rpm for 10min, discarding supernatant, air drying until ethanol is completely volatilized, and resuspending the precipitate with deionized water with the same volume as the original fermentation liquid. mu.L of the once resuspended and diluted hyaluronic acid solution was taken, 700. mu.L of an acetic acid buffer solution (sodium acetate concentration of 0.2mol/L and 0.15mol/L NaCl, pH adjusted to about 6.0 with acetic acid) was added thereto, 2ml of a CTAB solution (2.5g/L CTAB dissolved in 0.5mol/L NaOH solution) was added thereto, and the reaction was carried out for 5 minutes, followed by measuring OD400The value is obtained.
Fermentation results show that the yield of hyaluronic acid of Cg/pXYJ-hasA and Cg delta zwf/pXYJ-hasA is 0.3g/L and 2.2g/L respectively, so that the knockout of the glucose 6-phosphate dehydrogenase gene zwf plays an important role in improving the yield of hyaluronic acid.
The invention further provides application of the recombinant corynebacterium glutamicum delta zwf in construction of recombinant corynebacterium glutamicum with high hyaluronic acid yield.
The invention provides a recombinant corynebacterium glutamicum for high yield of hyaluronic acid with medium molecular weight, which is constructed by over-expressing endogenous UDP-glucose dehydrogenase gene udgA (SEQ ID NO:2) in recombinant corynebacterium glutamicum delta zwf.
The invention also provides a construction method of the recombinant corynebacterium glutamicum for high yield of the hyaluronic acid with the medium molecular weight, which comprises the following steps:
s21, constructing a recombinant plasmid pXYJ-hasA by the method described above;
s22, carrying out PCR by taking the genome of Corynebacterium glutamicum MB001 as a template and udgA-F and udgA-R as primers to obtain a UDP-glucose dehydrogenase gene udgA fragment of about 1200bp, and purifying a PCR product;
s23, carrying out single enzyme digestion on the plasmid pXYJ-hasA by SalI, connecting a udgA fragment to the plasmid pXYJ-hasA by using a Gibson Assembly kit, and naming the obtained recombinant plasmid as pXYJ-hasA-udgA;
s24, transforming pXYJ-hasA-udgA (conditions of an electrotransformation method are the same as the above) into the recombinant corynebacterium glutamicum delta zwf, screening positive clones, and naming the obtained recombinant bacterium as Cg delta zwf/pXYJ-hasA-udgA.
Wherein, the primer sequences are as follows:
hasA-F：5’-acagctatgacatgattacgcgaaccacgcaatgcgtctc-3’
hasA-R：5’-gcctgcaggtcgactcgtctcggttggcagtgac-3’
udgA-F：5’-caaccgagacaaaggaggacacatatgaaaattgccgtcgcagg-3’
udgA-R：5’-ccgccaaaacagccaagctgttagtcacgctggaaaatatcacgtgtataaact-3’。
the invention also provides a recombinant corynebacterium glutamicum high-yield and high-molecular-weight hyaluronic acid, which is constructed by simultaneously overexpressing an endogenous bifunctional UDP-N-acetylglucosamine pyrophosphorylase gene glmU (SEQ ID NO:3), a phosphoglucomutase gene glmM (SEQ ID NO:4) and a glucosamine-6-phosphate synthase gene glmS (SEQ ID NO:5) in the recombinant corynebacterium glutamicum delta zwf.
The invention also provides a construction method of the recombinant corynebacterium glutamicum for high-yield high-molecular-weight hyaluronic acid, which comprises the following steps:
s31, constructing a recombinant plasmid pXYJ-hasA by the method described above;
s32, carrying out PCR by taking the genome of Corynebacterium glutamicum MB001 as a template and glmU-F and glmU-R as primers to obtain a bifunctional UDP-N-acetylglucosamine pyrophosphorylase gene glmU fragment of about 1500bp and purifying a PCR product;
s33, carrying out PCR by taking the genome of Corynebacterium glutamicum MB001 as a template and glmM-F and glmM-R as primers to obtain a phosphoglucomutase gene glmM fragment of about 1400bp and purifying a PCR product;
s34, performing PCR by taking the genome of Corynebacterium glutamicum MB001 as a template and glmS-F and glmS-R as primers to obtain a glucosamine-6-phosphate synthetase gene glmS fragment of about 1900bp, and purifying a PCR product;
s35, performing single enzyme digestion on the plasmid pXYJ-hasA by KpnI, connecting glmU, glmM and glmS fragments to the pXYJ-hasA by using a Gibson Assembly kit, and naming the obtained recombinant plasmid as pXYJ-hasA-glmUMS;
s36, transforming pXYJ-hasA-glmUMS (conditions of an electrotransformation method are the same as above) into the recombinant corynebacterium glutamicum delta zwf, screening positive clones, and naming the obtained recombinant bacterium as Cg delta zwf/pXYJ-hasA-glmUMS.
Wherein, the primer sequences are as follows:
glmU-F：5’-ttggcaggatccccgggtacaaggatttgagataatcttgagcg-3’
glmU-R：5’-ggccaagatcttagccttcctggttgtggacg-3’
glmM-F：5’-ggaaggctaagatcttggccaggccgtgca-3’
glmM-R：5’-tttctcgatcattagacttctgcaaccactgcag-3’
glmS-F：5’-cagaagtctaatgatcgagaaaaagttgttgtaaagtcatgc-3’
glmS-R：5’-gctgaattcgagctcggtacttattcgacggtgacagactttgcc-3’。
the invention also provides a recombinant corynebacterium glutamicum for high yield and low molecular weight hyaluronic acid, which is constructed by over-expressing an exogenous hyaluronidase synthase gene Hyal (SEQ ID NO:6) in the recombinant corynebacterium glutamicum Cg delta zwf/pXYJ-hasA-udgA.
The invention also provides a construction method of the recombinant corynebacterium glutamicum for high yield of the low molecular weight hyaluronic acid, which comprises the following steps:
s41, designing a codon-optimized hyaluronidase synthetase gene Hyal (SEQ ID NO:6) based on the amino acid sequence of the hyaluronidase of the leech, and entrusting Wuxi Qinglan biological technology limited company to carry out gene synthesis; carrying out PCR by taking the artificially synthesized Hyal gene as a template and using Hyal-F and Hyal-R as primers to obtain a Hyal fragment of about 1500bp, and purifying a PCR product;
s42, carrying out double digestion on a plasmid pEC-S (purchased from Addgene) with a signal peptide by EcoRI/XbaI, connecting a Hyal fragment to the pEC-S by using a Gibson Assembly kit, and naming the obtained recombinant plasmid as pEC-Hyal;
s43, transforming pEC-hyal (the conditions of the electrotransformation method are the same as the above) into recombinant Corynebacterium glutamicum Cg delta zwf/pXYJ-hasA-udgA, screening positive clones, and naming the obtained recombinant bacteria as Cg delta zwf/pXYJ-hasA-udgA/pEC-hyal.
Wherein, the primer sequences are as follows:
hyal-F：5’-ctacttccactgcacaaagcatgaaggagattgcggtcacg-3’
hyal-R：5’-gcctgcaggtcgactctagattactttttgcaggcctctacattgg-3’。
the invention also provides application of the recombinant corynebacterium glutamicum capable of producing hyaluronic acid with high yield in fermentation production of hyaluronic acid.
Fermenting the recombinant corynebacterium glutamicum in a 2L fermentation tank, wherein the liquid loading is 1L, the inoculation amount is 1-10 v/v%, and the fermentation medium is as follows: glucose 30-100g/L、(NH4)2SO410-50g/L of corn steep liquor and 10-30g/L, KH of corn steep liquor2PO40.5-2g/L、K2HPO40.5-2g/L、MgSO41-5g/L、FeSO4·7H2O 0.01-1g/L、MnSO4·H20.01-1g/L of O, 0.5-2g/L of glutamine and 0.1-2g/L of arginine; the temperature is controlled between 28 ℃ and 32 ℃, the pH is controlled between 6.8 and 7.3, and the rotating speed is 600-; when fermenting for 3-5h, adding 0.5-10.0mM lactose to induce protein expression and hyaluronic acid production, wherein the fermentation period is 36-72h, and after fermentation, obtaining fermentation liquor containing hyaluronic acid.
Preferably, the fermentation medium is: glucose 50g/L, (NH)4)2SO430g/L of corn steep liquor and 20g/L, KH of corn steep liquor2PO41g/L、K2HPO40.5g/L、MgSO45g/L、FeSO4·7H2O0.01g/L、MnSO4·H20.01g/L of O, 1.5g/L of glutamine and 0.5g/L of arginine. The temperature in the fermentation process is controlled at 32 ℃, the rotating speed is 1000 r/min, and the pH is controlled at 7.0 by using NaOH solution. And (3) adding 10.0mM lactose to induce the expression of protein and the production of hyaluronic acid during fermentation for 3h, fermenting for 48h, and obtaining fermentation liquor containing hyaluronic acid after the fermentation is finished.
The invention has the following advantages:
the invention utilizes the food safety level microorganism corynebacterium glutamicum which can not secrete endotoxin, simplifies the post-extraction process, constructs the corynebacterium glutamicum recombinant strain of high-yield hyaluronic acid by a genetic engineering technology, and can meet the requirements of food, cosmetics and pharmaceuticals without complex separation.
The recombinant corynebacterium glutamicum capable of producing hyaluronic acid with high yield constructed by the invention can produce hyaluronic acid with different molecular weights (3000 Da-200 ten thousand Da), and the yield of the hyaluronic acid with different molecular weights can reach 7g/L-20g/L, which exceeds the yield of the prior art, so that the production cost of the hyaluronic acid is reduced.
Thirdly, the recombinant corynebacterium glutamicum Cg delta zwf/pXYJ-hasA-udgA of the high-yield medium-molecular-weight hyaluronic acid constructed by the invention can produce the hyaluronic acid with the molecular weight of about 30 ten thousand Da by fermentation, and the yield can reach 9-10 g/L; the recombinant corynebacterium glutamicum Cg delta zwf/pXYJ-hasA-glmUMS of high-molecular-weight hyaluronic acid can produce hyaluronic acid with the molecular weight of about 200 ten thousand Da through fermentation, and the yield can reach 6-7 g/L; the recombinant corynebacterium glutamicum Cg delta zwf/pXYJ-hasA-udgA/pEC-hyal for high yield of low molecular weight hyaluronic acid can produce hyaluronic acid with the molecular weight of about 3000Da by fermentation, and the yield can reach 15-20 g/L.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise indicated, the examples follow conventional experimental conditions, such as the Molecular Cloning handbook, Sambrook et al (Sambrook J & Russell DW, Molecular Cloning: a Laboratory Manual,2001), or the conditions as recommended by the manufacturer's instructions.
EXAMPLE 1 knockout of the Corynebacterium glutamicum glucose 6-phosphate dehydrogenase Gene zwf
1. Carrying out PCR by taking the genome DNA of Corynebacterium glutamicum MB001 as a template and zwf-up-F and zwf-up-R as primers to obtain a homologous fragment zwf-up of about 1000bp at the upstream of zwf of a glucose 6-phosphate dehydrogenase gene and purifying a PCR product;
2. carrying out PCR by taking the genome DNA of Corynebacterium glutamicum MB001 as a template and zwf-down-F and zwf-down-R as primers to obtain a homologous fragment zwf-down of about 1000bp at the downstream of zwf and purifying a PCR product;
3. the Corynebacterium glutamicum suicide plasmid pK18mobsacB (Journal of Biotechnology 104(2003)287-299) was double digested with EcoRI/XbaI, the zwf-up and zwf-down fragments were further ligated to the digested pK18mobsacB using the Gibson Assembly kit (NEB), and the resulting recombinant plasmid was named pK 18-zwf;
4. pK18-zwf was electrotransformed into Corynebacterium glutamicum MB001 using an electroporation apparatus (Berle) under conditions of a voltage of 2.5KV, a resistance of 200. omega. and a capacitance of 25. mu.F (cuvette width of 2 mm). A first selection of recombinant bacteria was performed on 25mg/L kanamycin LB plates. Positive recombinants were picked and further cultured overnight in liquid LB medium, and then subjected to secondary screening on a sucrose LB plate containing 100 g/L. Colony PCR is carried out by using zwf-up-F and zwf-down-R as primers, a recombinant of the zwf gene is knocked out, a fragment with the size of 2Kb can be amplified, and the recombinant bacterium is named as C.
Wherein, the primer sequences are as follows:
zwf-up-F：5’-acagctatgacatgattacgggcgattcctacgacgctca-3’
zwf-up-R：5’-taaattatggcacgatggtagtgtcacgatcc-3’
zwf-down-F：5’-taccatcgtgccataatttaggggcaaaaaatgatctttgaact-3’
zwf-down-R：5’-tgcatgcctgcaggtcgactgtgcgcgtactacatcaaccatag-3’。
example 2 verification of the Effect of the knock-out of the glucose 6-phosphate dehydrogenase Gene zwf on hyaluronic acid production
Based on the amino acid sequence of hyaluronic acid synthetase of streptococcus zooepidemicus, a codon-optimized hyaluronic acid synthetase gene hasA (SEQ ID NO:7) is designed, and the Wuxi Qinglan biological technology limited company is entrusted to carry out gene synthesis; PCR was carried out using the artificially synthesized hasA gene fragment as a template and hasA-F (5'-acagctatgacatgattacgcgaaccacgcaatgcgtctc-3') and hasA-R (5'-gcctgcaggtcgactcgtctcggttggcagtgac-3') as primers to obtain a hasA fragment of about 1400bp, and purification of the PCR product was carried out. Plasmid pXYJ-12 (purchased from Addge) carrying the lacY gene (SEQ ID NO:8) was double digested with EcoRI/XbaI, and the hasA fragment was ligated to pXYJ-12 using Gibson Assembly kit (NEB), and the resulting recombinant plasmid was named pXYJ-hasA. The plasmid pXYJ-hasA was transformed with a wild type Corynebacterium glutamicum MB001 and a recombinant strain C. glutamicum. DELTA. zwf by an electrotransfer method (conditions described in example 1), and the obtained recombinant strains were named Cg/pXYJ-hasA and Cg. DELTA. zwf/pXYJ-hasA, respectively.
Cg/pXYJ-hasA and Cg Δ zwf/pXYJ-hasA were fermented in a shake flask, and the hyaluronic acid production was examined. The fermentation medium is as follows: glucose 40g/L, (NH)4)2SO430g/L of corn steep liquor and 20g/L, KH of corn steep liquor2PO41g/L、K2HPO40.5g/L、MgSO45g/L、FeSO4·7H2O0.01g/L、MnSO4·H20.01g/L of O, 1.5g/L of glutamine and 0.5g/L of arginine. The temperature of the fermentation process is controlled at 32 ℃, and the rotating speed is 200 r/min. And when the fermentation is carried out for 3 hours, adding 10.0mM lactose to induce the expression of the protein and the production of hyaluronic acid, and detecting the content of the hyaluronic acid in the fermentation liquor after the fermentation is carried out for 72 hours.
The detection method of the hyaluronic acid comprises the following steps: taking 1mL of fermentation liquor, adding equivalent 0.1% sodium dodecyl sulfate solution (SDS), standing at room temperature for 30min, centrifuging the fermentation liquor at 12000rpm for 10min, collecting supernatant, adding 2 times volume of anhydrous ethanol, standing at 4 deg.C for 1h, and fully precipitating. Centrifuging at 4 deg.C and 12000rpm for 10min, discarding supernatant, air drying until ethanol is completely volatilized, and resuspending the precipitate with deionized water with the same volume as the original fermentation liquid. mu.L of the once resuspended and diluted hyaluronic acid solution was taken, 700. mu.L of an acetic acid buffer solution (sodium acetate concentration of 0.2mol/L and 0.15mol/L NaCl, pH adjusted to about 6.0 with acetic acid) was added thereto, 2ml of a CTAB solution (2.5g/L CTAB dissolved in 0.5mol/L NaOH solution) was added thereto, and the reaction was carried out for 5 minutes, followed by measuring OD400The value is obtained.
Fermentation results show that the yield of hyaluronic acid of Cg/pXYJ-hasA and Cg delta zwf/pXYJ-hasA is 0.3g/L and 2.2g/L respectively, so that the knockout of the glucose 6-phosphate dehydrogenase gene zwf plays an important role in improving the yield of hyaluronic acid.
Example 3 construction of recombinant Corynebacterium glutamicum producing high yields of medium molecular weight hyaluronic acid
1. The recombinant plasmid pXYJ-hasA was constructed in the same manner as described in example 2;
2. carrying out PCR by taking the genome of Corynebacterium glutamicum MB001 as a template and udgA-F and udgA-R as primers to obtain a UDP-glucose dehydrogenase gene udgA fragment of about 1200bp, and purifying a PCR product;
3. carrying out single enzyme digestion on the plasmid pXYJ-hasA by SalI, connecting a udgA fragment to the plasmid pXYJ-hasA by using a Gibson Assembly kit, and naming the obtained recombinant plasmid as pXYJ-hasA-udgA;
4. pXYJ-hasA-udgA was transformed into the recombinant Corynebacterium glutamicum.DELTA.zwf of example 1 by the electroporation method (conditions of the electroporation method are the same as those described in example 1), positive clones were selected, and the obtained recombinant strain was named Cg.DELTA.zwf/pXYJ-hasA-udgA.
Wherein, the primer sequences are as follows:
hasA-F：5’-acagctatgacatgattacgcgaaccacgcaatgcgtctc-3’
hasA-R：5’-gcctgcaggtcgactcgtctcggttggcagtgac-3’
udgA-F：5’-caaccgagacaaaggaggacacatatgaaaattgccgtcgcagg-3’
udgA-R：5’-ccgccaaaacagccaagctgttagtcacgctggaaaatatcacgtgtataaact-3’。
and (3) fermenting Cg delta zwf/pXYJ-hasA-udgA in a 2L fermentation tank, wherein the liquid loading amount is 1L, the inoculation amount is 10 v/v%, and the yield of the hyaluronic acid is detected. The fermentation medium is as follows: glucose 50g/L, (NH)4)2SO430g/L of corn steep liquor and 20g/L, KH of corn steep liquor2PO41g/L、K2HPO40.5g/L、MgSO45g/L、FeSO4·7H2O 0.01g/L、MnSO4·H20.01g/L of O, 1.5g/L of glutamine and 0.5g/L of arginine. The temperature in the fermentation process is controlled at 32 ℃, the rotating speed is 1000 r/min, and the pH is controlled at 7.0 by using NaOH solution. When the fermentation is carried out for 3 hours, 10.0mM lactose is added to induce the expression of protein and the production of hyaluronic acid, and the yield of hyaluronic acid is detected after the fermentation is carried out for 48 hours. The yield of hyaluronic acid reaches 9.6 g/L. The molecular weight of hyaluronic acid was measured by gel filtration chromatography (PL aqua gel-OH column, column temperature 25 ℃, mobile phase 0.2mol/L NaCl, flow rate 1ml/min, detector is differential refractometer), and the average molecular weight reached 30 ten thousand daltons.
Example 4 construction of recombinant Corynebacterium glutamicum producing high-molecular-weight hyaluronic acid
1. The recombinant plasmid pXYJ-hasA was constructed in the same manner as described in example 2;
2. carrying out PCR by taking the genome of Corynebacterium glutamicum MB001 as a template and glmU-F and glmU-R as primers to obtain a bifunctional UDP-N-acetylglucosamine pyrophosphorylase gene glmU fragment of about 1500bp and purifying a PCR product;
3. carrying out PCR by taking the genome of Corynebacterium glutamicum MB001 as a template and glmM-F and glmM-R as primers to obtain a phosphoglucomutase gene glmM fragment of about 1400bp and purifying a PCR product;
4. performing PCR by using the genome of Corynebacterium glutamicum MB001 as a template and glmS-F and glmS-R as primers to obtain a glucosamine-6-phosphate synthetase gene glmS fragment of about 1900bp and purifying a PCR product;
5. the plasmid pXYJ-hasA is subjected to single enzyme digestion by KpnI, glmU, glmM and glmS fragments are connected to the pXYJ-hasA by utilizing a Gibson Assembly kit, and the obtained recombinant plasmid is named as pXYJ-hasA-glmUMS;
6. pXYJ-hasA-glmUMS was transformed into the recombinant Corynebacterium glutamicum.DELTA.zwf of example 1 by the electroporation method (conditions of electroporation are the same as those described in example 1), positive clones were selected, and the obtained recombinant strain was named Cg.DELTA.zwf/pXYJ-hasA-glmUMS.
Wherein, the primer sequences are as follows:
glmU-F：5’-ttggcaggatccccgggtacaaggatttgagataatcttgagcg-3’
glmU-R：5’-ggccaagatcttagccttcctggttgtggacg-3’
glmM-F：5’-ggaaggctaagatcttggccaggccgtgca-3’
glmM-R：5’-tttctcgatcattagacttctgcaaccactgcag-3’
glmS-F：5’-cagaagtctaatgatcgagaaaaagttgttgtaaagtcatgc-3’
glmS-R：5’-gctgaattcgagctcggtacttattcgacggtgacagactttgcc-3’。
cg delta zwf/pXYJ-hasA-glmUMS was fermented in a 2L fermentation tank, the liquid loading was 1L, the inoculation amount was 10 v/v%, and the hyaluronic acid yield was examined. The fermentation conditions and the detection method were as described in example 3. The yield of hyaluronic acid reaches 6.8g/L, and the average molecule of hyaluronic acid reaches 200 ten thousand daltons.
Example 5 construction of recombinant Corynebacterium glutamicum for high production of Low molecular weight hyaluronic acid
1. Based on the amino acid sequence of the hyaluronidase of the leech, a codon-optimized hyaluronidase synthetase gene Hyal (SEQ ID NO:6) is designed, and the Wuxi Qinglan biological technology limited company is entrusted to carry out gene synthesis; carrying out PCR by taking the artificially synthesized Hyal gene as a template and using Hyal-F and Hyal-R as primers to obtain a Hyal fragment of about 1500bp, and purifying a PCR product;
2. plasmid pEC-S (purchased from Addgene) with signal peptide is subjected to double digestion by EcoRI/XbaI, Hyal fragment is connected to pEC-S by utilizing Gibson Assembly kit, and the obtained recombinant plasmid is named as pEC-Hyal;
3. pEC-hyal was transformed into the recombinant Corynebacterium glutamicum Cg. DELTA. zwf/pXYJ-hasA-udgA of example 3 by electroporation (conditions of electroporation are as described in example 1), positive clones were selected, and the resulting recombinant strain was named Cg. DELTA. zwf/pXYJ-hasA-udgA/pEC-hyal.
Wherein, the primer sequences are as follows:
hyal-F：5’-ctacttccactgcacaaagcatgaaggagattgcggtcacg-3’
hyal-R：5’-gcctgcaggtcgactctagattactttttgcaggcctctacattgg-3’。
and (3) fermenting Cg delta zwf/pXYJ-hasA-udgA/pEC-hyal in a 2L fermentation tank, wherein the liquid loading amount is 1L, the inoculation amount is 10 v/v%, and the hyaluronic acid yield is detected. The fermentation conditions and the detection method were as described in example 3. The yield of hyaluronic acid reaches 18.2g/L, and the average molecule reaches 3000 daltons.
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Claims (4)
1. A method for constructing recombinant corynebacterium glutamicum with high hyaluronic acid yield by using c.glutamicum Δ zwf, which is characterized by comprising the following steps:
1) construction of Glutamicum. DELTA. zwf
Knocking out a glucose 6-phosphate dehydrogenase gene zwf in Corynebacterium glutamicum (Corynebacterium glutamicum) by a genetic engineering means, and constructing to obtain C.glutamicum delta zwf;
the starting strain of the C.glutamicum delta zwf is Corynebacterium glutamicum MB 001;
2) construction of recombinant Corynebacterium glutamicum for high hyaluronic acid yield
And (3) overexpressing a hyaluronic acid synthase gene hasA with a sequence shown as SEQ ID NO:7 from streptococcus zooepidemicus in the C.glutamicum delta zwf to construct the recombinant corynebacterium glutamicum with high hyaluronic acid yield.
2. The method according to claim 1, wherein the specific construction method of C.glutamicum Δ zwf comprises the following steps:
s11, carrying out PCR by taking the genome DNA of Corynebacterium glutamicum MB001 as a template and zwf-up-F and zwf-up-R as primers to obtain upstream homologous fragment zwf-up of glucose 6-phosphate dehydrogenase gene zwf and purifying PCR products;
s12, carrying out PCR by taking the genome DNA of Corynebacterium glutamicum MB001 as a template and zwf-down-F and zwf-down-R as primers to obtain a zwf downstream homologous fragment zwf-down and carrying out PCR product purification;
s13, carrying out double enzyme digestion on the suicide plasmid pK18mobsacB of corynebacterium glutamicum by EcoRI/XbaI, connecting zwf-up and zwf-down fragments to the pK18mobsacB subjected to enzyme digestion by using a Gibsonassemby kit in one step, and naming the obtained recombinant plasmid as pK 18-zwf;
s14, converting pK18-zwf into Corynebacterium glutamicum MB001, and screening positive clones to obtain the corynebacterium glutamicum MB 001;
wherein the primer sequences are as follows:
zwf-up-F：5’-acagctatgacatgattacgggcgattcctacgacgctca-3’
zwf-up-R：5’-taaattatggcacgatggtagtgtcacgatcc-3’
zwf-down-F：5’-taccatcgtgccataatttaggggcaaaaaatgatctttgaact-3’
zwf-down-R：5’-tgcatgcctgcaggtcgactgtgcgcgtactacatcaaccatag-3’。
3. the method according to claim 1 or 2, wherein the recombinant corynebacterium glutamicum for high production of hyaluronic acid is constructed by a method comprising:
1) based on the amino acid sequence of hyaluronic acid synthetase of streptococcus zooepidemicus, a codon-optimized hyaluronic acid synthetase gene hasA is designed, the sequence is shown as SEQ ID NO. 7, PCR amplification is carried out by taking an artificially synthesized hasA gene as a template and hasA-F and hasA-R as primers, and an amplification product is purified to obtain a purified hasA fragment; carrying out double enzyme digestion on the vector pXYJ-12 carrying the lactose permease gene LacY by EcoRI/XbaI, connecting the purified hasA fragment to the vector pXYJ-12 by using a Gibson Assembly kit, and naming the obtained recombinant plasmid as pXYJ-hasA;
2) transforming pXYJ-hasA into the C.glutamicum delta zwf, and screening positive clones to obtain the pXYJ-hasA;
wherein the primer sequences are as follows:
hasA-F：5’-acagctatgacatgattacgcgaaccacgcaatgcgtctc-3’
hasA-R：5’-gcctgcaggtcgactcgtctcggttggcagtgac-3’。
4. use of recombinant Corynebacterium glutamicum produced in high yields of hyaluronic acid and constructed according to the method of any of claims 1 to 3, in the fermentative production of hyaluronic acid.
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