WO2020248359A1 - 一种高产透明质酸的重组菌及其构建方法与应用 - Google Patents

一种高产透明质酸的重组菌及其构建方法与应用 Download PDF

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WO2020248359A1
WO2020248359A1 PCT/CN2019/101569 CN2019101569W WO2020248359A1 WO 2020248359 A1 WO2020248359 A1 WO 2020248359A1 CN 2019101569 W CN2019101569 W CN 2019101569W WO 2020248359 A1 WO2020248359 A1 WO 2020248359A1
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gene
recombinant
expression
hyaluronic acid
pdapb
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于慧敏
成方宇
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清华大学
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C12N15/77—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Corynebacterium; for Brevibacterium
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
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    • C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
    • C12Y101/01022—UDP-glucose 6-dehydrogenase (1.1.1.22)
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    • C12Y204/01—Hexosyltransferases (2.4.1)
    • C12Y204/01212—Hyaluronan synthase (2.4.1.212)

Definitions

  • the invention belongs to the technical field of genetic engineering, and specifically relates to a recombinant bacteria with high hyaluronic acid production and a construction method and application thereof.
  • High molecular weight HA can be used as a filler in clinical surgery; medium molecular weight HA is used in cosmetics and skin care products; small molecular HA can be made into oral health products to supplement the sugar amines and sugar acids needed by the human body and enhance metabolism ,anti aging.
  • Corynebacterium glutamicum (Corynebacterium glutamicum) is a type of aerobic bacteria with short rod or small rod-shaped cells, which is Gram-positive, and is a GRAS (generally recognized as safe) strain recognized by the U.S. Video Drug Administration. Corynebacterium glutamicum itself does not produce any endotoxin and exotoxin, and is very suitable for the production of food safety grade and medical safety grade products. Yu Huimin and others successfully constructed HA-producing recombinant Corynebacterium glutamicum with a yield of 6.0g/L or more (CN103937734A); and knocked out the side effects in the HA-producing recombinant Corynebacterium glutamicum in 2017.
  • the purpose of the present invention is to provide a recombinant bacteria (or genetically engineered bacteria) with high hyaluronic acid production, which is to transfer the gene containing hyaluronic acid synthase and UDP-glucose dehydrogenase into Corynebacterium glutamicum , And enhance the expression of UDP-glucose dehydrogenase gene.
  • the hyaluronic acid synthase gene can be shasA
  • the UDP-glucose dehydrogenase can be CgHasB
  • the gene can be Cg10360 (or CghasB).
  • the method for down-regulating the expression level is gene knockout, mutation or introduction of antisense RNA.
  • the method for enhancing the expression of the UDP-glucose dehydrogenase gene is to place the UDP-glucose dehydrogenase gene under the control of two promoters, for example, one of which is PdapB, PdapA, Psod or Ptac promoter child.
  • the lactate dehydrogenase gene of the recombinant bacteria is inactivated.
  • the method for inactivating the lactate dehydrogenase gene can be gene knockout, and the method for knockout can be the method in Chinese Patent CN107354119A.
  • the Corynebacterium glutamicum ATCC13032 can be selected.
  • the method of using recombinant vectors in Chinese patent CN103937734A can be used to transfer the UDP-glucose dehydrogenase gene.
  • the hydrogenase gene is simultaneously expressed under the control of two promoters, and the recombinant bacteria lactate dehydrogenase gene is inactivated;
  • the method for down-regulating the expression of fructose bisphosphate aldolase gene or pyruvate dehydrogenase gene is: introducing antisense RNA expression cassette fragments and/or gene mutations; the bacterial lactate dehydrogenase gene
  • the method of inactivation is gene knockout, for example, the method in Chinese patent CN107354119A is selected.
  • the method of gene mutation can select the start codon mutation.
  • the method for enhancing the expression of the UDP-glucose dehydrogenase gene (CghasB) of Corynebacterium glutamicum that is, on the basis of the expression plasmid described in the Chinese patent CN103937734A, the CghasB gene is placed under two promoters for expression.
  • one of the promoters is the Ptac promoter contained in the plasmid PXMJ19, and the other promoter can be derived from the genome of Corynebacterium glutamicum, such as the promoter of the 4-hydroxytetrahydropyridine reductase gene (dapB), or it can be used Ptac promoter.
  • the specific construction method is as follows:
  • the gene shasA is transcribed by the Ptac promoter
  • the gene CghasB is transcribed by the Ptac promoter and the PdapB promoter at the same time.
  • the primer sequences used are as follows:
  • PdapB-F CTGTAAGGATCCCCGGGTACCCGCGTGAACGTTTCGTGC;
  • PdapB-R TATGTGTCCTCCTTTGGTACCTATGCTCCTTCATTTTCGTGG.
  • the method for preparing hyaluronic acid by recombinant bacteria can choose:
  • the genetically engineered bacteria liquid obtained above is connected to the fermentation medium according to the volume percentage of 1-20%, the inducer is added after culturing for 1-5 hours, and the culture is continued to obtain a fermentation broth containing hyaluronic acid.
  • the culture conditions in step (1) are: temperature 25-40°C, rotation speed 100-300rpm/min, time 10-30h; culture conditions in step (2): temperature 25-40°C, rotation speed 100-300rpm/min, pH It is 6.0-8.0, and the cultivation time is 24-72h.
  • a method of down-regulating the expression of fructose bisphosphate aldolase gene (fba) in Corynebacterium glutamicum can be selected to weaken the glycolytic pathway and strengthen HA synthesis.
  • the plasmid was used as a template, and PaFT-F and PaFT-R were used as primers for PCR reaction and purification to amplify the expression cassette of the fba gene antisense RNA.
  • the plasmid PXMJ19-A-PdapB-B was digested with MauBI.
  • the digested product and the expression cassette of the antisense RNA of the fba gene were subjected to a Gibson ligation reaction to obtain the plasmid PXMJ19-A-PdapB-B-aF.
  • the primer sequences used are as follows:
  • the recombinant plasmid PXMJ19-A-PdapB-B-aF was transformed into C.glu- ⁇ ldh using electrotransformation method, coated on LB plates (containing chloramphenicol), and resistant clones were picked for culture, and PCR verification was performed , The genetically engineered strain C.glu- ⁇ ldh/A-PdapB-B-aF transformed with the PXMJ19-A-PdapB-B-aF plasmid was obtained.
  • a method of down-regulating the expression of the pyruvate dehydrogenase gene can be selected to weaken the tricarboxylic acid cycle and strengthen HA synthesis.
  • the genome of Corynebacterium glutamicum was used as a template, and aE-F and aE-R were used as primers for PCR reaction and purification to amplify the antisense RNA fragment of the aceE gene (as shown in SEQ ID NO. 2 in the sequence table).
  • the plasmid PXMJ19 was double digested with SalI/EcorI.
  • the digested product and the antisense RNA fragment of the aceE gene are subjected to Gibson ligation reaction to obtain an expression cassette plasmid containing the antisense RNA of the aceE gene transcribed from the Ptac promoter.
  • the plasmid was used as a template, and PaET-F and PaET-R were used as primers for PCR reaction and purification to amplify an expression cassette of aceE gene antisense RNA.
  • aE-R AAAACAGCCAAGCTGAATTCGCGTTACCGTCGTTGGATTCG;
  • PaET-R GAGCAAAAACAGGAAGGCAATGTAGAAACGCAAAAAGGCCA.
  • the recombinant plasmid PK18-aEG was transformed into C.glu- ⁇ ldh using electrotransformation method, and LB plate (containing kanamycin) was spread, and resistant clones were picked for culture, and PCR verification was performed. Then, the positive clones were spread on LB plates containing 150g/L for secondary screening. After sequencing, the strain with the start codon mutated to GTG was selected, and C.glu- ⁇ ldh-aEG was obtained.
  • the primer sequences used are as follows:
  • aEG-down-R TGCCTGCAGGTCGACTCTAGATGATGGCTGCGTTCCAGC.
  • the recombinant plasmid PXMJ19-A-PdapB-B-aF-aE was transformed into C.glu- ⁇ ldh-aEG using electrotransformation, coated on LB plates (containing chloramphenicol), and resistant clones were picked for culture , Perform PCR verification, and obtain genetically engineered bacteria C.glu- ⁇ ldh-aEG/A-PdapB-B-aF-aE transformed with PXMJ19-A-PdapB-B-aF-aE plasmid.
  • the beneficial effects of the present invention are: the present invention adopts molecular biology methods and technologies, through metabolic engineering regulation strategies, on the basis of knocking out the by-product lactic acid synthesis pathway, strengthening HA synthesis pathway, weakening glycolysis pathway and tricarboxylic acid Circulation to construct a recombinant Corynebacterium glutamicum with high hyaluronic acid production.
  • the Corynebacterium glutamicum host of the present invention is non-pathogenic to humans and animals, and is a food-grade safe microorganism. Its hyaluronic acid yield is high. It is cultured in a feed fermentation tank with an output as high as 28g/L or more. It has the highest level of industrial production; the new strain has a good industrial application prospect.
  • Figure 1 shows the PCR verification of the additional promoter used in CghasB: Lane M is the DNA molecular weight standard; Lane 1 is the control without additional promoter; Lane 2 is the PdapA promoter; Lane 3 is the PdapB promoter; Lane 4 is the Psod Promoter; Lane 5 is the Ptac promoter.
  • Figure 2 shows C.glu- ⁇ ldh/AB, C.glu- ⁇ ldh/A-PdapA-B, C.glu- ⁇ ldh/A-PdapB-B, C.glu- ⁇ ldh/A-Psod-B and C.glu -The HA production and the relative expression of CghasB of ⁇ ldh/A-Ptac-B in shake flask fermentation.
  • Figure 3 shows the HA production and relative expression of fba of C.glu- ⁇ ldh/A-Ptac-B and C.glu- ⁇ ldh/A-Ptac-B-aF in shake flask fermentation.
  • Figure 4 shows the sequencing result of the mutation of the start codon ATG of aceE to GTG.
  • Figure 5 shows C.glu- ⁇ ldh/A-Ptac-B-aF, C.glu- ⁇ ldh-aEG/A-Ptac-B-aF, C.glu- ⁇ ldh/A-Ptac-B-aF-aE and C .glu- ⁇ ldh-aEG/A-Ptac-B-aF-aE HA production and relative enzyme activity of pyruvate dehydrogenase in shake flask fermentation.
  • Figure 7 is the recombinant Corynebacterium glutamicum C.glu- ⁇ ldh/AB, C.glu- ⁇ ldh/A-PdapB-B, C.glu- ⁇ ldh/A-PdapB-B-aF and C.glu- ⁇ ldh-aEG /A-PdapB-B-aF-aE in the shake flask fermentation HA production, and C.glu- ⁇ ldh-aEG/A-PdapB-B-aF-aE in the fermenter fed fermentation HA production.
  • this example is modified on the basis of the recombinant plasmid PXMJ19-AB (see Chinese patent CN103937734A), that is, inserting additional genes between shasA and CghasB
  • the promoter allows CghasB to be transcribed by the Ptac promoter and this additional promoter at the same time.
  • the additional promoters are PdapB (promoter of 4-hydroxytetrahydropyridine reductase gene), PdapA (promoter of dihydropicoline synthase gene), Psod (superoxide dismutase gene) Promoter) and Ptac promoter.
  • PdapB promoter of 4-hydroxytetrahydropyridine reductase gene
  • PdapA promoter of dihydropicoline synthase gene
  • Psod superoxide dismutase gene
  • Ptac promoter Ptac promoter
  • the genome of Corynebacterium glutamicum was used as a template, and PdapB-F and PdapB-R were used as primers to carry out PCR reaction and purification to amplify the promoter fragment of dapB gene.
  • the primer was synthesized by Tianyi Huiyuan Co., Ltd., dissolved in sterile water and diluted to 10 ⁇ M for use.
  • the pre-made buffer used for PCR amplification was purchased from Vazayme Company.
  • the amplification reaction system is:
  • Thermal cycling conditions are 94°C for 3min; 94°C for 15s, 60°C for 15s, 72°C for 150s, 35 cycles; 72°C for 10min.
  • Plasmid PXMJ19-AB was digested with KpnI (TaKaRa) for 30 minutes; the digested product and PCR product were purified by DNA purification kit (Biomega), and then the two fragments were ligated using Gibson ligation kit (Vazayme) Cyclization; the ligation product is transformed into E.coli host bacteria TOP10 competent cells (Solabao), and coated with LB medium (peptone 10g/L, yeast powder 5g/L, NaCl 10g/L, pH 7.0) solid plate ( Containing 5mg/L chloramphenicol); select resistant clones and extract a small amount of plasmids to obtain recombinant plasmid PXMJ19-A-PdapB-B.
  • recombinant plasmids PXMJ19-A-PdapA-B, PXMJ19-A-Psod-B and PXMJ19-A-Ptac-B can be prepared.
  • primers B-F on shasA and primers A-R on CghasB the additional promoter fragments can be verified.
  • the results are shown in Figure 1.
  • the primer sequence is as follows:
  • A-R CTAGTTTCACTCGTTCTTCATCAATG
  • the transformation method is: add 5 ⁇ L of recombinant plasmid and 100 ⁇ L of competent cells to a 1.5mL centrifuge tube, mix well and add a 0.1cm electro-rotor cup , Ice bath for 30 minutes; adjust the voltage of the electroporator to 1.8kV, load the electroporator cup into the electroporator, press the shock button; after the electric shock, add 1mL of recovery medium to the electroporator cup, resuspend the cells, and transfer to 1.5mL Centrifuge tube, heat shock at 46°C for 6min, 30°C, 200rpm shaking for 2h; take 300 ⁇ L of bacterial solution and spread it on LB solid medium (containing 5 ⁇ g/mL chloramphenicol), place it in a 30°C incubator and invert it for 40 hours.
  • LB solid medium containing 5 ⁇ g/mL chloramphenicol
  • the four recombinant strains and C.glu- ⁇ ldh/AB were cultured in shake flasks to produce HA.
  • the culture method is: inoculate the recombinant bacteria in LB liquid medium (containing 5 ⁇ g/mL chloramphenicol), culture at 30°C, 200rpm for 16h, insert into the fermentation medium at a ratio of 5%, at 28°C, 200rpm Cultivate for 48h. IPTG (final concentration 1mM) was added at 3h. After the completion of the culture, the fermentation broth containing hyaluronic acid was obtained by centrifugation at room temperature at 8000 rpm.
  • the formula of the fermentation medium is: glucose 40g/L, (NH 4 ) 2 SO 4 30g/L, corn flour 20g/L, KH 2 PO 4 1g/L, K 2 HPO 4 0.5g/L, MnSO 4 ⁇ 7H 2 O 10mg/L, FeSO 4 ⁇ 7H 2 O 10mg/L, MgSO 4 ⁇ 7H 2 O 0.5g/L.
  • C.glu- ⁇ ldh/A-PdapB-B had the highest HA production in shake flask fermentation, which increased from 6.9g/L to 7.6g/L.
  • this example further uses antisense RNA technology to weaken the reaction catalyzed by the fba gene expression enzyme in glycolysis.
  • the genome of Corynebacterium glutamicum was used as a template, and aF-F and aF-R were used as primers to perform PCR reaction and purification to amplify the antisense RNA fragment of the fba gene.
  • the conditions used for PCR were the same as in Example 1.
  • Plasmid PXMJ19 was double digested with SalI/EcorI.
  • the digested product and the antisense RNA fragment of the fba gene were subjected to a Gibson ligation reaction to obtain an expression cassette plasmid containing the antisense RNA of the fba gene transcribed from the Ptac promoter, and the E. coli host bacteria TOP10 competence was transformed by the method in Example 1. cell.
  • the plasmid was used as a template, and PaFT-F and PaFT-R were used as primers for PCR reaction and purification to amplify the expression cassette of the fba gene antisense RNA.
  • the plasmid PXMJ19-A-PdapB-B was digested with MauBI.
  • the digested product and the expression cassette of the antisense RNA of the fba gene were subjected to Gibson ligation reaction to obtain the plasmid PXMJ19-A-PdapB-B-aF and transformed into E. coli host bacteria TOP10 competent cells.
  • the plasmid PXMJ19-A-PdapB-B-aF was electrotransformed into C.glu- ⁇ ldh using the method in Example 1, and positive clones were picked for PCR verification.
  • the recombinant Corynebacterium glutamicum C.glu- ⁇ ldh/A-PdapB-B-aF can be obtained.
  • C.glu- ⁇ ldh/A-PdapB-B and C.glu- ⁇ ldh/A-PdapB-B-aF were also cultured in shake flasks in the same manner as in Example 1.
  • the introduction of the antisense RNA of the fba gene can attenuate its expression and increase the HA production to 8.1 g/L.
  • this example further uses antisense RNA technology and start codon mutation to weaken the expression of pyruvate dehydrogenase gene aceE, down-regulate the tricarboxylic acid cycle, and enhance HA synthesis.
  • the genome of Corynebacterium glutamicum was used as a template, and aE-F and aE-R were used as primers to carry out PCR reaction and purification to amplify the antisense RNA fragment of aceE gene.
  • the PCR reaction and purification were performed with PaET-F and PaET-R as primers to amplify the expression cassette of the aceE gene antisense RNA.
  • the plasmid PXMJ19-A-PdapB-B-aF was digested with XhoI.
  • the digested product and the expression cassette of aceE gene antisense RNA were subjected to Gibson ligation reaction to obtain plasmid PXMJ19-A-PdapB-B-aF-aE and transformed into E.coli host bacteria TOP10 competent cells.
  • homologous recombination double exchange was used to construct aceE start codon mutant strain.
  • the sequence of about 500bp upstream and downstream, and the product was purified; the plasmid PK18mobsacB was double-enzyme digested with EcorI/XbaI, and the product was purified.
  • the digested plasmid, the upstream fragment and the downstream fragment were subjected to Gibson ligation reaction to obtain the recombinant plasmid pK18-aEG.
  • the pK18-aEG was transferred into C.glu- ⁇ ldh by the electrotransformation method in Example 1, and the first screening was performed with kanamycin.
  • the obtained positive recombinants were cultured in LB liquid medium for 16 hours, and then spread on A secondary screening was performed on an LB plate containing 150g/L sucrose, and positive clones were picked and amplified by PCR and sequenced using two primers, aEG-up-F and aEG-down-R. As shown in Figure 4, the start codon of the aceE gene was changed from ATG to GTG to obtain the recombinant strain C.glu- ⁇ ldh-aEG
  • the recombinant plasmid PXMJ19-A-PdapB-B-aF was electro-transformed into C.glu- ⁇ ldh-aEG using the method in Example 1, and positive clones were picked for PCR verification.
  • the recombinant Corynebacterium glutamicum C.glu- ⁇ ldh-aEG/A-PdapB-B-aF can be obtained.
  • the recombinant plasmid PXMJ19-A-PdapB-B-aF-aE was transferred into C.glu- ⁇ ldh and C.glu- ⁇ ldh-aEG to obtain C.glu- ⁇ ldh/A-PdapB-B-aF -aE and C.glu- ⁇ ldh-aEG/A-PdapB-B-aF-aE.
  • These three strains and C.glu- ⁇ ldh/A-PdapB-B-aF were shaken flask cultured together with the method in Example 1 to produce HA. The results are shown in Fig. 5, all three methods can increase HA yield, among which C.glu- ⁇ ldh-aEG/A-PdapB-B-aF-aE yields the highest, reaching 9.0g/L.
  • Example 4 Using C.glu- ⁇ ldh-aEG/A-PdapB-B-aF-aE fed-batch fermentation to produce HA
  • the recombinant strain C.glu- ⁇ ldh-aEG/A-PdapB-B-aF-aE constructed in Example 3 was inoculated into LB liquid medium (containing 5 ⁇ g/mL chloramphenicol) and cultured at 30°C and 200 rpm 16h, insert into the fermentation medium (5L fermentor, working volume 2L) at a ratio of 5%.
  • the extraction method and determination method of HA are the same as in Example 1.
  • the results of the assay showed that the recombinant strain C.glu- ⁇ ldh-aEG/A-PdapB-B-aF-aE under fed-batch fermentation had a HA yield of more than 28g/L ( Figure 6), which is the highest level reported so far.
  • the molecular weight of the product is high-pressure liquid chromatography, the chromatographic column is Shodex SB-806M OHpak (8 ⁇ 300mm), the mobile phase is 0.2MNaCl, and the flow rate is 1.0mL/min.
  • the molecular weight standard was purchased from Lifecore Biomedical Inc., USA. The results show that the weight average molecular weight can reach 0.2-0.3MDa.

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Abstract

涉及基因工程技术领域的一种高产透明质酸的重组菌及其构建方法与应用。该重组菌是将透明质酸合成途径中UDP-葡萄糖脱氢酶基因加强表达，同时弱化竞争途径构建而成。所述谷氨酸棒杆菌宿主对人和动物均无致病性，为食品级安全微生物；高产透明质酸的重组谷氨酸棒杆菌的透明质酸产量高，高达28g/L以上，具有良好的产业化应用前景。

Description

一种高产透明质酸的重组菌及其构建方法与应用 技术领域
本发明属于基因工程技术领域，具体涉及一种高产透明质酸的重组菌及其构建方法与应用。
背景技术
透明质酸(Hyaluronic acid,HA)是一类广泛存在于人和动物体内的糖胺聚糖，其分子成线性，重复单元由D-葡萄糖醛酸(D-glucuronic acid,GlcA)和N-乙酰-氨基葡萄糖胺(N-acetyl-D-glucosamine,GlcNAc)二糖单位组成，分子量可达10 4～10 7Da。HA在人体中起着软组织润滑、促进伤口愈合，参与免疫反应等作用。HA的商业用途也十分广泛。高分子量HA可以作为填充剂用于临床手术中；中分子量HA则用于化妆品和护肤品领域中；小分子HA可以制成口服保健品，来补充人体所需的糖胺和糖酸，增强新陈代谢，延缓衰老。
随着生物技术的快速发展，透明质酸的制备方法已经逐渐由动物组织提取法变更为微生物发酵法。目前我国的HA生产菌株主要是兽疫链球菌，其产量可达10g/L。国外也在不断地对链球菌生产HA的技术进行研究(Pourzardosht,N.,Rasaee,M.J.,2017.Improved Yield of High Molecular Weight Hyaluronic Acid Production in a Stable Strain of Streptococcus zooepidemicus via the Elimination of the Hyaluronidase-Encoding Gene.Mol.Biotechnol.59,192–199.)但是链球菌本身具有潜在的致病性，这在一定程度上限制了其发展。另一方面，培养链球菌的培养基中含有血清、脑心浸取液等昂贵原料，其生产成本也随之提高。开发更加安全且经济实用的重组菌株逐渐被重视
中国专利ZL 98812773.3于2001年公开了“透明质酸合成酶基因及其应用”，将透明质酸合成酶基因引入重组菌，即可实现透明质酸的异源合成。丹麦的Novozyme公司在枯草芽孢杆菌中重构了HA合成途径，并申请了中国专利“低分子量透明质酸的产生”(CN101384724)。江南大学进一步利用代谢工程手段构建了产不同分子量透明质酸的重组枯草芽孢杆菌(CN105087456A)。Kaur等人则在乳酸乳球菌中实现了HA的合成，产量达3.03g/L(Kaur,M.,Jayaraman,G.,2016.Hyaluronan production and molecular weight is enhanced in  pathway-engineered strains of lactate dehydrogenase-deficient Lactococcuslactis.Metab.Eng.Commun.3,15–23)。
谷氨酸棒杆菌(Corynebacteriumglutamicum)是一类好氧细菌，细胞呈短杆或小棒状，为革兰氏阳性，并且是美国视频药物管理局认定的GRAS(一般认为安全)菌株。谷氨酸棒杆菌本身不会产生任何内毒素和外毒素，非常适合用于生产食品安全级和医药安全级的产物。于慧敏等人于2014年成功构建了生产HA的重组谷氨酸棒杆菌，产量达6.0g/L以上(CN103937734A)；并于2017年在生产HA的重组谷氨酸棒杆菌中敲除了副产物乳酸合成途径，使产量提升至22g/L以上(CN107354119A)。陈振等人则在重组谷氨酸棒杆菌中，通过敲除磷酸戊糖途径，使中等分子量的透明质酸产量达到9.6g/L，并构建了生产小分子HA的重组菌株(CN106190939A)。
发明内容
本发明的目的在于提供一种高产透明质酸的重组菌(或者说基因工程菌)，其是将含有透明质酸合成酶基因和UDP-葡萄糖脱氢酶基因转入到谷氨酸棒杆菌中，并且将UDP-葡萄糖脱氢酶基因加强表达得到。
上述重组菌中，所述透明质酸合成酶基因可以为shasA，所述UDP-葡萄糖脱氢酶可以为CgHasB，其基因可以为Cg10360(或CghasB)，具体可见中国专利CN103937734A。
上述重组菌中，进一步的，可以选择下调果糖二磷酸醛缩酶基因的表达量。
上述重组菌中，再进一步的，可以选择下调丙酮酸脱氢酶基因的表达量。
上述重组菌中，下调表达量的方法为基因敲除、突变或引入反义RNA。
上述重组菌中，所述将UDP-葡萄糖脱氢酶基因加强表达的方法为将UDP-葡萄糖脱氢酶基因置于两个启动子控制下，例如，其中一个为PdapB、PdapA、Psod或Ptac启动子。
上述重组菌中，所述重组菌的乳酸脱氢酶基因失活。
上述重组菌中，所述乳酸脱氢酶基因失活的方法可以选择基因敲除，敲除的方法可以选择中国专利CN107354119A中的方法。
上述重组菌中，所述谷氨酸棒杆菌可以选择谷氨酸棒杆菌ATCC13032。
本发明的目的还在于提供上述重组菌在制备透明质酸的中的应用。
本发明的目的也在于提供一种高产透明质酸的重组菌的构建方法，包括步骤 如下：
(1)将含有透明质酸合成酶基因和UDP-葡萄糖脱氢酶基因转入到谷氨酸棒杆菌中，例如可以选择中国专利CN103937734A中利用重组载体的方法进行转入，将UDP-葡萄糖脱氢酶基因同时置于两个启动子控制之下进行表达，并将重组菌乳酸脱氢酶基因失活；
或者进一步的选择，
(2)下调果糖二磷酸醛缩酶基因的表达量；
或者进一步的选择，
(3)下调丙酮酸脱氢酶基因的表达量。
上述构建方法中，所述下调果糖二磷酸醛缩酶基因或丙酮酸脱氢酶基因的表达量的方法为：引入反义RNA表达盒片段和/或基因突变；所述菌乳酸脱氢酶基因失活的方法为基因敲除，例如选择中国专利CN107354119A中的方法。
所述基因突变的方法可以选择起始密码子突变。
一种高产透明质酸的重组菌的构建方法，具体的，可以选择以下方法：
加强谷氨酸棒杆菌UDP-葡萄糖脱氢酶基因(CghasB)表达量的方法：即在中国专利CN103937734A所述的表达质粒基础上，将CghasB基因同时置于两个启动子下进行表达。例如，其中一个启动子是质粒PXMJ19所含有的Ptac启动子，另一个启动子可以来源于谷氨酸棒杆菌基因组，如4-羟基四氢吡啶还原酶基因(dapB)的启动子，也可以使用Ptac启动子。其具体构建方法如下：
以谷氨酸棒杆菌的基因组为模板，以PdapB-F和PdapB-R为引物进行PCR反应并纯化，扩增出dapB基因的启动子片段。用KpnI对质粒PXMJ19-AB(中国专利CN103937734A)进行单酶切。酶切产物和dapB启动子片段进行Gibson连接反应，得到重组质粒PXMJ19-A-PdapB-B。该质粒中，基因shasA被Ptac启动子转录，基因CghasB同时被Ptac启动子和PdapB启动子转录。所用引物序列如下：
PdapB-F：CTGTAAGGATCCCCGGGTACCCGCGTGAACGTTTCGTGC；
PdapB-R：TATGTGTCCTCCTTTGGTACCTATGCTCCTTCATTTTCGTGG。
进一步地，将重组质粒PXMJ19-A-PdapB-B使用电转化方法转入乳酸脱氢酶(LDH)失活的谷氨酸棒杆菌C.glu-Δldh(中国专利CN107354119A)，涂布 LB平板(含氯霉素)，挑取抗性克隆进行培养，进行PCR验证，获得转化有PXMJ19-A-PdapB-B质粒的重组菌C.glu-Δldh/A-PdapB-B。
重组菌制备透明质酸的方法，可以选择：
(1)将C.glu-Δldh/AB和C.glu-Δldh/A-PdapB-B接入LB液体培养基中进行培养，得到基因工程菌菌液；
(2)按照1-20％的体积百分比将上述得到的基因工程菌菌液接入发酵培养基中，培养1-5h后加入诱导剂，继续培养，得到含有透明质酸的发酵液。
上述发酵培养基的组成为：糖类20-100g/L，无机氮源10-50g/L，有机氮源2-40g/L，KH 2PO 4 0.1-5g/L，K 2HPO 4·12H 2O 0.5-5g/L，MgSO 4·7H 2O 0.1-8g/L，MnSO 4·H 2O 0.002-0.1g/L，FeSO 4·7H 2O 0.002-0.1g/L g/L，pH 6.0-8.0。
步骤(1)中培养条件为：温度25-40℃，转速100-300rpm/min，时间10-30h；步骤(2)中培养条件为：温度25-40℃，转速100-300rpm/min，pH为6.0-8.0，继续培养时间24-72h。
步骤(2)中所述诱导剂为异丙基硫代半乳糖苷或乳糖，加入量为0.05-5.0g/L。
上述发酵培养基中的糖类为葡萄糖、蔗糖、果糖、麦芽糖、淀粉或淀粉水解液；无机氮源为硫酸铵、氯化铵、硝酸铵、硝酸钠或硝酸钾；有机氮源为蛋白胨、酵母膏、酵母粉、牛肉膏、玉米浆、玉米浆粉或大豆粉。
进一步的，可以选择下调谷氨酸棒杆菌中的果糖二磷酸醛缩酶基因(fba)表达量的方法，来弱化糖酵解途径，强化HA合成。
方法是在重组质粒PXMJ19-A-PdapB-B上引入基因fba的反义RNA(aF)表达盒片段。
其具体构建方法如下：
以谷氨酸棒杆菌的基因组为模板，以aF-F和aF-R为引物进行PCR反应并纯化，扩增出fba基因的反义RNA片段(如序列表SEQIDNO.1所示)。用SalI/EcorI对质粒PXMJ19进行双酶切。酶切产物和fba基因的反义RNA片段进行Gibson连接反应，得到含有由Ptac启动子转录fba基因反义RNA的表达盒质粒。以该质粒为模板，以PaFT-F和PaFT-R为引物进行PCR反应并纯化，扩增出fba基因反义RNA的表达盒。用MauBI对质粒PXMJ19-A-PdapB-B进行单酶切。酶切 产物和fba基因反义RNA的表达盒进行Gibson连接反应，得到质粒PXMJ19-A-PdapB-B-aF。所用引物序列如下：
aF-F：CTTGCATGCCTGCAGGTCGACATTTCGAGGTTCTCGTCGATTGG；
aF-R：AAAACAGCCAAGCTGAATTCCTCCACCGGTGGTGCAGA；
PaFT-F：CGCTTCGCCTTCGCGCGCGTAAATCACTGCATAATTCGTGTCGC；
PaFT-R：TCAGCTTGCAATTCGCGCGCGTGTAGAAACGCAAAAAGGCCA。
进一步地，将重组质粒PXMJ19-A-PdapB-B-aF使用电转化方法转入C.glu-Δldh中，涂布LB平板(含氯霉素)，挑取抗性克隆进行培养，进行PCR验证，获得转化有PXMJ19-A-PdapB-B-aF质粒的基因工程菌C.glu-Δldh/A-PdapB-B-aF。
进一步的，可以选择下调丙酮酸脱氢酶基因(aceE)表达量的方法，来弱化三羧酸循环，强化HA合成。
方法是在重组质粒PXMJ19-A-PdapB-B-aF上引入基因aceE的反义RNA(aE)表达盒片段，或者，进一步的将基因组上aceE的起始密码子ATG突变为GTG。
其具体构建方法如下：
以谷氨酸棒杆菌的基因组为模板，以aE-F和aE-R为引物进行PCR反应并纯化，扩增出aceE基因的反义RNA片段(如序列表SEQIDNO.2所示)。用SalI/EcorI对质粒PXMJ19进行双酶切。酶切产物和aceE基因的反义RNA片段进行Gibson连接反应，得到含有由Ptac启动子转录aceE基因反义RNA的表达盒质粒。以该质粒为模板，以PaET-F和PaET-R为引物进行PCR反应并纯化，扩增出aceE基因反义RNA的表达盒。
用XhoI对质粒PXMJ19-A-PdapB-B-aF进行单酶切。酶切产物和aceE基因反义RNA的表达盒进行Gibson连接反应，得到质粒PXMJ19-A-PdapB-B-aF-aE。所用引物序列如下：
aE-F：CTTGCATGCCTGCAGGTCGACAGACCCATGGACACAGTTGGG；
aE-R：AAAACAGCCAAGCTGAATTCGCGTTACCGTCGTTGGATTCG；
PaET-F：TTCCCTTTTTTGCGGCATTTAAATCACTGCATAATTCGTGTCGC；
PaET-R：GAGCAAAAACAGGAAGGCAATGTAGAAACGCAAAAAGGCCA。
另一方面，以谷氨酸棒杆菌的基因组为模板，以aEG-up-F和aEG-up-R为 引物进行PCR反应并纯化，扩增出aceE基因上游片段；以aEG-down-F和aEG-down-R为引物进行PCR反应并纯化，扩增出aceE基因下游片段。用XbaI/EcorI对质粒PK18mobsacB进行双酶切。酶切产物和aceE基因的上下游片段进行Gibson连接反应，得到PK18-aEG。将重组质粒PK18-aEG使用电转化方法转入C.glu-Δldh中，涂布LB平板(含卡那霉素)，挑取抗性克隆进行培养，进行PCR验证。再将阳性克隆涂布于含有150g/L的LB平板进行二次筛选。经过测序筛选出起始密码子突变为GTG的菌株，得到C.glu-Δldh-aEG。所用引物序列如下：
aEG-up-F：CTATGACCATGATTACGAATTCATCACATCTCGCGGGAAACT；
aEG-up-R：GATCGGCCACTTCCACACCT；
aEG-down-F：AGGTGTGGAAgTGGCCGATC；
aEG-down-R：TGCCTGCAGGTCGACTCTAGATGATGGCTGCGTTCCAGC。
进一步地，将重组质粒PXMJ19-A-PdapB-B-aF-aE使用电转化方法转入C.glu-Δldh-aEG中，涂布LB平板(含氯霉素)，挑取抗性克隆进行培养，进行PCR验证，获得转化有PXMJ19-A-PdapB-B-aF-aE质粒的基因工程菌C.glu-Δldh-aEG/A-PdapB-B-aF-aE。
本发明的有益效果为：本发明采用分子生物学方法和技术，通过代谢工程调控的策略，在敲除副产物乳酸合成途径的基础上，强化HA合成途径，弱化糖酵解途径和三羧酸循环，构建得到高产透明质酸的重组谷氨酸棒杆菌。本发明的谷氨酸棒杆菌宿主对人和动物均无致病性，为食品级安全微生物，其透明质酸产量高，在发酵罐中补料发酵培养，产量高达28g/L以上，为现有工业生产的最高水平；新菌株具有良好的产业化应用前景。
附图说明
图1为强化CghasB所用额外启动子的PCR验证：其中，泳道M为DNA分子量标准；泳道1为无额外启动子的对照；泳道2为PdapA启动子；泳道3为PdapB启动子；泳道4为Psod启动子；泳道5为Ptac启动子。
图2为C.glu-Δldh/AB、C.glu-Δldh/A-PdapA-B、C.glu-Δldh/A-PdapB-B、C.glu-Δldh/A-Psod-B和C.glu-Δldh/A-Ptac-B在摇瓶发酵中的HA产量和CghasB相对表达量。
图3为C.glu-Δldh/A-Ptac-B和C.glu-Δldh/A-Ptac-B-aF在摇瓶发酵中的HA产量和fba相对表达量。
图4为aceE起始密码子ATG突变为GTG的测序结果。
图5为C.glu-Δldh/A-Ptac-B-aF、C.glu-Δldh-aEG/A-Ptac-B-aF、C.glu-Δldh/A-Ptac-B-aF-aE和C.glu-Δldh-aEG/A-Ptac-B-aF-aE在摇瓶发酵中的HA产量和丙酮酸脱氢酶相对酶活。
图6为重组菌株C.glu-Δldh-aEG/A-PdapB-B-aF-aE在5L发酵中补料培养生产HA过程中，HA产量和菌干重随时间的变化。
图7为重组谷氨酸棒杆菌C.glu-Δldh/AB、C.glu-Δldh/A-PdapB-B、C.glu-Δldh/A-PdapB-B-aF和C.glu-Δldh-aEG/A-PdapB-B-aF-aE在摇瓶发酵的HA产量，以及C.glu-Δldh-aEG/A-PdapB-B-aF-aE在发酵罐补料发酵的HA产量。
具体实施方式
下面结合附图和具体实施例对本发明作进一步说明。如未特别指明，实施例中所用的生化试剂均为市售试剂，实施例中所用的技术手段为本领域技术人员书中的常规手段。
实施例1构建CghasB加强表达菌株并对比其HA生产能力
为了强化谷氨酸棒杆菌HA合成中关键酶基因CghasB的表达量，本实施例在重组质粒PXMJ19-AB(具体见中国专利CN103937734A)的基础上进行改造，即在基因shasA和CghasB之间插入额外的启动子，使得CghasB可以同时被Ptac启动子和这个额外的启动子转录。在本实施例中，额外的启动子选取PdapB(4-羟基四氢吡啶还原酶基因的启动子)，PdapA(二氢甲基吡啶合成酶基因的启动子)，Psod(超氧化物歧化酶基因的启动子)和Ptac启动子。以PdapB为例，重组质粒的构建方法如下：
以谷氨酸棒杆菌的基因组为模板，以PdapB-F和PdapB-R为引物进行PCR反应并纯化，扩增出dapB基因的启动子片段。引物由天一辉远有限公司合成，用无菌水溶解并稀释至10μM使用。PCR扩增所用的预制缓冲液购自Vazayme公司。扩增反应体系为：
Figure PCTCN2019101569-appb-000001
Figure PCTCN2019101569-appb-000002
热循环条件为94℃ 3min；94℃ 15s，60℃ 15s，72℃ 150s，35个循环；72℃，10min。质粒PXMJ19-AB用KpnI(TaKaRa公司)进行单酶切30min；所得酶切产物和PCR产物通过DNA纯化试剂盒(Biomega公司)进行纯化，然后使用Gibson连接试剂盒(Vazayme公司)使两个片段连接环化；连接产物转化E.coli宿主菌TOP10感受态细胞(索莱宝公司)，涂布LB培养基(蛋白胨10g/L，酵母粉5g/L，NaCl 10g/L，pH 7.0)固体平板(含5mg/L氯霉素)；挑选抗性克隆，小量提取质粒，得到重组质粒PXMJ19-A-PdapB-B。同理可以制备重组质粒PXMJ19-A-PdapA-B、PXMJ19-A-Psod-B和PXMJ19-A-Ptac-B。利用shasA上的引物B-F和CghasB上的引物A-R可以验证出额外启动子的片段，其结果如图1所示。其中引物序列如下：
B-F：CTGAGCTTCCTGCTGAGCCC
A-R：CTAGTTTCACTCGTTCTTCATCAATG
将4个重组质粒转入C.glu-Δldh中(具体见中国专利CN107354119A)，转化方法为：向一个1.5mL离心管中加入5μL重组质粒以及100μL感受态细胞，混匀后加入0.1cm电转杯，冰浴30min；调节电穿孔仪电压为1.8kV，将电转杯装入电穿孔仪，按下电击键；电击结束后，向电转杯中加入1mL复苏培养基，重悬细胞，转入1.5mL离心管，46℃热激6min，30℃、200rpm震荡培养2h；取300μL菌液涂布于LB固体培养基(含5μg/mL氯霉素)，置于30℃培养箱中倒置培养40小时，挑取单菌落，进行PCR验证。即可得到重组谷氨酸棒杆菌C.glu-Δldh/A-PdapB-B、C.glu-Δldh/A-PdapA-B、C.glu-Δldh/A-Psod-B和C.glu-Δldh/A-Ptac-B。
将构建的4株重组菌和C.glu-Δldh/AB进行摇瓶培养生产HA。培养方法为：将重组菌接种于LB液体培养基(含5μg/mL氯霉素)中，30℃、200rpm下培养16h，以5％的比例接入发酵培养基中，在28℃、200rpm下培养48h。3h时加入 IPTG(终浓度1mM)。培养结束后常温8000rpm离心即得含透明质酸的发酵液。
发酵培养基的配方为：葡萄糖40g/L，(NH 4) 2SO 430g/L，玉米粉20g/L，KH 2PO 41g/L，K 2HPO 40.5g/L，MnSO 4·7H 2O 10mg/L，FeSO 4·7H 2O 10mg/L，MgSO 4·7H 2O 0.5g/L。
取上述所得发酵液1mL加入1mL 0.1％w/v的SDS溶液混匀，室温温浴20分钟；12000rpm离心10min，将上清液转移至新的10mL EP管中，加入2倍体积的冰乙醇，4℃放置1h；然后12000rpm离心10min，除去上清后室温下放置，待乙醇完全挥发后用去离子水重悬。取300μL重悬后的溶液，加入700μL乙酸缓冲液(0.2mol/L醋酸钠、0.15mol/L氯化钠，用乙酸调节pH至6.0)和2mL 2.5g/L的CTAB溶液(0.5mol NaOH溶解)，反应5min后测定OD 400。
如图2所示，额外启动子的引入都可以提升CghasB的表达量，同时提升HA的产量。其中，C.glu-Δldh/A-PdapB-B在摇瓶发酵中HA产量最高，由6.9g/L提升至7.6g/L。
实施例2构建fba弱化菌株并对比其HA生产能力
在实施例1的基础上，本实施例进一步利用反义RNA技术来弱化糖酵解中fba基因表达酶催化的反应。以谷氨酸棒杆菌的基因组为模板，以aF-F和aF-R为引物进行PCR反应并纯化，扩增出fba基因的反义RNA片段。PCR所用条件同实施例1。
质粒PXMJ19用SalI/EcorI进行双酶切。酶切产物和fba基因的反义RNA片段进行Gibson连接反应，得到含有由Ptac启动子转录fba基因反义RNA的表达盒质粒，并以实施例1中的方法转化E.coli宿主菌TOP10感受态细胞。以该质粒为模板，以PaFT-F和PaFT-R为引物进行PCR反应并纯化，扩增出fba基因反义RNA的表达盒。用MauBI对质粒PXMJ19-A-PdapB-B进行单酶切。酶切产物和fba基因反义RNA的表达盒进行Gibson连接反应，得到质粒PXMJ19-A-PdapB-B-aF并转化E.coli宿主菌TOP10感受态细胞。
将质粒PXMJ19-A-PdapB-B-aF以实施例1中的方法电转化至C.glu-Δldh中，挑取阳性克隆，进行PCR验证。即可得到重组谷氨酸棒杆菌C.glu-Δldh/A-PdapB-B-aF。同样以实施例1中的方法摇瓶培养C.glu-Δldh/A-PdapB-B和C.glu-Δldh/A-PdapB-B-aF。如图3所示，fba基因反义 RNA的引入可以减弱其表达，并且HA产量提升至8.1g/L。
实施例3构建aceE弱化菌株并对比其HA生产能力
在实施例2的基础上，本实施例进一步利用反义RNA技术和起始密码子突变来弱化丙酮酸脱氢酶基因aceE表达量，下调三羧酸循环，强化HA合成。以谷氨酸棒杆菌的基因组为模板，以aE-F和aE-R为引物进行PCR反应并纯化，扩增出aceE基因的反义RNA片段。再以实施例2中的方法，用PaET-F和PaET-R为引物进行PCR反应并纯化，扩增出aceE基因反义RNA的表达盒。用XhoI对质粒PXMJ19-A-PdapB-B-aF进行单酶切。酶切产物和aceE基因反义RNA的表达盒进行Gibson连接反应，得到质粒PXMJ19-A-PdapB-B-aF-aE并转化E.coli宿主菌TOP10感受态细胞。
同时利用同源重组双交换构建aceE起始密码子突变菌株。挑取野生谷氨酸棒杆菌为模板，以aEG-up-F/aEG-up-R和aEG-down-F/aEG-down-R为引物分别扩增其基因组中aceE基因起始密码子的上下游约500bp的序列，并对产物进行纯化；利用EcorI/XbaI对质粒PK18mobsacB进行双酶切，并对产物进行纯化。将酶切后的质粒，上游片段和下游片段进行Gibson连接反应，获得重组质粒pK18-aEG。将pK18-aEG以实施例1的电转方式转入C.glu-Δldh中，利用卡那霉素进行第一次筛选，将得到的阳性重组子于LB液体培养基中培养16h，然后涂布于含有150g/L蔗糖的LB平板上进行二次筛选，挑取阳性克隆并利用aEG-up-F和aEG-down-R两条引物进行PCR扩增并测序。如图4所示，aceE基因的起始密码子由ATG变为GTG，获得重组菌株C.glu-Δldh-aEG
进一步，将重组质粒PXMJ19-A-PdapB-B-aF以实施例1中的方法电转化至C.glu-Δldh-aEG中，挑取阳性克隆，进行PCR验证。即可得到重组谷氨酸棒杆菌C.glu-Δldh-aEG/A-PdapB-B-aF。以同样的方法将将重组质粒PXMJ19-A-PdapB-B-aF-aE转入C.glu-Δldh和C.glu-Δldh-aEG中，得到C.glu-Δldh/A-PdapB-B-aF-aE和C.glu-Δldh-aEG/A-PdapB-B-aF-aE。将这3株菌和C.glu-Δldh/A-PdapB-B-aF一同以实施例1中的方法摇瓶培养生产HA。结果如图5所示，三种方法都可以使HA产量提升，其中C.glu-Δldh-aEG/A-PdapB-B-aF-aE产量最高，达9.0g/L。
实施例4使用C.glu-Δldh-aEG/A-PdapB-B-aF-aE补料发酵生产HA
将实施例3中所构建的重组菌株C.glu-Δldh-aEG/A-PdapB-B-aF-aE接种于LB液体培养基(含5μg/mL氯霉素)中，30℃、200rpm下培养16h，以5％的比例接入发酵培养基中(5L发酵罐，工作体积2L)。在30℃、搅拌桨600rpm、通气1vvm、pH＝7.2的条件下下培养3h，加入IPTG(终浓度0.1mM)，继续培养48h，期间流加碳源，维持糖浓度在5-10g/L，培养结束后常温8000rpm离心即得含透明质酸的发酵液。发酵培养基组成同实施例1。
HA的提取方法和测定方法同实施例1。测定结果显示，重组菌株C.glu-Δldh-aEG/A-PdapB-B-aF-aE在补料发酵下，HA产量达28g/L以上(图6)，是目前所报道的最高水平。产物分子量采用高压液相色谱，色谱柱为Shodex SB-806M OHpak(8×300mm)，流动相为0.2MNaCl，流速为1.0mL/min。分子量标准购自Lifecore Biomedical Inc.，USA。结果表明，重均分子量可以达到0.2-0.3MDa。
将重组谷氨酸棒杆菌C.glu-Δldh/AB、C.glu-Δldh/A-PdapB-B、C.glu-Δldh/A-PdapB-B-aF和C.glu-Δldh-aEG/A-PdapB-B-aF-aE在摇瓶发酵的HA产量(实验方法同实施例1-3)，以及将C.glu-Δldh-aEG/A-PdapB-B-aF-aE在发酵罐补料发酵的HA产量(实验方法同实施例4)，其结果如图7所示。

Claims (10)

  1. 一种高产透明质酸的重组菌，其是将含有透明质酸合成酶基因和UDP-葡萄糖脱氢酶基因转入到谷氨酸棒杆菌中，并且将UDP-葡萄糖脱氢酶基因加强表达得到。
  2. 根据权利要求1所述的重组菌，其特征在于，所述重组菌下调果糖二磷酸醛缩酶基因的表达量。
  3. 根据权利要求1或2所述的重组菌，其特征在于，所述重组菌下调丙酮酸脱氢酶基因的表达量。
  4. 根据权利要求2或3所述的重组菌，其特征在于，下调表达量的方法为基因敲除、突变或引入反义RNA。
  5. 权利要求1所述的重组菌，其特征在于，所述将UDP-葡萄糖脱氢酶基因加强表达的方法为将UDP-葡萄糖脱氢酶基因置于两个启动子控制下，例如，其中一个为PdapB、PdapA、Psod或Ptac启动子。
  6. 根据权利要求1-5任一项所述的重组菌，其特征在于，所述重组菌的乳酸脱氢酶基因失活。
  7. 根据权利要求6所述的重组菌，其特征在于，所述谷氨酸棒杆菌为谷氨酸棒杆菌ATCC13032。
  8. 权利要求1-7任一项所述的重组菌在制备透明质酸的中的应用。
  9. 一种高产透明质酸的重组菌的构建方法，其特征在于，包括步骤如下：
    (1)将含有透明质酸合成酶基因和UDP-葡萄糖脱氢酶基因转入到谷氨酸棒杆菌中，将UDP-葡萄糖脱氢酶基因同时置于两个启动子控制之下进行表达，并将重组菌乳酸脱氢酶基因失活；
    或者进一步的选择，
    (2)下调果糖二磷酸醛缩酶基因的表达量；
    或者进一步的选择，
    (3)下调丙酮酸脱氢酶基因的表达量。
  10. 根据权利要求9所述的构建方法，其特征在于，所述下调果糖二磷酸醛缩酶基因或丙酮酸脱氢酶基因的表达量的方法为：引入反义RNA表达盒片段和/或基因突变，所述菌乳酸脱氢酶基因失活的方法为基因敲除。
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