WO2021077580A1 - 一种高效合成高纯度透明质酸及其寡聚糖的重组谷氨酸棒状杆菌 - Google Patents

一种高效合成高纯度透明质酸及其寡聚糖的重组谷氨酸棒状杆菌 Download PDF

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WO2021077580A1
WO2021077580A1 PCT/CN2019/126013 CN2019126013W WO2021077580A1 WO 2021077580 A1 WO2021077580 A1 WO 2021077580A1 CN 2019126013 W CN2019126013 W CN 2019126013W WO 2021077580 A1 WO2021077580 A1 WO 2021077580A1
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hyaluronic acid
corynebacterium glutamicum
udp
fermentation
seq
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PCT/CN2019/126013
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English (en)
French (fr)
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康振
陈坚
胡立涛
堵国成
王阳
李江华
李佳莲
张天萌
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华熙生物科技股份有限公司
江南大学
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Priority to BR112022007733A priority Critical patent/BR112022007733A2/pt
Priority to AU2019471428A priority patent/AU2019471428A1/en
Priority to KR1020227016442A priority patent/KR20220097413A/ko
Priority to EP19950145.3A priority patent/EP4050096A4/en
Priority to US17/755,191 priority patent/US20220380819A1/en
Priority to JP2022524109A priority patent/JP2023504769A/ja
Publication of WO2021077580A1 publication Critical patent/WO2021077580A1/zh

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Definitions

  • the invention relates to a recombinant Corynebacterium glutamicum for efficiently synthesizing high-purity hyaluronic acid and its oligosaccharides, and belongs to the technical field of bioengineering.
  • Hyaluronic acid is a linear acid mucopolysaccharide formed by polymerization of disaccharide units composed of N-acetylglucosamine and glucuronic acid.
  • Ultra-high molecular weight hyaluronic acid has good viscoelasticity, moisturizing and anti-inflammatory functions. It can be used as a viscoelastic agent in ophthalmic surgery and for intra-articular injection therapy.
  • High molecular weight hyaluronic acid has good moisturizing and lubricating effects and can be used in the cosmetics field.
  • High-purity hyaluronic acid and hyaluronic acid oligosaccharides have anti-tumor effects, promote wound healing, promote bone and angiogenesis, and immune regulation.
  • India there was a global hyaluronic acid market of 7.2 billion U.S. dollars, and it is predicted that the global value will reach 10.8 billion U.S. dollars in 2020.
  • the source of hyaluronic acid on the market is mainly obtained through fermentation of the natural hyaluronic acid-producing strain Streptococcus zooepidemicus.
  • Streptococcus zooepidemicus is a pathogenic strain that can cause many diseases
  • the hyaluronic acid produced cannot meet the requirements of the neighbourhoods such as medicine and food.
  • the purity of hyaluronic acid produced is low, which reduces the quality of the product.
  • hyaluronic acid synthase in some engineered strains to synthesize hyaluronic acid, such as Bacillus subtilis and Corynebacterium glutamicum.
  • Bacillus subtilis itself is prone to cell lysis, and the DNA released by cell lysis will cause the contamination of hyaluronic acid products.
  • Corynebacterium glutamicum has a thicker cell wall and strong tolerance. It has better cells than Bacillus subtilis. stability.
  • Corynebacterium glutamicum synthesizes more extracellular polysaccharides outside the cell. These polysaccharides not only compete for the substrate of the hyaluronic acid synthesis pathway, but also increase the difficulty of downstream hyaluronic acid purification and reduce the quality of hyaluronic acid products.
  • the first object of the present invention is to provide a Corynebacterium glutamicum that silences or knocks out the extracellular polysaccharide genes cg0420 and/or cg0424, and expresses hyaluronic acid synthase;
  • the cg0420 gene contains SEQ ID NO.1
  • the cg0424 contains the nucleotide sequence shown in SEQ ID NO. 2;
  • the hyaluronic acid synthase is shown in (a), (b) or (c):
  • the Corynebacterium glutamicum silences or knocks out the extracellular polysaccharide genes cg0420 (SEQ ID NO. 1) and cg0424 (SEQ ID NO. 2), and express hyaluronic acid synthase.
  • the hyaluronic acid synthase is derived from Streptococcus pyogenes (SEQ ID NO. 3).
  • the Corynebacterium glutamicum also enhances the UDP-N-acetylglucosamine and/or UDP-glucuronic acid pathway.
  • the UDP-N-acetylglucosamine pathway includes: glutamine-fructose-6-phosphate aminotransferase, phosphoglucose mutase, UDP-N-acetylglucosamine pyrophosphorylase/ Glucose-1-phosphate acetyltransferase bifunctional enzyme.
  • the UDP-glucuronic acid pathway includes: phosphoglucose mutase, glucose-6-phosphate urinamidase, and UDP-glucose dehydrogenase.
  • the phosphoglucose mutase pgm (SEQ ID NO. 4), glucose-6-phosphouriamidase GalU (SEQ ID NO. 5), UDP-glucose dehydrogenase Ugd (SEQ ID NO. 4) ID NO.6), glutamine-fructose-6-phosphate aminotransferase GlmS (SEQ ID NO.7), phosphoglucose mutase GlmM (SEQ ID NO.8), UDP-N-acetylglucosamine pyrophosphate
  • the amylase/glucose-1-phosphate acetyltransferase bifunctional enzyme GlmU (SEQ ID NO. 9) is derived from Pseudomonas putida KT2440.
  • the Corynebacterium glutamicum described above enhances the expression of at least one gene among pgM, ugd, galU, glms, glmM, and glmU.
  • the Corynebacterium glutamicum heterologously expresses the hyaluronic acid synthase gene hasA derived from Streptococcus pyogenes in the industrially safe strain Corynebacterium glutamicum ; And knock out the extracellular polysaccharide genes cg0420 and cg0424 of Corynebacterium glutamicum to remove the heteropolysaccharide on the cell surface; and construct the expression cassette to enhance the expression of the pathway genes pgM, ugd, galU, glms, glmM, glmU, and enhance the hyaluronic acid Synthetic substrate-synthesis of UDP-N-acetylglucosamine and UDP-glucuronic acid.
  • any of the above-mentioned Corynebacterium glutamicum also expresses Vitreoscilla hemoglobin VHb (SEQ ID NO. 10) to improve the bacterial cell performance of recombinant Corynebacterium glutamicum in a micro-oxygen environment. The ability to grow and synthesize hyaluronic acid.
  • the second objective of the present invention is to provide a method for constructing the Corynebacterium glutamicum, the method comprising: (1) knocking out the extracellular polysaccharide synthesis genes cg0420 and cg0424 step by step or simultaneously by constructing a knockout box; 2) Connect the gene encoding hyaluronic acid synthase to at least one gene of pgM, ugd, galU, glmS, glmM, and glmU with the vector, and transform it into the target strain cell.
  • the vector may be pXMJ19, pECXK99E, pEC-XT99A, pEKEx1, pEKEx2, pVWEx1, pVWEx2, pZ8-1, pECTAC-K99, pAPE12 (the above vectors are disclosed in Eggling, L. and Bott, M .,Handbook of corynebacterium glutamicum.2005, Boca Raton:Taylor&Francis.616p).
  • the method is to link pgm, galU, and ugd to the vector pXMJ19.
  • the method is glmS, glmM, glmU and the vector pECXK99E.
  • the third object of the present invention is to provide a method for producing hyaluronic acid, the method is to use the glutamic acid rod fermentation.
  • the fermentation is at 25-35°C for 24-72h.
  • the method further adds hyaluronic acid hydrolase or hyaluronic acid lyase in the early stage of fermentation; the added amount of hyaluronic acid hydrolase or hyaluronic acid lyase is 500-50000 U/ mL.
  • the fourth object of the present invention is to provide the application of the Corynebacterium glutamicum in the preparation of hyaluronic acid and its derivative products.
  • the present invention heterologously expresses the hyaluronic acid synthase gene hasA derived from Streptococcus pyogenes in the industrially safe strain Corynebacterium glutamicum, thereby constructing a synthetic pathway for hyaluronic acid.
  • the heteropolysaccharide on the cell surface is removed and the purity of hyaluronic acid is improved.
  • the Vitreoscilla hemoglobin VHb was also expressed in the recombinant Corynebacterium glutamicum to improve the growth of the recombinant Corynebacterium glutamicum and the hyaluronic acid production in a micro-oxygen environment. Synthesis ability. By knocking out cg0420, the yield and purity of hyaluronic acid have been improved to a certain extent. From 18g/L, the purity of the crude product is 75%, and the yield of hyaluronic acid is increased by 27.8%. The yield is 23g/L and the purity of the crude product is increased to 86%.
  • knocking out cg0420 On the basis of knocking out cg0420, knocking out cg0424, the production of hyaluronic acid increased by 58.3%, the yield reached 28.5g/L, and the purity of the crude product reached 95%.
  • double knockout express VHb to realize recombinant glutamate bar.
  • the production capacity of Bacillus hyaluronic acid reached 40g/L, which was 40.3% higher than that of the original strain.
  • 6000U/mL hyaluronic acid hydrolase was added externally to realize the production of hyaluronic acid oligosaccharides and finally hyaluronic acid.
  • the production capacity reached 72g/L, which was 152.6% higher than the original strain, and was 2.5 times that of the highest-producing strain reported so far.
  • Figure 1 The metabolic pathways and related enzymes for the production of hyaluronic acid by recombinant Corynebacterium glutamicum.
  • Figure 2 Diagram of the yield and product purity of hyaluronic acid of recombinant Corynebacterium glutamicum.
  • Figure 3 5L fermenter yield graph of hyaluronic acid of recombinant Corynebacterium glutamicum.
  • Figure 4 The output diagram of the 5L fermentor with the hyaluronic acid of the recombinant Corynebacterium glutamicum added with the hyaluronic acid hydrolase exogenously.
  • Figure 5 Mass spectrum of the disaccharide unit of hyaluronic acid produced by recombinant Corynebacterium glutamicum.
  • LB medium yeast powder 5g/L, peptone 10g/L, sodium chloride 10g/L
  • BHI Brain Heart Extract 17g/L, Sorbitol 21g/L
  • Fermentation medium glucose: 40g/L, corn steep liquor: 20g/L, KH 2 PO 4 : 15g/L, K 2 HPO 4 : 5g/L, MgSO 4 :1g/L.
  • Determination of the production of hyaluronic acid Dilute the appropriate fermentation broth by 10 times, centrifuge at 10,000 rpm for 10 minutes, take the supernatant and add 4 times the volume of pre-cooled ethanol, put -20 alcohol for 6 hours, centrifuge at 10,000 rpm for 10 minutes, discard the supernatant, add water to resuspend and restore to Centrifuge at 10,000 rpm for 5 min to take the supernatant, discard the precipitate, add 4 times the volume of pre-cooled ethanol, put -20 alcohol for 6 hours, centrifuge at 10,000 rpm for 10 min, discard the supernatant, add water to resuspend and restore to the original volume, centrifuge at 10,000 rpm for 5 min to take Supernatant, discard the pellet. Dilute the supernatant 50 times, and the final dilution factor is 500 times. When the sample is measured, the sample is diluted according to the linear effective range, and then measured by the sulfuric acid catabolism method.
  • Carbazole sulfate method for determination add 1ml sample to a glass tube containing 5mL borax sulfuric acid (4.77g borax dissolved in 500mL concentrated H2SO4), mix well, boil in a boiling water bath for 15 minutes, and cool on ice. Add 250 ⁇ L of carbazole reagent (dissolve 0.125g of carbazole in 100mL of absolute ethanol), mix well and cook for another 15min in a boiling water bath. The blank control is under the same conditions, only the sample is replaced with an equal volume of distilled water, and the absorbance at the wavelength of 530nm is measured.
  • Determination of the molecular weight of hyaluronic acid The fermentation supernatant is subjected to repeated alcohol precipitation to remove impurities to obtain higher purity hyaluronic acid, and its molecular weight is determined by HPLC.
  • the structure of hyaluronic acid was determined by LC-MS in the fermentation supernatant.
  • the impurities were removed by repeated alcohol precipitation to obtain hyaluronic acid of higher purity.
  • the sample was lysed by hyaluronidase overnight at 37°C to remove the hyaluronic acid. Cleavage into disaccharide units.
  • the Corynebacterium glutamicum suicide plasmid pK18mobsacB was double digested with EcoRI/BamhI, and the 0420-up and 0420-down fragments were ligated to the digested pK18mobsacB in one step with the Gibson Assembly kit, and the obtained recombinant plasmid was named pK18 -0420;
  • the plasmid pK18-0420 was transformed into Corynebacterium glutamicum ATCC13032 by electroporation using an electroporator, the electric shock conditions were voltage 1.5KV, 5ms, (shock cup width 1mm), click twice.
  • the first screening of recombinant bacteria was carried out on BHI plates containing 25mg/L of kanamycin. The positive recombinants were picked and cultured in liquid LB medium overnight, and then subjected to secondary screening on BHI plates containing 100g/L sucrose.
  • the recombinants with the 0420 gene knocked out can amplify 1Kb fragments, and the recombinant bacteria is named C.glutamicum ⁇ 0420.
  • the gene cg0424 was also knocked out by the above method, and the strains that knocked out cg0424 alone and knocked out cg0420 and cg0424 at the same time were named C.glutamicum ⁇ 0420 and C.glutamicum ⁇ 0420& ⁇ 0424, respectively.
  • primer sequences used are as follows:
  • PCR was performed with HasA-F and HasA-R as primers, and the fragment shown in SEQ ID NO.10 was amplified and the PCR product was purified;
  • the Corynebacterium glutamicum suicide plasmid pK18mobsacB was double digested with EcoRI/BamhI, and the U-up, D-down and VHb fragments were ligated to the digested pK18mobsacB in one step with the Gibson Assembly kit, and the obtained recombinant plasmid was named Is pK18-VHB;
  • the plasmid pK18-VHB was transformed into the C.glutamicum ⁇ 0420, C.glutamicum ⁇ 0424, C.glutamicum ⁇ 0420 ⁇ 0424 and wild-type strains constructed in step (1) by electroporation using an electroporator.
  • the electric shock conditions were voltage 1.5KV, 5ms, ( The width of the shock cup is 1mm), click twice.
  • the first screening of recombinant bacteria was carried out on BHI plates containing 25mg/L of kanamycin. The positive recombinants were picked and cultured in liquid LB medium overnight, and then subjected to secondary screening on BHI plates containing 100g/L sucrose.
  • the recombinant with HasA gene can amplify a 1.3Kb fragment, and the recombinant bacteria is named C.glutamicum-HasA.
  • Gene VHb was also integrated by the above method, and the final strains obtained were C.glutamicum ⁇ 0420-HasA-VHB, ⁇ 0424-HasA-VHB, ⁇ 0420& ⁇ 0424-HasA-VHB and WT-HasA-VHB.
  • D-down-F GTTACCGACGGTTTCTTTCATATTCCAAGCCGGAGAATTTCC (SEQ ID NO.17)
  • D-down-R CTATGACCATGATTACGAA ATGAAAGAAACCGTCGGTAAC (SEQ ID NO.18)
  • HasA-F AAGGAGCGTATATCATGCCTATTTTCAAGAAGACT (SEQ ID NO.19)
  • HasA-R AATAGGCATGATATACGCTCCTTTTATTTAAAAATAGTAACTTTTTTTCTAG (SEQ ID NO.20)
  • Pseudomonas putida (KT2440) was inoculated into 3ml LB liquid medium, cultured at 30°C and 220rpm for 24h, the bacteria were collected, and genomic DNA was extracted by the cell genome extraction kit.
  • Design primers pgm-F/pgm-R, galU-F/galU-R, ugd-F/ugd-R, glmS-F/glmS-R, glmM-F/glmM-R, glmU-F/glmU-R
  • the extracted Pseudomonas putida genomic DNA was used as a template, and pgm, ugd, galU, glmU, glmS and glmM genes were amplified by PCR amplification system and program.
  • the plasmid pXMJ19 and pECXK99E were digested with restriction enzyme sites to obtain linear plasmid pXMJ19, pECXK99E with amplified fragments pgm, ugd, galU and linear plasmid pXMJ19 with glmU, glmS, glmM and linear plasmid pECXK99E for Gibson assembly reaction.
  • the Gibson assembly reaction system transforms JM109 competent cells. The transformants were selected for plasmid sequencing reaction and compared with the sequence.
  • the recombinant plasmids pXMJ19-pgm-ugd-galU and pECX99E-glmU-glmM-glmS were successfully constructed and transformed into Corynebacterium glutamicum ATCC13032(C.glutamicum ⁇ 0420).
  • -HasA-VHB, ⁇ 0424-HasA-VHB, ⁇ 0420& ⁇ 0424-HasA-VHB and WT-HasA-VHB are named WT, ⁇ 0420, ⁇ 0424 and ⁇ 0420& ⁇ 0424, respectively.
  • Embodiment 2 Recombinant Corynebacterium glutamicum produces hyaluronic acid
  • the constructed recombinant Corynebacterium glutamicum strains: WT, ⁇ 0420, ⁇ 0424 and ⁇ 0420& ⁇ 0424 were inoculated with single clones in 5ml BHI medium, 200rpm, 30°C overnight culture. After 10h, the inoculum was transferred to a 250ml triangular shake flask (25ml fermentation medium) at 1% of the inoculum. Incubate at 200rpm and 28°C. After three hours of cultivation, IPTG with a final concentration of 0.25Mm is added to induce gene expression. The fermentation cycle is 48 hours.
  • Embodiment 3 5L fermenter fermentation culture of recombinant Corynebacterium glutamicum
  • Corynebacterium glutamicum strain Corynebacterium glutamicum HasA-VGB/pXMJ19-pgm-ugd-galU, pECX99E-glmU-glmM-glmS (knockout cg0420, cg0424) inoculated into 5ml BHI medium, 200rpm , Cultivate overnight at 30°C. After 10 hours, the inoculum was transferred to a 250 ml triangular shake flask (25ml fermentation medium) at a 1% inoculum. Incubate at 200 rpm and 28°C for 10 hours, and inoculate the fermenter with 10% of the inoculum.
  • the glucose content in the fermenter is maintained at about 10 g/L by feeding glucose, and the pH is controlled by feeding NaOH to the middle.
  • the final concentration of 6000 U/mL hyaluronic acid hydrolase was added exogenously after 20 hours of fermentation, and the fermentation cycle was 72 hours.
  • the OD was at 48 hours without the addition of hyaluronic acid hydrolase.
  • hyaluronic acid accumulates rapidly from 24 to 48 hours, and the output reaches 40g/L at 60 hours.
  • Example 2 According to the same strategy as in Example 1, the coding gene Cgl1118 (NC_003450.3) Cgl0452 (NC_003450.3) of another competitive precursor pathway was knocked out, and the recombinant plasmid constructed according to the steps (2) and (3) of Example 1 was transformed into Knockout Cgl1118 (NC_003450.3) Cgl0452 (NC_003450.3) cells were fermented according to the method of Example 2 or 3. The results showed that the hyaluronic acid production of the strain was significantly reduced after these genes were knocked out. The level of yield is only 3.1g/L. The main reason is that knocking out these genes affects the growth of the bacteria, causing the strain to grow slowly. The OD value of fermentation for 48h is 34, and the OD is only the wild type cultured under the same conditions. Half of the strain.

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Abstract

公开了一种高效合成高纯度透明质酸及其寡聚糖的重组谷氨酸棒状杆菌，属于生物工程技术领域。构建的重组谷氨酸棒状杆菌生产的透明质酸的产量达到40g/L，粗产品纯度达到95％。通过外源添加透明质酸水解酶并优化发酵条件，可以获得特定分子量的透明质酸寡糖，并实现透明质酸产量的进一步提升，使产量达到72g/L。

Description

一种高效合成高纯度透明质酸及其寡聚糖的重组谷氨酸棒状杆菌 技术领域
本发明涉及一种高效合成高纯度透明质酸及其寡聚糖的重组谷氨酸棒状杆菌，属于生物工程技术领域。
背景技术
透明质酸是由N-乙酰氨基葡萄糖和葡萄糖醛酸组成的双糖单位聚合而成的直链酸性黏多糖。超高分子量透明质酸具有较好的粘弹性，保湿性和抑制炎性等功能，可用于眼科手术中的粘弹剂和用于关节腔内注射治疗等。高分子量透明质酸具有良好的保湿，润滑的作用可被用于化妆品领域。高纯度的透明质酸和透明质酸寡糖具有抗肿瘤，促进伤口愈合，促进骨和血管生成，免疫调节等作用。2016年全球有72亿美元的透明质酸市场，预测2020年全球额达到108亿美元。
目前市场上的透明质酸来源主要通过天然产透明质酸菌株兽疫链球菌发酵生产获得。但由于兽疫链球菌是致病菌株，能够引起许多疾病，所以生产的透明质酸难以满足医药，食品等邻域的要求，此外生产的透明质酸纯度低，降低产品的品质。为了解决这方面的问题，人们利用基因工程技术在一些工程菌株中异源表达透明质酸合酶来合成透明质酸，比如枯草芽孢杆菌和谷氨酸棒杆菌。但是枯草芽孢杆菌本身容易发生细胞裂解，细胞裂解释放的DNA会造成透明质酸产品的污染，相比较而言谷氨酸棒杆菌细胞壁厚、耐受性强，有比枯草芽孢杆菌更好的细胞稳定性。但是谷氨酸棒杆菌会在细胞外合成较多的胞外多糖。这些多糖不但竞争透明质酸合成途径的底物，还增加下游透明质酸纯化的难度，降低透明质酸产品的品质。
发明内容
本发明的第一个目的是提供一种谷氨酸棒杆菌，沉默或敲除胞外多糖基因cg0420和/或cg0424，并表达透明质酸合酶；所述cg0420基因含有SEQ ID NO.1所示的核苷酸序列；所述cg0424含有SEQ ID NO.2所示的核苷酸序列；所述透明质酸合酶为(a)、(b)或(c)所示：
(a)来源于酿脓链球菌(Streptococcus pyogenes)且氨基酸序列与SEQ ID NO.3有至少90％同源性的酶；
(b)含有SEQ ID NO.3所示氨基酸序列的酶；
(c)在(a)或(b)中经过取代或缺失且具有透明质酸合酶活性的由(a)或(b)衍生的蛋白质。
在一种实施方式中，所述谷氨酸棒杆菌沉默或敲除胞外多糖基因cg0420(SEQ ID NO.1) 和cg0424(SEQ ID NO.2)，并表达透明质酸合酶。
在一种实施方式中，所述透明质酸合酶来源于酿脓链球菌(Streptococcus pyogenes)(SEQ ID NO.3)。
在一种实施方式中，所述谷氨酸棒杆菌还强化UDP-N-乙酰葡萄糖胺和/或UDP-葡萄糖醛酸途径。
在一种实施方式中，所述UDP-N-乙酰葡萄糖胺途径包括：谷氨酰胺-果糖-6-磷酸氨基转移酶、磷酸葡萄糖变位酶、UDP-N-乙酰葡萄糖胺焦磷酸化酶/葡萄糖-1-磷酸乙酰转移酶双功能酶。
在一种实施方式中，所述UDP-葡萄糖醛酸途径包括：磷酸葡萄糖变位酶、葡萄糖-6-磷酸尿酰胺转移酶、UDP-葡萄糖脱氢酶。
在一种实施方式中，所述磷酸葡萄糖变位酶pgm(SEQ ID NO.4)，葡萄糖-6-磷酸尿酰胺转移酶GalU(SEQ ID NO.5)，UDP-葡萄糖脱氢酶Ugd(SEQ ID NO.6)，谷氨酰胺-果糖-6-磷酸氨基转移酶GlmS(SEQ ID NO.7)，磷酸葡萄糖变位酶GlmM(SEQ ID NO.8)，UDP-N-乙酰葡萄糖胺焦磷酸化酶/葡萄糖-1-磷酸乙酰转移酶双功能酶GlmU(SEQ ID NO.9)来源于恶臭假单胞菌(Pseudomonas putida KT2440)。
在一种实施方式中，上述任一所述的谷氨酸棒杆菌强化pgM，ugd，galU，glms，glmM，glmU中至少一个基因的表达。
在一种实施方式中，所述谷氨酸棒杆菌是在工业安全菌株谷氨酸棒杆菌(Corynebacterium glutamicum)中异源表达来源于酿脓链球菌(Streptococcus pyogenes)的透明质酸合酶基因hasA；并敲除谷氨酸棒杆菌胞外多糖基因cg0420和cg0424以去除细胞外表的杂多糖；并构建表达框强化途径基因pgM，ugd，galU，glms，glmM，glmU的表达，加强透明质酸的合成底物——UDP-N-乙酰葡萄糖胺和UDP-葡萄糖醛酸的的合成。
在一种实施方式中，上述任一所述的谷氨酸棒杆菌还表达了透明颤菌血红蛋白VHb(SEQ ID NO.10)，以提高重组谷氨酸棒杆菌在微氧环境下菌体的生长和透明质酸的合成能力。本发明的第二个目的是提供所述谷氨酸棒杆菌的构建方法，所述方法包括：(1)通过构建敲除框，分步或同时敲除胞外多糖合成基因cg0420和cg0424；(2)将编码透明质酸合酶的基因与pgM，ugd，galU，glmS，glmM，glmU中的至少一个基因与载体连接，并转化至目的菌株细胞中。
在一种实施方式中，所述载体可以为pXMJ19、pECXK99E、pEC-XT99A、pEKEx1、pEKEx2、pVWEx1、pVWEx2、pZ8-1、pECTAC-K99、pAPE12(上述载体公开于Eggeling,L. and Bott,M.,Handbook of corynebacterium glutamicum.2005,Boca Raton:Taylor&Francis.616p)。
在一种实施方式中，所述方法是将pgm、galU、ugd与载体pXMJ19连接。
在一种实施方式中，所述方法是glmS、glmM、glmU与载体pECXK99E。
本发明的第三个目的是提供一种生产透明质酸的方法，所述方法是应用所述谷氨酸棒杆发酵。
在一种实施方式中，所述发酵是在25～35℃发酵24-72h。
在一种实施方式中，所述方法还在发酵的前期添加透明质酸水解酶或者透明质酸裂解酶；所述的透明质酸水解酶或者透明质酸裂解酶的添加量为500-50000U/mL。
本发明的第四个目的是提供所述谷氨酸棒杆菌在制备透明质酸及其衍生产品方面的应用。
有益效果：本发明在工业安全菌株谷氨酸棒杆菌(Corynebacterium glutamicum)中异源表达来源于酿脓链球菌(Streptococcus pyogenes)的透明质酸合酶基因hasA，构建了透明质酸的合成途径。通过敲除谷氨酸棒杆菌胞外多糖基因cg0420和cg0424，去除细胞外表的杂多糖提高了透明质酸的纯度。通过构建表达框强化途径基因pgM，ugd，galU，glms，glmM，glmU的表达，加强透明质酸的合成底物UDP-N-乙酰葡萄糖胺和UDP-葡萄糖醛酸的的合成，以解决透明质酸合成过程中底物供应不足的问题，从而提高重组谷氨酸棒杆菌透明质酸的合成能力。为了解决发酵过程中溶氧不足的问题，还在重组谷氨酸棒杆菌中表达了透明颤菌血红蛋白VHb，以提高重组谷氨酸棒杆菌在微氧环境下菌体的生长和透明质酸的合成能力。通过敲除cg0420透明质酸产量和纯度都得到了一定程度的提升，从18g/L，粗产品纯度75％,透明质酸产量提高了27.8％产量达到23g/L粗产品纯度提高到86％，在敲除cg0420的基础上敲除cg0424，透明质酸的产量提高了58.3％产量达到了28.5g/L同时粗产品纯度达到95％，在双敲除的基础上表达VHb实现重组谷氨酸棒杆菌透明质酸的生产能力达到40g/L较原始菌株产量提高了40.3％，发酵过程通过外源添加6000U/mL的透明质酸水解酶，实现透明质酸寡糖的生产，最终实现透明质酸的生产能力达到72g/L，较原始菌株提高了152.6％，是目前报道最高产菌株的2.5倍。
附图说明
图1：重组谷氨酸棒杆菌生产透明质酸的代谢途径及相关的酶类。
图2：重组谷氨酸棒杆菌的透明质酸的产量及产品纯度图。
图3：重组谷氨酸棒杆菌的透明质酸的5L发酵罐产量图。
图4：外源添加透明质酸水解酶重组谷氨酸棒杆菌的透明质酸的5L发酵罐产量图。
图5：重组谷氨酸棒杆菌生产的透明质酸的二糖单位的质谱图。
具体实施方式
菌株：谷氨酸棒杆菌(Corynebacterium glutamicum ATCC 13032)，质粒：pXMJ19,pEC-XK99E,pK18mobSacB
LB培养基:酵母粉5g/L，蛋白胨10g/L，氯化钠10g/L
BHI:脑心浸出液17g/L，山梨醇21g/L
发酵培养基：葡萄糖：40g/L，玉米浆干粉:20g/L，KH 2PO 4：15g/L,K 2HPO 4:5g/L,MgSO 4:1g/L.
透明质酸的产量测定：取适当发酵液稀释10倍，10000rpm离心10min取上清加4倍体积的预冷乙醇，放-20醇沉6hours，10000rpm离心10min弃上清，加水重悬并恢复至原体积，10000rpm离心5min取上清，弃沉淀，并加4倍体积的预冷乙醇，放-20醇沉6hours，10000rpm离心10min弃上清，加水重悬并恢复至原体积，10000rpm离心5min取上清，弃沉淀。取上清稀释50倍，最终稀释倍数为500倍。样品测定时，根据线性有效范围稀释样品，然后通过硫酸咔挫法测定。
硫酸咔唑法进行测定：向装有5mL硼砂硫酸(500mL浓H2SO4中溶解4.77g硼砂)的玻璃管中加入1ml样品，混匀后在沸水浴中煮15min，冰上冷却。加入250μL咔唑试剂(100mL无水乙醇中溶解0.125g咔唑)，混匀后沸水浴中再煮15min。空白对照为相同条件下，仅将样品替换为等体积蒸馏水，测定波长530nm处的吸光值。用不同浓度(l0、20、30、40、50μg mL -1)D-葡萄糖醛酸标准品进行反应，绘制标准曲线，再将样品吸光值带入标准曲线，计算透明质酸的含量。标准曲线方程：y＝126.88x-9.2639,R 2＝0.9991(x,吸光度A530；y,样品中葡萄糖醛酸含量(μg mL -1))。软骨素产量计算公式：透明质酸含量(g/L)＝(标准曲线测出的浓度*稀释倍数*2.067)/1000。样品测定时，根据线性有效范围稀释样品。
透明质酸的分子量测定：发酵上清，通过反复醇沉去除杂质，获得较高纯度的透明质酸，通过HPLC对其进行分子量测定。
透明质酸的结构LC-MS测定发酵上清，通过反复醇沉去除杂质，获得较高纯度的透明质酸，然后样品在37℃的条件下通过透明质酸酶过夜裂解处理，将透明质酸裂解为二糖单位。然后在样品中加入九倍体积的无水甲醇，离心去除杂质和未水解的透明质酸后，经过有机膜过滤出去不溶杂质后，通过液相质谱联用LC-MS分析二糖单位的结构。
实施例1:重组谷氨酸棒杆菌的构建
(1)基因cg0420和cg0424的敲除
以谷氨酸棒杆菌ATCC13032的基因组DNA为模板，以0420-up-F和0420-up-R为引物 进行PCR，获得cg0420上游约500bp的片段0420-up并进行PCR产物纯化；
以谷氨酸棒杆菌ATCC13032的基因组DNA为模板，以0420-down-F和0420-down-R为引物进行PCR，获得cg0420下游约500bp的片段0420-down并进行PCR产物纯化；
将谷氨酸棒杆菌自杀性质粒pK18mobsacB用EcoRI/BamhI进行双酶切，用Gibson Assembly试剂盒将0420-up和0420-down片段一步连接到酶切后的pK18mobsacB上，获得的重组质粒命名为pK18-0420；
采用电穿孔仪将质粒pK18-0420通过电转化转入到谷氨酸棒杆菌ATCC13032中，电击条件为电压1.5KV，5ms，(电击杯宽度为1mm)，点击二次。在含25mg/L的卡那霉素BHI平板上进行重组菌的第一次筛选。挑取阳性重组子进一步在液体LB培养基里过夜培养，然后在含有100g/L的蔗糖BHI平板上进行二次筛选。利用0420-up-F和0420-down-R为引物进行菌落PCR，敲除0420基因的重组子能够扩增出1Kb大小的片段，该重组菌命名为C.glutamicum Δ0420。基因cg0424，也用上述方法敲除，获得单独敲除cg0424和同时敲除cg0420和cg0424的菌株分别命名为C.glutamicum Δ0420和C.glutamicum Δ0420&Δ0424。
其中，所用引物序列如下：
0420-UP-F：GCAGGTCGACTCTAGAGGATCCAAGTTTCGAACCATGCTTGAAC(SEQ ID NO.11)
0420-UP-R:GATCTGATTCTTCGCACCAATAGGCGACATACCGTTTCTAACTGCTCAG(SEQ ID NO.12)
0420-down-F:CTGAGCAGTTAGAAACGGTATGTCGCCTATTGGTGCGAAGAATCAGATC(SEQ ID NO.13)
0420-down-R：CTATGACCATGATTACGAATTCTGGACCCTAAACTGAGCAGTGA(SEQ ID NO.14)
(2)基因HasA、VHb的整合
以谷氨酸棒杆菌ATCC13032的基因组DNA为模板，以U-up-F和U-up-R为引物进行PCR，扩增500bp的片段U-up并进行PCR产物纯化；
以谷氨酸棒杆菌ATCC13032的基因组DNA为模板，以D-down-F和D-down-R为引物进行PCR，扩增获得约500bp的片段D-down并进行PCR产物纯化；
以透明颤菌(Vitreoscilla stercoraria DSM 513)的基因组DNA为模板，以HasA-F和HasA-R为引物进行PCR，扩增获得SEQ ID NO.10所示的片段并进行PCR产物纯化；
将谷氨酸棒杆菌自杀性质粒pK18mobsacB用EcoRI/BamhI进行双酶切，用Gibson Assembly试剂盒将U-up、D-down和VHb片段一步连接到酶切后的pK18mobsacB上，获得 的重组质粒命名为pK18-VHB；
采用电穿孔仪将质粒pK18-VHB通过电转化转入到步骤(1)构建的C.glutamicumΔ0420，C.glutamicum Δ0424，C.glutamicumΔ0420 Δ0424和野生型菌株中，电击条件为电压1.5KV，5ms，(电击杯宽度为1mm)，点击二次。在含25mg/L的卡那霉素BHI平板上进行重组菌的第一次筛选。挑取阳性重组子进一步在液体LB培养基里过夜培养，然后在含有100g/L的蔗糖BHI平板上进行二次筛选。利用U-up-F和D-down-R为引物进行菌落PCR，整合上HasA基因的重组子能够扩增出1.3Kb大小的片段，该重组菌命名为C.glutamicum-HasA。基因VHb，也用上述方法整合，最后获得的菌株分别为C.glutamicum△0420-HasA-VHB，△0424-HasA-VHB，△0420&△0424-HasA-VHB和WT-HasA-VHB。
U-up-F:GCAGGTCGACTCTAGAGGATCCTTAGAAGAACTGCTTCTGAAT(SEQ ID NO.15)
U-up-R:AATAGGCATGATATACGCTCCTTCGAACACGGCGACACTGAAC(SEQ ID NO.16)
D-down-F:GTTACCGACGGTTTCTTTCATATTCCAAGCCGGAGAATTTCC(SEQ ID NO.17)
D-down-R:CTATGACCATGATTACGAA ATGAAAGAAACCGTCGGTAAC(SEQ ID NO.18)
HasA-F:AAGGAGCGTATATCATGCCTATTTTCAAGAAGACT(SEQ ID NO.19)
HasA-R:AATAGGCATGATATACGCTCCTTTTATTTAAAAATAGTAACTTTTTTTCTAG(SEQ ID NO.20)
(3)重组质粒pXMJ19-pgm-galU-ugd和pECXK99E-glmS-glmM-glmU构建
恶臭假单胞菌(Pseudomonas putida KT2440)接种于3ml LB液体培养基，在30℃220rpm培养24h，收集菌体，通过细胞基因组提取试剂盒提取基因组DNA。设计引物pgm-F/pgm-R，galU-F/galU-R，ugd-F/ugd-R，glmS-F/glmS–R，glmM-F/glmM–R，glmU-F/glmU-R以提取的恶臭假单胞菌基因组DNA为模板，通过PCR扩增体系和程序，扩增获取pgm，ugd，galU，glmU、glmS和glmM基因。选取酶切位点对质粒pXMJ19，pECXK99E质粒酶切，获得线性质粒pXMJ19，pECXK99E以扩增的片段pgm,ugd,galU和线性质粒pXMJ19与glmU、glmS，glmM与线性质粒pECXK99E进行吉布森组装反应。吉布森组装反应体系转化JM109感受态细胞。挑选转化子进行质粒测序反应，与序列比对，重组质粒pXMJ19-pgm-ugd-galU，pECX99E-glmU-glmM-glmS构建成功，并将其通过电转化转入Corynebacterium glutamicum ATCC13032(C.glutamicum△0420-HasA-VHB，△0424-HasA-VHB，△0420&△0424-HasA-VHB 和WT-HasA-VHB)分别命名为WT，△0420，△0424和△0420&△0424。
表1 重组质粒pXMJ19-pgm-ugd-galU构建所用引物
Figure PCTCN2019126013-appb-000001
表2 重组质粒pXMJ19-glmU-glmM-glmS构建所用引物
Figure PCTCN2019126013-appb-000002
实施例2:重组谷氨酸棒杆菌生产透明质酸
将构建的重组谷氨酸棒杆菌菌株：WT，△0420，△0424和△0420&△0424接单克隆于5ml BHI培养基中，200rpm，30℃过夜培养。10h后,按1％的接种量转接于250ml三角摇瓶(装液量为25ml发酵培养基)中。置于200rpm，28℃培养，培养三小时后并添加终浓度为0.25Mm的IPTG进行诱导基因表达，发酵周期为48小时，发酵结束后取上清加入四倍体积的乙醇进行醇沉，去除一些杂质，反复两次醇沉后，通过硫酸咔挫法对透明质酸含量进行测定，从图2可以看出通过敲除cg0420，cg0424透明质酸产量和纯度都有一定程度的提升，双敲除菌株摇瓶水平透明质酸产量达到6.9g/L纯度达到95％,。
实施例3:重组谷氨酸棒杆菌的5L发酵罐发酵培养
将构建的重组谷氨酸棒杆菌菌株：Corynebacterium glutamicum HasA-VGB/pXMJ19-pgm-ugd-galU，pECX99E-glmU-glmM-glmS(敲除cg0420，cg0424)接单克隆于5ml BHI培养基中，200rpm，30℃过夜培养。10h后，按1％的接种量转接于250 ml三角摇瓶(装液量为25ml发酵培养基)中。置于200rpm，28℃培养10h，以10％的接种量接种于发酵罐中，发酵过程中通过流加葡萄糖维持发酵罐中的葡萄糖含量在10g/L左右，通过流加NaOH，控制pH为中性，并在发酵20h时外源添加终浓度为6000U/mL的透明质酸水解酶，发酵周期为72h，从图3可以看出，在不添加透明质酸水解酶的情况下OD在48h时达到最高，透明质酸从24到48小时时快速积累，60小时时产量达到40g/L。从图4可以看出菌体在4-36小时为对数生长期，菌体快速生长，36小时时进入稳定期，且透明质酸在24小时到60小时时快速积累，且与图3相比发酵周期延长了12个小时，透明质酸产量也大幅度增长，72小时时透明质酸产量达到72g/L，较不添加水解酶提高了32g/L，OD也有一定增高。。
对比例：
按照实施例1相同的策略敲除另一竞争前体途径的编码基因Cgl1118(NC_003450.3)Cgl0452(NC_003450.3)，将按照实施例1步骤(2)、(3)构建的重组质粒转化至敲除Cgl1118(NC_003450.3)Cgl0452(NC_003450.3)的细胞中，按照实施例2或3的方法进行发酵，结果显示敲除这些基因后菌株的透明质酸产量得到大幅度的下降，摇瓶水平的产量仅为3.1g/L，主要原因是敲除这些基因影响了菌体的生长，导致菌株生长变得缓慢，发酵48h的OD值为34，,OD仅为相同条件下培养的野生型菌株的一半。
虽然本发明已以较佳实施例公开如上，但其并非用以限定本发明，任何熟悉此技术的人，在不脱离本发明的精神和范围内，都可做各种的改动与修饰，因此本发明的保护范围应该以权利要求书所界定的为准。

Claims (10)

  1. 一种谷氨酸棒杆菌，其特征在于，沉默或敲除胞外多糖基因cg0420和/或cg0424，并表达透明质酸合酶；所述透明质酸合酶为(a)、(b)或(c)所示：
    (a)来源于酿脓链球菌(Streptococcus pyogenes)且氨基酸序列与SEQ ID NO.3有至少90％同源性的酶；
    (b)含有SEQ ID NO.3所示氨基酸序列的酶；
    (c)在(a)或(b)中经过取代或缺失且具有透明质酸合酶活性的由(a)或(b)衍生的蛋白质。
  2. 根据权利要求1所述的谷氨酸棒杆菌，其特征在于，强化UDP-N-乙酰葡萄糖胺和/或UDP-葡萄糖醛酸途径。
  3. 根据权利要求2所述的谷氨酸棒杆菌，其特征在于，所述UDP-N-乙酰葡萄糖胺途径包括：谷氨酰胺-果糖-6-磷酸氨基转移酶、磷酸葡萄糖变位酶、UDP-N-乙酰葡萄糖胺焦磷酸化酶/葡萄糖-1-磷酸乙酰转移酶双功能酶；所述UDP-葡萄糖醛酸途径包括：磷酸葡萄糖变位酶、葡萄糖-6-磷酸尿酰胺转移酶、UDP-葡萄糖脱氢酶。
  4. 根据权利要求1～3任一所述的谷氨酸棒杆菌，其特征在于，还表达了透明颤菌来源的血红蛋白VHb。
  5. 根据权利要求1～4任一所述的谷氨酸棒杆菌，其特征在于，将编码UDP-N-乙酰葡萄糖胺和/或UDP-葡萄糖醛酸途径的基因及编码血红蛋白VHb的基因与载体连接，在谷氨酸棒杆菌中表达；所述载体包括以下任一种：pXMJ19、pECXK99E、pEC-XT99A、pEKEx1、pEKEx2、pVWEx1、pVWEx2、pZ8-1、pECTAC-K99、pAPE12。
  6. 构建权利要求1～5任一所述谷氨酸棒杆菌的方法，其特征在于，步骤包括：(1)通过构建敲除框，分步或同时敲除胞外多糖合成基因cg0420和cg0424；(2)将编码透明质酸合酶的基因、编码血红蛋白VHb的基因及pgM，ugd，galU，glmS，glmM，glmU中的至少一个基因与载体连接，并转化至谷氨酸棒杆菌细胞中。
  7. 一种生产透明质酸的方法，其特征在于，应用权利要求1～5任一所述谷氨酸棒杆进行发酵；所述发酵是在含有碳源、氮源、无机盐、金属离子和氧气培养环境中，于25～35℃发酵24-72h。
  8. 根据权利要求7所述的方法，其特征在于，在发酵的前期添加透明质酸水解酶或者透明质酸裂解酶；所述的透明质酸水解酶或者透明质酸裂解酶的添加量为500-50000U/mL。
  9. 根据权利要求7或8所述的方法，其特征在于，发酵过程补加葡萄糖。
  10. 权利要求1～4任一所述谷氨酸棒杆菌在制备透明质酸及其衍生产品方面的应用。
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