WO2022088263A1 - 一种高效生产琥珀酸的重组大肠杆菌及其构建方法 - Google Patents

一种高效生产琥珀酸的重组大肠杆菌及其构建方法 Download PDF

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WO2022088263A1
WO2022088263A1 PCT/CN2020/128254 CN2020128254W WO2022088263A1 WO 2022088263 A1 WO2022088263 A1 WO 2022088263A1 CN 2020128254 W CN2020128254 W CN 2020128254W WO 2022088263 A1 WO2022088263 A1 WO 2022088263A1
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escherichia coli
gene
recombinant escherichia
succinic acid
fermentation
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French (fr)
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刘立明
唐文秀
沈忱
罗秋玲
陈修来
刘佳
高聪
宋伟
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江南大学
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Definitions

  • the invention relates to a recombinant Escherichia coli for efficiently producing succinic acid and a construction method thereof, belonging to the technical field of bioengineering.
  • Succinic acid scientific name succinic acid
  • succinic acid is an important C4 platform compound.
  • succinic acid has a wide range of applications in food, chemistry, medicine and other fields.
  • the traditional production method of succinic acid is chemical synthesis method, mainly including paraffin oxidation method, methyl chloroacetate cyanidation hydrolysis method and vanadium pentoxide catalytic hydrogenation method, etc., but due to problems such as the reduction of petroleum resources and the increasingly serious environmental pollution, The disadvantages of chemical synthesis methods are increasingly apparent.
  • the production of succinic acid by fermentation can get rid of the dependence on non-renewable strategic resource petroleum, use renewable resources, fix carbon dioxide to reduce the greenhouse effect, and show a good development prospect.
  • the most studied succinic acid-producing strains are: Actinomyces succinic acid-producing, Anaerobic spirochetes succinic acid-producing and Escherichia coli.
  • Actinobacillus succinate-producing bacteria are usually selected from nature and carry out directional transformation, which can tolerate high concentrations of succinate. Carbon source, the maximum yield of 48h fermentation can reach 110g/L.
  • Anaerobes succinates can utilize a wide range of fermentation substrates, such as glucose, lactose, glycerol, etc.
  • the research results of Samuelov et al. It can reach 1.2mol/1.0mol glucose, and the maximum yield is 65.0g/L, but the fermentation of this strain requires strict anaerobic environment, which is difficult to achieve in industrial application.
  • Escherichia coli As a model strain, Escherichia coli has a clear genetic background and is easy to operate. Various molecular biology techniques can be used to transform strains. Therefore, the use of Escherichia coli to ferment succinic acid has become a hot spot, and many research progress has been made. Vemuri et al. Recombinant Escherichia coli AFP111 was fermented in two stages for 76h, the final concentration of succinic acid could reach 99.2g/L, the yield could reach 1.1g/g glucose, and the production intensity could reach 1.3g/L/h; Zhang Xueli et al.
  • Recombinant Escherichia coli HX024 was obtained through the construction of a sexual evolution strategy, and a one-step anaerobic fermentation was used for 96 hours.
  • the final succinic acid yield reached 95.9 g/L, and the yield reached 1 g/g glucose.
  • Zhu Liwen et al. optimized the expression of ppc and pck genes and enhanced CO2 fixation.
  • Path the recombinant Escherichia coli AFP111 strain was fermented for 96h, and the succinic acid yield reached 90.7g/L;
  • Zhang Jianguo et al. optimized the glucose absorption and metabolism pathway, and at the same time knocked out the by-product acetic acid encoding gene, fermented for 65h, and the final succinate yield reached 98.92g/L ;
  • Escherichia coli fermentation is relatively low.
  • by-products such as lactic acid, formic acid, acetic acid and ethanol in the fermentation broth.
  • the metabolism of cofactors is unbalanced and cannot tolerate high concentrations of products and substrate glucose.
  • Osmotic pressure and glucose absorption and utilization rate are too fast, leading to problems such as metabolic imbalance, product yield and low production intensity; in order to obtain high-performance production strains, it is usually necessary to use traditional breeding methods, various omics analysis and molecular biology transformation. combined method.
  • the present invention provides a recombinant Escherichia coli for efficiently producing succinic acid, which adopts Red homologous recombination technology to knock out the pyruvate formate lyase gene pflB-focA gene, Lactate dehydrogenase gene ldhA gene, phosphotransacetylase gene pta gene, and overexpress the key enzymes phosphoenolpyruvate carboxykinase pck and phosphite dehydrogenase ptxD in the succinate synthesis pathway.
  • the first object of the present invention is to provide a recombinant Escherichia coli for efficiently producing succinic acid.
  • the transacetylase encodes one or more of the genes pta, and overexpresses the phosphoenolpyruvate kinase Pck and the phosphite dehydrogenase PtxD.
  • nucleotide sequence of the gene encoding the phosphoenolpyruvate kinase is as shown in SEQ ID NO.1
  • nucleotide sequence of the gene encoding the phosphite dehydrogenase is as shown in SEQ ID NO.1 NO.2 is shown.
  • phosphoenolpyruvate carboxykinase pck and phosphite dehydrogenase ptxD are expressed by plasmid pTrcHisA.
  • nucleotide sequence of the pyruvate formate lyase encoding gene pflB-focA is shown in SEQ ID NO.3; the nucleotide sequence of the lactate dehydrogenase encoding gene ldhA is shown in SEQ ID NO.4; The nucleotide sequence of the phosphotransacetylase encoding gene pta is shown in SEQ ID NO.5.
  • the host of the recombinant Escherichia coli is Escherichia coli FMME-N-5, which was deposited in the China Center for Type Culture Collection on August 27, 2020, and the deposit address is Wuhan University, Wuhan, China, and the deposit number is CCTCC NO:M 2020454.
  • the second object of the present invention is to provide a kind of construction method of described recombinant Escherichia coli, and the steps of described construction method are:
  • the third object of the present invention is to provide the application of the Escherichia coli in the production of succinic acid. sour fermentation broth.
  • the formula of the fermentation medium is: glucose 30-50g/L, corn steep liquor 15-25g/L, (NH 4 ) 2 SO 4 2-4g/L, K 2 HPO 4 1.2-2.0g/ L, KH 2 PO 4 0.5-1.0 g/L, MgSO 4 ⁇ 7H 2 O 0.2-0.5 g/L, NaCl 1-2 g/L.
  • the aerobic stage is changed to the anaerobic stage.
  • converting the aerobic stage to the anaerobic stage is carried out by introducing CO 2 gas or adding 10-20 g/L bicarbonate.
  • the glucose concentration is controlled to be 5-15 g/L.
  • the inoculum size of the aerobic-anaerobic two-stage fermentation is 6-12% by volume percentage; the fermentation temperature is 35-38°C.
  • the fermentation time of the aerobic-anaerobic two-stage fermentation is 50-96h.
  • a pH neutralizer is added, and the pH neutralizer is one or a mixture of Na 2 CO 3 , K 2 CO 3 , NaOH, KOH, CaCO 3 , and basic magnesium carbonate.
  • an osmotic pressure protective agent is added, and the osmotic pressure protective agent is one or more of proline, methionine, cysteine, and betaine. kind of mix.
  • phosphite dehydrogenase can catalyze salt phosphate to generate one molecule of phosphate, while consuming one molecule of NAD + to generate one molecule of NADH, the phosphate produced by this reaction has no effect on cell growth and ptxD activity. Inhibition, and the reaction catalyzed by ptxD does not directly compete with cellular metabolites, so the expression of ptxD increases the supply of NADH without affecting the main metabolic pathway for succinate production.
  • the present invention adopts the Red homologous recombination strategy to knock out the relevant enzymes encoded by by-products affecting the production of succinate, including pyruvate formate lyase, lactate dehydrogenase, and phosphotransacetylase; It reduces the accumulation of by-products and is conducive to the accumulation of succinic acid; at the same time, over-expression of the pathway enzyme phosphoenolpyruvate carboxykinase and over-expression of phosphite dehydrogenase effectively increases the production of succinic acid, and finally the engineering strain is obtained E.coli FMME-N-5( ⁇ focA-pflB- ⁇ ldhA- ⁇ pta)-pck-ptxD, the yield of succinic acid reached 137g/L at 96h of fermentation, the yield of succinate reached 1g/g glucose, and the production intensity was 1.43g/L /h, the by-products lactic acid and formic acid do not accumulate,
  • Escherichia coli FMME-N-5 the Escherichia coli has been deposited in the China Center for Type Culture Collection on August 27, 2020, the deposit number is CCTCC NO: M 2020454, and the deposit address is Wuhan, China, Wuhan University .
  • Fig. 1 is the gel verification diagram of knockout pyruvate formate lyase
  • Fig. 2 is the verification chart of knockout lactate dehydrogenase gel
  • Fig. 3 is knockout phosphoric acid transacetylation gel verification diagram
  • Figure 4 is a structural diagram of a recombinant vector expressing phosphoenolpyruvate carboxykinase and phosphite dehydrogenase;
  • Fig. 5 is the result of fed-batch fermentation of E.coli FMME-N-5 ( ⁇ focA-pflB- ⁇ ldhA- ⁇ pta)-pck-ptxD fermenter for 96h fermentation of engineering strain.
  • sequence information of SEQ ID NO.1 is the phosphoenolpyruvate carboxykinase pck nucleotide sequence derived from Actinobacillus succinates;
  • SEQ ID NO.2 sequence information is the phosphite dehydrogenase ptxD nucleotide sequence of Pseudomonas stutzeri;
  • SEQ ID NO.3 sequence information is the nucleotide sequence of pyruvate formate lyase encoding gene pflB-focA;
  • SEQ ID NO.4 sequence information is the nucleotide sequence of lactate dehydrogenase encoding gene ldhA;
  • SEQ ID NO.5 sequence information is the nucleotide sequence of phosphotransacetylase encoding gene pta;
  • the cell density was expressed by the absorbance value detected at a wavelength of 600 nm by a spectrophotometer.
  • Fermentation broth pretreatment Centrifuge the fermentation broth at 12000 r/min for 5 min to take the supernatant. Dilute it to an appropriate multiple, and use an M-100 biosensor analyzer to detect the glucose concentration of the fermentation broth.
  • High performance liquid chromatography fermentation broth pretreatment: take the fermentation broth and centrifuge at 12000 r/min for 5 min to take the supernatant. After appropriate dilution, the yields of succinic acid, lactic acid, formic acid and acetic acid were detected by high performance liquid chromatography (HPLC).
  • HPLC high performance liquid chromatography
  • the instrument is a Waters e2695 reversed-phase high performance liquid chromatograph, and the chromatographic column adopts Bio-Rad HPX 87H; the mobile phase is 5 mmoL/L H 2 SO 4 ; the flow rate is set to 0.6 mL/min; the detector is an ultraviolet detector, and the detection wavelength is 210 nm, and the column temperature was 35°C.
  • Example 1 Knockout of the gene encoding pyruvate formate lyase
  • Gene editing fragments include upstream and downstream homology arm regions and resistance selection cassettes.
  • the designed sequence such as SEQ ID NO.6/SEQ ID NO.7 primer pair pf1B-focA-S/pf1B-focA-A amplifies the resistance screening gene Kan to obtain the pf1B-focA knockout box fragment.
  • the pKD46 plasmid was transformed into the expression host E.coli FMME-N-5 competent cells, and the recombinant strain E.coli FMME-N-5-pKD46 was obtained by colony PCR screening, and then the obtained pflB-focA was knocked out
  • the frame fragment was transferred into recombinant strain E.coli FMME-N-5-pKD46 competent cells by electroporation, and positive transformants were obtained by screening with 50 ⁇ g/mL Kan resistance plate.
  • thermosensitive plasmid pCP20 was used to induce the expression of FLP recombinase to eliminate the Kan resistance gene, and the temperature-sensitive plasmids pKD46 and pCP20 were eliminated by culturing and transferring at 42°C for 3 times.
  • FMME-N-5 ( ⁇ focA-pflB).
  • pflB-focA-S TTACTCCGTATTTGCATAAAAACCATGCGAGTTACGGGCCTATAAGTGTAGGCTGGAGCTGCTTC
  • pflB-focA-A ATAGATTGAGTGAAGGTACGAGTAATAACGTCCTGCTGCTGTTCTCATATGAATATCCTCCTTAG
  • Gene editing fragments include upstream and downstream homology arm regions and resistance selection cassettes.
  • the designed sequence such as SEQ ID NO.8/SEQ ID NO.9 primer pair ldhA-S/ldhA-A amplifies the resistance screening gene Kan to obtain the ldhA knockout box fragment.
  • the pKD46 plasmid was transformed into the expression host E.coli FMME-N-5- ⁇ focA-pflB competent cells, and the recombinant strain E.coli FMME-N-5- ⁇ focA-pflB-pKD46 was obtained by colony PCR screening, and then The obtained ldhA knockout box fragment was electroporated into recombinant strain E.coli FMME-N-5- ⁇ focA-pflB-pKD46 competent cells, and positive transformants were obtained by screening with 50 ⁇ g/mL Kan resistance plate.
  • thermosensitive plasmid pCP20 was used to induce the expression of FLP recombinase to eliminate the Kan resistance gene, and the temperature-sensitive plasmids pKD46 and pCP20 were eliminated by culturing and transferring at 42°C for 3 times. E.coli FMME was successfully obtained. -N-5( ⁇ focA-pflB- ⁇ ldhA).
  • ldhA-A TTAAACCAGTTCGTTCGGGCAGGTTTCGCCTTTTTCCAGATTGCTCATATGAATATCCTCCTTAG
  • Gene editing fragments include upstream and downstream homology arm regions and resistance selection cassettes.
  • the designed sequence such as SEQ ID NO.10/SEQ ID NO.11 primer pair pta-S/pta-A amplifies the resistance screening gene Kan to obtain the pta knockout box fragment.
  • the pKD46 plasmid was transformed into the expression host E.coli FMME-N-5- ⁇ focA-pflB- ⁇ ldhA competent cells, and the recombinant strain E.coli FMME-N-5- ⁇ focA-pflB- ⁇ ldhA-pKD46 was obtained by colony PCR screening, Then, the obtained pta knockout box fragment was transferred into recombinant strain E.coli FMME-N-5- ⁇ focA-pflB- ⁇ ldhA-pKD46 competent cells by electroporation, and positive results were obtained by screening with 50 ⁇ g/mL Kan resistance plate Turn.
  • thermosensitive plasmid pCP20 was used to induce the expression of FLP recombinase to eliminate the Kan resistance gene, and the temperature-sensitive plasmids pKD46 and pCP20 were eliminated by culturing and transferring at 42°C for 3 times.
  • FMME-N-5 ( ⁇ focA-pflB- ⁇ ldhA- ⁇ pta).
  • Gene editing fragments include upstream and downstream homology arm regions and resistance selection cassettes.
  • the designed sequence such as SEQ ID NO.12/SEQ ID NO.13 primer pair pta-ackA-S/pta-ackA-A amplifies the resistance screening gene Kan to obtain the pta-ackA knockout box fragment.
  • the pKD46 plasmid was transformed into the expression host E.coli FMME-N-5- ⁇ focA-pflB- ⁇ ldhA- ⁇ pta competent cells, and the recombinant strain E.coli FMME-N-5- ⁇ focA-pflB- ⁇ ldhA- was obtained by colony PCR screening pKD46, and then the obtained pta knockout box fragment was electroporated into recombinant strain E.coli FMME-N-5- ⁇ focA-pflB- ⁇ ldhA-pKD46 competent cells, and 50 ⁇ g/mL Kan resistance plate was used for screening Positive transformants were obtained.
  • thermosensitive plasmid pCP20 was used to induce the expression of FLP recombinase to eliminate the Kan resistance gene, and the temperature-sensitive plasmids pKD46 and pCP20 were eliminated by culturing and transferring at 42°C for 3 times.
  • .coli FMME-N-5 ( ⁇ focA-pflB- ⁇ ldhA- ⁇ pta-ackA).
  • pta-ackA-S ATGTCGAGTAAGTTAGTACTGGTTCTGAACTGCGGTAGTTCTTCAGTGTAGGCTGGAGCTGCTTC
  • pta-ackA-A TCAGGCAGTCAGGCGGCTCGCGTCTTGCGCGATAACCAGTTCTTCCATATGAATATCCTCCTTAG
  • the phosphoenolpyruvate carboxykinase pck used in the present invention is derived from Actinobacillus succinates, and the genomic DNA of Actinobacillus succinates is extracted;
  • sequences such as SEQ ID NO.14/SEQ ID NO.15 primer pair pck-S/pck-A were designed respectively. PCR amplification system and program to amplify the pck gene.
  • the pck obtained by PCR amplification was recovered by agarose gel nucleic acid electrophoresis.
  • the recovered product and the expression vector pTrcHisA were double digested with the restriction enzymes BamH I and XhoI for 3 h, and the digested product was coagulated by agarose nucleic acid electrophoresis.
  • the size of DNA and linearized plasmid were 1617 and 4405 bp, respectively, and then ligated with T4 DNA ligase overnight at 16°C, transformed into JM109 competent cells, and single colony was picked for PCR verification.
  • the positive transformants were sequenced and the alignment was correct. , which proved that the expression vector was constructed successfully, and the plasmid was named pTrcHisA-pck.
  • the phosphite dehydrogenase ptxD used in the present invention is derived from Pseudomonas stutzeri, and the gene Xu DNA of Pseudomonas aeruginosa is extracted;
  • the primer pair ptxD-S/ptxD-A such as SEQ ID NO.16/SEQ ID NO.17, was designed respectively, and the extracted genomic DNA of Pseudomonas stutzeri was used as the template, using standard The PCR amplification system and program were used to amplify the ptxD gene.
  • ptxD-S ACTCGAGATGCTGCCGAAACTGGTGATCACG
  • the ptxD obtained by PCR amplification was recovered by agarose gel nucleic acid electrophoresis.
  • the recovered product and the expression vector pTrcHisA-pck were double digested with restriction enzymes XhoI and Hind III for 3 h, and the digested product was digested with agarose nucleic acid.
  • the size of DNA and linearized plasmid were 1011 and 4370bp, respectively, and then ligated with T4 DNA ligase overnight at 16°C, transformed into JM109 competent cells, and single colony was picked for PCR verification, and the positive transformants were sequenced.
  • the expression vector is constructed successfully, and the plasmid is named pTrcHisA-pck-ptxD.
  • the recombinant plasmid was electroporated into the expression host strain E.coli FMME-N-5 ( ⁇ focA-pflB- ⁇ ldhA- ⁇ pta-ackA) to obtain the recombinant strain E.coli FMME-N-5 ( ⁇ focA-pflB- ⁇ ldhA- ⁇ pta-ackA )-pck-ptxD.
  • Example 7 Recombinant strain E. coli FMME-N-5 ( ⁇ focA-pflB- ⁇ ldhA- ⁇ pta-ackA)-pck-ptxD fermentor fed-batch fermentation
  • the fermentation medium on the fermenter is as follows: the fermentation medium is: the fermentation medium is glucose 35g/L, corn steep liquor 20g/L, (NH 4 ) 2 SO 4 3g/L, K 2 HPO 4 1.4g/L, KH 2 PO 4 0.6g/L, MgSO 4 ⁇ 7H 2 O 0.5g/L, NaCl 2g/L; feed medium: glucose 800g/L;
  • the E.coli FMME-N-5- ⁇ focA-pflB- ⁇ ldhA- ⁇ pta-pck-ptxD recombinant strain was picked for two-stage fermentation in a 7..5L fermenter.
  • the single clone was inoculated into 25mL (50mL shake flask) of LB medium as the primary seed solution, and cultured at 38°C and 200rpm for 8.5h. Take 200 ⁇ l of the first-grade seed solution and inoculate it into 50mL/500mL seed medium; cultivate at 38°C and 200rpm for 7.5h to obtain the second-class seed solution.
  • the initial liquid filling volume of the fermenter is 4L
  • the seed inoculation volume is 10%
  • the fermentation conditions in the aerobic stage are: the culture temperature is 38°C, the ventilation rate is 1vvm, the initial stirring speed is 600r/min, and the pH of the ammonia water is controlled at 7.0.
  • the dissolved oxygen is controlled at the level of 20%.
  • the stirring speed is 200r/min, and 800g is added.
  • glucose and control the feeding rate to control the pH of the fermentation broth ⁇ 10g/L, use basic magnesium carbonate to control pH 6.5 in the anaerobic stage, and the fermentation period is a total of 96h;
  • the results are shown in Figure 5.
  • the recombinant strain E.coli FMME-N-5- ⁇ focA-pflB- ⁇ ldhA- ⁇ pta-pck-ptxD produced succinic acid with a yield of 137 g/L, and the succinic acid yield reached 137 g/L.
  • the acid yield reaches 1g/g glucose, the production intensity is 1.43g/L/h, the by-products lactic acid and formic acid do not accumulate, and the acetic acid is only 1-2g/L;
  • the technology of the present invention uses genetic engineering technology to knock out the genes encoding related by-products: pyruvate formate lyase focA-pflB, lactate dehydrogenase ldhA, phosphotransacetylase pta, overexpression pathway enzyme phosphoenol Pyruvate carboxykinase pck and phosphite dehydrogenase ptxD balance cofactor metabolism to effectively increase succinate production.

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Abstract

公开了一种高效生产琥珀酸的重组大肠杆菌及其构建方法，敲除E.coli 产琥珀酸菌株FMME-N-5的副产物编码基因得到菌株E.coli FMME-N-5(ΔfocA-pflB-ΔldhA-Δpta-ackA)；将质粒pTrcHisA-pck-ptxD导入该菌株中过表达产琥珀酸放线杆菌磷酸烯醇式丙酮酸羧激酶pck和施式芽孢杆菌亚磷酸酯脱氢酶ptxD，筛选获得高效生产琥珀酸的工程菌E.coli FMME-N-5(ΔfocA-pflB-ΔldhA-Δpta-ackA)-pck-ptxD；该工程菌株在7.5L发酵罐上采用两阶段发酵策略，发酵96h，琥珀酸产量达到了137g/L，琥珀酸得率达到1g/g葡萄糖、生产强度为1.43g/L/h，副产物乳酸、甲酸不积累，乙酸1-2g/L。

Description

一种高效生产琥珀酸的重组大肠杆菌及其构建方法 技术领域
本发明涉及一种高效生产琥珀酸的重组大肠杆菌及其构建方法，属于生物工程技术领域。
背景技术
琥珀酸，学名丁二酸，是一种重要的C4平台化合物。作为合成通用化学品的起始原料，琥珀酸在食品、化学、医药以及其他领域有广泛的应用，琥珀酸被美国能源部列为12种最有潜力大宗生物基化学品中的首位。
琥珀酸的传统生产方法是化学合成法，主要有石蜡氧化法、氯乙酸甲酯氰化水解法和五氧化二钒催化加氢法等，但由于石油资源的减少和环境污染日益严重等问题，化学合成方法的弊端日益显现。而通过发酵法生产琥珀酸，能够摆脱对不可再生的战略资源石油的依赖，利用可再生资源，固定二氧化碳减轻温室效应，展现出良好的发展前景。目前研究较多的产琥珀酸的菌种有：产琥珀酸放线杆菌、产琥珀酸厌氧螺菌和大肠杆菌。产琥珀酸放线杆菌通常是从自然界中筛选，进行定向改造，能够耐受高浓度的琥珀酸盐，Guettler M等人利用突变株产琥珀酸放线杆菌FZ53生产琥珀酸产量最高，以葡萄糖为碳源，发酵48h产量可以达到最高产量110g/L，关于产琥珀酸放线杆菌菌种的研究较少，需要进一步的对其生理特性、发酵性能和遗传背景进行研究。产琥珀酸厌氧螺菌可以利用的发酵底物比较广泛，例如葡萄糖、乳糖、甘油等，Samuelov等人的研究结果表明，在最适条件下，产琥珀酸厌氧螺菌琥珀酸的产率可以达到 1.2mol/1.0mol葡萄糖，最高产量为65.0g/L，但是该菌株发酵需要严格的厌氧环境，工业化应用中很难实现。大肠杆菌作为模式菌株，遗传背景清晰，易操作，能够采用各种分子生物学的技术对菌种进行改造，所以采用大肠杆菌发酵琥珀酸已经成为一个热点，研究也取得了众多进展，Vemuri等利用重组大肠杆菌AFP111进行两阶段法发酵76h，琥珀酸的最终浓度可达到99.2g/L，得率达到1.1g/g葡萄糖，生产强度达到1.3g/L/h；张学礼等人运用基因工程及适应性进化策略构建得到重组大肠杆菌HX024，采用一步厌氧法发酵96h，最终琥珀酸产量达到95.9g/L，得率达到1g/g葡萄糖，朱丽雯等人组合优化表达ppc和pck基因，强化CO2固定路径，重组大肠杆菌AFP111菌株发酵96h，琥珀酸产量达到90.7g/L；张建国等人通过优化葡萄糖吸收代谢路径，同时敲除副产物乙酸编码基因，发酵65h，最终琥珀酸产量达到98.92g/L；
目前大肠杆菌发酵的生产效率较低，发酵液中通常有乳酸、甲酸、乙酸、乙醇等副产物，发酵过程辅因子代谢不平衡、不能耐受高浓度的产物浓度以及底物葡萄糖浓度、高浓度渗透压和葡萄糖吸收利用速度过快导致代谢失衡、产品得率及生产强度低等问题；为了获得高性能的生产菌株，通常需要利用传统选育手段、各种组学分析与分子生物学改造相结合的方法。
发明内容
为解决上述问题，本发明提供一种高效生产琥珀酸的重组大肠杆菌，采用Red同源重组技术对宿主菌FMME-N-5敲除大肠杆菌中编码丙酮酸甲酸裂解酶基因pflB-focA基因、乳酸脱氢酶基因ldhA基因、磷酸转乙酰基酶基因pta基因、并过表达琥珀酸合成路径中的关键酶磷酸式烯醇式丙酮酸羧激酶pck和亚磷酸酯脱氢酶ptxD。
本发明的第一个目的是提供一种高效生产琥珀酸的重组大肠杆菌，所述重组大肠杆菌敲除了大肠杆菌中丙酮酸甲酸裂解酶编码基因pflB-focA、乳酸脱氢酶编码基因ldhA、磷酸转乙酰基酶编码基因pta中的一种或多种，并过量表达 了磷酸烯醇式丙酮酸所激酶Pck和亚磷酸酯脱氢酶PtxD。
进一步地，编码所述的磷酸烯醇式丙酮酸激酶的基因的核苷酸序列如SEQ ID NO.1所示，编码所述的亚磷酸酯脱氢酶的基因的核苷酸序列如SEQ ID NO.2所示。
进一步地，所述磷酸烯醇式丙酮酸羧激酶pck和亚磷酸酯脱氢酶ptxD是通过质粒pTrcHisA进行表达。
进一步地，所述丙酮酸甲酸裂解酶编码基因pflB-focA的核苷酸序列如SEQ ID NO.3所示；乳酸脱氢酶编码基因ldhA的核苷酸序列如SEQ ID NO.4所示；磷酸转乙酰基酶编码基因pta的核苷酸序列如SEQ ID NO.5所示。
进一步地，所述重组大肠杆菌的宿主为大肠杆菌FMME-N-5，于2020年8月27日保藏于中国典型培养物保藏中心，保藏地址为中国武汉武汉大学，保藏编号为CCTCC NO:M 2020454。
本发明的第二个目的是提供一种所述重组大肠杆菌的构建方法，所述构建方法的步骤为：
(1)构建pflB-focA、ldhA、pta基因敲除框片段；依次将基因敲除框片段转入带有pKD46质粒的宿主菌，筛选获得敲除目标基因的菌株；
(2)扩增获得磷酸烯醇式丙酮酸羧激酶pck和亚磷酸酯脱氢酶ptxD基因片段；将基因片段连接到表达载体上，然后将连接有基因片段的表达载体转入步骤(1)的菌株中，得到所述的重组大肠杆菌。
本发明的第三个目的是提供所述大肠杆菌在生产琥珀酸的应用，所述的应用是采用所述的重组大肠杆菌在发酵培养基中进行有氧-厌氧两阶段发酵，得到含有琥珀酸的发酵液。
进一步地，所述的发酵培养基的配方为：葡萄糖30-50g/L，玉米浆15-25g/L，(NH 4) 2SO 4 2-4g/L，K 2HPO 4 1.2-2.0g/L，KH 2PO 4 0.5-1.0g/L，MgSO 4·7H 2O 0.2-0.5g/L，NaCl 1-2g/L。
进一步地，所述的有氧-厌氧两阶段发酵是在菌体OD 600＝52-60时，将有氧阶段转为厌氧阶段。
进一步地，将有氧阶段转为厌氧阶段是通过通入CO 2气体或者添加10-20g/L碳酸氢盐的方式进行。
进一步地，在厌氧阶段，控制葡萄糖浓度为5-15g/L。
进一步地，所述的有氧-厌氧两阶段发酵的接种量，按体积百分比计，为6-12％；发酵温度为35-38℃。
进一步地，所述的有氧-厌氧两阶段发酵的发酵时间为50-96h。
进一步地，在厌氧阶段，添加pH中和剂，所述pH中和剂为Na 2CO 3、K 2CO 3、NaOH、KOH、CaCO 3、碱式碳酸镁一种或几种混合。
进一步地，在有氧-厌氧两阶段发酵48h后，添加渗透压保护剂，所述渗透压保护剂为脯氨酸、甲硫氨酸、半胱氨酸、甜菜碱中的一种或几种混合。
在本发明中，亚磷酸酯脱氢酶(ptxD)可以催化盐磷酸盐生成一分子磷酸盐，同时消耗一分子NAD +生成一分子NADH，该反应产生的磷酸盐对细胞生长和ptxD活性不会产生抑制，且ptxD催化的反应不会直接竞争细胞代谢物，因此表达ptxD增加NADH供应的同时，不会对琥珀酸生成的主要代谢路径产生影响。
本发明的有益效果：
本发明采用Red同源重组策略敲除影响琥珀酸生成的副产物编码的相关酶，包括丙酮酸甲酸裂解酶、乳酸脱氢酶、磷酸转乙酰基酶；在不影响菌体生长速度的同时会减少副产物的积累，同时有利于琥珀酸的积累；同时过量表达路径酶磷酸烯醇式丙酮酸羧激酶和过量表达亚磷酸酯脱氢酶，有效提高了琥珀酸的产量，最终得到的工程菌株E.coli FMME-N-5(ΔfocA-pflB-ΔldhA-Δpta)-pck-ptxD，发酵96h琥珀酸产量达到了137g/L，琥珀酸得率达到1g/g葡萄糖、生产强度为1.43g/L/h，副产物乳酸、甲酸不积累，乙酸低于10g/L；本发明构 建菌株有利于琥珀酸的工业化生产。
生物材料保藏
大肠杆菌(Escherichia coli)FMME-N-5，所述大肠杆菌已于2020年8月27日保藏于中国典型培养物保藏中心，保藏编号为CCTCC NO:M 2020454，保藏地址为中国武汉，武汉大学。
附图说明
图1为敲除丙酮酸甲酸裂解酶胶验证图；
图2为敲除乳酸脱氢酶胶验证图；
图3为敲除磷酸转乙酰基胶验证图；
图4为表达磷酸烯醇式丙酮酸羧激酶和亚磷酸酯脱氢酶重组载体结构图；
图5为构建工程菌株发酵96h E.coli FMME-N-5(ΔfocA-pflB-ΔldhA-Δpta)-pck-ptxD发酵罐补料分批发酵结果。
具体实施方式
下面结合具体实施例对本发明作进一步说明，以使本领域的技术人员可以更好地理解本发明并能予以实施，但所举实施例不作为对本发明的限定。
序列表中未相关核苷酸序列信息：
(1)SEQ ID NO.1序列信息为产琥珀酸放线杆菌来源的磷酸烯醇式丙酮酸羧激酶pck核苷酸序列；
(2)SEQ ID NO.2序列信息为施氏假单胞菌的亚磷酸酯脱氢酶ptxD核苷酸序列；
(3)SEQ ID NO.3序列信息为丙酮酸甲酸裂解酶编码基因pflB-focA的核苷酸序列；
(4)SEQ ID NO.4序列信息为乳酸脱氢酶编码基因ldhA的核苷酸序列；
(5)SEQ ID NO.5序列信息为磷酸转乙酰基酶编码基因pta的核苷酸序列；
菌体浓度的测定：
取适量发酵液用2mol/L的盐酸中和后，菌体密度以分光光度计600nm波长下检测的吸光值来表示。
葡萄糖的测定：
发酵液预处理：取发酵液12000r/min离心5min取上清。稀释至适宜倍数，用M-100生物传感分析仪检测发酵液葡萄糖浓度。
有机酸的测定：
高效液相色谱法：发酵液预处理：取发酵液12000r/min离心5min取上清。稀释适宜倍数后，利用高效液相色谱仪(HPLC)检测琥珀酸、乳酸、甲酸、乙酸的产量。仪器为Waters e2695反相高效液相色谱仪，色谱柱采用Bio-Rad HPX 87H；流动相为5mmoL/L H 2SO 4；流速设定为0.6mL/min；检测器为紫外检测器，检测波长为210nm，柱温为35℃。
实施例1：敲除丙酮酸甲酸裂解酶编码基因
(1)为了实现编码丙酮酸甲酸裂解酶的基因的敲除以减少副产物加酸的量，借助Red同源重组技术构建基因组编辑片段。基因编辑片段包括上下游同源臂区域、抗性筛选盒。以质粒pKD4为模板，设计序列如SEQ ID NO.6/SEQ ID NO.7引物对pflB-focA-S/pflB-focA-A扩增抗性筛选基因Kan获得pflB-focA敲除框片段。
(2)将pKD46质粒转化进入表达宿主E.coli FMME-N-5感受态细胞中，通过菌落PCR筛选得到重组菌株E.coli FMME-N-5-pKD46，然后将得到的pflB-focA敲除框片段采用电转的方法转入重组菌株E.coli FMME-N-5-pKD46感受态细胞中，采用50μg/mL的Kan抗性平板筛选得到阳性转化子。最后借助温敏型质粒pCP20热诱导FLP重组酶表达消除Kan抗性基因，42℃培养转接3次，消除温敏型质粒pKD46和pCP20；成功得到了敲除丙酮酸甲酸裂解酶的E.coli FMME-N-5(ΔfocA-pflB)。
引物序列信息：5’→3’方向
pflB-focA-S：TTACTCCGTATTTGCATAAAAACCATGCGAGTTACGGGCCTATAAGTGTAGGCTGGAGCTGCTTC
pflB-focA-A：ATAGATTGAGTGAAGGTACGAGTAATAACGTCCTGCTGCTGTTCTCATATGAATATCCTCCTTAG
实施例2：敲除乳酸脱氢酶表达基因
(1)E.coli FMME-N-5(ΔfocA-pflB)的构建同实施例1
(2)为了进一步减少副产物乳酸的量以对编码乳酸脱氢酶的基因进行敲除，借助Red同源重组技术构建基因组编辑片段。基因编辑片段包括上下游同源臂区域、抗性筛选盒。以质粒pKD4为模板，设计序列如SEQ ID NO.8/SEQ ID NO.9引物对ldhA-S/ldhA-A扩增抗性筛选基因Kan获得ldhA敲除框片段。
(3)将pKD46质粒转化进入表达宿主E.coli FMME-N-5-ΔfocA-pflB感受态细胞中，通过菌落PCR筛选得到重组菌株E.coli FMME-N-5-ΔfocA-pflB-pKD46，然后将得到的ldhA敲除框片段采用电转的方法转入重组菌株E.coli FMME-N-5-ΔfocA-pflB-pKD46感受态细胞中，采用50μg/mL的Kan抗性平板筛选得到阳性转化子。最后借助温敏型质粒pCP20热诱导FLP重组酶表达消除Kan抗性基因，42℃培养转接3次，消除温敏型质粒pKD46和pCP20；成功得到了敲除乳酸脱氢酶的E.coli FMME-N-5(ΔfocA-pflB-ΔldhA)。
引物序列信息：5’→3’方向
ldhA-S：ATGAACTCGCCGTTTTATAGCACAAAACAGTACGACAAGAAGTACGTGTAGGCTGGAGCTGCTTC
ldhA-A：TTAAACCAGTTCGTTCGGGCAGGTTTCGCCTTTTTCCAGATTGCTCATATGAATATCCTCCTTAG
实施例3：敲除磷酸转乙酰基酶表达基因
(1)E.coli FMME-N-5(ΔfocA-pflB-ΔldhA)的构建同实施例2
(2)为了进一步减少副产物乙酸的量又保证细胞生长，以对编码磷酸专乙酰基的基因进行敲除，借助Red同源重组技术构建基因组编辑片段。基因编辑片段包括上下游同源臂区域、抗性筛选盒。以质粒pKD4为模板，设计序列如SEQ ID NO.10/SEQ ID NO.11引物对pta-S/pta-A扩增抗性筛选基因Kan获得pta敲除框片段。
将pKD46质粒转化进入表达宿主E.coli FMME-N-5-ΔfocA-pflB-ΔldhA感受态细胞中，通过菌落PCR筛选得到重组菌株E.coli FMME-N-5-ΔfocA-pflB-ΔldhA-pKD46，然后将得到的pta敲除框片段采用电转的方法转入重组菌株E.coli FMME-N-5-ΔfocA-pflB-ΔldhA-pKD46感受态细胞中，采用50μg/mL的Kan抗性平板筛选得到阳性转化子。最后借助温敏型质粒pCP20热诱导FLP重组酶表达消除Kan抗性基因，42℃培养转接3次，消除温敏型质粒pKD46和pCP20；成功得到了敲除磷酸转乙酰基酶的E.coli FMME-N-5(ΔfocA-pflB-ΔldhA-Δpta)。
引物序列信息：5’→3’方向
pta-S：GTGTCCCGTATTATTATGCTGATCCCTACCGGAACCAGCGTCGGTCGTGTAGGCTGGAGCTGCTTC
pta-A：TACACCATCGCGCTGACTGCGATTCAGTCTGCACAGCAGCAGCAGTAACATATGAATATCCTCCTTAG
实施例4：敲除磷酸转乙酰基-乙酸激酶表达基因
(1)E.coli FMME-N-5(ΔfocA-pflB-ΔldhA-Δpta)的构建同实施例3
(2)为了进一步减少副产物乙酸的量，借助Red同源重组技术构建基因组编辑片段。基因编辑片段包括上下游同源臂区域、抗性筛选盒。以质粒pKD4为模板，设计序列如SEQ ID NO.12/SEQ ID NO.13引物对pta-ackA-S/pta-ackA-A扩增抗性筛选基因Kan获得pta-ackA敲除框片段。
将pKD46质粒转化进入表达宿主E.coli FMME-N-5-ΔfocA-pflB-ΔldhA-Δpta 感受态细胞中，通过菌落PCR筛选得到重组菌株E.coli FMME-N-5-ΔfocA-pflB-ΔldhA-pKD46，然后将得到的pta敲除框片段采用电转的方法转入重组菌株E.coli FMME-N-5-ΔfocA-pflB-ΔldhA-pKD46感受态细胞中，采用50μg/mL的Kan抗性平板筛选得到阳性转化子。最后借助温敏型质粒pCP20热诱导FLP重组酶表达消除Kan抗性基因，42℃培养转接3次，消除温敏型质粒pKD46和pCP20；成功得到了敲除磷酸转乙酰基-乙酸激酶的E.coli FMME-N-5(ΔfocA-pflB-ΔldhA-Δpta-ackA)。
引物序列信息：5’→3’方向
pta-ackA-S：ATGTCGAGTAAGTTAGTACTGGTTCTGAACTGCGGTAGTTCTTCAGTGTAGGCTGGAGCTGCTTC
pta-ackA-A：TCAGGCAGTCAGGCGGCTCGCGTCTTGCGCGATAACCAGTTCTTCCATATGAATATCCTCCTTAG
实施例5：表达载体pTrcHisA-pck的构建
本发明所用的磷酸烯醇式丙酮酸羧激酶pck来源于产琥珀酸放线杆菌，提取产琥珀酸放线杆菌的基因组DNA；
根据已公布的基因组信息序列，分别设计序列如SEQ ID NO.14/SEQ ID NO.15引物对pck-S/pck-A，以提取的产琥珀酸放线杆菌基因组DNA为模板，采用标准的PCR扩增体系和程序，扩增获取pck基因。
pck-S:GGGATCCATGACTGACTTAAACAAACTCGTT
pck-A:CCGCTCGAGAATACGAAAACCTGGCCGCGGTT
PCR扩增获取的pck，采用琼脂糖凝胶核酸电泳切胶回收，回收产物与表达载体pTrcHisA分别利用限制性内切酶BamH I和XhoI进行双酶切3h，酶切产物采用琼脂糖核酸电泳凝胶回收，DNA和线性化质粒大小分别为1617、4405bp，然后采用T4 DNA连接酶16℃过夜连接，转化至JM109感受态细胞中，挑取单菌落PCR验证，阳性转化子进行测序，比对正确，证明表达载体构建成 功，质粒命名为pTrcHisA-pck。
实施例6：表达载体pTrcHisA-pck-ptxD的构建
本发明所用的亚磷酸酯脱氢酶ptxD来源于施氏假单胞菌，提取铜绿假单胞菌的基因徐DNA；
根据已公布的基因组信息序列，分别设计序列如SEQ ID NO.16/SEQ ID NO.17引物对ptxD-S/ptxD-A，以提取的施氏假单胞菌的基因组DNA为模板，采用标准的PCR扩增体系和程序，扩增获取ptxD基因。
引物序列信息：5’→3’方向:
ptxD-S：ACTCGAGATGCTGCCGAAACTGGTGATCACG
ptxD-A：AAGCTTAATCGTGCGGCGACCAAGCCGAAA
PCR扩增获取的ptxD，采用琼脂糖凝胶核酸电泳切胶回收，回收产物与表达载体pTrcHisA-pck分别利用限制性内切酶XhoI和Hind III进行双酶切3h，酶切产物采用琼脂糖核酸电泳凝胶回收，DNA和线性化质粒大小分别为1011、4370bp，然后采用T4 DNA连接酶16℃过夜连接，转化至JM109感受态细胞中，挑取单菌落PCR验证，阳性转化子进行测序，比对正确，证明表达载体构建成功，质粒命名为pTrcHisA-pck-ptxD。将重组质粒电转入表达宿主菌E.coli FMME-N-5(ΔfocA-pflB-ΔldhA-Δpta-ackA)，得到重组菌株E.coli FMME-N-5(ΔfocA-pflB-ΔldhA-Δpta-ackA)-pck-ptxD。
实施例7：重组菌株E.coli FMME-N-5(ΔfocA-pflB-ΔldhA-Δpta-ackA)-pck-ptxD发酵罐补料分批发酵
发酵罐上发酵培养基如下：发酵培养基为：发酵培养基为葡萄糖35g/L，玉米浆20g/L，(NH 4) 2SO 4 3g/L，K 2HPO 4 1.4g/L，KH 2PO 4 0.6g/L，MgSO 4·7H 2O 0.5g/L，NaCl 2g/L；补料培养基：葡萄糖800g/L；
挑取E.coli FMME-N-5-ΔfocA-pflB-ΔldhA-Δpta-pck-ptxD重组菌株进行 7..5L发酵罐两阶段发酵。单克隆接种于25mL(50mL摇瓶)的LB培养基作为一级种子液，置于38℃ 200rpm的条件下培养8.5h。取一级种子液200μl接种至50mL/500mL的种子培养基中；38℃、200rpm培养7.5h得到二级种子液。发酵罐的初始装液量为4L，种子接种量为10％，有氧阶段发酵条件为：培养温度为38℃，通气量为1vvm，初始搅拌转速为600r/min，氨水控制pH为7.0，整个有氧阶段溶氧控制在20％的水平，当菌体浓度生长至OD 600＝55-60时，转为厌氧发酵阶段；厌氧阶段：停止通气，搅拌转速为200r/min，补加800g/L葡萄糖并控制补料速率控制发酵液的pH<10g/L，厌氧阶段使用碱式碳酸镁控制pH＝6.5，发酵周期为总共96h；
根据琥珀酸的产量测定，结果如图5所示，发酵96h，重组菌株E.coli FMME-N-5-ΔfocA-pflB-ΔldhA-Δpta-pck-ptxD生产琥珀酸产量达到了137g/L,琥珀酸得率达到1g/g葡萄糖、生产强度为1.43g/L/h，副产物乳酸、甲酸不积累，乙酸仅有1-2g/L；
以上结果说明，本发明技术采用基因工程技术，敲除相关副产物编码基因：丙酮酸甲酸裂解酶focA-pflB、乳酸脱氢酶ldhA、磷酸转乙酰基酶pta、过量表达路径酶磷酸烯醇式丙酮酸羧激酶pck和亚磷酸酯脱氢酶ptxD平衡辅因子代谢能够有效提高琥珀酸的产量。
以上所述实施例仅是为充分说明本发明而所举的较佳的实施例，本发明的保护范围不限于此。本技术领域的技术人员在本发明基础上所作的等同替代或变换，均在本发明的保护范围之内。本发明的保护范围以权利要求书为准。

Claims (10)

  1. 一种高效生产琥珀酸的重组大肠杆菌，其特征在于，所述重组大肠杆菌敲除了大肠杆菌中丙酮酸甲酸裂解酶编码基因pflB-focA、乳酸脱氢酶编码基因ldhA、磷酸转乙酰基酶编码基因pta中的一种或多种，并过量表达了磷酸烯醇式丙酮酸所激酶Pck和亚磷酸酯脱氢酶PtxD。
  2. 根据权利要求1所述的重组大肠杆菌，其特征在于，编码所述的磷酸烯醇式丙酮酸激酶的基因的核苷酸序列如SEQ ID NO.1所示，编码所述的亚磷酸酯脱氢酶的基因的核苷酸序列如SEQ ID NO.2所示。
  3. 根据权利要求1所述的重组大肠杆菌，其特征在于，所述磷酸烯醇式丙酮酸羧激酶pck和亚磷酸酯脱氢酶ptxD是通过质粒pTrcHisA进行表达。
  4. 根据权利要求1所述的重组大肠杆菌，其特征在于，所述重组大肠杆菌的宿主为大肠杆菌FMME-N-5，于2020年8月27日保藏于中国典型培养物保藏中心，保藏地址为中国武汉武汉大学，保藏编号为CCTCC NO:M 2020454。
  5. 一种权利要求1-4任一项所述重组大肠杆菌的构建方法，其特征在于，所述构建方法的步骤为：
    (1)构建pflB-focA、ldhA、pta基因敲除框片段；依次将基因敲除框片段转入带有pKD46质粒的宿主菌，筛选获得敲除目标基因的菌株；
    (2)扩增获得磷酸烯醇式丙酮酸羧激酶pck和亚磷酸酯脱氢酶ptxD基因片段；将基因片段连接到表达载体上，然后将连接有基因片段的表达载体转入步骤(1)的菌株中，得到所述的重组大肠杆菌。
  6. 权利要求1-4任一项所述的大肠杆菌在生产琥珀酸的应用，其特征在于，所述的应用是采用所述的重组大肠杆菌在发酵培养基中进行有氧-厌氧两阶段发酵，得到含有琥珀酸的发酵液。
  7. 根据权利要求6所述的应用，其特征在于，所述的有氧-厌氧两阶段发 酵是在菌体OD 600＝52-60时，将有氧阶段转为厌氧阶段。
  8. 根据权利要求6所述的应用，其特征在于，将有氧阶段转为厌氧阶段是通过通入CO 2气体或者添加10-20g/L碳酸氢盐的方式进行。
  9. 根据权利要求6所述的应用，其特征在于，在厌氧阶段，控制葡萄糖浓度为5-15g/L。
  10. 根据权利要求6所述的应用，其特征在于，所述的有氧-厌氧两阶段发酵的接种量，按体积百分比计，为6-12％；发酵温度为35-38℃。
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