WO2021120237A1 - 一株固定co 2产苹果酸的大肠杆菌工程菌的构建及应用 - Google Patents

一株固定co 2产苹果酸的大肠杆菌工程菌的构建及应用 Download PDF

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WO2021120237A1
WO2021120237A1 PCT/CN2019/127756 CN2019127756W WO2021120237A1 WO 2021120237 A1 WO2021120237 A1 WO 2021120237A1 CN 2019127756 W CN2019127756 W CN 2019127756W WO 2021120237 A1 WO2021120237 A1 WO 2021120237A1
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gene
escherichia coli
malic acid
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刘立明
胡贵鹏
陈修来
马丹蕾
刘佳
罗秋玲
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Jiangnan University
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Definitions

  • the invention relates to the construction and application of an Escherichia coli engineered bacteria that fixes CO 2 to produce malic acid, and belongs to the field of fermentation engineering.
  • CO 2 storage technologies to enhance CO 2 capture or reduce CO 2 release.
  • Traditional carbon dioxide storage technologies based on physical and chemical methods include carbon dioxide capture (such as post-combustion and oxyfuel combustion), carbon dioxide separation (such as adsorption and membrane separation), and carbon dioxide storage (such as saline aquifers, offshore geological structures).
  • carbon dioxide capture such as post-combustion and oxyfuel combustion
  • carbon dioxide separation such as adsorption and membrane separation
  • carbon dioxide storage such as saline aquifers, offshore geological structures.
  • the mitigation of carbon dioxide is of great significance.
  • these methods have some obvious shortcomings, such as high energy consumption, high operating costs, or the production of degradation products that are harmful to human health and the environment.
  • microbial CO 2 storage is a green and environmentally friendly way to alleviate the greenhouse effect, and it can also produce high value-added chemicals at the same time.
  • CO 2 fixation by heterotrophic microorganisms can be divided into three levels: (i) directly enhance the endogenous carboxylation reaction in heterotrophic microorganisms; (ii) construct artificially synthesized CO 2 fixation branches in heterotrophic microorganisms and connect them to Central carbon metabolism; (iii) Transform heterotrophic microorganisms so that they can grow with CO 2 as the sole carbon source.
  • the significant defect of the first level is that only a few special compounds can be produced , and CO 2 cannot be fixed for products without carboxylation reaction in the synthesis pathway.
  • the third level has not been fully realized at present, and the best research progress is obtained.
  • L-malic acid is an important four-carbon platform compound and has been listed as one of the basic compounds by the US Department of Energy. Its application fields involve food, medicine, chemical and other industries. In the food field, L-malic acid has become the third-most used food sour agent after citric acid and lactic acid; in the pharmaceutical industry, L-malic acid directly participates in human metabolism and has the effect of anti-fatigue and protecting liver, kidney and heart. And to reduce the toxic and side effects of anti-cancer drugs; in the chemical industry, L-malic acid is used in the production of daily cosmetics, metal cleaning and trimming, fabric finishing, electroless plating, etc. L-malic acid is an important intermediate metabolite in the tricarboxylic acid cycle. Malic acid cannot be detected in the fermentation broth of wild-type E. coli (below the detection limit of HPLC).
  • the first object of the present invention is to provide an engineered strain of Escherichia coli capable of fixing CO 2 to produce malic acid.
  • the engineered Escherichia coli strain has been knocked out of the fumarate reductase gene (frdBC) and the fumarase gene (fumB).
  • lactate dehydrogenase gene lactate dehydrogenase gene (ldhA) and alcohol dehydrogenase gene (adhE), and overexpression of formate dehydrogenase (FDH), acetyl coenzyme A synthetase (ACS), acylated acetaldehyde dehydrogenase (ACDH) , Formaldehyde lyase (FLS), dihydroxyacetone kinase (DHAP), malic enzyme (ME) and phosphorous oxidoreductase (PTXD).
  • FDH formate dehydrogenase
  • ACS acetyl coenzyme A synthetase
  • ACDH acylated acetaldehyde dehydrogenase
  • FLS Formaldehyde lyase
  • DHAP dihydroxyacetone kinase
  • ME malic enzyme
  • PTXD phosphorous oxidoreductase
  • the nucleotide sequence of the fumarate reductase gene is consistent with the gene of accession number: NP_418577.1 or NP_418576.1 on NCBI.
  • the nucleotide sequence of the fumarase gene is consistent with the gene of accession number: NP_418546.1 on NCBI.
  • the nucleotide sequence of the lactate dehydrogenase gene is consistent with the gene of accession number: NP_415898.1 on NCBI.
  • the nucleotide sequence of the alcohol dehydrogenase gene is consistent with the gene of accession number: NP_415757.1 on NCBI.
  • the Escherichia coli engineered bacteria is obtained by using Escherichia coli MG1655 as a host; the Escherichia coli MG1655 ( 700926 (TM ) is a wild-type E. coli purchased on ATCC.
  • the nucleotide sequence of the formate dehydrogenase gene is consistent with the gene of accession number: CP001666.1 on NCBI.
  • nucleotide sequence of the acetyl-CoA synthase gene is consistent with the gene of accession number: NP_418493.1 on NCBI.
  • the gene sequence of the acylated acetaldehyde dehydrogenase is shown in SEQ ID NO.1.
  • the gene sequence of formaldehyde lyase is shown in SEQ ID NO.2.
  • the gene sequence of dihydroxypyruvate kinase is shown in SEQ ID NO.3.
  • the nucleotide sequence of the malic enzyme gene is consistent with the gene of accession number: NP_418493.1 on NCBI.
  • nucleotide sequence of the phosphorous oxidoreductase gene is shown in SEQ ID NO.4.
  • the formate dehydrogenase, acetyl-CoA synthase, acylated acetaldehyde dehydrogenase, formaldehyde lyase and dihydroxyacetone kinase are gradually connected by homotaylase assembly. It was overexpressed on the vector pER, and the final plasmid was named pER-CF5A.
  • the malic enzyme gene and the phosphite oxidoreductase gene are connected to the vector pCDR by homo-tailase assembly for overexpression, and the final plasmid is named pCDR-ME-PTXD.
  • the second object of the present invention is to provide the application of the E. coli engineered bacteria in the fermentation production of malic acid.
  • the application is performed after activating the engineered Escherichia coli bacteria at a temperature of 30-37°C, a rotation speed of 700-800 rpm, an oxygen ventilation rate of 0.8-1.2 vvm, and a pH of 6.5-7.0. Aerobic culture is 12-18h; then, the oxygen ventilation is adjusted to 0vvm, the rotation speed is adjusted to 180-200rpm, and nitrogen is introduced for 10-20min (1vvm), the fermentation time is 60-80h under anaerobic conditions, and the pH is maintained neutral.
  • the fermentation medium used in the fermentation contains glucose 40-50g/L, Na 2 HPO 3 ⁇ 5H 2 O 20-50 mM, KHCO 3 30-50 mM, Na 2 HPO 4 ⁇ 12H 2 O 15.11 g/L, KH 2 PO 4 3g/L NH 4 Cl 1g/L, NaCl 0.5g/L, each L of medium contains 1mL trace element liquid; trace element liquid: FeCl 3 ⁇ 6H 2 O 2.4g/L, CoCl 2 ⁇ 6H 2 O 0.3g /L, CuCl 2 0.15g/L, ZnCl 2 ⁇ 4H 2 O 0.3g/L, NaMnO 4 0.3g/L, H 3 BO 3 0.075g/L, MnCl 2 ⁇ 4H 2 O 0.495g/L, soluble in Prepared in 0.1M HCl.
  • the invention constructs an engineered strain that can reduce the accumulation of malic acid by knocking out the fumarate reductase gene and the fumaric acid enzyme gene.
  • the synthesis of malate by blocking pyruvate as the node is one of the shortest paths found so far. When constructing this path, the synthesis pathway of the main by-product of the pyruvate node needs to be blocked.
  • the invention achieves the purpose of increasing the accumulation of precursor pyruvate by knocking out the lactate dehydrogenase gene and the alcohol dehydrogenase gene.
  • the present invention takes Escherichia coli MG1655 as the starting strain and utilizes metabolic engineering methods to construct the CO 2 fixation pathway, the malate synthesis pathway, block the path of malate metabolism, and block the branch pathway of pyruvate, thereby obtaining the large intestine producing malate.
  • Bacillus genetically engineered bacteria After 72 hours of fermentation, the yield of malic acid reached 39 g/L, and the yield of malic acid to glucose was 1.53 mol/mol.
  • the fermentation process is anaerobic fermentation, with high product yield and large output. At present, there is no report on the production of malic acid by the CO 2 fixation method in the present invention.
  • the present invention combines CO 2 fixation with malic acid production, not only can provide a new solution for effectively mitigating the greenhouse effect, but also can provide new ideas for the production of malic acid at the same time, turning waste materials into useful substances.
  • Figure 1 Construction of the path for CO 2 to synthesize malic acid in E. coli engineered bacteria.
  • FIG. 2 FrdBC gene knockout verification electrophoresis diagram [lanes 1-18 all indicate transformants with successfully knocked out frdBC gene, lane 19 (marked “+”) indicates the colony PCR result of wild-type E. coli, lane 19 (marked "-”) is the blank control group].
  • FIG. 3 FumB gene knockout verification electrophoresis graph [Swiss 1-11, 13-14 all indicate transformants with successfully knocked out fumB gene, lanes 12, 15-16 are transformants with unsuccessfully knocked out fumB gene, and No. 17 Lane (marked with "+”) represents the colony PCR result of wild-type E. coli, lane 18 (marked with "-”) is the blank control group].
  • FIG. 4 The electrophoresis diagram of the ldhA gene knockout verification [lane 3 is a transformant that has not successfully knocked out the fumB gene, and lanes 8-10 show a transformant that has successfully knocked out the ldhA gene].
  • FIG. 5 Electrophoresis diagram of adhE gene knockout verification [lanes 1-4, 6-14, and 16 all indicate transformants with successfully knocked out adhE gene, and lane 17 (marked with “+”) indicates the colony PCR results of wild-type E. coli , Lane 18 (marked with "-”) is the blank control group].
  • FIG. 6 Map of recombinant plasmid pER-CF5A.
  • FIG. 7 Map of recombinant plasmid pCDR-ME-PTXD.
  • Fig. 8 The yield-time curve of malic acid produced by E. coli fermentation.
  • Figure 9 The yield of malic acid produced by E. coli fermentation.
  • pKD4, pKD46 and pCP20 plasmids were purchased from BioVector NTCC.
  • the pER plasmid is a plasmid obtained by transforming the promoter region with pETM6 (purchased from addgene, #49795)
  • the pCDR plasmid is a plasmid obtained by transforming the promoter region with pCDM4 (purchased from addgene, #49796).
  • 20mL M9 medium containing 5-10g/L glucose and 20-50mM NaHCO 3
  • Gas chromatographic column RTX-QBOND (30m; 0.32mm inner diameter, film thickness 10mm (Bellefont RESTEK, Pennsylvania, USA).
  • the carrier gas is helium
  • the chromatographic column is at a constant temperature of 80°C
  • the flow rate is 15mL/min
  • the inlet pressure is 68.8 kPa.
  • a and B are respectively the CO 2 concentration of the headspace of the serum bottle before and after the culture in the serum bottle.
  • the headspace volume is 5 mL
  • the dry cell weight in the serum bottle is C mg
  • the culture time is 2 hours.
  • Example 1 Construction of Escherichia coli engineering strain capable of fixing CO 2 to produce malic acid
  • the knockout primers QCfrdBC-S and QCfrdBC-A were designed (Table 1), and the pKD4 plasmid was used as a template to amplify the frdBC knockout frame and gel recovery.
  • the pKD4 plasmid contains two FRT sites (which can be folded under the action of flipase to eliminate the DNA sequence between FRT sites), and between the two FRT sites is kanamycin (kan, as a gene knockout Screen the coding gene of stress), namely FRT-kan-FRT.
  • the electrotransformed competent cells were spread on an LB solid medium plate containing kanamycin (50 g/mL), and cultured upside down for 12-24 h. After the single bacteria grow out of the plate, use the verification primers YZfrdBC-S and YZfrdBC-A (Table 1) to screen positive transformants.
  • the pCP20 plasmid was transformed into the positive transformant to eliminate the kanamycin resistance gene, and then the YZfrdBC-S and YZfrdBC-A primers were used for verification.
  • the electrophoretic band size of the successfully knocked-out transformant was 529bp, and the control group without knockout
  • the electrophoresis band size is 1917bp ( Figure 2).
  • the strain that successfully knocked out the frdBC gene in MG1655 was named GH0101.
  • the knockout primers QCfumB-S and QCfumB-A were designed (Table 1), and the pKD4 plasmid was used as a template to amplify the fumB knockout frame and gel recovery.
  • the fumB knockout box was electrotransformed into E. coli GH0101 competent cells containing pKD46 plasmid (the electrotransformation voltage and time were 1800V and 5ms, respectively).
  • the electrotransformed competent cells were spread on an LB solid medium plate containing kanamycin (50 g/mL), and cultured upside down for 12-24 h. After the single bacteria grow out of the plate, use the verification primers YZfumB-S and YZfumB-A (Table 1) to screen positive transformants.
  • the pCP20 plasmid was transformed into the positive transformant to eliminate the kanamycin resistance gene, and then the YZfumB-S and YZfumB-A primers were used for verification.
  • the electrophoretic band size of the successfully knocked-out transformant was 506bp, and the control group without knockout
  • the electrophoresis band size is 1940bp ( Figure 3).
  • the strain that successfully knocked out the fumB gene in GH0101 was named GH0201.
  • the knockout primers QCldhA-S and QCldhA-A were designed according to the ldhA gene sequence of Escherichia coli MG1655 in the NCBI database, and the pKD4 plasmid was used as a template to amplify the ldhA knockout frame and gel recovery.
  • the knockout box of ldhA was electrotransformed into E. coli GH0201 competent cells containing pKD46 plasmid (the electrotransformation voltage and time were 1800V and 5ms, respectively).
  • the electrotransformed competent cells were spread on an LB solid medium plate containing kanamycin (50 g/mL), and cultured upside down for 12-24 h. After the single bacteria grow out of the plate, use the verification primers YZldhA-S and YZldhA-A (Table 1) to screen positive transformants.
  • the pCP20 plasmid was transformed into the positive transformant to eliminate the kanamycin resistance gene, and then the YZldhA-S and YZldhA-A primers were used for verification.
  • the electrophoretic band size of the successfully knocked-out transformant was 744bp, and the control group without knockout
  • the electrophoresis band size is 2132bp ( Figure 4).
  • the strain that successfully knocked out the ldhA gene in GH0201 was named GH0301.
  • the knockout primers QCadhE-S and QCadhE-A were designed (Table 1), and the pKD4 plasmid was used as a template to amplify the adhE knockout frame and gel recovery.
  • the adhE knockout box was electrotransformed into E. coli GH0301 competent cells containing pKD46 plasmid (the electrotransformation voltage and time were 1800V and 5ms, respectively).
  • the electrotransformed competent cells were spread on an LB solid medium plate containing kanamycin (50 g/mL), and cultured upside down for 12-24 h. After the single bacteria grow on the plate, use the verification primers YZadhE-S and YZadhE-A (Table 1) to screen positive transformants.
  • the pCP20 plasmid was transformed into a positive transformant to eliminate the kanamycin resistance gene, and then YZadhE-S and YZadhE-A primers were used for verification.
  • the electrophoretic band size of the successfully knocked-out transformant was 352bp, and the control group without knockout The electrophoresis band size is 2676bp ( Figure 5).
  • the strain that successfully knocked out the adhE gene in GH0301 was named GH0401.
  • the amplification primers FDH-S and FDH-A (Table 1) were designed according to the gene sequence of Clostridium jondalae formate dehydrogenase provided in the NCBI database, and the gene sequence of FDH protein was amplified using the Clostridium jondalae genome as a template. After the gel is recovered, it is connected to the plasmid pER (BglII and XhoI) by one-step homologous recombination to obtain the recombinant plasmid pER-FDH; the gene sequence of the pER plasmid is shown in SEQ ID NO.5. According to the E.
  • the amplification primers ACS-S and ACS-A were designed to amplify the ACS protein coding gene sequence using the E. coli MG1655 genome as a template.
  • a one-step homologous recombination method was used to connect to the plasmid pER (BglII and XhoI) to obtain the recombinant plasmid pER-ACS.
  • the fragments of the ACDH, FLS and DHAK encoding genes were obtained by gene synthesis, and then ligated to the plasmid pER (BglII and XhoI) by enzyme digestion and ligation.
  • Recombinant plasmids pER-ACDH, pER-FLS and pER-DHAK using homo-tail enzyme assembly technology [ACS Synth Biol 1,256-266 (2012)], the above five plasmids (pER-FDH, pER-ACS, pER-ACDH , PER-FLS and pER-DHAK) gradually assembled into a plasmid pER-CF5A.
  • the same tail enzymes used are BlnI and SpeI, and the positions of the restriction sites are shown in Figure 6.
  • the amplification primers ME-S and ME-A were designed to amplify the coding malic enzyme using the genome of C. acetobutylicum as a template.
  • the recombinant plasmid pCDR-ME was obtained by ligating the recombinant plasmid pCDR (BglII and XhoI) to the plasmid pCDR (BglII and XhoI) by one-step homologous recombination; the gene sequence of the pCDR plasmid is shown in SEQ ID NO.6.
  • the fragment of the PTXD encoding gene is obtained, and then ligated to the plasmid pCDR (BglII and XhoI) by restriction enzyme connection to obtain the recombinant plasmid pCDR-PTXD; the same-tail enzyme assembly technology is used to combine the above two plasmids (pCDR-ME and pCDR-PTXD) are assembled into a plasmid pCDR-ME-PTXD.
  • the same-tail enzymes used are BlnI and SpeI, and the positions of the restriction sites are shown in Figure 7.
  • the two plasmids pER-CF5A and pCDR-ME-PTXD obtained above were transformed into E. coli GH0401 competent cells and spread on a double-resistant plate containing spectinomycin and ampicillin.
  • the obtained transformant is The E. coli genetically engineered bacteria of the present invention is named GH0407.
  • the pER empty plasmid and pCDR-ME-PTXD were transformed into E. coli GH0401 competent cells to obtain engineered strain GH0402 as a control strain to verify the effect of heterologous CO 2 fixation pathway (HFLS, Figure 1) on malic acid synthesis.
  • HFLS heterologous CO 2 fixation pathway
  • Example 2 Fermentation to produce malic acid using engineered Escherichia coli GH0402 and GH0407
  • Plate activation medium and activation culture conditions are LB medium, activation conditions: 37°C incubator inverted for 12 hours.
  • the fermentation medium used for fermentation contains: glucose 50g/L, Na 2 HPO 3 ⁇ 5H 2 O 20mM, KHCO 3 50mM, Na 2 HPO 4 ⁇ 12H 2 O 15.11g/L, KH 2 PO 4 3g/L NH 4 Cl 1g/L, NaCl 0.5g/L, trace element liquid 1mL/L; trace element liquid: FeCl 3 ⁇ 6H 2 O 2.4g/L, CoCl 2 ⁇ 6H 2 O 0.3g/L, CuCl 2 0.15g/L, ZnCl 2 ⁇ 4H 2 O 0.3g/L, NaMnO 4 0.3g/L, H 3 BO 3 0.075g/L, MnCl 2 ⁇ 4H 2 O 0.495g/L, using 0.1M HCl as a solvent.
  • the final malic acid yield in the supernatant of GH0407 fermentation broth measured by high performance liquid chromatography (HPLC) was 39 g/L ( Figure 8), and the yield of malic acid to glucose reached 1.53 mol/mol (the highest reported so far) ( Figure 9) ), the CO 2 fixation rate of the engineered strain GH0407 is 41 mg gDCW -1 h -1 , which is higher than the CO 2 fixation rate of most autotrophic cyanobacteria (6-25 mg gDCW -1 h -1 ) (Table 2).
  • a rapid and reversible reaction occurs between HCO 3 - and CO 2 in the fermentation broth.
  • HCO 3 - can also be used as an acid-base neutralizer to control the pH of the fermentation broth. Therefore, by adding KHCO 3 to the medium, CO can be created. 2 Environment.

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Abstract

提供了一株固定CO 2产苹果酸的大肠杆菌工程菌的构建及应用，属于发酵领域。所述工程菌是对大肠杆菌Escherichia coli MG1655进行基因工程改造获得的菌；所述基因工程改造为敲除富马酸还原酶基因、富马酸酶基因、乳酸脱氢酶基因和乙醇脱氢酶基因，并过量表达甲酸脱氢酶、乙酰辅酶A合成酶、酰化乙醛脱氢酶、甲醛裂合酶、二羟基丙酮激酶、苹果酸酶和亚磷酸氧化还原酶，获得菌株GH0407。上述菌株用于发酵生产苹果酸，以CO 2和葡萄糖作为共底物厌氧发酵72h，苹果酸产量达到39g/L，得率为1.53mol/mol，原始出发菌株不积累苹果酸。

Description

一株固定CO 2产苹果酸的大肠杆菌工程菌的构建及应用 技术领域
本发明涉及一株固定CO 2产苹果酸的大肠杆菌工程菌的构建及应用，属于发酵工程领域。
背景技术
大气CO 2浓度的不断提升影响了全球气候变化，因此迫切需要发展有效的CO 2封存技术，以增强CO 2的捕获或者减少CO 2的释放。传统的基于物理和化学方法的二氧化碳封存技术，包括二氧化碳捕获(如后燃烧和氧燃料燃烧)、二氧化碳分离(如吸附和膜分离)和二氧化碳储存(如盐碱含水层、近海地质构造)，对于减缓二氧化碳具有重要意义。然而，这些方法存在一些明显的不足，如能耗大，运行成本高，或产生对人体健康和环境都有害的降解产物。相比之下，微生物CO 2封存是缓解温室效应的一种绿色环保途径，还可以同时生产高附加值化学品。
异养微生物固定CO 2可分为三个层次：(i)直接增强异养微生物中的内源性羧化反应；(ii)在异养微生物中构建人工合成的CO 2固定支路并连接到中心碳代谢；(iii)改造异养微生物使其能够以CO 2作为唯一碳源生长。第一层次的显著缺陷是只能生产少数特殊的化合物，对于合成途径没有羧化反应的产物来说无法固定CO 2，而第三层次目前还没有完全实现，目前最好的研究进展是获得了一株半自养型大肠杆菌。因此，为了更好的利用异养微生物固定CO 2生产化学品，需要人工构建新的CO 2固定途径，并且连接到糖酵解上游，从而达到CO 2固定效率高，产物谱广的目的。
L-苹果酸是一种重要的四碳平台化合物，已被美国能源部列为基础化合物之一，其应用领域涉及食品、医药、化工等行业。在食品领域，L-苹果酸已成为继柠檬酸、乳酸之后，用量排第三位的食品酸味剂；在医药行业中，L-苹果酸直接参与人体新陈代谢，具有抗疲劳保护肝肾心脏的作用以及降低抗癌药物的毒副作用等；在化工领域，L-苹果酸被用于日用化妆品的生产，金属的清洗和修整，织物整理，化学镀层等。L-苹果酸是三羧酸循环中的重要中间代谢物，野生型大肠杆菌的发酵液中不能检测到苹果酸(低于HPLC检测限)。
发明内容
本发明的第一个目的是提供一种能够固定CO 2生产苹果酸的大肠杆菌工程菌株，所述大肠杆菌工程菌株被敲除富马酸还原酶基因(frdBC)、富马酸酶基因(fumB)、乳酸脱氢酶基因(ldhA)和乙醇脱氢酶基因(adhE)，并过量表达甲酸脱氢酶(FDH)、乙酰辅酶A合成酶(ACS)、酰化乙醛脱氢酶(ACDH)、甲醛裂合酶(FLS)、二羟基丙酮激酶(DHAP)、苹果酸酶(ME)和亚磷酸氧化还原酶(PTXD)。
在本发明的一种实施方式中，所述富马酸还原酶基因的核苷酸序列与NCBI上accession number:NP_418577.1或NP_418576.1的基因一致。
在本发明的一种实施方式中，所述富马酸酶基因的核苷酸序列与NCBI上accession number: NP_418546.1的基因一致。
在本发明的一种实施方式中，所述乳酸脱氢酶基因的核苷酸序列与NCBI上accession number:NP_415898.1的基因一致。
在本发明的一种实施方式中，所述乙醇脱氢酶基因的核苷酸序列与NCBI上accession number:NP_415757.1的基因一致。
在本发明的一种实施方式中，所述大肠杆菌工程菌是以Escherichia coli MG1655为宿主得到的；所述Escherichia coli MG1655(
Figure PCTCN2019127756-appb-000001
700926 TM)是ATCC上购买的野生型大肠杆菌。
在本发明的一种实施方式中，所述甲酸脱氢酶基因的核苷酸序列与NCBI上accession number:CP001666.1的基因一致。
在本发明的一种实施方式中，所述乙酰辅酶A合成酶基因的核苷酸序列与NCBI上accession number:NP_418493.1的基因一致。
在本发明的一种实施方式中，所述酰化乙醛脱氢酶的基因序列如SEQ ID NO.1所示。
在本发明的一种实施方式中，甲醛裂合酶的基因序列如SEQ ID NO.2所示。
在本发明的一种实施方式中，二羟基丙酮酸激酶的基因序列如SEQ ID NO.3所示。
在本发明的一种实施方式中，所述苹果酸酶基因的核苷酸序列与NCBI上accession number:NP_418493.1的基因一致。
在本发明的一种实施方式中，所述亚磷酸氧化还原酶基因的核苷酸序列如SEQ ID NO.4所示。
在本发明的一种实施方式中，所述甲酸脱氢酶、乙酰辅酶A合成酶、酰化乙醛脱氢酶、甲醛裂合酶和二羟基丙酮激酶是由同尾酶组装的方式逐步连接到载体pER上进行过量表达，最终质粒命名为pER-CF5A。所述苹果酸酶基因和亚磷酸氧化还原酶基因是由同尾酶组装的方式连接到载体pCDR上进行过量表达，最终质粒命名为pCDR-ME-PTXD。
本发明的第二个目的是提供所述大肠杆菌工程菌在发酵生产苹果酸方面的应用。在本发明的一种实施方式中，所述应用是将大肠杆菌工程菌活化后，于温度30-37℃、转速700-800rpm、氧气通气量0.8-1.2vvm、pH＝6.5-7.0条件下进行好氧培养12-18h；然后，将氧气通气量调至0vvm，转速调整至180-200rpm，并通入氮气10-20min(1vvm)，厌氧条件下发酵时间60-80h，维持pH中性。可选地，所述应用是将大肠杆菌工程菌活化后，于温度37℃、转速800rpm、氧气通气量1vvm、pH＝6.5-7.0条件下进行好氧培养；其次，将氧气通气量调至0vvm，转速调整至200rpm，并通入氮气10-20min(1vvm)，厌氧条件下发酵时间72h，采用250g/LKHCO 3作为酸碱中和剂以维持pH＝7。
所述发酵使用的发酵培养基含有葡萄糖40-50g/L，Na 2HPO 3·5H 2O 20-50mM，KHCO 3 30-50mM，Na 2HPO 4·12H 2O 15.11g/L、KH 2PO 4 3g/L NH 4Cl 1g/L、NaCl 0.5g/L，每L培养基含有1mL微量元素液；微量元素液：FeCl 3·6H 2O 2.4g/L、CoCl 2·6H 2O 0.3g/L、CuCl 2 0.15g/L、ZnCl 2·4H 2O 0.3g/L、NaMnO 4 0.3g/L、 H 3BO 3 0.075g/L、MnCl 2·4H 2O 0.495g/L，溶于0.1M HCl中配制。
本发明通过敲除富马酸还原酶基因和富马酸酶基因构建了一株可以少了积累苹果酸的工程菌株。以阻断丙酮酸为节点合成苹果酸是目前发现的最短路径之一，构建该路径时，需要将丙酮酸节点的主要副产物合成途径进行阻断。本发明通过敲除乳酸脱氢酶基因和乙醇脱氢酶基因，以达到增加前体丙酮酸积累的目的。
本发明以Escherichia coli MG1655为出发菌株，利用代谢工程手段，通过构建CO 2固定途径、苹果酸合成途径、阻断苹果酸代谢去路以及和阻断丙酮酸谢支路，从而得到生产苹果酸的大肠杆菌基因工程菌。在发酵72h后苹果酸的产量达到39g/L，苹果酸对葡萄糖得率为1.53mol/mol。该发酵过程为厌氧发酵，产品得率高、产量大，目前未见本发明中CO 2固定途径生产苹果酸的报道。
本发明将CO 2固定与苹果酸生产相结合，不仅能够对有效缓解温室效应提供新的方案，而且可以同时为苹果酸的生产提供新的思路，将废弃物质变成有用物质。
附图说明
图1大肠杆菌工程菌中构建CO 2合成苹果酸的路径。
图2 frdBC基因敲除验证电泳图【1-18号泳道均表示成功敲除frdBC基因的转化子，19号泳道(标注“+”)表示野生型大肠杆菌的菌落PCR结果，19号泳道(标注“-”)为空白对照组】。
图3 fumB基因敲除验证电泳图【1-11、13-14号泳道均表示成功敲除fumB基因的转化子，12、15-16号泳道是未成功敲除fumB基因的转化子，17号泳道(标注“+”)表示野生型大肠杆菌的菌落PCR结果，18号泳道(标注“-”)为空白对照组】。
图4 ldhA基因敲除验证电泳图【3号泳道是未成功敲除fumB基因的转化子，8-10号泳道表示成功敲除ldhA基因的转化子】。
图5 adhE基因敲除验证电泳图【1-4、6-14、16号泳道均表示成功敲除adhE基因的转化子，17号泳道(标注“+”)表示野生型大肠杆菌的菌落PCR结果，18号泳道(标注“-”)为空白对照组】。
图6重组质粒pER-CF5A的图谱。
图7重组质粒pCDR-ME-PTXD的图谱。
图8大肠杆菌发酵生产苹果酸产量-时间曲线。
图9大肠杆菌发酵生产苹果酸得率。
具体实施方式
苹果酸检测方法(高效液相色谱条件)：色谱柱：Aminex HPX-87H(7.8×300mm)，流动相：5mM H 2SO 4，柱温：35℃，检测波长：210nm，进样量：10μl，流速：0.6ml/min。
商业化质粒产品的购买来源：pKD4、pKD46以及pCP20质粒购自BioVector NTCC。pER质粒是pETM6(购自addgene，#49795)改造启动子区域后获得的质粒，pCDR质粒是pCDM4(购自addgene，#49796) 改造启动子区域后获得的质粒。
CO 2固定速率的检测及计算方法：(1)检测方法：首先，使用LB培养基将大肠杆菌培养至对数生长中期(OD 600＝0.4-0.8)；其次，收集对数中期细胞，并使用20mL M9培养基(含5-10g/L葡萄糖和20-50mM NaHCO 3)重悬至OD 600＝3-5；再次，将20mL的细胞悬液转移至25mL的血清瓶中培养2h；最后，注入浓盐酸释放细胞悬液中的总无机碳，用气相色谱仪测定血清瓶顶空气体中CO 2的浓度。气相色谱柱：RTX-QBOND(30m；0.32mm内径，膜厚度10mm(美国宾夕法尼亚州贝尔方特RESTEK公司)。载气为氦气，色谱柱恒温80℃,流速15mL/min，进样口压力68.8kPa。
(2)计算方法：CO 2固定速率＝(B-A)mg/mL×5mL/(C mg×2h)
注：A和B分别为血清瓶中培养前后血清瓶顶空的CO 2浓度，顶空体积为5mL，血清瓶中细胞干重为C mg，培养时间为2h。
实施例1：构建能够固定CO 2生产苹果酸的大肠杆菌工程菌株
(1)E.coli MG1655富马酸还原酶基因frdBC的敲除
根据NCBI数据库中Escherichia coli MG1655的frdBC基因序列设计敲除引物QCfrdBC-S和QCfrdBC-A(表1)，以pKD4质粒为模板扩增frdBC的敲除框并进行胶回收。备注：pKD4质粒包含两个FRT位点(在翻转酶作用下可以折叠从而消除FRT位点之间的DNA序列)，两个FRT位点之间是卡那霉素(kan，作为基因敲除的筛选压力)的编码基因，即FRT-kan-FRT。在敲除基因时，设计引物扩增出FRT-kan-FRT的DNA片段。需要指出的是，设计的两条扩增引物分别包含靶基因的上下游39-49bp，即我们最终获得的DNA片段是“靶基因上游39-49 bp-FRT-kan-FRT-靶基因上游39-49bp”，这个DNA片段称为靶基因的敲除框。将frdBC的敲除框通过电转化的形式导入含有pKD46质粒的E.coli MG1655感受态细胞中(电转化电压和时间分别为1800V和5ms)。将电转的感受态细胞涂布于含卡那霉素(50g/mL)的LB固体培养基平板上，倒置培养12-24h。待平板长出单菌落后，使用验证引物YZfrdBC-S和YZfrdBC-A(表1)筛选阳性转化子。
将pCP20质粒转化至阳性转化子以消除卡那霉素抗性基因，再使用YZfrdBC-S和YZfrdBC-A引物进行验证，成功敲除的转化子电泳条带大小为529bp，没有敲除的对照组电泳条带大小为1917bp(图2)。在MG1655体内成功敲除frdBC基因的菌株命名为GH0101。
(2)E.coli GH0101富马酸酶基因fumB的敲除
根据NCBI数据库中Escherichia coli MG1655的fumB基因序列设计敲除引物QCfumB-S和QCfumB-A(表1)，以pKD4质粒为模板扩增fumB的敲除框并进行胶回收。将fumB的敲除框通过电转化的形式导入含有pKD46质粒的E.coli GH0101感受态细胞中(电转化电压和时间分别为1800V和5ms)。将电转的感受态细胞涂布于含卡那霉素(50g/mL)的LB固体培养基平板上，倒置培养12-24h。待平板长出单菌落后，使用验证引物YZfumB-S和YZfumB-A(表1)筛选阳性转化子。
将pCP20质粒转化至阳性转化子以消除卡那霉素抗性基因，再使用YZfumB-S和YZfumB-A引物进行验证，成功敲除的转化子电泳条带大小为506bp，没有敲除的对照组电泳条带大小为1940bp(图3)。在GH0101体内成功敲除fumB基因的菌株命名为GH0201。
(3)E.coli GH0201乳酸脱氢酶基因ldhA的敲除
根据NCBI数据库中Escherichia coli MG1655的ldhA基因序列设计敲除引物QCldhA-S和QCldhA-A(表1)，以pKD4质粒为模板扩增ldhA的敲除框并进行胶回收。将ldhA的敲除框通过电转化的形式导入含有pKD46质粒的E.coli GH0201感受态细胞中(电转化电压和时间分别为1800V和5ms)。将电转的感受态细胞涂布于含卡那霉素(50g/mL)的LB固体培养基平板上，倒置培养12-24h。待平板长出单菌落后，使用验证引物YZldhA-S和YZldhA-A(表1)筛选阳性转化子。
将pCP20质粒转化至阳性转化子以消除卡那霉素抗性基因，再使用YZldhA-S和YZldhA-A引物进行验证，成功敲除的转化子电泳条带大小为744bp，没有敲除的对照组电泳条带大小为2132bp(图4)。在GH0201体内成功敲除ldhA基因的菌株命名为GH0301。
(4)E.coli GH0301乙醇脱氢酶基因adhE的敲除
根据NCBI数据库中Escherichia coli MG1655的adhE基因序列设计敲除引物QCadhE-S和QCadhE-A(表1)，以pKD4质粒为模板扩增adhE的敲除框并进行胶回收。将adhE的敲除框通过电转化的形式导入含有pKD46质粒的E.coli GH0301感受态细胞中(电转化电压和时间分别为1800V和5ms)。将电转的感受态细胞涂布于含卡那霉素(50g/mL)的LB固体培养基平板上，倒置培养12-24h。待平板长出单菌落后，使用验证引物YZadhE-S和YZadhE-A(表1)筛选阳性转化子。
将pCP20质粒转化至阳性转化子以消除卡那霉素抗性基因，再使用YZadhE-S和YZadhE-A引物进行验证，成功敲除的转化子电泳条带大小为352bp，没有敲除的对照组电泳条带大小为2676bp(图5)。在GH0301体内成功敲除adhE基因的菌株命名为GH0401。
(5)FDH、ACS、ACDH、FLS和DHAK蛋白的过表达
根据NCBI数据库中提供的永达尔梭菌甲酸脱氢酶基因序列设计扩增引物FDH-S与FDH-A(表1)，以永达尔梭菌基因组为模板，扩增出FDH蛋白的基因序列，胶回收后采用一步同源重组的方式连接到质粒pER(BglII和XhoI)上得到重组质粒pER-FDH；pER质粒的基因序列如SEQ ID NO.5所示。根据NCBI数据库中提供的大肠杆菌MG1655乙酰CoA合成酶基因序列设计扩增引物ACS-S与ACS-A(表1)，以大肠杆菌MG1655的基因组为模板，扩增ACS蛋白的编码基因序列，胶回收后采用一步同源重组的方式连接到质粒pER(BglII和XhoI)上得到重组质粒pER-ACS。
根据文献提供的ACDH、FLS和DHAK基因序列，采用基因合成的方式，分别得到ACDH、FLS和DHAK编码基因的片段，然后以酶切连接的方式连接到质粒pER上(BglII和XhoI)，分别得到重组质粒pER-ACDH、pER-FLS和pER-DHAK；采用同尾酶组装技术【ACS Synth Biol 1,256-266(2012)】，分别将 上述五个质粒(pER-FDH、pER-ACS、pER-ACDH、pER-FLS和pER-DHAK)逐步组装成一个质粒pER-CF5A。采用的同尾酶为BlnI和SpeI，酶切位点的位置见图6。
(6)ME和PTXD蛋白的过表达
根据NCBI数据库中提供的丙酮丁醇梭菌苹果酸酶基因序列设计扩增引物ME-S与ME-A(表1)，以丙酮丁醇梭菌的基因组为模板，扩增出编码苹果酸酶的基因片段，并采用一步同源重组的方式连接到质粒pCDR(BglII和XhoI)上得到重组质粒pCDR-ME；pCDR质粒的基因序列如SEQ ID NO.6所示。采用基因合成的方式，得到PTXD编码基因的片段，然后以酶切连接的方式连接到质粒pCDR(BglII和XhoI)上，得到重组质粒pCDR-PTXD；采用同尾酶组装技术，将上述两个质粒(pCDR-ME和pCDR-PTXD)组装成一个质粒pCDR-ME-PTXD。采用的同尾酶为BlnI和SpeI，酶切位点的位置见图7。
将上述得到的两个质粒pER-CF5A和pCDR-ME-PTXD转化至E.coli GH0401感受态细胞中，涂布于含有壮观霉素和氨苄青霉素的双抗性平板上，获得的转化子即为本发明所述的大肠杆菌基因工程菌，命名为GH0407。此外，将pER空质粒与pCDR-ME-PTXD转化至E.coli GH0401感受态细胞获得工程菌株GH0402作为对照菌株，以验证异源CO 2固定途径(HFLS，图1)对苹果酸合成的影响。
实施例2：利用工程大肠杆菌GH0402和GH0407发酵生产苹果酸
平板活化培养基及活化培养条件：平板活化培养基是LB培养基，活化条件：37℃培养箱倒置12h。发酵使用的发酵培养基含有：葡萄糖50g/L、Na 2HPO 3·5H 2O 20mM、KHCO 3 50mM、Na 2HPO 4·12H 2O 15.11g/L、KH 2PO 4 3g/L NH 4Cl 1g/L、NaCl 0.5g/L、微量元素液1mL/L；微量元素液：FeCl 3·6H 2O 2.4g/L、CoCl 2·6H 2O 0.3g/L、CuCl 2 0.15g/L、ZnCl 2·4H 2O 0.3g/L、NaMnO 4 0.3g/L、H 3BO 3 0.075g/L、MnCl 2·4H 2O 0.495g/L，以0.1M HCl为溶剂。将工程大肠杆菌GH0402和GH0407于平板活化后，挑取单菌落于液体LB种子培养基中，37℃、200rpm培养12h(OD 600＝3-4)。种子培养完成后，按2％(v/v)的接种量接种至发酵培养基中，于温度37℃、转速800rpm、氧气通气量1vvm、pH＝7.0条件下培养16h后关闭氧气通量，并通入氮气10-20min(氮气通量：1vvm)除去残留氧气，厌氧条件下继续发酵时间72h。整个过程采用250g/L KHCO 3作为酸碱中和剂以维持pH＝7。
采用高效液相色谱(HPLC)测得GH0407发酵液上清中最终的苹果酸产量为39g/L(图8)，苹果酸对葡萄糖得率达到1.53mol/mol(为目前报道最高)(图9)，工程菌株GH0407的CO 2固定速率为41mg gDCW -1h -1，比多数自养蓝藻的CO 2固定速率(6-25mg gDCW -1h -1)更高(表2)。发酵液中的HCO 3 -与CO 2之间会发生快速的可逆反应，同时HCO 3 -还可以作为酸碱中和剂控制发酵液的pH，因此，通过向培养基中添加KHCO 3可以创造CO 2环境。
我们设置了两个对照组：(i)发酵液中不添加KHCO 3，采用NaOH作为酸碱中和剂；(ii)以不含CO 2固定途径(即不含pER-CF5A质粒)的菌株GH0402作为对照组。结果表明，当使用NaOH替代KHCO 3作为酸碱中和剂时(即不提供CO 2环境时)，工程菌株GH0407苹果酸的产量仅为2.3g/L，对葡萄糖得率 仅为0.14mol/mol；对照组GH0402发酵上清液中最终的苹果酸产量为22g/L，对葡萄糖得率为1.13mol/mol。从对照组的数据可以看出，CO 2确实提高了苹果酸的产量和得率。
表1基因敲除引物与蛋白过量表达引物序列
引物名称 引物序列
QCfrdBC-S ATGGCTGAGATGAAAAACCTGAAAATTGAGGTGGTGCGCTATAACCCGGGTGTAGGCTGGAGCTGCTTC
QCfrdBC-A TTACCAGTACAGGGCAACAAACAGGATTACGATGGTGGCAACCACAGTTATGGGAATTAGCCATGGTCC
YZfrdBC-S TGGAGTACAGCGACGTGAAG
YZfrdBC-A GGAATACGCGACCAATGAAG
QCfumB-S ATGTCAAACAAACCCTTTATCTACCAGGCACCTTTCCCGATGGGGAAAGGTGTAGGCTGGAGCTGCTTC
QCfumB-A TTACTTAGTGCAGTTCGCGCACTGTTTGTTGACGATTTGCTGGAAGAAGATGGGAATTAGCCATGGTCC
YZfumB-S TGTGAGCGTATCGTGCGTC
YZfumB-A CGTGAAATTACAATCGCAAAC
QCldhA-S ATGAAACTCGCCGTTTATAGCACAAAACAGTACGACAAGAAGTACCTGCGTGTAGGCTGGAGCTGCTTC
QCldhA-A TTAAACCAGTTCGTTCGGGCAGGTTTCGCCTTTTTCCAGATTGCTTAAGATGGGAATTAGCCATGGTCC
YZldhA-S AACCCACAGCCCGAGCGT
YZldhA-A GGCTTACCGTTTACGCTTTCC
QCadhE-S ATGGCTGTTACTAATGTCGCTGAACTTAACGCACTCGTAGAGCGTGTAAGTGTAGGCTGGAGCTGCTTC
QCadhE-A TTAAGCGGATTTTTTCGCTTTTTTCTCAGCTTTAGCCGGAGCAGCTTCTATGGGAATTAGCCATGGTCC
YZadhE-S TCATCACCGCACTGACTAT
YZadhE-A TCCTTAACTGATCGGCATT
FDH-S agatatacatatggcagatctGATGAAAAGTATACTAACTACTTGTCCTTATTGT
FDH-A ggtttctttaccagactcgagTTAAGCGTCTTTACGCATACTCTTTT
ACS-S agatatacatatggcagatctGATGAGCCAAATTCACAAACACACC
ACS-A ggtttctttaccagactcgagTTACGATGGCATCGCGATAGC
ME-S agatatacatatggcagatctGATGAATAATTTAAAAGGTTTAGAATTACTAAGAA
ME-A ggtttctttaccagactcgagTTATCTATAGTATGGTTCCCAAATTTCA
表2微生物CO 2固定速率的比较
Figure PCTCN2019127756-appb-000002

Claims (10)

  1. 一种固定CO 2生产苹果酸的大肠杆菌工程菌株，其特征在于，所述大肠杆菌工程菌株被敲除富马酸还原酶基因(frdBC)、富马酸酶基因(fumB)、乳酸脱氢酶基因(ldhA)和乙醇脱氢酶基因(adhE)，并过量表达甲酸脱氢酶(FDH)、乙酰辅酶A合成酶(ACS)、酰化乙醛脱氢酶(ACDH)、甲醛裂合酶(FLS)、二羟基丙酮激酶(DHAP)、苹果酸酶(ME)和亚磷酸氧化还原酶(PTXD)。
  2. 根据权利要求1所述的一种固定CO 2生产苹果酸的大肠杆菌工程菌株，其特征在于，所述大肠杆菌工程菌是以Escherichia coli MG1655为宿主。
  3. 根据权利要求1或2所述的一种固定CO 2生产苹果酸的大肠杆菌工程菌株，其特征在于，所述甲酸脱氢酶、乙酰辅酶A合成酶、酰化乙醛脱氢酶、甲醛裂合酶和二羟基丙酮激酶是由同尾酶组装的方式逐步连接到载体上进行过量表达。
  4. 根据权利要求3所述的一种固定CO 2生产苹果酸的大肠杆菌工程菌株，其特征在于，所述载体的核苷酸序列如SEQ ID NO.5所示。
  5. 根据权利要求1-4任一所述的一种固定CO 2生产苹果酸的大肠杆菌工程菌株，其特征在于，所述苹果酸酶基因和亚磷酸氧化还原酶基因是由同尾酶组装的方式连接到载体上进行过量表达。
  6. 根据权利要求5所述的一种固定CO 2生产苹果酸的大肠杆菌工程菌株，其特征在于，所述载体的核苷酸序列如SEQ ID NO.6所示。
  7. 根据权利要求1-6任一所述的一种固定CO 2生产苹果酸的大肠杆菌工程菌株，其特征在于，所述富马酸还原酶基因的核苷酸序列与NCBI上accession number:NP_418577.1或NP_418576.1的基因一致，所述富马酸酶基因的核苷酸序列与NCBI上accession number:NP_418546.1的基因一致，所述乳酸脱氢酶基因的核苷酸序列与NCBI上accession number:NP_415898.1的基因一致，所述乙醇脱氢酶基因的核苷酸序列与NCBI上accession number:NP_415757.1的基因一致，所述甲酸脱氢酶基因的核苷酸序列与NCBI上accession number:CP001666.1的基因一致，所述乙酰辅酶A合成酶基因的核苷酸序列与NCBI上accession number:NP_418493.1的基因一致，所述酰化乙醛脱氢酶的基因序列如SEQ ID NO.1所示，甲醛裂合酶的基因序列如SEQ ID NO.2所示，二羟基丙酮酸激酶的基因序列如SEQ ID NO.3所示，所述苹果酸酶基因的核苷酸序列与NCBI上accession number:NP_418493.1的基因一致，所述亚磷酸氧化还原酶基因的核苷酸序列如SEQ ID NO.4所示。
  8. 权利要求1-7任一所述的一种固定CO 2生产苹果酸的大肠杆菌工程菌株在发酵生产苹果酸方面的应用。
  9. 根据权利要求8所述的应用，其特征在于，将大肠杆菌工程菌活化后，于温度30-37℃、转速700-800rpm、氧气通气量0.8-1.2vvm、pH＝6.5-7.0条件下进行好氧培养12-18h；然后，将氧气通气量调至0vvm，转速调整至180-200rpm，并以1vvm的速度通入氮气10-20min，厌氧条件下发酵时间60-80h，维持pH中性。
  10. 根据权利要求9所述的应用，其特征在于，发酵使用的发酵培养基含有葡萄糖40-50g/L，Na 2HPO 3·5H 2O 20-50mM，KHCO 330-50mM，Na 2HPO 4·12H 2O 15.11g/L、KH 2PO 43g/LNH 4Cl 1g/L、NaCl 0.5g/L，每L培养基含有1mL微量元素液；微量元素液：FeCl 3·6H 2O 2.4g/L、CoCl 2·6H 2O 0.3g/L、CuCl 20.15g/L、ZnCl 2·4H 2O 0.3g/L、NaMnO 40.3g/L、H 3BO 30.075g/L、MnCl 2·4H 2O 0.495g/L，溶于0.1M HCl中配制。
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