苹果酸脱氢酶及异柠檬酸脱氢酶扩增对E.coli产琥珀酸的影响
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摘要
本文以代谢工程的理论为指导,通过对大肠杆菌代谢途径的分析,选取在厌氧条件下表达活性降低的苹果酸脱氢酶和异柠檬酸脱氢酶,研究其在厌氧条件下对大肠杆菌琥珀酸产量的影响。通过扩增mdh和icd基因并将其分别与表达载体相连,增大苹果酸脱氢酶和异柠檬酸脱氢酶的活性,从而提高琥珀酸产量。主要得到以下结果:
通过Genbank查得苹果酸脱氢酶基因mdh和异柠檬酸脱氢酶icd的基因序列,克隆扩增编码苹果酸脱氢酶的mdh基因片段和编码异柠檬酸脱氢酶的icd基因片段;
利用同源重组连接试剂盒将扩增得到的mdh基因整合到原核表达载体pET28a(+)上,构建了表达mdh基因的质粒pIFmdh;
通过双酶切和连接的手段将扩增得到的icd基因与表达载体pET28a(+)相连,构建得到表达icd基因的重组质粒pIFicd;
将构建得到的重组质粒pIFmdh和pIFicd转入野生型大肠杆菌W1485中,分别构建了重组菌TUC1(pIFmdh)和重组菌TUC2(pIFicd);
对TUC1和TUC2进行发酵,并通过色谱测定重组菌株琥珀酸产量。重组菌TUC1琥珀酸产率达0.22mol/mol葡萄糖,比野生型W1485提高47%;重组菌TUC2琥珀酸产率为0.195mol/mol葡萄糖,比野生型W1485提高30%。
In this paper, we take the theory of metabolic engineering as a guide, through the analysis of the metabolic pathways of E.coli, we select malate dehydrogenase and isocitrate dehydrogenase, which activity reduced in anaerobic conditions, and study its impact of E.coli in succinic production under anaerobic conditions.We amplificate mdh and icd gene, and associated them with expression vector. We enhancing the succinic acid production by increasing malate dehydrogenase and isocitrate dehydrogenase activity. The results of this study are as follows:
We search through Genbank for malate dehydrogenase and isocitrate dehydrogenase gene sequence, clonal expansion mdh gene, which encoding the malate dehydrogenase, and clonal expansion icd gene, which encoding the isocitrate dehydrogenase;
With the use of homologous recombination connection kit, we integrated the amplified mdh gene into the prokaryotic expression vector pET28a(+), and constructed expression plasmid pIFmdh with mdh gene;
We construct recombinant plasmid pIFicd through the double digestion and connection of amplified icd gene and expression vector pET28a(+);
We transduct the recombinant plasmid pIFmdh and pIFicd into wild type E.coli W1485, construct the recombinant bacteria TUC1(pIFmdh) and TUC2(pIFicd);
We carry out fermentation of recombinant strains TUC1 and TUC2 and use chromatography to detect their succinate production. The succinate production rate of recombinant strain TUC1 is 0.22mol/mol glucose, raise 47% than the wild type W1485; the succinate production rate of recombinant strain TUC2 is 0.195mol/mol glucose, raise 30% than the wild type W1485.
通过Genbank查得苹果酸脱氢酶基因mdh和异柠檬酸脱氢酶icd的基因序列,克隆扩增编码苹果酸脱氢酶的mdh基因片段和编码异柠檬酸脱氢酶的icd基因片段;
利用同源重组连接试剂盒将扩增得到的mdh基因整合到原核表达载体pET28a(+)上,构建了表达mdh基因的质粒pIFmdh;
通过双酶切和连接的手段将扩增得到的icd基因与表达载体pET28a(+)相连,构建得到表达icd基因的重组质粒pIFicd;
将构建得到的重组质粒pIFmdh和pIFicd转入野生型大肠杆菌W1485中,分别构建了重组菌TUC1(pIFmdh)和重组菌TUC2(pIFicd);
对TUC1和TUC2进行发酵,并通过色谱测定重组菌株琥珀酸产量。重组菌TUC1琥珀酸产率达0.22mol/mol葡萄糖,比野生型W1485提高47%;重组菌TUC2琥珀酸产率为0.195mol/mol葡萄糖,比野生型W1485提高30%。
In this paper, we take the theory of metabolic engineering as a guide, through the analysis of the metabolic pathways of E.coli, we select malate dehydrogenase and isocitrate dehydrogenase, which activity reduced in anaerobic conditions, and study its impact of E.coli in succinic production under anaerobic conditions.We amplificate mdh and icd gene, and associated them with expression vector. We enhancing the succinic acid production by increasing malate dehydrogenase and isocitrate dehydrogenase activity. The results of this study are as follows:
We search through Genbank for malate dehydrogenase and isocitrate dehydrogenase gene sequence, clonal expansion mdh gene, which encoding the malate dehydrogenase, and clonal expansion icd gene, which encoding the isocitrate dehydrogenase;
With the use of homologous recombination connection kit, we integrated the amplified mdh gene into the prokaryotic expression vector pET28a(+), and constructed expression plasmid pIFmdh with mdh gene;
We construct recombinant plasmid pIFicd through the double digestion and connection of amplified icd gene and expression vector pET28a(+);
We transduct the recombinant plasmid pIFmdh and pIFicd into wild type E.coli W1485, construct the recombinant bacteria TUC1(pIFmdh) and TUC2(pIFicd);
We carry out fermentation of recombinant strains TUC1 and TUC2 and use chromatography to detect their succinate production. The succinate production rate of recombinant strain TUC1 is 0.22mol/mol glucose, raise 47% than the wild type W1485; the succinate production rate of recombinant strain TUC2 is 0.195mol/mol glucose, raise 30% than the wild type W1485.
引文
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[41] Vemuri GN, Eiteman MA, Altman E. Effects of growth mode and pyruvate carboxylase on succinic acid production by metabolically engineered strains of Escherichia coli. Appl Environ Microbiol, 2002, 68:1715-1727
[42] Vemuri GN, Eiteman MA, Altman E. Succinate production in dual-phase Escherichia coli fermentations depends on the time of transition from aerobic to anaerobic conditions. J Ind Microbiol Biotechnol, 2002, 28(6): 325-32
[43] Pan JG, Shin SA, Park CK. Pta ldhA double mutant Escherichia coli SS373 and the method of producing succinic acid therefrom. 2002, US patent 6,448,061
[44] Gokarn RR, Eiteman MA, Altman E. Metabolic analysis of Escherichia coli in the presence and absence of the carboxylating enzymes phosphoenolpyruvate carboxylase and pyruvate carboxylase. Appl Environ Microbiol, 2000, 66: 1844-1850
[45] Lee SJ, Lee DY, Kim TY, et al. Metabolic engineering of Escherichia coli for enhanced production of succinic acid, based on genome comparison and in silico gene knockout simulation. Appl Environ Microbiol, 2005, 71: 7880-7887
[46] Jantama K, Haupt M J, Svoronos S A, et al. Combining Metabolic Engineering and Metabolic Evolution to Develop Nonrecombinant Strains of Escherichia coli C That Produce Succinate and Malate. Biotechnology and Bioengineering, 2008, 99(5): 1140-1153
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[49] Sanchez AM, Bennett GN, San KY. Efficient succinic acid production from glucose through overexpression of pyruvate carboxylase in an Escherichia coli alcohol dehydrogenase and lactate dehydrogenase mutant. Biotechnol Prog. 2005, 21(2): 358-365
[50] Sanchez AM, Bennett GN, San KY. Novel pathway engineering design of the anaerobic central metabolic pathway in Escherichia coli to increase succinate yield and productivity. Metab Eng, 2005, 7: 229-239
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[2]中国医药公司上海化学试剂采购供应站编写,试剂手册,上海科学技术出版社,1985,1158
[3]邝生鲁,贡长生,琥珀酸的开发与应用前景,化工时刊,1991,第1期,24-26
[4]姚蒙正,程侣柏,王家儒编著。精细化工产品合成原理,北京:中国石化出版社,2000.145-147
[5]司航主编,化工产品手册,有机化工原料,化学工业出版社,1985,230-232
[6] Wollenberg RH, Frank P. Modified succinimides in fue composition, 1998, US patent 4,767,850
[7]宋光勋,冯光炷,李和平等,辛烯基琥珀酸淀粉酯的合成及其应用研究进展[J],粮油加工,2006(8):81-84
[8]张跃军,华平,磺基琥珀酸酯盐类表面活性剂的研究进展[J],江苏化工,2001年06期
[9]王庆昭,吴巍,赵学明.生物转化法制取琥珀酸及其衍生物的前景分析,化工进展, 2005,23(7):794-798
[10]徐春菊,绿色水处理剂聚环氧琥珀酸的合成及性能研究:[硕士学位论文],大连:大连理工大学,2006
[11]梁轶,顺酐的生产及市场,精细石油化工进展,2001,7:41-48
[12]吴靖,醇(醚)型磺基琥珀酸单酯二钠盐的应用[J],牙膏工业,2003年04期,27-29
[13]王庆昭,高产琥珀酸大肠杆菌的代谢工程:[博士学位论文],天津:天津大学,2006
[14] Guettler MV, Jain MK. Actinobacillus succinogenes sp. nov, a novel succinic acid producing strain from the bovine rumen. Int J Syst Bacteriol, 1999, 49: 207-216
[15] Kaneuchi C, Seki M, Komagata K. Production of succinic acid from citric acid and related acids by Lactobacillus strains, Appl Environ Microbiol, 1988, 54: 3053-3056
[16]沈菲.琥珀酸发酵条件的优化和菌种选育:[天津大学硕士学位论文];天津,天津大学,2003
[17] Guettler MV, Jain MK. Method for making succinic acid, Anaerobiospirillum succiniciproducens variants for use in process and methods for obtaining variants.1996, US Patent, 1996, 5521075
[18] Guettler MV, Jain MK. Method for making succinic acid, bacterial variants for use in the process, and methods for obtaining variants.1996, US Patent, 1996, 5573931
[19] Millard CS, Chao YP, Liao JC. Enhanced production of succinic acid by overexpression of phosphoenolpyruvate carboxylase Escherichia coli. Appl Enviro Microbio, 1996, 62(5): 1808-18102
[20] Zeikus JG, Jain MK, Elankovan P. Biotechnology of succinic acid production and market for derived industrial products. Appl Microbiol Biotechnol, 1999, 51: 545-552
[21] Lee PC, Lee WG, Kwon S, et al. Batch and continuous fermentation of succinic acid from whey by Anaerobiospirillum succiniciproducens. Appl Microbiol Biotechnol, 2000, 54: 23-27
[22] Lee PC, Lee WG, Lee SY, et al. Effects of medium components on the growth of Anaerobiospirillum succiniciproducens and succinic acid production. Process Biochemistry, 1999, 35: 49-55
[23] Guettler MV, Rumler D, Jain MK, Actinobacillus succinogenes sp. nov., a novel succinic acid producing strain from the bovine rumen, Int J Syst Bacteriol 1999,49: 207-216
[24] Guettler MV, Jain MK, Rumler D, Method for making succinic acid bacterial variants for use in the process, and methods for obtaining variants, 1996, US patent 5,573,931
[25] Van der Werf MJ, Guetter MV, Jain MK, et al. Environmental and physiological factors affecting the succinate product ratio during carbohydrate fermentation by Actinobacillus sp. 130Z. Arch Microbiol, 1997, 167:332-342
[26] Cannata JB, de Flombaum MAC, Phosphoenolpyruvate carboxykinase from bakers yeast, Kinetics of phosphoenolpyruvate formation, Biol Chem, 1974, 249:3356-3365
[27] Gutter MF, Kolenbrander HM, Formation of oxaloacetate by CO2 fixation on phosphoenolpyruvate, 1972.p. 117-168. In P. Boyer (ed.),The enzymes, 3rd ed,vol. 6. Academic Press,New York, N.Y
[28] Kim P, Laivenieks M, Vieille C , et al. Effect of Overexpression of Actinobacillus succinogenes phosphoenolpyruvate carboxykinase on succinate production in Escherichia coli.,Appl Environ Microbiol,2004,70: 1238-1241
[29] Glassner DA, Datta R, Process for the production and purification of succinic acid, 1992, US patent 5,521,075
[30] Samuelov NS, Lamed R, Lowe S, et al. Infuence of CO2-HCO3 levels and pH on growth, succinate production and enzyme activities of Anaerobiospirillum succiniciproducens, Appl Environ Microbiol, 1991, 57: 3013-3019
[31] Sokatch JR. Bacterial physiology and metabolism, Academic Press Inc. (London), Ltd., London, 1969
[32] Wendisch VF, Bott M, Eikmanns BJ. Metabolic Engineering of Escherichia coli and Corynebacterium Glutamicum for Biotechnological Production of Organic Acids and Amino Acids [J]. Curr Opin Microbiol, 2006, 9: 268–274.
[33] Lin H, Bennett GN, San KY. Effect of Carbon Sources Differing in OxidationState and Transport Route on Succinate Production in Metabolically Engineered Escherichia coli [J]. J. Ind. Microbiol. Biotechnol, 2005, 32(3): 87–93.
[34] Christophe C, Naruemol N R, Joachim W S, et al. Dynamic Modeling of the Central Carbon Metabolism of Escherichia coli [J]. Biotechnol. Bioeng., 2002, 79(1): 53–73.
[35]詹晓北,陈设,郑志永,大肠杆菌生产琥珀酸的代谢工程研究进展[J].化学工程学报,2007,7(4):840-846
[36]沈同王镜岩,生物化学第2版[M].北京高等教育出版社, 2002. 112–113.
[37]朱红裕李强,外源蛋白在大肠杆菌中的可溶性表达策略[J].过程工程学报, 2006, 6(1): 150–155.
[38] Bunch PK, Mat-Jan F, Lee N, et al. The ldh gene encoding the fermentative lactate dehydrogenase of Escherichia coli, Microbiology, 1997, 143:187-195
[39] Stols L, Kulkarni G, Harris BG, et al. Expression of Ascaris suum malic enzyme in a mutant Escherichia coli allows production of succinic acid from glucose. Appl Biochem Biotechnol, 1997, 63-65:153-158
[40] Chatterjee R, Millard CS, Champion K, et al. Mutation of the ptsG Gene results in increased production of succinate in fermentation of glucose by Escherichia coli. Appl Environ Microbiol, 2001, 67:148-154
[41] Vemuri GN, Eiteman MA, Altman E. Effects of growth mode and pyruvate carboxylase on succinic acid production by metabolically engineered strains of Escherichia coli. Appl Environ Microbiol, 2002, 68:1715-1727
[42] Vemuri GN, Eiteman MA, Altman E. Succinate production in dual-phase Escherichia coli fermentations depends on the time of transition from aerobic to anaerobic conditions. J Ind Microbiol Biotechnol, 2002, 28(6): 325-32
[43] Pan JG, Shin SA, Park CK. Pta ldhA double mutant Escherichia coli SS373 and the method of producing succinic acid therefrom. 2002, US patent 6,448,061
[44] Gokarn RR, Eiteman MA, Altman E. Metabolic analysis of Escherichia coli in the presence and absence of the carboxylating enzymes phosphoenolpyruvate carboxylase and pyruvate carboxylase. Appl Environ Microbiol, 2000, 66: 1844-1850
[45] Lee SJ, Lee DY, Kim TY, et al. Metabolic engineering of Escherichia coli for enhanced production of succinic acid, based on genome comparison and in silico gene knockout simulation. Appl Environ Microbiol, 2005, 71: 7880-7887
[46] Jantama K, Haupt M J, Svoronos S A, et al. Combining Metabolic Engineering and Metabolic Evolution to Develop Nonrecombinant Strains of Escherichia coli C That Produce Succinate and Malate. Biotechnology and Bioengineering, 2008, 99(5): 1140-1153
[47] Lin H, Bennett GN, San KY. Fed-batch culture of a metabolically engineered Escherichia coli strain designed for high-level succinate production and yield under aerobic conditions. Biotechnol Bioeng, 2005, 90, 775-779
[48] Lin H, Bennett GN, San KY. Metabolic engineering of aerobic succinate production systems in Escherichia coli to improve process productivity and achieve the maximum theoretical succinate yield. Metab Eng, 2005, 7: 116-127
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