大肠杆菌dapA基因的敲除及其对蛋氨酸产量影响的研究
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摘要
本实验采用重叠引物PCR技术,应用高保真DNA聚合酶和带有EcoRⅠ酶切位点的引物,人工扩增合成了两端为dapA基因同源序列(各50bp),中间为氯霉素抗性筛选基因的片段DPSCAT。将所得线性化重组片段,经电转化,转入E.coli K12/pKD46感受态细胞中。经过双抗性固体培养基筛选和基因组PCR鉴定,重组片段成功与目的基因发生同源置换,获得一株重组菌株。
通过对Escherichia coli K-12菌株和重组菌株进行蛋氨酸含量的测定。在相同培养条件下,对发酵培养基上清液分别进行高效液相色谱分析,测定发酵液上清中蛋氨酸的含量。重组菌株中蛋氨酸的含量是Escherichia coli K-12菌株的3.61倍,即蛋氨酸的产量提高了2.61倍。利用该重组系统,构建了dapA基因缺失的大肠杆菌基因工程菌株,并获得一株蛋氨酸产量提高菌株。
在家禽日粮中,L-蛋氨酸是第一限制性氨基酸,参与机体内肾上腺素、胆碱、肌酸的合成,活性甲基的转移及肝脏内磷脂的代谢等生理功能。我国蛋氨酸市场广阔,因此提高微生物发酵法生产L-蛋氨酸的效率,降低生产成本,减小工业污染对加快我国饲料业、养殖业等工业的发展和整个社会的健康、可持续发展具有重要的战略意义。
L-methionine(L-Met), an essential amino acid, involves in synthesis of adrenalin, bilineurine and creatine in vivo, and the transfer of active methyls, the metabolism of phospholipids in livers as well. With the development of feed industry in China, the demand for L-Met is increasing. However, domestic supply is insufficient and import of L-Met is indispensable. Microbial fermentation, which is an important method to produce other amino acids, is expected to be an efficient way to produce L-Met. As is known, the method has the remarkable features of lowering costs and pollution, and promoting the development of feed industry and animal husbandry nationwide.
Dihydrodipicolinate synthase (DHDPS) is coded by dapA gene. And DHDPS is the enzyme that catalysts the first unique step of L-lysine biosynthesis in plants and microorganisms. It catalyzes the aldol condensation of L-aspartate-β-semialdehyde and pyruvate to dihydropicolinic acid via a Schiff base formation between pyruvate and a lysine residue. The functional enzyme is a homotetramer consisting of a dimer of dimers. DHDPS is member of dihydrodipicolinate synthase family that comprises several pyruvate-dependent class I aldolases that use the same catalytic step to catalyze different reactions in different pathways. The present study focuses on deleting dapA gene in E.coli to provide more precursors for high-yield of L-Met by means of gene targeting technique. The study might be a reference for producing other metabolites from microorganisms.
A recombination system has been developed for efficient chromosome engineering in Escherichia coli by using electroporated linear DNA. A defectiveλprophage supplies functions that protect and recombine an electroporated linear DNA substrate in the bacterial cell. The use of recombination eliminates the requirement for standard cloning as all novel joints are engineered by chemical synthesis in vitro and the linear DNA is efficiently recombined into place in vivo. The technology and manipulations required are simple and straightforward. A temperature-dependent repressor tightly controls prophage expression, and, thus, recombination functions can be transiently supplied by shifting cultures to 42°C for 15 min. The efficient prophage recombination system does not require host RecA function and depends primarily on Exo, Beta, and Gam functions expressed from the defectiveλprophage. The defective prophage can be moved to other strains and can be easily removed from any strain. Gene disruptions and modifications of both the bacterial chromosome and bacterial plasmids are possible. This system will be especially useful for the engineering of large bacterial plasmids such as those from bacterial artificial chromosome libraries. pKD46 has an optimized ribosome-binding site for efficient translation of gam and expresses gam, bet, and exo from the arabinose-inducible ParaB promoter. It is also a temperaturesensitive replicon to allow for its easy elimination.
L-lysine branch is one of Asp metabolic pathways in Escherichia coli. Its presence certainly affect the yield of another amino acid, L-methionine, which shares the same precursor as L-lysine. dapA codes the first key enzyme for L-lysine synthesis. Dihydrodipicolinate synthase (DHDPS) is a key enzyme in lysine biosynthesis. It catalyzes the aldol condensation of L-aspartate-beta- semialdehyde and pyruvate to dihydropicolinic acid via a Schiff base formation between pyruvate and a lysine residue. The functional enzyme is a homotetramer consisting of a dimer of dimers. DHDPS is member of dihydrodipicolinate synthase family that comprises several pyruvate-dependent class I aldolases that use the same catalytic step to catalyze different reactions in different pathways.
E.coli K12 with pKD46 plasmid (K12/pKD46)was involved in the experiment. Induced by arabinose, Red recombinases fromλbacteriophage were expressed, which provided the host with recombination ability. In overlapping-primer PCR, High-fidelity DNA polymerase and primers with EcoRⅠcleavage sites were used. DPSCAT fragments with 50bp of dapA gene at two ends and chloramphenicol-resistance gene in between were amplified. The linear fragment were cloned into pMD18-T vectors and then transformed into E.coli strain DH5α. By extracting plasmids, enzyme cutting identification, the results were as good as expected. By sequencing analysis, it indicated that the homogolous rate was 99% which confirmed the accuracy of the results.
DPSCAT fragments cloned into vectors were amplified. The linear fragments were recombined and transformed into E.coli K12/pKD46 by electroporation. The positive strains of L-Lys deletion mutant were screened and identified.
The L-Met in the samples of wild type E.coli K-12 and respectively were compared quantitatively by HPLC. The results indicate that the L-Met of recombinants was as 3.61 times as that of wild type E.coli K-12. The strains with dapA deletion were obtained and were characterized as L-Met high-yield.
通过对Escherichia coli K-12菌株和重组菌株进行蛋氨酸含量的测定。在相同培养条件下,对发酵培养基上清液分别进行高效液相色谱分析,测定发酵液上清中蛋氨酸的含量。重组菌株中蛋氨酸的含量是Escherichia coli K-12菌株的3.61倍,即蛋氨酸的产量提高了2.61倍。利用该重组系统,构建了dapA基因缺失的大肠杆菌基因工程菌株,并获得一株蛋氨酸产量提高菌株。
在家禽日粮中,L-蛋氨酸是第一限制性氨基酸,参与机体内肾上腺素、胆碱、肌酸的合成,活性甲基的转移及肝脏内磷脂的代谢等生理功能。我国蛋氨酸市场广阔,因此提高微生物发酵法生产L-蛋氨酸的效率,降低生产成本,减小工业污染对加快我国饲料业、养殖业等工业的发展和整个社会的健康、可持续发展具有重要的战略意义。
L-methionine(L-Met), an essential amino acid, involves in synthesis of adrenalin, bilineurine and creatine in vivo, and the transfer of active methyls, the metabolism of phospholipids in livers as well. With the development of feed industry in China, the demand for L-Met is increasing. However, domestic supply is insufficient and import of L-Met is indispensable. Microbial fermentation, which is an important method to produce other amino acids, is expected to be an efficient way to produce L-Met. As is known, the method has the remarkable features of lowering costs and pollution, and promoting the development of feed industry and animal husbandry nationwide.
Dihydrodipicolinate synthase (DHDPS) is coded by dapA gene. And DHDPS is the enzyme that catalysts the first unique step of L-lysine biosynthesis in plants and microorganisms. It catalyzes the aldol condensation of L-aspartate-β-semialdehyde and pyruvate to dihydropicolinic acid via a Schiff base formation between pyruvate and a lysine residue. The functional enzyme is a homotetramer consisting of a dimer of dimers. DHDPS is member of dihydrodipicolinate synthase family that comprises several pyruvate-dependent class I aldolases that use the same catalytic step to catalyze different reactions in different pathways. The present study focuses on deleting dapA gene in E.coli to provide more precursors for high-yield of L-Met by means of gene targeting technique. The study might be a reference for producing other metabolites from microorganisms.
A recombination system has been developed for efficient chromosome engineering in Escherichia coli by using electroporated linear DNA. A defectiveλprophage supplies functions that protect and recombine an electroporated linear DNA substrate in the bacterial cell. The use of recombination eliminates the requirement for standard cloning as all novel joints are engineered by chemical synthesis in vitro and the linear DNA is efficiently recombined into place in vivo. The technology and manipulations required are simple and straightforward. A temperature-dependent repressor tightly controls prophage expression, and, thus, recombination functions can be transiently supplied by shifting cultures to 42°C for 15 min. The efficient prophage recombination system does not require host RecA function and depends primarily on Exo, Beta, and Gam functions expressed from the defectiveλprophage. The defective prophage can be moved to other strains and can be easily removed from any strain. Gene disruptions and modifications of both the bacterial chromosome and bacterial plasmids are possible. This system will be especially useful for the engineering of large bacterial plasmids such as those from bacterial artificial chromosome libraries. pKD46 has an optimized ribosome-binding site for efficient translation of gam and expresses gam, bet, and exo from the arabinose-inducible ParaB promoter. It is also a temperaturesensitive replicon to allow for its easy elimination.
L-lysine branch is one of Asp metabolic pathways in Escherichia coli. Its presence certainly affect the yield of another amino acid, L-methionine, which shares the same precursor as L-lysine. dapA codes the first key enzyme for L-lysine synthesis. Dihydrodipicolinate synthase (DHDPS) is a key enzyme in lysine biosynthesis. It catalyzes the aldol condensation of L-aspartate-beta- semialdehyde and pyruvate to dihydropicolinic acid via a Schiff base formation between pyruvate and a lysine residue. The functional enzyme is a homotetramer consisting of a dimer of dimers. DHDPS is member of dihydrodipicolinate synthase family that comprises several pyruvate-dependent class I aldolases that use the same catalytic step to catalyze different reactions in different pathways.
E.coli K12 with pKD46 plasmid (K12/pKD46)was involved in the experiment. Induced by arabinose, Red recombinases fromλbacteriophage were expressed, which provided the host with recombination ability. In overlapping-primer PCR, High-fidelity DNA polymerase and primers with EcoRⅠcleavage sites were used. DPSCAT fragments with 50bp of dapA gene at two ends and chloramphenicol-resistance gene in between were amplified. The linear fragment were cloned into pMD18-T vectors and then transformed into E.coli strain DH5α. By extracting plasmids, enzyme cutting identification, the results were as good as expected. By sequencing analysis, it indicated that the homogolous rate was 99% which confirmed the accuracy of the results.
DPSCAT fragments cloned into vectors were amplified. The linear fragments were recombined and transformed into E.coli K12/pKD46 by electroporation. The positive strains of L-Lys deletion mutant were screened and identified.
The L-Met in the samples of wild type E.coli K-12 and respectively were compared quantitatively by HPLC. The results indicate that the L-Met of recombinants was as 3.61 times as that of wild type E.coli K-12. The strains with dapA deletion were obtained and were characterized as L-Met high-yield.
引文
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[2] Spunt, S. Trink, B., Pai, S. I.et al. Absence of TSG101 transcript abnormalities in human cancers. Oncogene,1998, 16: 2815-2818.
[3] Tsiagbe V K, Cook M E, HarperA E, et al. Enhanced immune responses in broiler chicks fed methionine-supplemented diets [ J ].Poult Sci, 1987, 66 (7) : 1147-1154.
[4] Hillova J, Hill M, Belehradek J Jr, et al. Loss of the oncogene from human H-ras-1-transfected NIH/3T3 cells grown in the presence of excess methionine.[J]Natl Cancer Inst. 1986 ,77(3): 721-732.
[5] Swick, R. A. , Pierson E. E. M. Effect of methionine sources and dietary acidulants on resistance of broiler chickens to heat stress conditions. Poult. Sci. 1988, 68(1): 208.
[6] 燕磊, 杨维仁,杨在宾, 等. 不同水平瘤胃保护性蛋氨酸对小尾寒羊氮代谢及生产性能的影响. 2005, 26(6): 27-30.
[7] 石程,程存归. 固一液相转移催化法合成蛋氨酸. 化学工程师,1999,2: 5-6.
[8] 黄光斗,鲁国彬,黄征青,等. 蛋氨酸的合成及研究进展[J].化工时刊, 2003,17 (3):10-12.
[9] Geiger Friedhelm, Halsberghe Baudouin, Hasselbach Hans-Joachim,et al. ·Process for the preparation of D,L-methionine or the salt thereof [P],US:5990349,1999-11-23.
[10] Geiger Friedhelm, Halsberghe Baudouin, Hasselbach Hans-Joachim.et al. Process for the preparation of D,L-methionine or the salt thereof [P], US:5770769,1998-06-23.
[11] Hasseberg Hans-Albrecht, Huthmacher Klaus, Rautenberg Stephan, et al. Weigel Horst Method for the continuous preparation of methionine or methionine derivatives [P], US:5672749,1997-09-30.
[12] Earl Pierson,Mario Giella,Max Tishler.Synthesis of DL-Methionine . Chemistry,1947 , 1450.
[13] Livak KJ ,LaRossa RA ,Falco SC ,et al. Microbiological identification and characterization of an amino acid biosynthetic enzyme as the site of sulfonylurea herbicide action. ACS Symposium Series [ACS SYMP. SER.]. 1987,1450.
[14] 张伟国,钱和.氨基酸生产技术及其应用.北京:中国轻工业出版社,1997, 100~107.
[15] 刘云,许其寿.氨基酸发酵生产的研究进展[J]. 氨基酸和生物资源,1999, 21 (4) :19~23.
[16] 陈三增. 氨基酸的制造技术[J]. 发酵科技通讯,2002 ,31 (4 ): 41~43.
[17] Kinoshita S, Udaka S, Shimono M. Amino acid fermentation. Production of L-glutamic acid by various microorganisms. J Gen Appl Microbiol 1957,3:193–205.
[18] Kase H, Nakayama K. Isolation and characterization of S-adenosylmethionine requiring mutants and role of Sadenosylmethionine in the regulation of methionine biosynthesis in Corynebacterium glutamicum. Agric Biol Chem 1975,39(1):161–168.
[19] Nakayama K, Araki K, Kase H. Microbial production of essential amino acid with Corynebacterium glutamicum mutants. Adv Exp Med Biol 1978, 105:649–661
[20] Banik AK, Majumdar SK. Studies on methionine fermentation. Part I. Selection of mutants of Micrococcus glutamicus and optimum conditions for methionine production. Indian J Exp Biol 1974, 12:363–365.
[21] Banik AK, Majumdar SK. Effect of minerals on production of methionine by Micrococcus glutamicus. Indian J Exp Biol 1975,13:510–512.
[22] Mondal S, Chatterjee SP. Enhancement of methionine production by methionine analogue resistant mutants of Brevibacterium heali. Acta Biotechnol 1994, 14:199–204.
[23] Mondal S, Das YB, Chatterjee SP. L-methionine production by double auxotrophic mutants of an ethionine resistant strain of Brevibacterium heali. Acta Biotechnol1994, 14:61–66.
[24] Mondal S, Das YB, Chatterjee SP. Methionine production by microorganisms. Folia Microbiol 1996, 41:465–472.
[25] Morinaga Y, Tani Y, Yamada H. L-Methionine production ethionine resistant mutant of facultative methylotroph Pseudomonas FM 518. Agric Biol Chem 1982, 46(2):473–480.
[26] Roy SK, Biswas SR, Mishra AK, Nanda G. Production and purification of methionine from a multianalog reistant mutant B-6 US-215 of Bacillus megaterium B71. J Micro Biotech 1989, 4:35–41.
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