产核黄素枯草芽孢杆菌hprK基因敲除及其发酵特性的研究
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摘要
在枯草芽孢杆菌中,HPr蛋白既参与葡萄糖的跨膜运输,也参与碳分解代谢物阻遏作用(CCR)。己糖磷酸转移酶系统(PTS)是枯草芽孢杆菌主要的葡萄糖跨膜运输系统,HPr蛋白在15位组氨酸磷酸化,形成HPr-His15-P,作为磷酸基团由磷酸烯醇丙酮酸传递给酶Ⅱa的中间载体。同时,碳分解代谢物激活双功能的HPr激酶/HPr-Ser46-P磷酸酶(HprK/P)的激酶活性,HPr激酶催化HPr蛋白46位的丝氨酸磷酸化,形成HPr-Ser46-P,作为碳分解代谢物调节蛋白(CcpA)的辅助因子,与CcpA蛋白形成复合物,在细胞的CCR效应中,起主要的调控作用。
重组枯草芽孢杆菌24A1是一株CcpA缺陷的核黄素生产菌株。由于CcpA的缺陷,破坏了细胞的碳分解代谢物调控机制,降低了葡萄糖所引起的CCR效应的强度,使核黄素发酵能够在高葡萄糖浓度下进行。但是,高浓度葡萄糖所引起的胞内碳分解代谢物水平的升高,仍然能够激活HPr激酶,使HPr蛋白46位丝氨酸被磷酸化。高浓度葡萄糖的存在使HPr激酶的活性持续存在,必然导致大部分HPr蛋白以HPr-Ser46-P的形式存在。非磷酸化的HPr的缺乏,可能会在一定程度上影响PTS系统的正常功能,降低细胞对葡萄糖的吸收速率。
本文采用插入失活方法敲除了枯草芽孢杆菌24A1的hprK基因,构建了CcpA、HprK/P双缺陷的菌株B. subtilisZHc/pMX45。初步摇瓶发酵显示,B. subtilis ZHc/pMX45可以耐受12%的葡萄糖,与24A1/pMX45相比,在10%葡萄糖浓度下,核黄素积累量提高了19.2%,达到4.374mg/mL。
In bacillus subtilis the HPr protein participate either in the glucose transportation,or in the carbon catabolite repression effect.the phosphoenol pyruvate:sugar phosphotransferase system (PEP-PTS) is the main glucose transportation system in B.subtilis.The HPr protein is phosphorylated at His-15 forming HPr-His15-P transferring phosphate group from HPr to EIIa.While HprK/P phosphorylate HPr at Ser46 forming HPr-Ser46-P as Co-effector of CcpA.
The Knockout of CcpA gene increases the amount of FBP in vivo. And FBP could activate HPr kinase.So when CcpA is deleted, most part of the HPr will be phosphorylated at Ser-46. HPr-Ser46-P cannot participate in the transportation of glucose.Absorpton of glucose is blocked.
In this study, obstacle of glucose absorption is relieved by disruption of hprk gene.The obtained B.subtilisZHc/pMX45 can tolerate glucose at the concentration of 12%.When the glucose concentration is 10%, production of riboflavin reaches the peak value of 4.374mg/ml, 19.2% higher than that of B.subtilis24A1/pMX45 at the glucose concentration of 8%.
重组枯草芽孢杆菌24A1是一株CcpA缺陷的核黄素生产菌株。由于CcpA的缺陷,破坏了细胞的碳分解代谢物调控机制,降低了葡萄糖所引起的CCR效应的强度,使核黄素发酵能够在高葡萄糖浓度下进行。但是,高浓度葡萄糖所引起的胞内碳分解代谢物水平的升高,仍然能够激活HPr激酶,使HPr蛋白46位丝氨酸被磷酸化。高浓度葡萄糖的存在使HPr激酶的活性持续存在,必然导致大部分HPr蛋白以HPr-Ser46-P的形式存在。非磷酸化的HPr的缺乏,可能会在一定程度上影响PTS系统的正常功能,降低细胞对葡萄糖的吸收速率。
本文采用插入失活方法敲除了枯草芽孢杆菌24A1的hprK基因,构建了CcpA、HprK/P双缺陷的菌株B. subtilisZHc/pMX45。初步摇瓶发酵显示,B. subtilis ZHc/pMX45可以耐受12%的葡萄糖,与24A1/pMX45相比,在10%葡萄糖浓度下,核黄素积累量提高了19.2%,达到4.374mg/mL。
In bacillus subtilis the HPr protein participate either in the glucose transportation,or in the carbon catabolite repression effect.the phosphoenol pyruvate:sugar phosphotransferase system (PEP-PTS) is the main glucose transportation system in B.subtilis.The HPr protein is phosphorylated at His-15 forming HPr-His15-P transferring phosphate group from HPr to EIIa.While HprK/P phosphorylate HPr at Ser46 forming HPr-Ser46-P as Co-effector of CcpA.
The Knockout of CcpA gene increases the amount of FBP in vivo. And FBP could activate HPr kinase.So when CcpA is deleted, most part of the HPr will be phosphorylated at Ser-46. HPr-Ser46-P cannot participate in the transportation of glucose.Absorpton of glucose is blocked.
In this study, obstacle of glucose absorption is relieved by disruption of hprk gene.The obtained B.subtilisZHc/pMX45 can tolerate glucose at the concentration of 12%.When the glucose concentration is 10%, production of riboflavin reaches the peak value of 4.374mg/ml, 19.2% higher than that of B.subtilis24A1/pMX45 at the glucose concentration of 8%.
引文
1 J.Stülke,W Hillen. Regulation of Carbon Catabolism in Bacillus Species. Annu Rev microbiol,2000, 54:849-80.
2 Jorg Stulke and Wolfgang Hillen.Carbon catabolite repression in bacteria.Current Opinion in Microbiology,1999,2:195-201
3 Matthew S. Moreno, B. L. Schneider, R. R. Maile, et al. Catabolite repression mediated by the CcpA protein in Bacillus subtilis: novel modes of regulation revealed by whole-genome analyses. Molecular Microbiol.,2001,39(5):1366-1381
4 Reinhold Brückner , Fritz Titgemeyer .Carbon catabolite repression in bacteria: choice of the carbon source and autoregulatory limitation of sugar utilization FEMS Microbiology Letters,2002, 209, 141-148.
5 Ludwig H, Rebhan N, Blencke, HM, Merzbacher, Control of the glycolytic gapA operon by the catabolite control protein A in Bacillus subtilis: a novel mechanism of CcpA-mediated regulation. Mol Microbiol 2002 Jul 45:543-53.
6 Kim H, Roux A, Sonenshein AL. Direct and indirect roles of CcpA in regulation of Bacillus subtilis .Krebs cycle genes,Mol Microbiol ,2002, Jul ,45:179-90.
7 Deutscher. J, E. Küster, U. Bergstedt, et al. Protein kinase-dependent HPr/CcpA interaction links glycolytic activity to carbon catabolite repression in gram-positive bacteria. Molecular Microbiol,1995,15:1049-1053
8 Gagnon, G., C. Vadeboncoeur, L. Gauthier, and M. Frenette. Regulation of ptsH and ptsI gene expression in Streptococcus salivarius ATCC 25975. Mol. Microbiol.1995
9 Grundy, F. J., D. A. Waters, S. H. Allen, Regulation of the Bacillus subtilis acetate kinase gene by CcpA. J. Bacteriol, 1993
10 Kim, J. H., M. I. Voskuil, and G. H. Chambliss. NADP, corepressor for the Bacillus subtilis catabolite control protein CcpA. Proc. Natl. Acad. Sci. USA 1998,95:9590-9595
11 Isabelle Martin-Verstraete, Josef Deutscher, and Anne Galinier.Phosphorylation of HPr and Crh by HprK, Early Steps in the Catabolite Repression Signalling Pathway for the Bacillus subtilis Levanase Operon. Journal of Bacteriology, May 1999, Vol. 181, No. 9,p. 2966-2969
12 Alexandra Kraus, wolfgang Hillen. Analysis of CcpA mutations defective in carbon catabolite repression Bacillus magaterium, FEMS Microbiology Letter ,1997,153221-226.
13 J. Deutscher,U. Kuster, C. A.Alpert,and W. Hengstenberg. Bacterial phosphoenolpyruvate-dependent phosphotransferase system: P-ser-HPr and its possible regulatory function. Biochemistry ,1984,23:4455-4460.
14 Faires N., S. Tobisch, S. Bachem, Martin-Verstraete. The catabolite control protein CcpA controls ammonium assimilation in Bacillus subtilis. J. Mol. Microbiol. Biotechnol, 1999.1:141~148.
15 Jones B. E., V. Dossonet, E. Küster, et al. Binding of the catabolite repressor protein CcpA to its DNA target is regulated by phosphorylation of its corepressor HPr. J. Biol. Chem,1997,272:26530-26535
16 Law J., G. Buist, A. Haandrikman, et al . A system to generate chromosomal mutations in Lactococcus lactis which allows fast analysis of targeted genes. J. Bacteriol,1995, 177:7011-7018
17 Miwa Y., A. Nakata, A. Ogiwara, et al. Evaluation and characterization of catabolite-responsive elements (cre) of Bacillus subtilis. Nucleic Acids Res,1995, 28:1206-1210
18 Galinier, A., Deutscher, J. & Martin-Verstraete.Phosphorylation of either Crh or HPr mediates binding of CcpA to the Bacillus subtilis xyn cre and catabolite repression of the xyn operon. J Mol Biol ,1999,286: 307-314.
19 Alexandra Kraus, wolfgang Hillen. Analysis of CcpA mutations defective in carbon catabolite repression Bacillus magaterium, FEMS Microbiology Letter ,1997,153,221-226.
20 S. Chauvaux, CcpA and HPr (ser-P) : mediators of catabolite repression in Bacillus subtilis, Forum in Mcrobiology, 14th , 518-522.
21 Sylvie Nessler, Sonia Fieulaine, Sandrine Poncet, et al.HPr Kinase/Phosphorylase, the Sensor Enzyme of Catabolite Repression in Gram-Positive Bacteria: Structural Aspects of the Enzyme and the Complex with Its Protein Substrate. Journal of Bacteriology, 2003, Vol. 185, No. 14,p. 4003-4010
22 Fujita Y., Y. Miwa, A. Galinier, and J. Deutscher. Specific recognition of the Bacillus subtilis gnt cis-acting catabolite-responsive element by a protein complex formed between CcpA and seryl-phosphorylated HPr. Mol. Microbiol. 1995,17:953-960
23 Mijakovic, I., S. Poncet, A. Galinier, et al. Pyrophosphate-producing protein dephosphorylation by HPr kinase/phosphorylase: a relic of early life? Proc. Natl. Acad. Sci. USA ,2002,99:13442-13447.
24 Galinier, A., J. Haiech, M.-C. Kilhoffer, The Bacillus subtilis crh gene encodes a HPr-like protein involved in catabolite repression. Proc. Natl. Acad. Sci. USA , 1997. 94: 8439- 8444.
25 Deutscher, J., and M. H. Saier, Jr. ATP-dependent protein kinase-catalyzed phosphorylation of a seryl residue in HPr, a phosphate carrier protein of the phosphotransferase system in Streptococcus pyogenes. Proc. Natl. Acad. Sci. USA 1983,80:6790-6794
26 Vicente Mondero, Sandrine Poncet, Ivan Mijakovic, Sconia Fieulaine, Valerie Dossonnet, Isabelle Martin-Verstraete, Sylvie Nessler, Josef Deutscher. Mutations lowering the phosphatase acticity of HPr kinase/phosphatase switch off carbon metabolism. The EMBO Journal ,2001,Vol.20 No. 15 pp.3928-3937
27 J.萨姆布鲁克,D .W. 拉赛尔,分子克隆实验指南(第三版),科学出版社
28 Hueck C.J. , Hillen W. Catabolite repression in Bacillus subtilis: a global regulatory mechanism for the Gram-positive bacteria? Mol Microbiol 1995,15: 395-401.
29 Kravanja M., Engelmann R., Dossonnet V., et al. The hprK gene of Enterococcus faecalis encodes a novel bifunctional enzyme: the HPr kinase/phosphatase. Mol Microbiol, 1999,31: 59-66.
30 Saier, M.H., Jr, Chauvaux, S., Deutscher, J., Reizer, J. & Ye, J.-J. Protein phosphorylation and the regulation of carbon metabolism: comparisons in Gram-negative versus Gram-positive bacteria. Trends Biochem Sci ,1995,20: 267-271
31 韩聪, 张惟材, 游松等, 大肠杆菌 ptsG 基因敲除及其缺陷株生长特性研究, 生物工程学报, 20(1),16-20
2 Jorg Stulke and Wolfgang Hillen.Carbon catabolite repression in bacteria.Current Opinion in Microbiology,1999,2:195-201
3 Matthew S. Moreno, B. L. Schneider, R. R. Maile, et al. Catabolite repression mediated by the CcpA protein in Bacillus subtilis: novel modes of regulation revealed by whole-genome analyses. Molecular Microbiol.,2001,39(5):1366-1381
4 Reinhold Brückner , Fritz Titgemeyer .Carbon catabolite repression in bacteria: choice of the carbon source and autoregulatory limitation of sugar utilization FEMS Microbiology Letters,2002, 209, 141-148.
5 Ludwig H, Rebhan N, Blencke, HM, Merzbacher, Control of the glycolytic gapA operon by the catabolite control protein A in Bacillus subtilis: a novel mechanism of CcpA-mediated regulation. Mol Microbiol 2002 Jul 45:543-53.
6 Kim H, Roux A, Sonenshein AL. Direct and indirect roles of CcpA in regulation of Bacillus subtilis .Krebs cycle genes,Mol Microbiol ,2002, Jul ,45:179-90.
7 Deutscher. J, E. Küster, U. Bergstedt, et al. Protein kinase-dependent HPr/CcpA interaction links glycolytic activity to carbon catabolite repression in gram-positive bacteria. Molecular Microbiol,1995,15:1049-1053
8 Gagnon, G., C. Vadeboncoeur, L. Gauthier, and M. Frenette. Regulation of ptsH and ptsI gene expression in Streptococcus salivarius ATCC 25975. Mol. Microbiol.1995
9 Grundy, F. J., D. A. Waters, S. H. Allen, Regulation of the Bacillus subtilis acetate kinase gene by CcpA. J. Bacteriol, 1993
10 Kim, J. H., M. I. Voskuil, and G. H. Chambliss. NADP, corepressor for the Bacillus subtilis catabolite control protein CcpA. Proc. Natl. Acad. Sci. USA 1998,95:9590-9595
11 Isabelle Martin-Verstraete, Josef Deutscher, and Anne Galinier.Phosphorylation of HPr and Crh by HprK, Early Steps in the Catabolite Repression Signalling Pathway for the Bacillus subtilis Levanase Operon. Journal of Bacteriology, May 1999, Vol. 181, No. 9,p. 2966-2969
12 Alexandra Kraus, wolfgang Hillen. Analysis of CcpA mutations defective in carbon catabolite repression Bacillus magaterium, FEMS Microbiology Letter ,1997,153221-226.
13 J. Deutscher,U. Kuster, C. A.Alpert,and W. Hengstenberg. Bacterial phosphoenolpyruvate-dependent phosphotransferase system: P-ser-HPr and its possible regulatory function. Biochemistry ,1984,23:4455-4460.
14 Faires N., S. Tobisch, S. Bachem, Martin-Verstraete. The catabolite control protein CcpA controls ammonium assimilation in Bacillus subtilis. J. Mol. Microbiol. Biotechnol, 1999.1:141~148.
15 Jones B. E., V. Dossonet, E. Küster, et al. Binding of the catabolite repressor protein CcpA to its DNA target is regulated by phosphorylation of its corepressor HPr. J. Biol. Chem,1997,272:26530-26535
16 Law J., G. Buist, A. Haandrikman, et al . A system to generate chromosomal mutations in Lactococcus lactis which allows fast analysis of targeted genes. J. Bacteriol,1995, 177:7011-7018
17 Miwa Y., A. Nakata, A. Ogiwara, et al. Evaluation and characterization of catabolite-responsive elements (cre) of Bacillus subtilis. Nucleic Acids Res,1995, 28:1206-1210
18 Galinier, A., Deutscher, J. & Martin-Verstraete.Phosphorylation of either Crh or HPr mediates binding of CcpA to the Bacillus subtilis xyn cre and catabolite repression of the xyn operon. J Mol Biol ,1999,286: 307-314.
19 Alexandra Kraus, wolfgang Hillen. Analysis of CcpA mutations defective in carbon catabolite repression Bacillus magaterium, FEMS Microbiology Letter ,1997,153,221-226.
20 S. Chauvaux, CcpA and HPr (ser-P) : mediators of catabolite repression in Bacillus subtilis, Forum in Mcrobiology, 14th , 518-522.
21 Sylvie Nessler, Sonia Fieulaine, Sandrine Poncet, et al.HPr Kinase/Phosphorylase, the Sensor Enzyme of Catabolite Repression in Gram-Positive Bacteria: Structural Aspects of the Enzyme and the Complex with Its Protein Substrate. Journal of Bacteriology, 2003, Vol. 185, No. 14,p. 4003-4010
22 Fujita Y., Y. Miwa, A. Galinier, and J. Deutscher. Specific recognition of the Bacillus subtilis gnt cis-acting catabolite-responsive element by a protein complex formed between CcpA and seryl-phosphorylated HPr. Mol. Microbiol. 1995,17:953-960
23 Mijakovic, I., S. Poncet, A. Galinier, et al. Pyrophosphate-producing protein dephosphorylation by HPr kinase/phosphorylase: a relic of early life? Proc. Natl. Acad. Sci. USA ,2002,99:13442-13447.
24 Galinier, A., J. Haiech, M.-C. Kilhoffer, The Bacillus subtilis crh gene encodes a HPr-like protein involved in catabolite repression. Proc. Natl. Acad. Sci. USA , 1997. 94: 8439- 8444.
25 Deutscher, J., and M. H. Saier, Jr. ATP-dependent protein kinase-catalyzed phosphorylation of a seryl residue in HPr, a phosphate carrier protein of the phosphotransferase system in Streptococcus pyogenes. Proc. Natl. Acad. Sci. USA 1983,80:6790-6794
26 Vicente Mondero, Sandrine Poncet, Ivan Mijakovic, Sconia Fieulaine, Valerie Dossonnet, Isabelle Martin-Verstraete, Sylvie Nessler, Josef Deutscher. Mutations lowering the phosphatase acticity of HPr kinase/phosphatase switch off carbon metabolism. The EMBO Journal ,2001,Vol.20 No. 15 pp.3928-3937
27 J.萨姆布鲁克,D .W. 拉赛尔,分子克隆实验指南(第三版),科学出版社
28 Hueck C.J. , Hillen W. Catabolite repression in Bacillus subtilis: a global regulatory mechanism for the Gram-positive bacteria? Mol Microbiol 1995,15: 395-401.
29 Kravanja M., Engelmann R., Dossonnet V., et al. The hprK gene of Enterococcus faecalis encodes a novel bifunctional enzyme: the HPr kinase/phosphatase. Mol Microbiol, 1999,31: 59-66.
30 Saier, M.H., Jr, Chauvaux, S., Deutscher, J., Reizer, J. & Ye, J.-J. Protein phosphorylation and the regulation of carbon metabolism: comparisons in Gram-negative versus Gram-positive bacteria. Trends Biochem Sci ,1995,20: 267-271
31 韩聪, 张惟材, 游松等, 大肠杆菌 ptsG 基因敲除及其缺陷株生长特性研究, 生物工程学报, 20(1),16-20