几株基因工程链霉菌的构建
摘要
链霉菌是重要的抗生素生产菌,其代谢过程的深入研究可以从基因水平解释体内代谢物变化,以及对目标代谢产物的影响,从而为工业生产菌株的分子水平优化提供方向。
乙酸是链霉菌无氧代谢的末端产物,乙酸累积抑制菌体生长和外源蛋白的表达。乙酰-CoA是某些次级代谢产物的重要前体,在理论上阻断乙酰-CoA生成乙酸的途径有可能促进菌体生长,同时增加目标产物的产量。敲除糖酵解途径中的关键限速酶磷酸果糖激酶主要合成基因来使碳代谢流从糖酵解途径转移到磷酸戊糖途径,可研究两条代谢途径对葡萄糖代谢的影响,以及不同代谢途径的中间产物对次级代谢物质合成的作用,对初级代谢的进一步研究有指导意义。透明颤菌血红蛋白所具备的捕捉和释放氧气的能力使之在宿主细胞发酵时可以更好地满足宿主细胞氧气的需求。在链霉菌中表达透明颤菌血红蛋白,能够改善链霉菌对高氧的需要,促进体内代谢,在理论上可能提高发酵过程中目的产物的产量。
本文利用RED敲除原理分两步敲除链霉菌乙酸代谢途径关键酶pta-ackA基因和磷酸果糖激酶基因pfkA1。通过同源对比,扩增出关键酶基因。通过PCR、酶切、连接将其克隆到cosmid载体上,构建中间质粒。将质粒转化到大肠杆菌BW25113/pIJ790体内,然后将安普抗性标记片断目标基因置换,构建出敲除载体。通过原生质体转化方式,导入利迪链霉菌。并通过松弛培养,筛选得到链霉菌pta-ackA缺失菌株AS01和pfkA1缺失菌株AS02。
根据透明颤菌血红蛋白基因vgb碱基序列和链霉菌基因密码子偏好性设计PCR引物,对vgb基因的部分密码子加以突变。然后将vgb基因克隆到pIB139质粒强启动子的下游,构建出整合载体pIB139-vgb。通过原生质体转化的方式将整合载体转入链霉菌,通过抗性筛选获得链霉菌vgb表达菌株AS 03。
基因工程链霉菌的构建为研究基因功能,以及链霉菌体内代谢网络的变化奠定了很重要的基础。
Streptomyces is an important antibiotic producing bacteria, their metabolic processes of in-depth study can be explained from the genetic level in vivo metabolite changes and the impact on the target metabolite, so that industrial production strain optimization of the molecular orientation.
Acetic acid is the end product of anaerobic metabolism in Streptomyces. Accumulation of acetic acid inhibits the growth of mycelium and the expression of exogenous protein. Acetyl-CoA is an important secondary metabolites of some precursors, in theory block the production of acetic acid acetyl-CoA pathway could promote cell growth, while increasing the output of the target product. Knockout of the key rate-limiting enzyme phosphofructokinase gene pfkA1 of glycolytic pathway could promote carbon metabolic flux shift from the glycolytic pathway to the pentose phosphate pathway, thus we could research the two metabolic pathways of glucose metabolism, also different metabolic ways in the middle of the secondary metabolites of the synthetic product of the role of metabolic transformation of primary guide for further study. The fermentation process of Streptomyces lydicus needs high demand for oxygen. The ability of Vitreoscilla hemoglobin (VHb) to capture and release oxygen can better meet the needs of oxygen in the fermentation of host cells. Therefore, the expression of Vitreoscilla hemoglobin in Streptomyces can improve the needs of high oxygen and promote the body metabolism, and improve the production of purpose product
In this paper, two steps are taken to knock out the key gene pta-ackA and pfkA1, according to RED recombination principle. By homology comparison, the key gene was amplified. By PCR, enzyme digestion, the connection will be cloned into cosmid vector construct intermediate plasmid. The plasmid was transformed into E. coli BW25113/pIJ790 body, the resistance marker apr fragments, which were amplificated by PCR, were electrotransfered to E. coli BW25113/pIJ790 to disrupt the taget genes. Then constructed a knockout vector. The knockout vector introduced into Streptomyces by polyethylene glycol-mediated transformation of protoplasts. Through relaxation training, screened lack of Streptomyces pta-ackA deletion mutant strain AS01 and pfkA1 deletion mutant strain AS02.
According to Vitreoscilla hemoglobin gene vgb gene sequence, and codon preference of Streptomyces design PCR primers, the part of the vgb gene mutation in codon to be. Vgb gene fragment was then cloned into plasmid pIB139, the downstream of the strong promoter. Integration vector pIB139-vgb was constructed. And the integration vector was introduced into Streptomyces through protoplast transformation. Streptomyces AS03 with vgb expression was selected through antibiotic resistance.
Construction of genetic engineering Streptomyces laid a very important change in the basis to study gene function, and metabolic networks in Streptomyces.
乙酸是链霉菌无氧代谢的末端产物,乙酸累积抑制菌体生长和外源蛋白的表达。乙酰-CoA是某些次级代谢产物的重要前体,在理论上阻断乙酰-CoA生成乙酸的途径有可能促进菌体生长,同时增加目标产物的产量。敲除糖酵解途径中的关键限速酶磷酸果糖激酶主要合成基因来使碳代谢流从糖酵解途径转移到磷酸戊糖途径,可研究两条代谢途径对葡萄糖代谢的影响,以及不同代谢途径的中间产物对次级代谢物质合成的作用,对初级代谢的进一步研究有指导意义。透明颤菌血红蛋白所具备的捕捉和释放氧气的能力使之在宿主细胞发酵时可以更好地满足宿主细胞氧气的需求。在链霉菌中表达透明颤菌血红蛋白,能够改善链霉菌对高氧的需要,促进体内代谢,在理论上可能提高发酵过程中目的产物的产量。
本文利用RED敲除原理分两步敲除链霉菌乙酸代谢途径关键酶pta-ackA基因和磷酸果糖激酶基因pfkA1。通过同源对比,扩增出关键酶基因。通过PCR、酶切、连接将其克隆到cosmid载体上,构建中间质粒。将质粒转化到大肠杆菌BW25113/pIJ790体内,然后将安普抗性标记片断目标基因置换,构建出敲除载体。通过原生质体转化方式,导入利迪链霉菌。并通过松弛培养,筛选得到链霉菌pta-ackA缺失菌株AS01和pfkA1缺失菌株AS02。
根据透明颤菌血红蛋白基因vgb碱基序列和链霉菌基因密码子偏好性设计PCR引物,对vgb基因的部分密码子加以突变。然后将vgb基因克隆到pIB139质粒强启动子的下游,构建出整合载体pIB139-vgb。通过原生质体转化的方式将整合载体转入链霉菌,通过抗性筛选获得链霉菌vgb表达菌株AS 03。
基因工程链霉菌的构建为研究基因功能,以及链霉菌体内代谢网络的变化奠定了很重要的基础。
Streptomyces is an important antibiotic producing bacteria, their metabolic processes of in-depth study can be explained from the genetic level in vivo metabolite changes and the impact on the target metabolite, so that industrial production strain optimization of the molecular orientation.
Acetic acid is the end product of anaerobic metabolism in Streptomyces. Accumulation of acetic acid inhibits the growth of mycelium and the expression of exogenous protein. Acetyl-CoA is an important secondary metabolites of some precursors, in theory block the production of acetic acid acetyl-CoA pathway could promote cell growth, while increasing the output of the target product. Knockout of the key rate-limiting enzyme phosphofructokinase gene pfkA1 of glycolytic pathway could promote carbon metabolic flux shift from the glycolytic pathway to the pentose phosphate pathway, thus we could research the two metabolic pathways of glucose metabolism, also different metabolic ways in the middle of the secondary metabolites of the synthetic product of the role of metabolic transformation of primary guide for further study. The fermentation process of Streptomyces lydicus needs high demand for oxygen. The ability of Vitreoscilla hemoglobin (VHb) to capture and release oxygen can better meet the needs of oxygen in the fermentation of host cells. Therefore, the expression of Vitreoscilla hemoglobin in Streptomyces can improve the needs of high oxygen and promote the body metabolism, and improve the production of purpose product
In this paper, two steps are taken to knock out the key gene pta-ackA and pfkA1, according to RED recombination principle. By homology comparison, the key gene was amplified. By PCR, enzyme digestion, the connection will be cloned into cosmid vector construct intermediate plasmid. The plasmid was transformed into E. coli BW25113/pIJ790 body, the resistance marker apr fragments, which were amplificated by PCR, were electrotransfered to E. coli BW25113/pIJ790 to disrupt the taget genes. Then constructed a knockout vector. The knockout vector introduced into Streptomyces by polyethylene glycol-mediated transformation of protoplasts. Through relaxation training, screened lack of Streptomyces pta-ackA deletion mutant strain AS01 and pfkA1 deletion mutant strain AS02.
According to Vitreoscilla hemoglobin gene vgb gene sequence, and codon preference of Streptomyces design PCR primers, the part of the vgb gene mutation in codon to be. Vgb gene fragment was then cloned into plasmid pIB139, the downstream of the strong promoter. Integration vector pIB139-vgb was constructed. And the integration vector was introduced into Streptomyces through protoplast transformation. Streptomyces AS03 with vgb expression was selected through antibiotic resistance.
Construction of genetic engineering Streptomyces laid a very important change in the basis to study gene function, and metabolic networks in Streptomyces.
引文
[1]唐功利,陈海宝,代谢工程研究进展.有机化学, 2000. 20(5): p. 634-640.
[2] Borodina I, Siebring J, Zhang J, et al. Antibiotic overproduction in Streptomyces coelicolorA3(2) mediated by phosphofructokinase deletion. Journal of Biological Chemistry, 2008. 283(37): 25186-25199.
[3]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, 2004. 20(1): p. 16-20.
[4] Lee, S.Y.,大肠杆菌的高密度培养.国外医学:预防.诊断.治疗用生物制品分册, 1997. 20(2): p. 67-70.
[5] Patel SM, Stark BC, Hwang KW, et al. Cloning and expression of Vitreoscilla hemoglobin gene in Burkholderia species strain DNT for enhancement of 2,4-dinitrotoluene degradation .Biotechnology Progress, 2000, 16(1): 26-30.
[6]汪承刚,张菊红,谢妤等.甘蓝型油菜依赖焦磷酸的磷酸果糖激酶(PFK)基因片段克隆及hpRNAi载体构建.分子植物育种,2006.4(2):40-48.
[7]韩蓓,蔡亚君,胡晓敏等.球形芽胞杆菌C3-41磷酸果糖激酶的克隆、表达及基本生物活性.微生物学报,2008.5(48):602-607.
[8] Wakabayashi, S., H. Matsubara, and D.A. Webster. Primary sequence of a dimeric bacterial hemoglobin from vitreoscilla. Nature, 1986. 322(6078): p. 481-483.
[9]袁宁胡又佳朱春宝等,透明颤菌血红蛋白的dna改组研究.中国生物工程杂志, 2006. 26(11): p. 14-19.
[10] Bulow, L., et al. The metabolic effects of native and transgenic hemoglobins on plants. Trends in Biotechnology, 1999. 17(1): 21-24.
[11] Kallio, P.T. and J.E. Bailey. Intracellular expression of Vitreoscilla hemoglobin (VHb) enhances total protein secretion and improves the production of alpha-amylase and neutral protease in Bacillus subtilis. Biotechnology Progress, 1996. 12(1): 31-39.
[12] Dikshit KL, Dikshit RP, Webster DA. Study of Vitreoscilla globin (vgb) gene expression and promoter activity in E.coli through transcriptional fusion . Nucleic Acids Research, 1990, 18: 4149-4155.
[13] Khosla C, Bailey JE. The Vitreoscilla hemoglobin gene: molecular cloning, nucleotide sequence and genetic expression in Escherichia coli . Molecular and General Genetics, 1988, 214(1): 158-161.
[14] Volokhan, O, et al., An unexpected role for the putative 4'-phosphopantet- heinyl transferase-encoding gene nysF in the regulation of nystatin blosynthesis in Streptomyces noursei ATCC 11455. Fems Microbiology Letters, 2005. 249(1): 57-64.
[15] Farres J, Kallio PT. Improved cell growth in tobacco suspension cultures expressing Vitreoscilla hemoglobin . Biotechnology Progress, 2002, 18: 229-233.
[16] Feng L, Chen S, Sun M, et al. Expression of Vitreoscilla hemoglobin in Bacillusthuringiensis improve the cell density and insecticidal crystal proteins yield . Applied Microbiology and Biotechnology, 2007, 74: 390-397.
[17] Tsai PS, Hatzimanikatis V, Bailey JE. Effect of Vitreoscilla hemoglobin dosage on microaerobic Escherichia coli carbon and energy metabolism . Biotechnology and Bioengineering, 1996, 49: 139-150.
[18]黄培堂.分子克隆实验指南.北京:科学出版社, 2002.
[19]严光琳.从D2葡萄糖直接发酵生产维生素C前体22酮基262古龙酸.微生物学学报, 1991, 31(3): 198-205.
[20] Wahlbom CF, Eliasson A, Hahn-H¨agerdal B. Intracellular fluxes in a recombinant xylose-utilizing Saccharomyces cerevisiae cultivated anaerobically at different dilution rates and feed concentrations . Biotechnology and Bioengineering, 2001, 72: 289-296.
[21] Baltz RH, Genetic manipulation of antibiotic-producing Streptomyces. Trends Microbiol, 1998. 6(2): 76-83.
[22]许杨,涂追.丝状真菌基因敲除技术研究进展.食品与生物技术学报, 2007,26(1):120-126.
[23] Xiang L, Moore BS, Characterization of Benzoyl Coenzyme A Biosynthesis Genes in the Enterocin-Producing Bacterium“Streptomyces maritimus”. Journal of Bacteriology, 2003, 185(2): 399–404.
[24] Luzhetskyy A, Zhu L, Gibson M, et al., Generation of novel landomycins M and O through targeted gene disruption. Chenbiochem, 2005, 6(4): 675-678.
[25] Hopwood DA, Genetic contributions to understanding polyketide synthases. Che Rev, 1997, 97(7): 2465-2498.
[26]聂丽萍,张集慧,谭华荣,卷圈产色链霉菌尼可霉素生物合成基因—sanL的结构与功能研究.微生物学报,2001, 41(1): 59-64.
[27]陈芝,宋渊,文莹,李季伦,阿维链霉菌中aveD基因阻断对阿维菌素合成的影响.微生物学报,2001, 41(4): 440-446.
[28] Ostash B, Rix U, Rix LL, et al., Generation of new landomycins by combinatorial biosynthetic manipulation of the LndGT4 gene of the landomycin E cluster in S. globisporus. Chem Biol, 2004, 11(4): 547-555.
[29] Li W, Liu G, Tan H, Disruption of sabR affects Nikkomycin biosynthesis and morphogenesis in Streptomyces ansochromogenes. Biotechnology Letters, 2003, 25(18):1491-1497.
[30] Li W, Tan H, Structure and Function of sanV: A gene involved in Nikkomycin biosynthesis of Streptomyces ansochromogenes. Current Microbiology, 2003, 46(6): 403-407.
[31] Hartl DL, Conditional Targeted Deletions in Plasmodium falciparum. Senior Scholar Award in Global Infectious Disease, 2003 (http://64.71.129.47 /awrd. jsp?id=296).
[32] Bertolt G, Greg L et al. PCR-targeted Streptomyces gene replacement identifies a protein domain needed for biosynthesis of the sesquiterpene soil odor geosmin. PNAS 2003,100(4):1541-1546.
[33]谢丽萍,宫倩等.天蓝淡红链霉菌SIPI-1482中酮还原酶基因dnrU的阻断突变研究.生物技术通讯,2007,18(5):739-742.
[34]刘志勇,郭美锦等.龟裂链霉菌zwf2基因阻断提高土霉素生物合成.微生物学报,2008,48(1):21-25.
[35] Chen H, Olesen S G, Harrison P H M. Biosynthesis of streptolydigin: Origin of the oxygen atoms. Organic Letters, 2006. 8(23): 5329-5332.
[36] Li X B, Qiao B, Yuan Y J. Differential analysis of secondary metabolites by LC-MS following strain improvement of Streptomyces lydicus AS 4.2501. Biotechnology and Applied Biochemistry, 2006. 45: 107-118.
[37]辛艳丽,贾玉艳,王恒樑.利用Red系统快速改构含鼠β-酪蛋白基因的细菌人工染色体.中国生物工程杂志,2005.25(8):45-50.
[38] Gust B, Challis G L, Fowler K , et al. PCR-targeted Streptomyces gene replacement identifies a protein domain needed for biosynthesis of the sesquiterpene soil odor geosmin . Proceedings of the NationalAcademy of Sciences of the United States of America, 2003. 100(4): 1541-1546.
[39] Mellouli, L., et al., Efficient transformation procedure of a newly isolated Streptomyces sp TN58 strain producing antibacterial activities. Current Microbiology, 2004. 49(6): 400-406.
[40] Lotfi M, Ines K, Samiha S, et al. Efficient Transformation Procedure of a Newly Isolated Streptomyces sp.TN58 Strain Producing Antibacterial Activities. Current Microbiology, 2004, 49:400-406.
[41] MazyServais, C., D. Baczkowski, and J. Dusart, Electroporation of intact cells of Streptomyces parvulus and Streptomyces vinaceus. Fems Microbiology Letters, 1997. 151(2): 135-138.
[42] Nicholson JK, Lindon JC, Holmes E, Metabonomics: understanding the metabolic responses of living systems to pathophysiological stimuli via multivariate statistical analysis of biological NMR spectroscopic data. Xenobiotica, 1999, 29(11):1181-1189.
[43] Oliver F, Combining genomics, metabolome analysis, and biochemical modelling to understand metabolic networks. Compar Funct Genom. 2001 (2): 155-168.
[44] Taylor J, King RD, AltmannT, et al., Application of metabolomics to plant genotype discrimination using statistics and machine learning. Bioinformatics, 2002, 18(Suppl. 2): 241-248.
[45] Oliver F, Joachim K, et al. Metabolite profiling for plant functional genomics. Nature Biotechnology 2000 (18): 1157– 1161.
[2] Borodina I, Siebring J, Zhang J, et al. Antibiotic overproduction in Streptomyces coelicolorA3(2) mediated by phosphofructokinase deletion. Journal of Biological Chemistry, 2008. 283(37): 25186-25199.
[3]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, 2004. 20(1): p. 16-20.
[4] Lee, S.Y.,大肠杆菌的高密度培养.国外医学:预防.诊断.治疗用生物制品分册, 1997. 20(2): p. 67-70.
[5] Patel SM, Stark BC, Hwang KW, et al. Cloning and expression of Vitreoscilla hemoglobin gene in Burkholderia species strain DNT for enhancement of 2,4-dinitrotoluene degradation .Biotechnology Progress, 2000, 16(1): 26-30.
[6]汪承刚,张菊红,谢妤等.甘蓝型油菜依赖焦磷酸的磷酸果糖激酶(PFK)基因片段克隆及hpRNAi载体构建.分子植物育种,2006.4(2):40-48.
[7]韩蓓,蔡亚君,胡晓敏等.球形芽胞杆菌C3-41磷酸果糖激酶的克隆、表达及基本生物活性.微生物学报,2008.5(48):602-607.
[8] Wakabayashi, S., H. Matsubara, and D.A. Webster. Primary sequence of a dimeric bacterial hemoglobin from vitreoscilla. Nature, 1986. 322(6078): p. 481-483.
[9]袁宁胡又佳朱春宝等,透明颤菌血红蛋白的dna改组研究.中国生物工程杂志, 2006. 26(11): p. 14-19.
[10] Bulow, L., et al. The metabolic effects of native and transgenic hemoglobins on plants. Trends in Biotechnology, 1999. 17(1): 21-24.
[11] Kallio, P.T. and J.E. Bailey. Intracellular expression of Vitreoscilla hemoglobin (VHb) enhances total protein secretion and improves the production of alpha-amylase and neutral protease in Bacillus subtilis. Biotechnology Progress, 1996. 12(1): 31-39.
[12] Dikshit KL, Dikshit RP, Webster DA. Study of Vitreoscilla globin (vgb) gene expression and promoter activity in E.coli through transcriptional fusion . Nucleic Acids Research, 1990, 18: 4149-4155.
[13] Khosla C, Bailey JE. The Vitreoscilla hemoglobin gene: molecular cloning, nucleotide sequence and genetic expression in Escherichia coli . Molecular and General Genetics, 1988, 214(1): 158-161.
[14] Volokhan, O, et al., An unexpected role for the putative 4'-phosphopantet- heinyl transferase-encoding gene nysF in the regulation of nystatin blosynthesis in Streptomyces noursei ATCC 11455. Fems Microbiology Letters, 2005. 249(1): 57-64.
[15] Farres J, Kallio PT. Improved cell growth in tobacco suspension cultures expressing Vitreoscilla hemoglobin . Biotechnology Progress, 2002, 18: 229-233.
[16] Feng L, Chen S, Sun M, et al. Expression of Vitreoscilla hemoglobin in Bacillusthuringiensis improve the cell density and insecticidal crystal proteins yield . Applied Microbiology and Biotechnology, 2007, 74: 390-397.
[17] Tsai PS, Hatzimanikatis V, Bailey JE. Effect of Vitreoscilla hemoglobin dosage on microaerobic Escherichia coli carbon and energy metabolism . Biotechnology and Bioengineering, 1996, 49: 139-150.
[18]黄培堂.分子克隆实验指南.北京:科学出版社, 2002.
[19]严光琳.从D2葡萄糖直接发酵生产维生素C前体22酮基262古龙酸.微生物学学报, 1991, 31(3): 198-205.
[20] Wahlbom CF, Eliasson A, Hahn-H¨agerdal B. Intracellular fluxes in a recombinant xylose-utilizing Saccharomyces cerevisiae cultivated anaerobically at different dilution rates and feed concentrations . Biotechnology and Bioengineering, 2001, 72: 289-296.
[21] Baltz RH, Genetic manipulation of antibiotic-producing Streptomyces. Trends Microbiol, 1998. 6(2): 76-83.
[22]许杨,涂追.丝状真菌基因敲除技术研究进展.食品与生物技术学报, 2007,26(1):120-126.
[23] Xiang L, Moore BS, Characterization of Benzoyl Coenzyme A Biosynthesis Genes in the Enterocin-Producing Bacterium“Streptomyces maritimus”. Journal of Bacteriology, 2003, 185(2): 399–404.
[24] Luzhetskyy A, Zhu L, Gibson M, et al., Generation of novel landomycins M and O through targeted gene disruption. Chenbiochem, 2005, 6(4): 675-678.
[25] Hopwood DA, Genetic contributions to understanding polyketide synthases. Che Rev, 1997, 97(7): 2465-2498.
[26]聂丽萍,张集慧,谭华荣,卷圈产色链霉菌尼可霉素生物合成基因—sanL的结构与功能研究.微生物学报,2001, 41(1): 59-64.
[27]陈芝,宋渊,文莹,李季伦,阿维链霉菌中aveD基因阻断对阿维菌素合成的影响.微生物学报,2001, 41(4): 440-446.
[28] Ostash B, Rix U, Rix LL, et al., Generation of new landomycins by combinatorial biosynthetic manipulation of the LndGT4 gene of the landomycin E cluster in S. globisporus. Chem Biol, 2004, 11(4): 547-555.
[29] Li W, Liu G, Tan H, Disruption of sabR affects Nikkomycin biosynthesis and morphogenesis in Streptomyces ansochromogenes. Biotechnology Letters, 2003, 25(18):1491-1497.
[30] Li W, Tan H, Structure and Function of sanV: A gene involved in Nikkomycin biosynthesis of Streptomyces ansochromogenes. Current Microbiology, 2003, 46(6): 403-407.
[31] Hartl DL, Conditional Targeted Deletions in Plasmodium falciparum. Senior Scholar Award in Global Infectious Disease, 2003 (http://64.71.129.47 /awrd. jsp?id=296).
[32] Bertolt G, Greg L et al. PCR-targeted Streptomyces gene replacement identifies a protein domain needed for biosynthesis of the sesquiterpene soil odor geosmin. PNAS 2003,100(4):1541-1546.
[33]谢丽萍,宫倩等.天蓝淡红链霉菌SIPI-1482中酮还原酶基因dnrU的阻断突变研究.生物技术通讯,2007,18(5):739-742.
[34]刘志勇,郭美锦等.龟裂链霉菌zwf2基因阻断提高土霉素生物合成.微生物学报,2008,48(1):21-25.
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