谷氨酸棒状杆菌基因敲除系统的构建
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摘要
谷氨酸棒状杆菌(Corynebacterium glutamicum)是放线菌目棒状杆菌属高GC含量的革兰氏阳性细菌,在氨基酸工业生产中具有广泛应用。目前,代谢工程育种正在成为谷氨酸棒状杆菌氨基酸生产菌育种的主要手段,迫切需要开发各种表达载体和敲除载体。本研究主要是开发各种适用于谷氨酸棒状杆菌的敲除系统。主要研究结果如下:
(1)针对果聚糖蔗糖酶编码基因sacB,构建了含有不同启动子的sacB表达质粒,并测定比较了它们在谷氨酸棒状杆菌中的果聚糖蔗糖酶活性。实验证明,枯草芽孢杆菌sacB基因自身的启动子不能在谷氨酸棒状杆菌中有效表达sacB;PlacM启动子能够有效表达sacB基因,赋予谷氨酸棒状杆菌蔗糖敏感性。通过测定果聚糖蔗糖酶活性,发现在谷氨酸棒状杆菌中,PlacM启动子效率比sacB基因野生型启动子效率提高18倍左右。
(2)利用基于sacB的整合型载体pDXW-3成功敲除了C. glutamicum ATCC 13032的aceE基因。比较发现,pDXW-3载体比现在常用的pK18mobsacB/pK19mobsacB载体更加适合谷氨酸棒状杆菌染色体基因敲除。
(3)构建了基于同源重组-特异位点重组的谷氨酸棒状杆菌基因敲除系统。该系统包括一系列载体,如pDTW109、pDTW201和pDTW202等。pDTW109质粒含有大肠杆菌复制起点oriE、温敏的棒状杆菌复制子rep (TS)、PtacM启动子启动下的氯霉素抗性基因cat和重组酶编码基因cre。pDTW109可以在Escherichia coli-C. glutamicum中穿梭复制、表达Cre重组酶和通过提高温度培养来去除。pDTW201和pDTW202分别含有两侧连接loxP、loxPLE/loxPRE重组位点的kan盒,可以作为模板扩增kan盒片段。
(4)利用基于同源重组-特异位点重组的敲除系统敲除了C. glutamicum ATCC 14067染色体上的aceE基因,构建了菌株YTW-101。YTW-101胞内丙酮酸积累,可以作为构建以丙酮酸作为底物的氨基酸生产菌的出发菌株。进一步敲除了YTW-101染色体上的ilvA基因,构建了菌株YTW-102。YTW-102胞内不仅丙酮酸积累而且苏氨酸至2-酮丁酸的合成被阻断,因此可以作为构建L-缬氨酸或L-亮氨酸生产菌的出发菌株。
Corynebacterium glutamicum belongs to actinomycetales, is a high G+C content, gram-positive bacterium. It has been widely used for production of different amino acids. In recent years, metabolic engineering has been used to construct amino acid producer. Therefore, it is urgent to develop expression vectors and gene-deletion vector suitable for C. glutamicum. This study was focused on the construction of novel gene-deletion vectors of C. glutamicum. The main results are listed below:
(1) The expression of sacB gene coding levansucrase under different promoter was measured in C. glutamicum. It showed the native promoter of sacB failed to efficiently promote its expression in C. glutamicum; PlacM effectively promote its expression in C. glutamicum. The activity of levansucrase expressed in C.glutamicum showed PlacM is 18 times stronger than the native promoter of sacB in C. glutamicum.
(2) Using pDXW-3 as gene-deletion vectors, aceE gene of C. glutamicum ATCC 13032 was deleted successfully. Compared with the currently available pK18mobsacB/pK19mobsacB vectors, pDXW-3 is more suitable for gene deletion and allelic exchange by homologous recombination in C. glutamicum ATCC 13032.
(3) Based on the recombination/site-specific recombination system, a second novel gene deletion system in C. glutamicum was constructed. This system contains several vectors, such as pDTW109, pDTW201 and pDTW202. The vector pDTW109 harbors oriE for replication in Escherichia coli, rep (TS) for temperature-sensitive replication in C. glutamicum, cat under tacM promoter for chloromycetin resistance, cre for expressing recombinase. pDTW109 in C. glutamicum could be lost by culturing at high temperature. pDTW201 harbors a kan cassette flanking with two loxP sites, while pDTW202 harbors a kan cassette flanking with loxPLE/loxPRE.
(4) Using the recombination/site-specific recombination system, the aceE gene in C. glutamicum ATCC 14067 was deleted, resulting YTW-101. YTW-101 could accumulate pyruvate, and therefore be used as a candidate for constructing amino acid producer. The ilvA gene in YTW-101 was further deleted, resulting YTW-102. YTW-102 could be a good candidate for constructing L-valine and L-leucine producer.
(1)针对果聚糖蔗糖酶编码基因sacB,构建了含有不同启动子的sacB表达质粒,并测定比较了它们在谷氨酸棒状杆菌中的果聚糖蔗糖酶活性。实验证明,枯草芽孢杆菌sacB基因自身的启动子不能在谷氨酸棒状杆菌中有效表达sacB;PlacM启动子能够有效表达sacB基因,赋予谷氨酸棒状杆菌蔗糖敏感性。通过测定果聚糖蔗糖酶活性,发现在谷氨酸棒状杆菌中,PlacM启动子效率比sacB基因野生型启动子效率提高18倍左右。
(2)利用基于sacB的整合型载体pDXW-3成功敲除了C. glutamicum ATCC 13032的aceE基因。比较发现,pDXW-3载体比现在常用的pK18mobsacB/pK19mobsacB载体更加适合谷氨酸棒状杆菌染色体基因敲除。
(3)构建了基于同源重组-特异位点重组的谷氨酸棒状杆菌基因敲除系统。该系统包括一系列载体,如pDTW109、pDTW201和pDTW202等。pDTW109质粒含有大肠杆菌复制起点oriE、温敏的棒状杆菌复制子rep (TS)、PtacM启动子启动下的氯霉素抗性基因cat和重组酶编码基因cre。pDTW109可以在Escherichia coli-C. glutamicum中穿梭复制、表达Cre重组酶和通过提高温度培养来去除。pDTW201和pDTW202分别含有两侧连接loxP、loxPLE/loxPRE重组位点的kan盒,可以作为模板扩增kan盒片段。
(4)利用基于同源重组-特异位点重组的敲除系统敲除了C. glutamicum ATCC 14067染色体上的aceE基因,构建了菌株YTW-101。YTW-101胞内丙酮酸积累,可以作为构建以丙酮酸作为底物的氨基酸生产菌的出发菌株。进一步敲除了YTW-101染色体上的ilvA基因,构建了菌株YTW-102。YTW-102胞内不仅丙酮酸积累而且苏氨酸至2-酮丁酸的合成被阻断,因此可以作为构建L-缬氨酸或L-亮氨酸生产菌的出发菌株。
Corynebacterium glutamicum belongs to actinomycetales, is a high G+C content, gram-positive bacterium. It has been widely used for production of different amino acids. In recent years, metabolic engineering has been used to construct amino acid producer. Therefore, it is urgent to develop expression vectors and gene-deletion vector suitable for C. glutamicum. This study was focused on the construction of novel gene-deletion vectors of C. glutamicum. The main results are listed below:
(1) The expression of sacB gene coding levansucrase under different promoter was measured in C. glutamicum. It showed the native promoter of sacB failed to efficiently promote its expression in C. glutamicum; PlacM effectively promote its expression in C. glutamicum. The activity of levansucrase expressed in C.glutamicum showed PlacM is 18 times stronger than the native promoter of sacB in C. glutamicum.
(2) Using pDXW-3 as gene-deletion vectors, aceE gene of C. glutamicum ATCC 13032 was deleted successfully. Compared with the currently available pK18mobsacB/pK19mobsacB vectors, pDXW-3 is more suitable for gene deletion and allelic exchange by homologous recombination in C. glutamicum ATCC 13032.
(3) Based on the recombination/site-specific recombination system, a second novel gene deletion system in C. glutamicum was constructed. This system contains several vectors, such as pDTW109, pDTW201 and pDTW202. The vector pDTW109 harbors oriE for replication in Escherichia coli, rep (TS) for temperature-sensitive replication in C. glutamicum, cat under tacM promoter for chloromycetin resistance, cre for expressing recombinase. pDTW109 in C. glutamicum could be lost by culturing at high temperature. pDTW201 harbors a kan cassette flanking with two loxP sites, while pDTW202 harbors a kan cassette flanking with loxPLE/loxPRE.
(4) Using the recombination/site-specific recombination system, the aceE gene in C. glutamicum ATCC 14067 was deleted, resulting YTW-101. YTW-101 could accumulate pyruvate, and therefore be used as a candidate for constructing amino acid producer. The ilvA gene in YTW-101 was further deleted, resulting YTW-102. YTW-102 could be a good candidate for constructing L-valine and L-leucine producer.
引文
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2 Stackebrandt E, Rainey F A, Ward-Rainey N L. Proposal for a new hierarchic classification system, actinobacteria classis nov. Int J Syst Evol Micr, 1997, Apr: 479-491
3 Kalinowski J, Bathe B, Bartels D, et al. The complete Corynebacterium glutamicum ATCC 13032 genome sequence and its impact on the production of L-aspartate-derived amino acids and vitamins. J Biotechnol, 2003, 104(1-3): 5-25
4 Eggeling L, Bott M, 2005. Handbook of Corynebacterium glutamicum. Taylor & Francis, Boca Raton
5 Santamaria R, Gil J A, Mesas J M, et al. Characterization of an endogenous plasmid and development of cloning vectors and a transformation system in Brevibacterium lactofermentum. J Gen Microbiol, 1984, 130(9): 2237-2246
6 Ozaki A, Katsumata R, Oka T, et al. Functional expression of the genes of Escherichia coli in gram-positive Corynebacterium glutamicum. Mol Gen Genet, 1984, 196(1): 175-178
7 Tauch A, Gotker S, Puhler A, et al. The alanine racemase gene alr is an alternative to antibiotic resistance genes in cloning systems for industrial Corynebacterium glutamicum strains. J Biotechnol, 2002, 99(1): 79-91
8 Zupancic T J, Kittle J D, Baker B D, et al. Isolation of promoters from Brevibacterium flavum strain MJ233C and comparison of their gene expression levels in B. flavum and Escherichia coli. FEMS Microbiol Lett, 1995, 131(2): 121-126
9 Cadenas R F, Gil J A, Martin J F. Expression of Streptomyces genes encoding extracellular enzymes in Brevibacterium lactofermentum: secretion proceeds by removal of the same leader peptide as in Streptomyces lividans. Appl Microbiol Biotechnol, 1992, 38(3): 362-369
10 Knoppova M, Phensaijai M, Vesely M, et al. Plasmid vectors for testing in vivo promoter activities in Corynebacterium glutamicum and Rhodococcus erythropolis. Curr Microbiol, 2007, 55(3): 234-239
11 Burkovski A, 2008. Corynebacteria: Genomics and Molecular Biology. Caister Academic Press, Norfolk
12 Nesvera J, Patek M. Tools for genetic manipulations in Corynebacterium glutamicum and their applications. Appl Microbiol Biot, 2011, 90(5): 1641-1654
13 Tsuchiya M, Morinaga Y. Genetic Control Systems of Escherichia Coli Can Confer Inducible Expression of Cloned Genes in Coryneform Bacteria. Nat Biotech,1988, 6(4): 428-430
14 Schafer A, Tauch A, Jager W, et al. Small mobilizable multi-purpose cloning vectors derived from the Escherichia coli plasmids pK18 and pK19: selection of defined deletions in the chromosome of Corynebacterium glutamicum. Gene,1994, 145(1): 69-73
15 Derbise A, Lesic B, Dacheux D, et al. A rapid and simple method for inactivating chromosomal genes in Yersinia. FEMS Immunol Med Microbiol,2003, 38(2): 113-116
16 Rossi M S, Paquelin A, Ghigo J M, et al. Haemophore-mediated signal transduction across the bacterial cell envelope in Serratia marcescens: the inducer and the transported substrate are different molecules. Mol Microbiol,2003, 48(6): 1467-1480
17 Chaveroche M K, Ghigo J M, d'Enfert C. A rapid method for efficient gene replacement in the filamentous fungus Aspergillus nidulans. Nucleic Acids Res,2000, 28(22): E97
18 Husseiny M I, Hensel M. Rapid method for the construction of Salmonella enterica Serovar Typhimurium vaccine carrier strains. Infect Immun,2005, 73(3): 1598-1605
19 Lesic B, Rahme L G. Use of the lambda Red recombinase system to rapidly generate mutants in Pseudomonas aeruginosa. BMC Molecular Biology, 2008, 9: 20.
20 Yamamoto S, Izumiya H, Morita M, et al. Application of lambda Red recombination system to Vibrio cholerae genetics: simple methods for inactivation and modification of chromosomal genes. Gene, 2009, 438(1-2): 57-64
21 Ellis H M, Yu D G, Ditizio T, et al. High efficiency mutagenesis, repair, and engineering of chromosomal DNA using single-stranded oligonucleotides. Proc Natl Acad Sci U S A, 2001, 98(12): 6742-6746
22 Van Kessel J C, Marinelli L J, Hatfull G F. Innovation recombineering mycobacteria and their phages. Nat Rev Microbiol, 2008, 6(11): 851-857
23 Austin S, Ziese M, Sternberg N. A novel role for site-specific recombination in maintenance of bacterial replicons. Cell, 1981, 25(3): 729-736
24 Hoess R H, Ziese M, Sternberg N. P1 site-specific recombination: nucleotide sequence of the recombining sites. Proc Natl Acad Sci U S A, 1982, 79(11): 3398-3402
25 Langer S J, Ghafoori A P, Byrd M, et al. A genetic screen identifies novel non-compatible loxP sites. Nucleic Acids Res, 2002, 30(14): 3067-3077
26 Sauer B, Henderson N. Site-specific DNA recombination in mammalian cells by the Cre recombinase of bacteriophage P1. Proc Natl Acad Sci U S A, 1988, 85(14): 5166-5170
27 Albert H, Dale E C, Lee E, et al. Site-specific integration of DNA into wild-type and mutant lox sites placed in the plant genome. The Plant Journal, 1995, 7(4): 649-659
28 Gay P, Le Coq D, Steinmetz M, et al. Positive selection procedure for entrapment of insertion sequence elements in gram-negative bacteria. J Bacteriol, 1985, 164(2): 918-921
29 Jager W, Schafer A, Puhler A, et al. Expression of the Bacillus subtilis sacB gene leads to sucrose sensitivity in the gram-positive bacterium Corynebacterium glutamicum but not in Streptomyces lividans. J Bacteriol, 1992, 174(16): 5462-5465
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2 Stackebrandt E, Rainey F A, Ward-Rainey N L. Proposal for a new hierarchic classification system, actinobacteria classis nov. Int J Syst Evol Micr, 1997, Apr: 479-491
3 Kalinowski J, Bathe B, Bartels D, et al. The complete Corynebacterium glutamicum ATCC 13032 genome sequence and its impact on the production of L-aspartate-derived amino acids and vitamins. J Biotechnol, 2003, 104(1-3): 5-25
4 Eggeling L, Bott M, 2005. Handbook of Corynebacterium glutamicum. Taylor & Francis, Boca Raton
5 Santamaria R, Gil J A, Mesas J M, et al. Characterization of an endogenous plasmid and development of cloning vectors and a transformation system in Brevibacterium lactofermentum. J Gen Microbiol, 1984, 130(9): 2237-2246
6 Ozaki A, Katsumata R, Oka T, et al. Functional expression of the genes of Escherichia coli in gram-positive Corynebacterium glutamicum. Mol Gen Genet, 1984, 196(1): 175-178
7 Tauch A, Gotker S, Puhler A, et al. The alanine racemase gene alr is an alternative to antibiotic resistance genes in cloning systems for industrial Corynebacterium glutamicum strains. J Biotechnol, 2002, 99(1): 79-91
8 Zupancic T J, Kittle J D, Baker B D, et al. Isolation of promoters from Brevibacterium flavum strain MJ233C and comparison of their gene expression levels in B. flavum and Escherichia coli. FEMS Microbiol Lett, 1995, 131(2): 121-126
9 Cadenas R F, Gil J A, Martin J F. Expression of Streptomyces genes encoding extracellular enzymes in Brevibacterium lactofermentum: secretion proceeds by removal of the same leader peptide as in Streptomyces lividans. Appl Microbiol Biotechnol, 1992, 38(3): 362-369
10 Knoppova M, Phensaijai M, Vesely M, et al. Plasmid vectors for testing in vivo promoter activities in Corynebacterium glutamicum and Rhodococcus erythropolis. Curr Microbiol, 2007, 55(3): 234-239
11 Burkovski A, 2008. Corynebacteria: Genomics and Molecular Biology. Caister Academic Press, Norfolk
12 Nesvera J, Patek M. Tools for genetic manipulations in Corynebacterium glutamicum and their applications. Appl Microbiol Biot, 2011, 90(5): 1641-1654
13 Tsuchiya M, Morinaga Y. Genetic Control Systems of Escherichia Coli Can Confer Inducible Expression of Cloned Genes in Coryneform Bacteria. Nat Biotech,1988, 6(4): 428-430
14 Schafer A, Tauch A, Jager W, et al. Small mobilizable multi-purpose cloning vectors derived from the Escherichia coli plasmids pK18 and pK19: selection of defined deletions in the chromosome of Corynebacterium glutamicum. Gene,1994, 145(1): 69-73
15 Derbise A, Lesic B, Dacheux D, et al. A rapid and simple method for inactivating chromosomal genes in Yersinia. FEMS Immunol Med Microbiol,2003, 38(2): 113-116
16 Rossi M S, Paquelin A, Ghigo J M, et al. Haemophore-mediated signal transduction across the bacterial cell envelope in Serratia marcescens: the inducer and the transported substrate are different molecules. Mol Microbiol,2003, 48(6): 1467-1480
17 Chaveroche M K, Ghigo J M, d'Enfert C. A rapid method for efficient gene replacement in the filamentous fungus Aspergillus nidulans. Nucleic Acids Res,2000, 28(22): E97
18 Husseiny M I, Hensel M. Rapid method for the construction of Salmonella enterica Serovar Typhimurium vaccine carrier strains. Infect Immun,2005, 73(3): 1598-1605
19 Lesic B, Rahme L G. Use of the lambda Red recombinase system to rapidly generate mutants in Pseudomonas aeruginosa. BMC Molecular Biology, 2008, 9: 20.
20 Yamamoto S, Izumiya H, Morita M, et al. Application of lambda Red recombination system to Vibrio cholerae genetics: simple methods for inactivation and modification of chromosomal genes. Gene, 2009, 438(1-2): 57-64
21 Ellis H M, Yu D G, Ditizio T, et al. High efficiency mutagenesis, repair, and engineering of chromosomal DNA using single-stranded oligonucleotides. Proc Natl Acad Sci U S A, 2001, 98(12): 6742-6746
22 Van Kessel J C, Marinelli L J, Hatfull G F. Innovation recombineering mycobacteria and their phages. Nat Rev Microbiol, 2008, 6(11): 851-857
23 Austin S, Ziese M, Sternberg N. A novel role for site-specific recombination in maintenance of bacterial replicons. Cell, 1981, 25(3): 729-736
24 Hoess R H, Ziese M, Sternberg N. P1 site-specific recombination: nucleotide sequence of the recombining sites. Proc Natl Acad Sci U S A, 1982, 79(11): 3398-3402
25 Langer S J, Ghafoori A P, Byrd M, et al. A genetic screen identifies novel non-compatible loxP sites. Nucleic Acids Res, 2002, 30(14): 3067-3077
26 Sauer B, Henderson N. Site-specific DNA recombination in mammalian cells by the Cre recombinase of bacteriophage P1. Proc Natl Acad Sci U S A, 1988, 85(14): 5166-5170
27 Albert H, Dale E C, Lee E, et al. Site-specific integration of DNA into wild-type and mutant lox sites placed in the plant genome. The Plant Journal, 1995, 7(4): 649-659
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