一种新的基于Red重组及I-SecI体内切割的大肠杆菌基因组无痕删减方法
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摘要
目前常用的基因修饰方法是在Red同源重组介导下,电转线性PCR片段替换染色体上指定序列。因PCR过程错误掺入,该方法常常会在同源序列部位产生一些突变。为了避免此类突变,我们建立了一种新的无痕删除方法。首先将含有抗性标记(两侧带有I-Sec I识别位点)的线性DNA电转到Red重组感受态细胞内,用抗性基因替换基因组上指定序列;然后,将携带融合同源臂(两侧带有I-Sec I位点)的供体质粒导入上述细胞,诱导表达I-Sec I内切酶切割供体质粒释放同源片段,同时切除染色体上抗性基因产生双链断裂,通过分子间同源重组实现无痕删除。我们应用该方法连续删除了大肠杆菌DH1基因组上11个非必需区,使基因组减小10.59%。PCR测序证明所有删减区域同源臂未发生突变,基因组重测序证明指定区域被删除。删减菌的生长变化不大,但耐酸能力有所改变,并对番茄红素合成有不同影响。
Red-based recombineering has been widely used in Escherichia coli genome modification through electroporating PCR fragments into electrocompetent cells to replace target sequences. Some mutations in the PCR fragments may be brought into the homologous regions near the target. To solve this problem in markeless gene deletion we developed a novel method characterized with two-step recombination and a donor plasmid. First, generated by PCR a linear DNA cassette which comprises a I-Sec I site-containing marker gene and homologous arms was electroporated into cells for marker-substitution deletion of the target sequence. Second, after a donor plasmid carrying the I-Sec I site-containing fusion homologous arm was chemically transformed into the marker-containing cells, the fusion arms and the marker was simultaneously cleaved by I-Sec I endonuclease and the marker-free deletion was stimulated by double-strand break-mediated intermolecular recombination. Eleven nonessential regions in E. coli DH1 genome were sequentially deleted by our method, resulting in a 10.59% reduced genome size. These precise deletions were also verified by PCR sequencing and genome resequencing. Though no change in the growth rate on the minimal medium, we found the genome-reduced strains have some alteration in the acid resistance and for the synthesis of lycopene.
Red-based recombineering has been widely used in Escherichia coli genome modification through electroporating PCR fragments into electrocompetent cells to replace target sequences. Some mutations in the PCR fragments may be brought into the homologous regions near the target. To solve this problem in markeless gene deletion we developed a novel method characterized with two-step recombination and a donor plasmid. First, generated by PCR a linear DNA cassette which comprises a I-Sec I site-containing marker gene and homologous arms was electroporated into cells for marker-substitution deletion of the target sequence. Second, after a donor plasmid carrying the I-Sec I site-containing fusion homologous arm was chemically transformed into the marker-containing cells, the fusion arms and the marker was simultaneously cleaved by I-Sec I endonuclease and the marker-free deletion was stimulated by double-strand break-mediated intermolecular recombination. Eleven nonessential regions in E. coli DH1 genome were sequentially deleted by our method, resulting in a 10.59% reduced genome size. These precise deletions were also verified by PCR sequencing and genome resequencing. Though no change in the growth rate on the minimal medium, we found the genome-reduced strains have some alteration in the acid resistance and for the synthesis of lycopene.
引文
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[19]Yu BJ,Kang KH,Lee JH,et al.Rapid and efficient construction of markerless deletions in the Escherichia coli genome.Nucleic Acids Res,2008,36(14):e84.
[20]Masuda N,Church GM.Regulatory network of acid resistance genes in Escherichia coli.Mol Microbiol,2003,48(3):699-712.
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[24]Pey J,Valgepea K,Rubio A,et al.Integrating gene and protein expression data with genome-scale metabolic networks to infer functional pathways.BMC Syst Biol,2013,7:134.
[25]Leprince A,van Passel MW,dos Santos VAM.Streamlining genomes:toward the generation of simplified and stabilized microbial systems.Curr Opin Biotechnol,2012,23(5):651–658.
[2]Chen XZ,Zhou L,Tian KM,et al.Metabolic engineering of Escherichia coli:a sustainable industrial platform for bio-based chemical production.Biotechnol Adv,2013,31(8):1200-1223.
[3]Blattner FR,PlunkettⅢG,Bloch CA,et al.The complete genome sequence of Escherichia coli K-12.Science,1997,277(5331):1453–1462.
[4]Riley M,Abe T,Arnaud MB,et al.Escherichia coli K-12:a cooperatively developed annotation snapshot-2005.Nucleic Acids Res,2006,34(1):1–9.
[5]Mizoguchi H,Mori H,Fujio T.Escherichia coli minimum genome factory.Biotechnol Appl Biochem,2007,46(3):157–167.
[6]Kolisnychenko V,Plunkett III G,Herring CD,et al.Engineering a reduced Escherichia coli genome.Genome Res,2002,12(4):640-647.
[7]Pósfai G,Plunkett III G,Fehér T,et al.Emergent properties of reduced-genome Escherichia coli.Science,2006,312(5776):1044-1046.
[8]Mizoquchi H,Sawano Y,Kato JI,et al.Superpositioning of deletions promotes growth of Escherichia coli with a reduced genome.DNA Res,2008,15(5):277-284.
[9]Hirokawa Y,Kawano H,Tanaka-Masuda K,et al.Genetic manipulations restored the growth fitness of reduced-genome Escherichia coli.J Biosci Bioeng,2013,116(1):52-58.
[10]Yu J,Huang Y,Zhou CL,et al.Discovery of nonessential sequences in Escherichia coli DH1genome based on genomic comparison.Lett Biotechnol,2014,25(5):640-643(in Chinese).虞剑,黄勇,周长林,等.利用基因组比对方法寻找大肠杆菌DH1基因组中的非必需序列.生物技术通讯,2014,25(5):640-643.
[11]Chaveroche MK,Ghigo JM,d’Enfert C.A rapid method for efficient gene replacement in the filamentous fungus Aspergillus nidulans.Nucleic Acids Res,2000,28(22):E97.
[12]Jin Y,Watt RM,Danchin A,et al.Small noncoding RNA Gcv B is a novel regulator of acid resistance in Escherichia coli.BMC Genomics,2009,10:165.
[13]Yoon SH,Lee YM,Kim JE,et al.Enhanced lycopene production in Escherichia coli engineered to synthesize isopentenyl diphosphate and dimethylallyl diphosphate from mevalonate.Biotechnol Bioeng,2006,94(6):1025-1032.
[14]Sharan SK,Thomason LC,Kuznetsov SG,et al.Recombineering:a homologous recombination-based method of genetic engineering.Nat Protoc,2009,4(2):206-223.
[15]Kuhlman TE,Cox EC.Site-specific chromosomal integration of large synthetic constructs.Nucleic Acids Res,2010,38(6):e92.
[16]Datsenko KA,Wanner BL.One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products.Proc Natl Acad Sci USA,2000,97(12):6640-6645.
[17]Court DL,Sawitzke JA,Thomason LC.Genetic engineering using homologous recombination.Annu Rev Genet,2002,36:361-388.
[18]Hashimoto M,Ichimura T,Mizoguchi H,et al.Cell size and nucleoid organization of engineered Escherichia coli cells with a reduced genome.Mol Microbiol,2005,55(1):137-149.
[19]Yu BJ,Kang KH,Lee JH,et al.Rapid and efficient construction of markerless deletions in the Escherichia coli genome.Nucleic Acids Res,2008,36(14):e84.
[20]Masuda N,Church GM.Regulatory network of acid resistance genes in Escherichia coli.Mol Microbiol,2003,48(3):699-712.
[21]Hobbs EC,Astarita JL,Storz G.Small RNAs and small proteins involved in resistance to cell envelope stress and acid shock in Escherichia coli:analysis of a bar-coded mutant collection.J Bacteriol,2010,192(1):59-67.
[22]Kanjee U,Houry WA.Mechanisms of acid resistance in Escherichia coli.Annu Rev Microbiol,2013,67:65-81.
[23]Tang BC,Hao T,Yuan QQ,et al.Genome minimization method based on metabolic network analysis and its application to Escherichia coli.Chin J Biotech,2013,29(8):1173-1184(in Chinese).汤彬彩,郝彤,袁倩倩,等.一种基于代谢网络分析最小化基因组的方法及其在大肠杆菌中的应用.生物工程学报,2013,29(8):1173-1184.
[24]Pey J,Valgepea K,Rubio A,et al.Integrating gene and protein expression data with genome-scale metabolic networks to infer functional pathways.BMC Syst Biol,2013,7:134.
[25]Leprince A,van Passel MW,dos Santos VAM.Streamlining genomes:toward the generation of simplified and stabilized microbial systems.Curr Opin Biotechnol,2012,23(5):651–658.