Abstract: With the availability of the whole genome sequence of Escherichia coli or Corynebacterium glutamicum, strategies for directed DNA manipulation have developed rapidly. DNA manipulation plays an important role in understanding the function of genes and in constructing novel engineering bacteria according to requirement. DNA manipulation involves modifying the autologous genes and expressing the heterogenous genes. Two alternative approaches, using electroporation linear DNA or recombinant suicide plasmid, allow a wide variety of DNA manipulation. However, the over-expression of the desired gene is generally executed via plasmid-mediation. The current review summarizes the common strategies used for genetically modifying E. coli and C. glutamicum genomes, and discusses the technical problem of multi-layered DNA manipulation. Strategies for gene over-expression via integrating into genome are proposed. This review is intended to be an accessible introduction to DNA manipulation within the bacterial genome for novices and a source of the latest experimental information for experienced investigators.

遗传改造细菌基因组的策略：基因定点突变、基因失活和基因过表达

[1]Amador, E., Franciso, J., Castro, J.M., 2000. A Brevibacterium lactofermentum 16S rRNA gene used as target site for homologous recombination. FEMS Microbiol. Lett., 185(2):199-204.

[2]Adachi, Y., Fukuhara, C., 2012. TA strategy for rapid and efficient site-directed mutagenesis. Anal. Biochem., 431(1):66-68.

[3]Baba, T., Ara, T., Hasegawa, M., et al., 2006. Construction of Escherichia coli K-frame, single-gene knockout mutants: the Keio collection. Mol. Syst. Biol., 2:2006-2008.

[4]Berg, C.M., Berg, D.E., 1996. Transposable element tools for microbial genetics. In: Neidhardt, F.C., Curtiss III, R.C., Ingraham, J.L., et al. (Eds.), Escherichia coli and Salmonella. ASM Press, Washington, DC, USA, p.2588-2612.

[5]Barettino, D., Feigenbutz, M., Valcárcel, R., et al., 1994. Improved method for PCR-mediated site-directed mutagenesis. Nucleic Acids Res., 22(3):541-542.

[6]Boles, E., Miosga, T., 1995. A rapid and highly efficient method for PCR-based site-directed mutagenesis using only one new primer. Curr. Genet., 28(2):197-198.

[7]Brøns-Poulsen, J., Petersen, N., Horder, M., et al., 1998. An improved PCR based method for site directed mutagenesis using megaprimers. Mol. Cell. Probes, 12(6):345-348.

[8]Becker, J., Zelder, O., Häfner, S., et al., 2011. From zero to hero—design-based systems metabolic engineering of Corynebacterium glutamicum for L-lysine production. Metab. Eng., 13(2):159-168.

[9]Causey, T.B., Shanmugam, K.T., Yomano, L.P., et al., 2004. Engineering Escherichia coli for efficient conversion of glucose to pyruvate. PNAS, 101(8):2235-2240.

[10]Chapnik, N., Sherman, H., Frog, O., 2008. A one-tube site-directed mutagenesis method using PCR and primer extension. Anal. Biochem., 372(2):255-257.

[11]Chatellier, J., Mazza, A., Brousseau, R., et al., 1995. Codon-based combinatorial alanine scanning site-directed mutagenesis: design, implementation, and polymerase chain reaction screening. Anal. Biochem., 229(2):282-290.

[12]Chiu, J., March, P.E., Lee, R., et al., 2004. Site-directed, ligase-independent mutagenesis (SLIM): a single-tube methodology approaching 100% efficiency in 4 h. Nucleic Acids Res., 32(21):e174.

[13]Cornet, F., Mortier, I., Patte, J., et al., 1994. Plasmid pSC101 harbors a recombination site, psi, which is able to resolve plasmid multimers and to substitute for the analogous chromosomal Escherichia coli site dif. J. Bacteriol., 176(11):3188-3195.

[14]Correia, A., Martin, J.F., Castro, J.M., 1996. Targeted integration of foreign genes into repetitive sequences of the Brevibacterium lactofermentum chromosome. FEMS Microbiol. Lett., 142(2-3):259-264.

[15]Dai, Z.M., Zhu, X.J., Chen, Q., et al., 2007. PCR-suppression effect: kinetic analysis and application to representative or long molecule biased PCR-based amplification of complex samples. J. Biotechnol., 128(3):435-443.

[16]Datsenko, K.A., Wanner, B.L., 2000. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. PNAS, 97(12):6640-6645.

[17]Davis, D.P., Seqaloff, D.L., 2002. N-linked carbohydrates on G protein-coupled receptors: mapping sites of attachment and determining functional roles. Methods Enzymol., 343:137-156.

[18]Davis, M.D., Wonderling, R.S., Walker, S.C., et al., 1999. Analysis of the effects of charge cluster mutations in adeno-associated virus Rep68 protein in vitro. J. Virol., 73(3):2084-2093.

[19]Dean, D., 1981. A plasmid cloning vector for the direct selection of strains carrying recombinant plasmids. Gene, 15(1):99-102.

[20]Diatchenko, L., Lau, Y.F., Campbell, A.P., et al., 1996. Suppression subtractive hybridization: a method for generating differentially regulated or tissue-specific cDNA probes and libraries. PNAS, 93(12):6025-6030.

[21]Donnenberg, M.S., Kaper, J.B., 1991. Construction of an eae deletion mutant of enteropathogenic Escherichia coli by using a positive-selection suicide vector. Infect. Immun., 59(12):4310-4317.

[22]Evans, P.M., Liu, C., 2005. SiteFind: a software tool for introducing a restriction site as a marker for successful site-directed mutagenesis. BMC Mol. Biol., 6(1):22.

[23]Fushan, A.A., Drayna, D.T., 2009. MALS: an efficient strategy for multiple site-directed mutagenesis employing a combination of DNA amplification, ligation and suppression PCR. BMC Biotechnol., 9(1):83.

[24]Gay, P., Le Coq, D., Steinmetz, M., et al., 1983. Cloning structural gene sacB, which codes for exoenzyme levansucrase of Bacillus subtilis: expression of the gene in Escherichia coli. J. Bacteriol., 153(3):1424-1431.

[25]Georgi, T., Rittmann, D., Wendisch, V.F., 2005. Lysine and glutamate production by Corynebacterium glutamicum on glucose, fructose and sucrose: roles of malic enzyme and fructose-1,6-bisphosphatase. Metab. Eng., 7(4):291-301.

[26]Ho, S.N., Hunt, H.D., Horton, R.M., et al., 1989. Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene, 77(1):51-59.

[27]Hogrefe, H.H., Cline, J., Youngblood, G.L., et al., 2002. Creating randomized amino acid libraries with the QuikChange^® Multi Site-Directed Mutagenesis Kit. Biotechniques, 33(5):1158-1165.

[28]Holland, E.G., Acca, F.E., Belanger, K.M., et al., 2015. In vivo elimination of parental clones in general and site-directed mutagenesis. J. Immun. Methods, 417:67-75.

[29]Homilton, C.M., Aldea, M.M., Washburm, B.K., et al., 1989. New method for generating deletions and gene replacements in Escherichia coli. J. Bacteriol., 171(9):4617-4622.

[30]Horton, R.M., Cai, Z., Ho, S.N., et al., 1990. Gene splicing by overlap extension: tailor-made genes using the polymerase chain reaction. Biotechniques, 8(5):528-535.

[31]Hou, X.H., Chen, X.D., Zhang, Y., et al., 2012. L-Valine production with minimization of by-products’ synthesis in Corynebacterium glutamicum and Brevibacterium flavum. Amino Acids, 43(6):2301-2311.

[32]Hu, J., Li, Y., Zhang, H., et al., 2014. Construction of a novel expression system for use in Corynebacterium glutamicum. Plasmid, 75:18-26.

[33]Ikeda, M., Katsumata, R., 1998. A novel system with positive selection for the chromosomal integration of replicative plasmid DNA in Corynebacterium glutamicum. Microbiology, 144(Pt 7):1863-1868.

[34]Imai, Y., Matsushima, Y., Sugimura, T., et al., 1991. A simple and rapid method for generating a deletion by PCR. Nucleic Acids Res., 19(10):2785.

[35]Imaizumi, A., Takikawa, R., Chie, K., et al., 2005. Improved production of L-lysine by disruption of stationary phase-specific rmf gene in Escherichia coli. J. Biotechnol., 117(1):111-118.

[36]Inui, M., Suda, M., Okina, S., 2007. Transcriptional profiling of Corynebacterium glutamicum metabolism during organic acid production under oxygen deprivation conditions. Microbiology, 153(8):2491-2504.

[37]Johnston, C., Polard, P., Clavers, J.P., 2013. The DpnI/DpnII pneumococcal system, defense against foreign attack without compromising genetic exchange. Mobile Genet. Elem., 3(4):e25582.

[38]Jones, D.H., Winistorfer, S.C., 1991. Site-specific mutagenesis and DNA recombination by using PCR to generate recombinant circles in vitro or by recombination of linear PCR products in vivo. Methods, 2(1):2-10.

[39]Judson, N., Mekalanos, J.J., 2000. Transposon-based approaches to identify essential bacterial genes. Trends Microbiol., 8(11):521-526.

[40]Kalinowski, J., Bathe, B., Daniela, B., et al., 2003. The complete Corynebacterium glutamicum ATCC 13032 genome sequence and its impact on the production of L-aspartate-derived amino acids and vitamins. J. Biotechnol., 104(1-3):5-25.

[41]Karnik, A., Karnik, R., Grefen, C., 2013. SDM-Assist software to design site-directed mutagenesis primers introducing “silent” restriction sites. BMC Bioinformatics, 14(1):105-114.

[42]Kato, C., Ohimiya, R., Mizuno, T., 1998. A rapid method for disrupting gene in Escherichia coli genome. Biosci. Biotechnol. Biochem., 62(9):1826-1829.

[43]Ke, S.H., Madison, E.L., 1997. Rapid and efficient mutagenesis by single-tube “megaprimer” PCR method. Nucleic Acids Res., 25(16):3371-3372.

[44]Khare, V., Eckert, K.A., 2002. The proofreading 3'→5' exonuclease activity of DNA polymerases: a kinetic barrier to translesion DNA synthesis. Mutat. Res.-Fund. Mol. M., 510(1-2):45-54.

[45]Kim, N.S., 2015. Transposable elements and genomics. Genes Genom., 37(2):111-112.

[46]Kim, Y.G., Maas, S., 2000. Multiple site mutagenesis with high targeting efficiency in one cloning step. Biotechniques, 28:196-198.

[47]Kirsch, R.D., Joly, E., 1998. An improved PCR-mutagenesis strategy for two-site mutagenesis or sequence swapping between related genes. Nucleic Acids Res., 26(7):1848-1850.

[48]Lacks, S., Greenberg, B., 1977. Complementary specificity of restriction endonucleases of Diplococcus pneumoniae with respect to DNA methylation. J. Mol. Biol., 114(1):153-168.

[49]Li, J., Li, C.H., Xiao, W., et al., 2008. Site-directed mutagenesis by combination of homologous recombination and DpnI digestion of the plasmid template in Escherichia coli. Anal. Biochem., 373(2):389-391.

[50]Liang, X.Q., Pen, L.S., Li, K., et al., 2012. A method for multi-site-directed mutagenesis based on homologous recombination. Anal. Biochem., 427(1):99-101.

[51]Liew, K.S., Ho, W.S., Pang, S.L., et al., 2015. Development and characterization of microsatellite markers in sawih tree (Duabanga moluccana Blume) using ISSR-suppression PCR techniques. Physiol. Mol. Biol. Plants, 21(1):163-165.

[52]Ling, M.M., Robinson, B.H., 1997. Approaches to DNA mutagenesis: an overview. Anal. Biochem., 254(2):157-178.

[53]Liu, H., Naismith, J.H., 2008. An efficient one-step site-directed deletion, insertion, single and multiple-site plasmid mutagenesis protocol. BMC Biotechnol., 8(1):91.

[54]Lu, L., Patel, H., Bisser, J.J., 2002. Optimizing DpnI digestion conditions to detect replicated DNA. Biotechniques, 33(2):316-318.

[55]Luo, F.G., Du, X.L., Weng, T.T., et al., 2012. Efficient multi-site-directed mutagenesis directly from genomic template. J. Biosci., 37(Suppl. 1):965-969.

[56]Maloy, S.R., Nunn, W.D., 1981. Selection for loss of tetracycline resistance by Escherichia coli. J. Bacteriol., 145(2):1110-1112.

[57]Mandaci, S., 2011. Site-directed mutagenesis as the cornerstone of protein engineering: from basic biotechnology to industrial enzymes. Curr. Opin. Biotechnol., 22(Suppl. 1):S39.

[58]Martens, J.H., Barg, H., Warren, M., et al., 2002. Microbial production of vitamin B₁₂. Appl. Microbiol. Biotechnol., 58(3):278-285.

[59]Martin, A., Toselli, E., Rosier, M.F., et al., 1995. Rapid and high efficiency site-directed mutagenesis by improvement of the homologous recombination technique. Nucleic Acids Res., 23(9):1642-1643.

[60]Meetei, A.R., Rao, M.R.S., 1998. Generation of multiple site-specific mutations in a single polymerase chain reaction product. Anal. Biochem., 264(2):288-291.

[61]Mitchell, L.A., Cai, Y.Z., Taylor, M., et al., 2013. Multichange isothermal mutagenesis: a new strategy for multiple site-directed mutations in plasmid DNA. ACS Synth. Biol., 2(8):473-477.

[62]Montaldo, H.H., 2006. Genetic engineering applications in animal breeding. Electron. J. Biotechnol., 9(2):157-170.

[63]Muyrers, J.P.P., Zhang, Y.M., Stewart, A.F., 2001. Techniques: recombinogenic engineering—new options for cloning and manipulating DNA. Trends Biochem. Sci., 26(5):325-331.

[64]Nakashima, N., Miyazaki, K., 2014. Bacterial cellular engineering by genome editing and gene inactivation. Int. J. Mol. Sci., 15(2):2773-2793.

[65]Nossal, N.G., 1974. DNA synthesis on a double-stranded DNA template by the T4 bacteriophage DNA polymerase and the T4 gene 32 DNA unwinding protein. J. Biol. Chem., 249(17):5668-5676.

[66]Patel, D.H., Wi, S.G., Bae, H.J., 2009. Modification of overlap expression PCR: a mutagenic approach. Indian J. Biotechnol., 8(2):181-186.

[67]Peng, R.H., Xiong, A.S., Yao, Q.H., 2006. A direct and efficient PAGE-mediated overlap extension PCR method for gene multiple-site mutagenesis. Appl. Microbiol. Biotechnol., 73(1):234-240.

[68]Perlak, F.J., 1990. Single step large scale site-directed in vitro mutagenesis using multiple oligonucleotides. Nucleic Acids Res., 18(24):7457-7458.

[69]Philippe, N., Alcaraz, J.P., Coursange, E., et al., 2004. Improvement of pCVD442, a suicide plasmid for gene allele exchange in bacteria. Plasmid, 51(3):246-255.

[70]Picard, V., Ersdal-Badju, E., Lu, A., et al., 1994. A rapid and efficient one-tube PCR based mutagenesis technique using Pfu DNA polymerase. Nucleic Acids Res., 22(13):2587-2591.

[71]Poustka, A., Rackwitez, H.R., Frischauf, A.M., et al., 1984. Selective isolation of cosmid clones by homologous recombination in Escherichia coli. PNAS, 81(13):4129-4133.

[72]Qi, D., Scholthof, K.B.G., 2008. A one-step PCR-based method for rapid and efficient site-directed fragment deletion, insertion and substitution mutagenesis. J. Virol. Methods, 149(1):85-90.

[73]Reyrat, J.M., Pelicic, V., Gicquel, B., et al., 1998. Counterselectable markers: untapped tools for bacterial genetics and pathogenesis. Infect. Immun., 66(9):4011-4017.

[74]Saeedi, P., Moosaabadi, J.M., Sebtahmadi, S.S., et al., 2012. Site-directed mutagenesis in bacteriorhodopsin mutants and their characterization for bioelectrical and biotechnological equipment. Biotechnol. Lett., 34(3):455-462.

[75]Salerno, J.C., Jones, R.J., Erdogan, E., et al., 2005. A single-stage polymerase-based protocol for the introduction of deletions and insertions without subcloning. Mol. Biotechnol., 29(3):225-232.

[76]Sawitzke, J.A., Thomason, L.C., Bubunenko, M., et al., 2013. Recombineering: using drug cassettes to knock out genes in vivo. Methods Enzymol., 533:79-102.

[77]Schäfer, A., Tauch, A., Jäger, W., et al., 1994. Small mobilizable multi-purpose cloning vectors derived from the Escherichia coli plasmids pK18 and pK19: selection of defined deletions in the chromosome of Corynebacterium glutumicum. Gene, 145(1):69-73.

[78]Seraphin, B., Kandels-Lewis, S., 1996. An efficient PCR mutagenesis strategy without gel purification step that is amenable to automation. Nucleic Acids Res., 24(16):3276-3277.

[79]Seyfang, A., Jin, J.H., 2004. Multiple site-directed mutagenesis of more than 10 sites simultaneously and in a single round. Anal. Biochem., 324(2):285-291.

[80]Shankarappa, B., Vijayananda, K., 1992. SILMUT: a computer program for the identification of regions suitable for silent mutagenesis to introduce restriction enzyme recognition sequences. Biotechniques, 12(6):882-884.

[81]Siloto, R.M.P., Weselake, R.J., 2012. Site saturation mutagenesis: methods and applications in protein engineering. Biocatal. Agric. Biotechnol., 1(3):181-189.

[82]Siwek, W., Czapinska, H., Bochtler, M., et al., 2012. Crystal structure and mechanism of action of the N⁶-methyladenine-dependent type IIM restriction endonuclease R.DpnI. Nucleic Acids Res., 40(15):7563-7572.

[83]Stoynova, L., Solórzano, R., Collins, E.D., 2004. Generation of large deletion mutants from plasmid DNA. Biotechniques, 36:402-406.

[84]Sun, S.H., Huang, H., Qi, Y.C., et al., 2015. Complementary annealing mediated by exonuclease: a method for seamless cloning and conditioning site-directed mutagenesis. Biotechnol. Biotec. Eq., 29(1):105-110.

[85]Suzuki, N., Okai, N., Nonaka, H., et al., 2006. High-throughput transposon mutagenesis of Corynebacterium glutamicum and construction of a single-gene disruptant mutant library. Appl. Environ. Microbiol., 72(5):3750-3755.

[86]Tauch, A., Götker, S., Pühler, A., et al., 2002. The alanine racemase gene alr is an alternative to antibiotic resistance genes in cloning systems for industrial Corynebacterium glutamicum strains. J. Biotechnol., 99(1):79-91.

[87]Tian, J., Liu, Q., Dong, S., et al., 2010. A new method for multi-site-directed mutagenesis. Anal. Biochem., 406(1):83-85.

[88]Tilly, K., Elias, A.F., Bono, J.L., et al., 2000. DNA exchange and insertional inactivation in spirochetes. J. Mol. Microbiol. Biotechnol., 2(4):433-442.

[89]Tseng, W.C., Lin, J.W., Wei, T.Y., et al., 2008. A novel megaprimed and ligase-free, PCR-based, site-directed mutagenesis method. Anal. Biochem., 375(2):376-378.

[90]Tu, H.M., Sun, S.S.M., 1996. Generation of a combination of mutations by use of multiple mutagenic oligonucleotides. Biotechniques, 20(3):352-354.

[91]Turchin, A., Lawler, J.F., 1999. The primer generator: a program that facilitates the selection of oligonucleotides for site-directed mutagenesis. Biotechniques, 26(4):672-676.

[92]Urban, A., Nenkirchen, S., Jaeger, K.E., 1997. A rapid and efficient method for site-directed mutagenesis using one-step overlap extension PCR. Nucleic Acids Res., 25(11):2227-2228.

[93]Wan, H.S., Li, Y.W., Fan, Y., et al., 2012. A site-directed mutagenesis method particularly useful for creating otherwise difficult-to-make mutants and alanine scanning. Anal. Biochem., 420(2):163-170.

[94]Wäneskog, M., Bjerling, P., 2014. Multi-fragment site-directed mutagenic overlap extension polymerase chain reaction as a competitive alternative to the enzymatic assembly method. Anal. Biochem., 444:32-37.

[95]Wang, H.P., Zhou, N., Ding, F., et al., 2011. An efficient approach for site-directed mutagenesis using central overlapping primers. Anal. Biochem., 418(2):304-306.

[96]Wang, X.H., Pineau, C., Guschinskaya, N., et al., 2012. Cysteine scanning mutagenesis and disulfide mapping analysis of arrangement of GspC and GspD protomers within the Type 2 secretion system. J. Biol. Chem., 287(23):19082-19093.

[97]Weiner, M.P., Costa, G.L., Schoettlin, W., et al., 1994. Site-directed mutagenesis of double-stranded DNA by the polymerase chain reaction. Gene, 151(1-2):119-123.

[98]Wu, D.G., Guo, X.W., Lu, J., et al., 2013. A rapid and efficient one-step site-directed deletion, insertion, and substitution mutagenesis protocol. Anal. Biochem., 434(2):254-258.

[99]Xu, D.Q., Tan, Y.Z., Huan, X.J., et al., 2010. Construction of a novel shuttle vector for use in Brevibacterium flavum, an industrial amino acid producer. J. Microbiol. Methods, 80(1):86-92.

[100]Xu, J.Z., Zhang, J.L., Guo, Y.F., et al., 2013. Improvement of cell growth and production of L-lysine by genetically modified Corynebacterium. glutamicum during growth on molasses. J. Ind. Microbiol. Biotechnol., 40(12):1423-1432.

[101]Xu, J.Z., Han, M., Zhang, J.Z., et al., 2014a. Improvement of L-lysine production combines with minimization of by-products synthesis in Corynebacterium glutamicum. J. Chem. Technol. Biotechnol., 89(12):1924-1933.

[102]Xu, J.Z., Han, M., Zhang, J.Z., et al., 2014b. Metabolic engineering Corynebacterium glutamicum for the L-lysine production by increasing the flux into L-lysine biosynthetic pathway. Amino Acids, 46(9):2165-2175.

[103]Xu, J.Z., Xia, X.H., Zhang, J.Z., et al., 2014b. A method for gene amplification and simultaneous deletion in Corynebacterium glutamicum genome without any genetic markers. Plasmid, 72:9-17.

[104]Xu, J.Z., Zhang, J.L., Guo, Y.F., et al., 2015. Genetically modifying aspartate aminotransferase and aspartate ammonia-lyase affects metabolite accumulation in L-lysine producing strain derived from Corynebacterium glutamicum ATCC13032. J. Mol. Catal. B Enzym., 113:82-89.

[105]Xu, M.J., Zhang, R.Z., Liu, X.Y., et al., 2013. Improving the acidic stability of a β-mannanase from Bacillus subtilis by site-directed mutagenesis. Process Biochem., 48(8):1166-1173.

[106]Yu, D.G., Ellis, H.M., 2000. An efficient recombination system for chromosome engineering in Escherichia coli. PNAS, 97(11):5978-5983.

[107]Zhang, X.L., Jantama, K., Moore, J.C., et al., 2007. Production of L-alanine by metabolically engineered Escherichia coli. Appl. Microbiol. Biotechnol., 77(2):355-366.

[108]Zhang, Y.M., Buchholz, F., Muyrers, J.P.P., et al., 1998. A new logic for DNA engineering using recombination in Escherichia coli. Nat. Genet., 20(20):123-128.

[109]Zheng, L., Baumann, U., Reymond, J.L., 2004. An efficient one-step site-directed and site-saturation mutagenesis protocol. Nucleic Acids Res., 32(14):e115.

[110]Zhou, S., Causey, T.B., Hasona, A., et al., 2003. Production of optically pure D-lactic acid in mineral salts medium by metabolically engineered Escherichia coli W3110. Appl. Environ. Microbiol., 69(1):399-407.