2016 ; Vol.26-4: 725~729
|Author||Sang-Hyun Lee, Hyun Ju Kim, Yong-An Shin, Kyoung Heon Kim, Sang Jun Lee|
|Place of duty||Department of Biotechnology, Graduate School, Korea University, Seoul 02841, Republic of Korea,R&D Center, GS Caltex Corporation, Daejeon 34122, Republic of Korea|
|Title||Single Crossover-Mediated Markerless Genome Engineering in Clostridium acetobutylicum|
J. Microbiol. Biotechnol.2016 ;
|Abstract||A novel genome-engineering tool in Clostridium acetobutylicum was developed based on singlecrossover homologous recombination. A small-sized non-replicable plasmid, pHKO1, was
designed for efficient integration into the C. acetobutylicum genome. The integrated pHKO1
plasmid backbone, which included an antibiotic resistance gene, can be excised in vivo by Flp
recombinase, leaving a single flippase recognition target sequence in the middle of the
targeted gene. Since the pSHL-FLP plasmid, the carrier of the Flp recombinase gene, employed
the segregationally unstable pAMβ1 replicon, the plasmid was rapidly cured from the mutant
C. acetobutylicum. Consequently, our method makes it easier to engineer C. acetobutylicum.|
|Key_word||Clostridium acetobutylicum, single crossover, gene inactivation, markerless|
Al-Hinai MA, Fast AG, Papoutsakis ET. 2012. Novel system for efficient isolation of Clostridium double-crossover allelic exchange mutants enabling markerless chromosomal gene deletions and DNA integration. Appl. Environ. Microbiol. 78:8112-8121.
Awad MM, Bryant AE, Stevens DL, Rood JI. 1995. Virulence studies on chromosomal alpha-toxin and theta-toxin mutants constructed by allelic exchange provide genetic evidence for the essential role of alpha-toxin in Clostridium perfringensmediated gas gangrene. Mol. Microbiol. 15: 191-202.
Bi C, Jones SW, Hess DR, Tracy BP, Papoutsakis ET. 2011. SpoIIE is necessary for asymmetric division, sporulation, and expression of σF, σE, and σG but does not control solvent production in Clostridium acetobutylicum ATCC 824. J. Bacteriol. 193: 5130-5137.
Cartman ST, Kelly ML, Heeg D, Heap JT, Minton NP. 2012. Precise manipulation of the Clostridium difficile chromosome reveals a lack of association between the tcdC genotype and toxin production. Appl. Environ. Microbiol. 78: 4683-4690.
Gr e en E M, B oynton Z L, H arr s iL M, R udolph F B, Papoutsakis ET, Bennett GN. 1996. Genetic manipulation of acid formation pathways by gene inactivation in Clostridium acetobutylicum ATCC 824. Microbiology 142: 2079-2086
Hanahan D. 1983. Studies on transformation of Escherichia coli with plasmids. J. Mol. Biol. 166: 557-580.
Heap JT, Pennington OJ, Cartman ST, Carter GP, Minton NP. 2007. The ClosTron: a universal gene knock-out system for the genus Clostridium. J. Microbiol. Methods 70: 452-464.
Jones SW, Tracy BP, Gaida SM, Papoutsakis ET. 2011. Inactivation of σ F in Clostridium acetobutylicum ATCC 824 blocks sporulation prior to asymmetric division and abolishes σ E and σ G protein expression but does not block solvent formation. J. Bacteriol. 193: 2429-2440.
Kennedy CL, Krejany EO, Young LF, O'Connor JR, Awad MM, Boyd RL, et al. 2005. The alpha-toxin of Clostridium septicum is essential for virulence. Mol. Microbiol. 57: 1357-1366.
Kovach ME, Elzer PH, Hill DS, Robertson GT, Farris MA, Roop RM 2nd, Peterson KM. 1995. Four new derivatives of the broad-host-range cloning vector pBBR1MCS, carrying different antibiotic-resistance cassettes. Gene 166: 175-176.
Lee SH, Eom M-H, Kim S, Kwon MA, Kim J, Shin YA, Kim KH. 2015. Ex situ product recovery and strain engineering of Clostridium acetobutylicum f or enhanced p roduction o f butanol. Process Biochem. 50: 1683-1691.
Lee SH, Kwon MA, Choi S, Kim S, Kim J, Shin YA, Kim KH. 2015. A new shuttle plasmid that stably replicates in Clostridium acetobutylicum. J. Microbiol. Biotechnol. 25: 1702-1708.
Lesiak JM, Liebl W, Ehrenreich A. 2014. Development of an in vivo methylation system for the solventogen Clostridium saccharobutylicum NCP 262 and analysis of two endonuclease mutants. J. Biotechnol. 188: 97-99.
Mermelstein LD, Papoutsakis ET. 1993. In vivo methylation in Escherichia coli by the Bacillus subtilis phage phi 3T I methyltransferase to protect plasmids from restriction upon transformation of Clostridium acetobutylicum ATCC 824. Appl. Environ. Microbiol. 59: 1077-1081.
Mermelstein LD, Welker NE, Bennett GN, Papoutsakis ET. 1992. Expression of cloned homologous fermentative genes in Clostridium acetobutylicum ATCC 824. Nat. Biotechnol. 10:190-195.
O'Connor JR, Lyras D, Farrow KA, Adams V, Powell DR, Hinds J, et al. 2006. Construction and analysis of chromosomal Clostridium difficile mutants. Mol. Microbiol. 61: 1335-1351.
Ohse M, Takahashi K, Kadowaki Y, Kusaoke H. 1995. Effects of plasmid DNA sizes and several other factors on transformation of Bacillus subtilis ISW1214 with plasmid DNA by electroporation. Biosci. Biotechnol. Biochem. 59: 1433-1437.
Tracy BP, Jones SW, Papoutsakis ET. 2011. Inactivation of σ E and σG in Clostridium acetobutylicum illuminates their roles in clostridial-cell-form biogenesis, granulose synthesis, solventogenesis, and spore morphogenesis. J. Bacteriol. 193:1414-1426.
Zhang Y, Yu M, Yang ST. 2012. Effects of ptb knockout on butyric acid fermentation by Clostridium tyrobutyricum. Biotechnol. Prog. 28: 52-59.