枯草芽孢杆菌甘油激酶编码基因定点突变提升甘油利用水平的研究
Effect of site-specific mutagenesis of glycerol kinase coding gene on the glycerol utilization ofBacillus subtilis
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摘要:采用无痕等位基因置换方法,将枯草芽孢杆菌基因组上的甘油激酶编码基因 glpK第270位氨基酸残基M突变为I,构建了突变工程菌株枯草芽孢杆菌M270I,并分析了该突变菌株的生长特性.结果表明:对甘油激酶编码基因 glpK进行定点突变可有效提升枯草芽孢杆菌对甘油的利用水平,与出发菌株枯草芽孢杆菌168 Δ upp相比,突变菌株枯草芽孢杆菌M270I在M9甘油基本盐液体培养基中的比生长速率提升了11%,延滞期缩短了2~4 h,最大菌体生物量提升了16%.Abstract:By employing a site-specific mutagenesis strategy for glycerol kinase (encoding by glpK), the active-site residue Met270 was replaced with Ile in the Bacillus subtilisgenome. A mutant strain Bacillus subtilisM270I was constructed successfully by using the markerless gene replacement system. The results of growth analysis indicated that the mutant strain got a higher level of glycerol utilization. Compared with the control strain Bacillus subtilis168 Δ upp, the mutant strain exhibited higher specific growth rate (increased by 11%) and higher biomass yield (improved by 16%) with a shorten lag phase (shorten 2~4 h) in the M9 glycerol minimal medium.
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- [1]
CHOI W J.Glycerol-based biorefinery for fuels and chemicals[J].Recent Patents on Biotechnology,2008,2(3):173.
- [2]
ALMEIDA J R M,QUIRINO B F,FÁVARO L C L,et al.Biodiesel biorefinery:Opportunities and challenges for microbial production of fuels and chemicals from glycerol waste[J].Biotechno-logy for Biofuels,2012,5(1):48.
- [3]
GERVÁSIO P D S,MACK M,CONTIERO J.Glycerol:A promising and abundant carbon source for industrial microbiology[J].Biotechnology Advances,2009,27(1):30.
- [4]
CÉLINE F,EHRLICH S D,NOIROT P.A new mutation delivery system for genome-scale approaches inBacillus subtilis[J].Molecular Microbiology,2010,46(1):25.
- [5]
ANAGNOSTOPOULOS C A,SPIZIZEN J.Requirements for transformation inBacillus Subtilis[J].Journal of Bacteriology,1961,81(5):741.
- [6]
刘小红.甘油合成关键酶基因的克隆表达及其应用[D].汉中:陕西理工学院,2014.
- [7]
SOUSA M D,DANTAS I T,FELIX A K N,et al.Crude glycerol from biodiesel industry as substrate for biosurfactant production byBacillus subtilisATCC 6633[J].Brazilian Archives of Biology and Technology,2014,57(2):295.
- [8]
KARLA M,RAMÓN D A,GEORGINA H,et al.Coutilization of glucose and glycerol enhances the production of aromatic compounds in anEscherichia colistrain lacking the phosphoenolpyruvate:Carbohydrate phosphotransferase system[J].Microbial Cell Factories,2008,7(1):1.
- [9]
EMMANUELLE D,PASCALE S,SANDRINE P,et al.Antitermination by GlpP,catabolite repression via CcpA and inducer exclusion triggered by P-GlpK dephosphorylation controlBacillus subtilis glpFKexpression[J].Molecular Microbiology,2002,43(4):1039.
- [10]
HERRING C D,RAGHUNATHAN A,HONISCH C,et al.Comparative genome sequencing ofEscherichia coliallows observation of bacterial evolution on a laboratory timescale[J].Nature Genetics,2006,38(12):1406.
- [11]
胡海艳,甘祥武,黄秀敏,等.基于易错PCR的β-甘露聚糖酶体外分子定向进化研究[J].轻工学报,2020,35(4):8.
- [12]
付月灵,刘峙,张文蔚,等.植物病原细菌Ⅲ型效应子的双向作用[J].中国生物防治,2010(S1):95.
- [13]
SHI T,WANG G,WANG Z,et al.Establishment of a markerless mutation delivery system inBacillus subtilisstimulated by a double-strand break in the chromosome[J].PLoS ONE,2013,8(11):e81370.
- [14]
ARNOLD K,BORDOLI L,KOPP J,et al.The SWISS-MODEL workspace:A web-based environment for protein structure homology modelling[J].Bioinformatics,2006,22(2):195.
- [15]
ROBERT X,GOUET P.Deciphering key features in protein structures with the new ENDscript server[J].Nucleic Acids Research,2014,42(W1):W320.
- [16]
ORMÖ M,BYSTROM C E,REMINGTON S J.Crystal structure of a complex ofEscherichia coliglycerol kinase and an allosteric effector fructose 1,6-bisphosphate[J].Biochemistry,1998,37(47):16565.
- [1]
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