spo0A基因缺失对克劳氏芽孢杆菌发酵生产性能的影响
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摘要
通过基因敲除的方法构建spo0A基因缺失菌株,并评价spo0A基因缺失对克劳氏芽孢杆菌(Bacillus clausii)QL-1发酵生产性能的影响。经重叠延伸PCR和基因同源单交换技术,获得spo0A基因缺失突变株B. clausii QL-1△spo0A;通过系统比较spo0A基因敲除前后芽孢生成率、生物量及淀粉酶酶活的改变,发现B. clausii QL-1△spo0A菌株不产芽孢、生物量提高了24%,且B. clausii QL-1△spo0A发酵72 h淀粉酶酶活为1.58×105 U/g,淀粉酶活提高83.72%。说明克劳氏芽孢杆菌spo0A基因缺失对菌体生物量、芽孢生成率及代谢产物的生成等具有重要影响。
The spo0 A gene deletion strain was constructed by the gene knockout method, and the effect of spo0 A gene deletion on the fermentation performance of Bacillus clausii QL-1 was evaluated. Through overlap extension PCR and gene homologous single exchange technique, the spo0 A gene deletion mutant B. clausii QL-1△spo0 A was obtained. By systematically comparing the changes of the spore formation rate, biomass and amylase activity before and after spo0 A gene deletion, the results showed that strain B. clausii QL-1△spo0 A was asporulate, the biomass increased by24%, and the amylase activity of B. clausii QL-1△spo0 A was 1.58×105 U/g after fermentation for 72 h, which increased by 83.72%. It was found that B. clausii with spo0 A gene deletion had an important effect on the mycelia biomass, spore formation rate and metabolites generation.
The spo0 A gene deletion strain was constructed by the gene knockout method, and the effect of spo0 A gene deletion on the fermentation performance of Bacillus clausii QL-1 was evaluated. Through overlap extension PCR and gene homologous single exchange technique, the spo0 A gene deletion mutant B. clausii QL-1△spo0 A was obtained. By systematically comparing the changes of the spore formation rate, biomass and amylase activity before and after spo0 A gene deletion, the results showed that strain B. clausii QL-1△spo0 A was asporulate, the biomass increased by24%, and the amylase activity of B. clausii QL-1△spo0 A was 1.58×105 U/g after fermentation for 72 h, which increased by 83.72%. It was found that B. clausii with spo0 A gene deletion had an important effect on the mycelia biomass, spore formation rate and metabolites generation.
引文
[1] SELLA S R B R, VANDENBERGHE L P S. Life cycle and spore resistance of spore-forming Bacillus atrophaeus[J]. Microbiol Res, 2014, 169(2):931-939.
[2] Al-HINAI M A, JONES S W, PAPOUTSAKIS E T. The Clostridium sporulation programs:diversity and preservation of endospore differentiation[J]. Microbiol Mol Biol Rev Mmbr, 2015, 79(1):19-37.
[3] VEENING J W, HAMOEN L W, KUIPERS O P. Phosphatases modulate the bistable sporulation gene expression pattern in Bacillus subtilis[J].Mol Microbiol, 2010, 56(6):1481-1494.
[4] HIGGINS D, DWORKIN J. Recent progress in Bacillus subtilis sporulation[J]. FEMS Microbiol Rev, 2015, 36(1):131-148.
[5] ERRINGTON J. Regulation of endospore formation in Bacillus subtilis[J]. Nat Rev Microbiol, 2003, 1(2):117-129.
[6]刘燕,秦玉昌,潘宝海.枯草芽孢杆菌(Bacillus subtilis)在芽孢形成过程中的几个关键事件[J].生命科学,2005,17(4):360-363.
[7]孙静,陈建华.枯草芽孢杆菌形成芽孢时母细胞中基因表达的调控[J].生物技术,2007,17(3):79-83.
[8] CASTILLA V, MUNOZ D, VILLAR L, et al. Spo0A, the key transcriptional regulator for entrance into sporulation, is an inhibitor of DNA replication[J]. Embo J, 2006, 25(16):3890-3899.
[9] LEAUX J R, GROSSMAN A D. Isolation and characterization of kinC, a gene that encodes a senor kinase homologous to the sporulation senor kinase KinA and KinB in Bacillus subtilis[J]. J Bacteriol, 1995, 177(6):166-175.
[10] PETTIT L J, BROWNE H P. Functional genomics reveals that Clostridium difficile Spo0A coordinates sporulation, virulence and metabolism[J]. BMC G, 2014, 15(1):160-168.
[11] KATARINA M, ZUZANA C, NIELS B, et al. Morphogenic protein RodZ interacts with sporulation specific SpoIIE in Bacillus subtilis[J].Plos One, 2016, 11(7):159-167.
[12] GUO M S. Regulation of bacterial outer membrane homeostasis by the sigma-factor, sigma E[J]. Dissertations Theses-Gradworks, 2014, 10(2):147-153.
[13] AHMADPOUR F, YAKHCHALI B. Development of an asporogenic Bacillus cereus strain to improve keratinase production in exponential phase by switching sigma H on and sigma F off[J]. FEMS Microbiol Lett,2017, 12(2):259-268.
[14]余志强,杨明明,杨朝霞,等.同源重组法构建枯草杆菌spo A基因缺失突变株[J].武汉大学学报,2004,50(12):229-233.
[15]宋晓蕾,周芬芬,高琼,等. spo0A基因在艰难梭菌生长及芽孢形成中的作用[J].中国感染与化疗杂志,2017,17(12):33-36.
[16]魏娟,孙长.苏云金芽孢杆菌HD-73菌株基因敲除突变株的构建及特[J].微生物学报,2008,35(10):1581-1586.
[17] SANDOVAL N R, VENKATARAMANAN K P, GROTH T, et al. Wholegenome sequence of an evolved Clostridium pasteurianum strain reveals Spo0A deficiency responsible for increased butanol production and superior growth[J]. Biotechnol Biofuels, 2015, 8(1):227-244.
[2] Al-HINAI M A, JONES S W, PAPOUTSAKIS E T. The Clostridium sporulation programs:diversity and preservation of endospore differentiation[J]. Microbiol Mol Biol Rev Mmbr, 2015, 79(1):19-37.
[3] VEENING J W, HAMOEN L W, KUIPERS O P. Phosphatases modulate the bistable sporulation gene expression pattern in Bacillus subtilis[J].Mol Microbiol, 2010, 56(6):1481-1494.
[4] HIGGINS D, DWORKIN J. Recent progress in Bacillus subtilis sporulation[J]. FEMS Microbiol Rev, 2015, 36(1):131-148.
[5] ERRINGTON J. Regulation of endospore formation in Bacillus subtilis[J]. Nat Rev Microbiol, 2003, 1(2):117-129.
[6]刘燕,秦玉昌,潘宝海.枯草芽孢杆菌(Bacillus subtilis)在芽孢形成过程中的几个关键事件[J].生命科学,2005,17(4):360-363.
[7]孙静,陈建华.枯草芽孢杆菌形成芽孢时母细胞中基因表达的调控[J].生物技术,2007,17(3):79-83.
[8] CASTILLA V, MUNOZ D, VILLAR L, et al. Spo0A, the key transcriptional regulator for entrance into sporulation, is an inhibitor of DNA replication[J]. Embo J, 2006, 25(16):3890-3899.
[9] LEAUX J R, GROSSMAN A D. Isolation and characterization of kinC, a gene that encodes a senor kinase homologous to the sporulation senor kinase KinA and KinB in Bacillus subtilis[J]. J Bacteriol, 1995, 177(6):166-175.
[10] PETTIT L J, BROWNE H P. Functional genomics reveals that Clostridium difficile Spo0A coordinates sporulation, virulence and metabolism[J]. BMC G, 2014, 15(1):160-168.
[11] KATARINA M, ZUZANA C, NIELS B, et al. Morphogenic protein RodZ interacts with sporulation specific SpoIIE in Bacillus subtilis[J].Plos One, 2016, 11(7):159-167.
[12] GUO M S. Regulation of bacterial outer membrane homeostasis by the sigma-factor, sigma E[J]. Dissertations Theses-Gradworks, 2014, 10(2):147-153.
[13] AHMADPOUR F, YAKHCHALI B. Development of an asporogenic Bacillus cereus strain to improve keratinase production in exponential phase by switching sigma H on and sigma F off[J]. FEMS Microbiol Lett,2017, 12(2):259-268.
[14]余志强,杨明明,杨朝霞,等.同源重组法构建枯草杆菌spo A基因缺失突变株[J].武汉大学学报,2004,50(12):229-233.
[15]宋晓蕾,周芬芬,高琼,等. spo0A基因在艰难梭菌生长及芽孢形成中的作用[J].中国感染与化疗杂志,2017,17(12):33-36.
[16]魏娟,孙长.苏云金芽孢杆菌HD-73菌株基因敲除突变株的构建及特[J].微生物学报,2008,35(10):1581-1586.
[17] SANDOVAL N R, VENKATARAMANAN K P, GROTH T, et al. Wholegenome sequence of an evolved Clostridium pasteurianum strain reveals Spo0A deficiency responsible for increased butanol production and superior growth[J]. Biotechnol Biofuels, 2015, 8(1):227-244.