spoIIE基因缺失对克劳氏芽孢杆菌淀粉酶酶活的影响
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摘要
通过对克劳氏芽孢杆菌(Bacillus clausii) QL-1spoIIE基因敲除,研究了其对菌体芽孢形成、生物量以及淀粉酶酶活的影响。将重叠的spoIIE-Cmr片段电转化至B.clausii QL-1感受态细胞,仅通过一次同源单交换实现对spoIIE基因快速敲除,成功获得spoIIE基因缺失菌株B.clausii QL-1ΔspoIIE。发酵培养B.clausii QL-1ΔspoIIE与出发菌株相比,B.clausii QL-1ΔspoIIE芽孢生成率28 h降低为0.48%,20 h生物量提高了20%,84 h淀粉酶酶活是出发菌株的5.58倍,通过补料发酵B.clausii QL-1ΔspoIIE菌株80 h最大生物量达到7.2 mg·m L~(-1),84 h淀粉酶酶活达到690 U·m L~(-1),较未补料前提高了33.30%。经验证spoIIE基因为B.clausii QL-1芽孢形成关键基因,同时它的缺失有利于淀粉酶酶活的提高,这对后续构建芽孢杆菌类高产淀粉酶工业生产菌株具有重要意义。
The spoIIE gene was knocked out of Bacillus clausii QL-1,and the experiment studied the influence of bacteria sporulation biomass and amylase activity.Electrotransformation of overlapping spoIIE-Cmrfragments into B.clausii QL-1competent cell made rapid knockout of the spoIIE gene by only one time homologous single exchange.It successfully obtained spoIIE gene deletion strain B.clausii QL-1ΔspoIIE.Compared with the original strain,the sporulation rate of B.clausii QL-1ΔspoIIE decreased to 0.48% at 28 h and the biomass increased by 20% at 20 h.Amylase activity was 5.58 times that of the original strain at 84 h.The maximum biomass of B.clausii QL-1ΔspoIIE strain reached 7.2 mg·mL~(-1) by fed-batch fermentation at 80 h,and the amylase activity reached 690 U·mL~(-1) at 84 h,increased by 33.30%,compared with that before unfeeding.It was verified that the spoIIE gene was a key gene for the sporulation of B.clausii QL-1,and its deletion had a certain effect on the production of amylase,which was of great significance for the subsequent construction of industrial strains of enzyme activity Bacillus.
The spoIIE gene was knocked out of Bacillus clausii QL-1,and the experiment studied the influence of bacteria sporulation biomass and amylase activity.Electrotransformation of overlapping spoIIE-Cmrfragments into B.clausii QL-1competent cell made rapid knockout of the spoIIE gene by only one time homologous single exchange.It successfully obtained spoIIE gene deletion strain B.clausii QL-1ΔspoIIE.Compared with the original strain,the sporulation rate of B.clausii QL-1ΔspoIIE decreased to 0.48% at 28 h and the biomass increased by 20% at 20 h.Amylase activity was 5.58 times that of the original strain at 84 h.The maximum biomass of B.clausii QL-1ΔspoIIE strain reached 7.2 mg·mL~(-1) by fed-batch fermentation at 80 h,and the amylase activity reached 690 U·mL~(-1) at 84 h,increased by 33.30%,compared with that before unfeeding.It was verified that the spoIIE gene was a key gene for the sporulation of B.clausii QL-1,and its deletion had a certain effect on the production of amylase,which was of great significance for the subsequent construction of industrial strains of enzyme activity Bacillus.
引文
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[14]Hou X,Yu X,Du B,et al.A single amino acid mutation in Spo0A results in sporulation deficiency of Paenibacillus polymyxa SC2[J].Research in Microbiology,2016,167(6):472-479.
[15]Katarína M,Zuzana C,Niels B,et al.Morphogenic protein Rod Z Interacts with sporulation specific Spo IIE in Bacillus subtilis[J].Plos One,2016,11(7):159-167.
[16]Marguerite A.Cervin,Richard J.Lewis,James A.Brannigan et al.The Bacillus subtilis regulator Sin R inhibits spo II Gpromoter transcription in vitro without displacing RNA polymerase[J].Nucleic Acids Research,1998,26(16):3806-3812.
[17]T Salman,M Kamal,M Ahmed,et al.Medium optimization for the production of amylase by Bacillus subtilis RM16 in Shakeflask fermentation[J].Pakistan Journal of Pharmaceutical Sciences,2016,29(2):439.
[18]Bo Wu,Guo-Ku Hu,Hong Feng,et al.Cloning and expression of anα-amylase gene from Phanerochaete chrysosporium[J].Current Microbiology,2007,55(2):105-113.
[19]Takeko Kodama,Keiji Endo,Katsutoshi Ara,et al Effect of Bacillus subtilis spo0A mutation on cell wall lytic enzymesand extracellular proteases and prevention of cell lysis[J].Journal of Bioscience and Bioengineering,2007,103(1):13-21.
[20]李瑞芳,薛雯雯,黄亮,等.枯草芽孢杆菌感受态细胞的制备及质粒转化方法研究[J].生物技术通报,2011,35(5):227-230.
[2]杨慧,王振华,潘康成.芽孢杆菌产淀粉酶活性的研究[J].现代农业科技,2007(2):90-91.
[3]Kang Z,Yang S,Du G C,et al.Molecular engineering of secretory machinery components for high-level secretion of proteins in Bacillus species[J].Journal of Industrial Microbiology&Biotechnology,2014,41(11):1599-1607.
[4]Sella S R B R,Vandenberghe L P S.Life cycle and spore resistance of spore-forming Bacillus atrophaeus[J].Microbiological Research,2014,169(2):931-939.
[5]Laura J Pettit,Hilary P Browne,Lu Yu,et al.Functional genomics reveals that Clostridium difficile Spo0A coordinates sporulation virulence and metabolism[J].BMC Genomics,2014,15:160.
[6]刘燕,秦玉昌,潘宝海.枯草芽孢杆菌在(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.
[7]Sonenshein A L.Control of sporulation initiation in Bacillus subtilis[J].Current Opinion in Microbiology,2000,3(6):561-566.
[8]Boonstra M,Jong I G,Scholefield G,et al.Spo0A regulates chromosome copy number during sporulation by directly binding to the origin of replication in Bacillus subtilis[J].Molecular Microbiology,2013,87(4):925-938.
[9]Predich M,Nair G,Smith I.Bacillus subtilis early sporulation genes kin A,spo0F and spo0A are Transcribed by the RNApolymerase containing sigma H[J].Journal of Bacteriology,1992,174(9):2771-2778.
[10]Hibert D W,Piggot P J.Compartmentalization of gene expression during Bacillus subitilisspore formation[J].MicrobiolMolBiol Rev,2004,68(2):234-262.
[11]Irene S Tan,Kumaran S Ramamurthi.Spore formation in Bacillus subtilis[J].Environmental Microbiology Reports,2014,6(3):212-225.
[12]Kolek J,Diallo M,Vasylkivska M,et al.Comparison of expression of key sporulation,solventogenic and acetogenic genes in C.beijerinckii NRRL B-598 and its mutant strain overexpressing spo0A[J].Applied Microbiology&Biotechnology,2017,101(22):8279.
[13]刘燕,秦玉昌,潘宝海,等.芽孢形成中的sigma因子[J].生命科学,2005,17(4):355-359.
[14]Hou X,Yu X,Du B,et al.A single amino acid mutation in Spo0A results in sporulation deficiency of Paenibacillus polymyxa SC2[J].Research in Microbiology,2016,167(6):472-479.
[15]Katarína M,Zuzana C,Niels B,et al.Morphogenic protein Rod Z Interacts with sporulation specific Spo IIE in Bacillus subtilis[J].Plos One,2016,11(7):159-167.
[16]Marguerite A.Cervin,Richard J.Lewis,James A.Brannigan et al.The Bacillus subtilis regulator Sin R inhibits spo II Gpromoter transcription in vitro without displacing RNA polymerase[J].Nucleic Acids Research,1998,26(16):3806-3812.
[17]T Salman,M Kamal,M Ahmed,et al.Medium optimization for the production of amylase by Bacillus subtilis RM16 in Shakeflask fermentation[J].Pakistan Journal of Pharmaceutical Sciences,2016,29(2):439.
[18]Bo Wu,Guo-Ku Hu,Hong Feng,et al.Cloning and expression of anα-amylase gene from Phanerochaete chrysosporium[J].Current Microbiology,2007,55(2):105-113.
[19]Takeko Kodama,Keiji Endo,Katsutoshi Ara,et al Effect of Bacillus subtilis spo0A mutation on cell wall lytic enzymesand extracellular proteases and prevention of cell lysis[J].Journal of Bioscience and Bioengineering,2007,103(1):13-21.
[20]李瑞芳,薛雯雯,黄亮,等.枯草芽孢杆菌感受态细胞的制备及质粒转化方法研究[J].生物技术通报,2011,35(5):227-230.