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| Improving the Ability of Bacillus licheniformis to Produce Alkaline Protease by Inactivating Sec Pathway Repressor Protein and Extracellular Proteases |
HAO Man,HUI Wei,SHAO Lanying,SHI Chaoshuo,LU Fuping,ZHANG Huitu**( ) |
| Laboratory of Applied Microbiology and Enzyme Engineering, College of Biotechnology, Tianjin University of Science &Technology, Tianjin 300457, China |
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Abstract In order to investigate the influence of the Sec secretion pathway on alkaline protease production in Bacillus licheniformis, the molecular chaperone-blocking protein gene hrcA and the three extracellular protease genes epr, bpr, and vpr in the genome of B. licheniformis TCCC11470 (BLΔuppΔepsΔpgs) were sequentially deleted. By comparing the alkaline protease activities before and after gene deletion, it was found that the knockout strains TCCC11470ΔhrcA and TCCC11470ΔhrcAΔeprΔbprΔvpr reached alkaline protease activities of 18 521.2 U/mL and 20 048.5 U/mL after 42 hours, respectively, which were 27.9% and 38.5% higher than the control strain BLΔuppΔepsΔpgs (14 478.6 U/mL), respectively. These results indicate that optimizing the Sec secretion pathway can effectively enhance the enzymatic activity of alkaline protease, providing new insights and research strategies for the construction of industrial enzyme production hosts.
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Received: 13 July 2023
Published: 03 April 2024
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| [1] |
Okuda M, Sumitomo N, Takimura Y, et al. A new subtilisin family: nucleotide and deduced amino acid sequences of new high-molecular-mass alkaline proteases from Bacillus spp. Extremophiles, 2004, 8(3): 229-235.
doi: 10.1007/s00792-004-0381-8
|
|
|
| [2] |
de Souza P M, de Assis Bittencourt M L, Caprara C C, et al. A biotechnology perspective of fungal proteases. Brazilian Journal of Microbiology, 2015, 46(2): 337-346.
doi: 10.1590/S1517-838246220140359
pmid: 26273247
|
|
|
| [3] |
Verma A, Singh H, Anwar S, et al. Microbial keratinases: industrial enzymes with waste management potential. Critical Reviews in Biotechnology, 2017, 37(4): 476-491.
doi: 10.1080/07388551.2016.1185388
pmid: 27291252
|
|
|
| [4] |
Niu D D, Wang Z X. Development of a pair of bifunctional expression vectors for Escherichia coli and Bacillus licheniformis. Journal of Industrial Microbiology & Biotechnology, 2007, 34(5): 357-362.
|
|
|
| [5] |
Wang J J, Shih J C H. Fermentation production of keratinase from Bacillus licheniformis PWD-1 and a recombinant B. subtilis FDB-29. Journal of Industrial Microbiology and Biotechnology, 1999, 22(6): 608-616.
doi: 10.1038/sj.jim.2900667
|
|
|
| [6] |
王光强. 枯草芽孢杆菌中Sec分泌途径底物特性及非经典分泌途径的研究. 无锡: 江南大学, 2013.
|
|
|
| [6] |
Wang G Q. Research on the substrate characteirstics of sec pathway and non-classical protein secretion pathway in Bacillus subtilis. Wuxi: Jiangnan University, 2013.
|
|
|
| [7] |
de Keyzer J, van der Does C, Driessen A J M. The bacterial translocase: a dynamic protein channel complex. Cellular and Molecular Life Sciences CMLS, 2003, 60(10): 2034-2052.
doi: 10.1007/s00018-003-3006-y
|
|
|
| [8] |
Yuan G, Wong S L. Isolation and characterization of Bacillus subtilis groE regulatory mutants: evidence for orf39 in the dnaK operon as a repressor gene in regulating the expression of both groE and dnaK. Journal of Bacteriology, 1995, 177(22): 6462-6468.
pmid: 7592421
|
|
|
| [9] |
Sambrook J, Russell D W. Molecular cloning. laboratory manual. 3rd ed. New York: Cold Spring Harbor Laboratory Press, 2001.
|
|
|
| [10] |
叶棋浓. 现代分子生物学技术及实验技巧. 北京: 化学工业出版社, 2015.
|
|
|
| [10] |
Ye Q N. Current molecular biology techniques and tips. Beijing: Chemical Industry Press, 2015.
|
|
|
| [11] |
Zhou C X, Liu H, Yuan F Y, et al. Development and application of a CRISPR/Cas9 system for Bacillus licheniformis genome editing. International Journal of Biological Macromolecules, 2019, 122: 329-337.
doi: 10.1016/j.ijbiomac.2018.10.170
|
|
|
| [12] |
国家卫生和计划生育委员会, 国家食品药品监督管理总局. 中华人民共和国国家标准: 食品安全国家标准食品微生物学检验乳酸菌检验 GB 4789.35-2016. 北京: 中国标准出版社, 2017.
|
|
|
| [12] |
National Health and Family Planning Commission of the People’s Republic of China, National Medical Products Administration of the People’s Republic of China. National Standard (Mandatory) of the People’s Republic of China: Food microbiology inspection Lactic acid bacteria inspection. GB 4789.35-2016. Beijing: Standards Press of China, 2017.
|
|
|
| [13] |
中华人民共和国国家质量监督检验检疫总局, 中国国家标准化管理委员会. 中华人民共和国推荐性国家标准: 蛋白酶制剂 GB/T 23527-2009. 北京: 中国标准出版社, 2009.
|
|
|
| [13] |
General Administration of Quality Supervision, Inspection and Quarantine of the People’s Republic of China, Standardization Administration of the People’s Republic of China. National Standard (Recommended) of the People’s Republic of China: Proteinase preparations. GB/T 23527-2009. Beijing: Standards Press of China, 2009.
|
|
|
| [14] |
鲍方名. 巨大芽孢杆菌热休克系统的克隆和操纵子的分析. 南京: 南京师范大学, 2008.
|
|
|
| [14] |
Bao F M. Cloning and operon analysis of heat shock system of Bacillus megaterium. Nanjing: Nanjing Normal University, 2008.
|
|
|
| [15] |
Veith B, Herzberg C, Steckel S, et al. The complete genome sequence of Bacillus licheniformis DSM13, an organism with great industrial potential. Journal of Molecular Microbiology and Biotechnology, 2004, 7(4): 204-211.
|
|
|
| [16] |
Bashir F, Asgher M, Hussain F, et al. Development and characterization of cross-linked enzyme aggregates of thermotolerant alkaline protease from Bacillus licheniformis. International Journal of Biological Macromolecules, 2018, 113: 944-951.
doi: 10.1016/j.ijbiomac.2018.03.009
|
|
|
| [17] |
Schumann W. Production of recombinant proteins in Bacillus subtilis. Advances in Applied Microbiology, 2007, 62: 137-189.
|
|
|
| [18] |
张心怡, 沈镇炎, 段绪果, 等. 芽孢杆菌表达系统研究进展. 基因组学与应用生物学, 2020, 39(7): 3110-3118.
|
|
|
| [18] |
Zhang X Y, Shen Z Y, Duan X G, et al. The research progress on Bacillus expression system. Genomics and Applied Biology, 2020, 39(7): 3110-3118.
|
|
|
| [19] |
陈坤. 2709碱性蛋白酶的高产工程菌株构建及应用性能分析. 天津:天津科技大学. 2018.
|
|
|
| [19] |
Chen K. Construction of the engineering bacteria with high yield of alkaline protease 2709 and its application performance. Tianjin: Tianjin University of Science and Technology, 2018.
|
|
|
| [20] |
Zhou C X, Yang G C, Zhang L, et al. Construction of an alkaline protease overproducer strain based on Bacillus licheniformis 2709 using an integrative approach. International Journal of Biological Macromolecules, 2021, 193: 1449-1456.
doi: 10.1016/j.ijbiomac.2021.10.208
|
|
|
| [21] |
Wu S C, Ye R, Wu X C, et al. Enhanced secretory production of a single-chain antibody fragment from Bacillus subtilis by coproduction of molecular chaperones. Journal of Bacteriology, 1998, 180(11): 2830-2835.
pmid: 9603868
|
|
|
| [22] |
Zhang G Q, Bao P, Zhang Y, et al. Enhancing electro-transformation competency of recalcitrant Bacillus amyloliquefaciens by combining cell-wall weakening and cell-membrane fluidity disturbing. Analytical Biochemistry, 2011, 409(1): 130-137.
doi: 10.1016/j.ab.2010.10.013
|
|
|
| [23] |
Meddeb-Mouelhi F, Dulcey C, Beauregard M. High transformation efficiency of Bacillus subtilis with integrative DNA using glycine betaine as osmoprotectant. Analytical Biochemistry, 2012, 424(2): 127-129.
doi: 10.1016/j.ab.2012.01.032
pmid: 22387342
|
|
|
| [24] |
Wu X C, Lee W, Tran L, et al. Engineering a Bacillus subtilis expression-secretion system with a strain deficient in six extracellular proteases. Journal of Bacteriology, 1991, 173(16): 4952-4958.
doi: 10.1128/jb.173.16.4952-4958.1991
pmid: 1907264
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