该工作旨在研究枯草芽孢杆菌表达系统中不同元件之间的适配性,从而构建高效蛋白分泌载体,并进一步应用于提高壳聚糖酶Csn21c的胞外表达量。首先,构建包含不同启动子(P43、Pshuttle09)、核糖体结合位点(RBS1、RBS5)和信号肽(SnprB、SphoD、SymzC)的12个质粒,并以分别以3种蛋白(琼胶酶AgWH50C、壳聚糖酶Csn21c和脂肪酶Lip15)为指示蛋白比较不同元件组合的蛋白分泌水平,发现载体pP435Y*K(P43-RBS5-SymzC)和pP095P*K(Pshuttle09-RBS5-SphoD)优于其他载体。相较原载体(pP431N*K),载体pP435Y*K能使琼胶酶AgWH50C酶活性提高184.25%,脂肪酶Lip15酶活性提高52.8%,壳聚糖酶Csn21c酶活性提高164.62%。最后,通过比较5种不同培养基,确定了TB培养基为壳聚糖酶Csn21c的最适发酵培养基,使用TB培养基壳聚糖酶Csn21c的最高酶活力达到21.14 U/mL,较LB培养基的酶活力提高了128.31%。
The purpose of this work was to investigate the compatibility between different elements in the expression system of Bacillus subtilis, constructing an efficient protein secretion vector, which could be further applied to improve the extracellular expression level of chitosanase Csn21c.Firstly, 12 plasmids containing different promoters (P43, Pshuttle09), ribosomal binding sites (RBS1, RBS5), and signal peptides (SnprB, SphoD, SymzC) were constructed, and the protein secretion levels of different element combinations were compared with three proteins (agarase AgWH50C, chitosanase Csn21c, and lipase Lip15) as indicator proteins, and the vectors pP435Y*K (P43-RBS5-SymzC) and pP095P*K (Pshuttle09-RBS5-SphoD) was superior to other vectors.By using vector pP435Y*K, the activity of agar AgWH50C enzyme increased by 184.25%, the activity of lipase Lip15 enzyme increased by 52.8%, and the activity of chitosanase Csn21c enzyme increased by 164.62% compared with that of the original vector (pP431N*K).Finally, by comparing five different media, it was determined that the TB medium was the most suitable fermentation medium for chitosanase Csn21c, and the highest enzyme activity of chitosanase Csn21c in the TB medium reached 21.14 U/mL, which was 128.31% higher than that of LB medium.
[1] LI W F, ZHOU X X, LU P.Bottlenecks in the expression and secretion of heterologous proteins in Bacillus subtilis[J].Research in Microbiology, 2004, 155(8):605-610.
[2] SONG Y F, NIKOLOFF J M, ZHANG D W.Improving protein production on the level of regulation of both expression and secretion pathways in Bacillus subtilis[J].Journal of Microbiology and Biotechnology, 2015, 25(7):963-977.
[3] CHEN P T, SHAW J F, CHAO Y P, et al.Construction of chromosomally located T7 expression system for production of heterologous secreted proteins in Bacillus subtilis[J].Journal of Agricultural and Food Chemistry, 2010, 58(9):5392-5399.
[4] HERAVI K M, WENZEL M, ALTENBUCHNER J.Regulation of mtl operon promoter of Bacillus subtilis:Requirements of its use in expression vectors[J].Microbial Cell Factories, 2011, 10:83.
[5] BHAVSAR A P, ZHAO X, BROWN E D.Development and characterization of a xylose-dependent system for expression of cloned genes in Bacillus subtilis:Conditional complementation of a teichoic acid mutant[J].Applied and Environmental Microbiology, 2001, 67(1):403-410.
[6] LIU S L, DU K.Enhanced expression of an endoglucanase in Bacillus subtilis by using the sucrose-inducible sacB promoter and improved properties of the recombinant enzyme[J].Protein Expression and Purification, 2012, 83(2):164-168.
[7] LEE S J, PAN J G, PARK S H, et al.Development of a stationary phase-specific autoinducible expression system in Bacillus subtilis[J].Journal of Biotechnology, 2010, 149(1-2):16-20.
[8] WENZEL M, MÜLLER A, SIEMANN-HERZBERG M, et al.Self-inducible Bacillus subtilis expression system for reliable and inexpensive protein production by high-cell-density fermentation[J].Applied and Environmental Microbiology, 2011, 77(18):6419-6425.
[9] ZHANG X Z, CUI Z L, HONG Q, et al.High-level expression and secretion of methyl parathion hydrolase in Bacillus subtilis WB800[J].Applied and Environmental Microbiology, 2005, 71(7):4101-4103.
[10] YANG M M, ZHANG W W, JI S Y, et al.Generation of an artificial double promoter for protein expression in Bacillus subtilis through a promoter trap system[J].PLoS One, 2013, 8(2):e56321.
[11] SONG W, NIE Y, MU X Q, et al.Enhancement of extracellular expression of Bacillus naganoensis pullulanase from recombinant Bacillus subtilis:Effects of promoter and host[J].Protein Expression and Purification, 2016, 124:23-31.
[12] ZHANG X Z, CUI Z L, HONG Q, et al.High-level expression and secretion of methyl parathion hydrolase in Bacillus subtilis WB800[J].Applied and Environmental Microbiology, 2005, 71(7):4101-4103.
[13] XIA Y, CHEN W, ZHAO J X, et al.Construction of a new food-grade expression system for Bacillus subtilis based on theta replication plasmids and auxotrophic complementation[J].Applied Microbiology and Biotechnology, 2007, 76(3):643-650.
[14] VELLANOWETH R L, RABINOWITZ J C.The influence of ribosome-binding-site elements on translational efficiency in Bacillus subtilis and Escherichia coli in vivo[J].Molecular Microbiology, 1992, 6(9):1105-1114.
[15] SALIS H M, MIRSKY E A, VOIGT C A.Automated design of synthetic ribosome binding sites to control protein expression[J].Nature Biotechnology, 2009, 27(10):946-950.
[16] WANG J P, AI X L, MEI H, et al.High-throughput identification of promoters and screening of highly active promoter-5′-UTR DNA region with different characteristics from Bacillus thuringiensis[J].PLoS One, 2013, 8(5):e62960.
[17] MAO X Z, HUANG Z Y, SUN G Y, et al.High level production of diacetylchitobiose deacetylase by refactoring genetic elements and cellular metabolism[J].Bioresource Technology, 2021, 341:125836.
[18] DEGERING C, EGGERT T, PULS M, et al.Optimization of protease secretion in Bacillus subtilis and Bacillus licheniformis by screening of homologous and heterologous signal peptides[J].Applied and Environmental Microbiology, 2010, 76(19):6370-6376.
[19] ZHANG J J, KANG Z, LING Z M, et al.High-level extracellular production of alkaline polygalacturonate lyase in Bacillus subtilis with optimized regulatory elements[J].Bioresource Technology, 2013, 146:543-548.
[20] ZHU F M, JI S Y, ZHANG W W, et al.Development and application of a novel signal peptide probe vector with PGA as reporter in Bacillus subtilis WB700:Twenty-four tat pathway signal peptides from Bacillus subtilis were monitored[J].Molecular Biotechnology, 2008, 39(3):225-230.
[21] SNYDER A J, MUKHERJEE S, GLASS J K, et al.The canonical twin-arginine translocase components are not required for secretion of folded green fluorescent protein from the ancestral strain of Bacillus subtilis[J].Applied and Environmental Microbiology, 2014, 80(10):3219-3232.
[22] LIU N, MAO X Z, YANG M, et al.Gene cloning, expression and characterisation of a new β-agarase, AgWH50C, producing neoagarobiose from Agarivorans gilvus WH0801[J].World Journal of Microbiology & Biotechnology, 2014, 30(6):1691-1698.
[23] GAO L, SUN J N, SECUNDO F, et al.Cloning, characterization and substrate degradation mode of a novel chitinase from Streptomyces albolongus ATCC 27414[J].Food Chemistry, 2018, 261:329-336.
[24] RASOL R, RASHIDAH A R, NAZUHA R N, et al.Psychrotrophic lipase producers from Arctic soil and sediment samples[J].Polish Journal of Microbiology, 2014, 63(1):75-82.
[25] GUO N, SUN J N, WANG W, et al.Cloning, expression and characterization of a novel chitosanase from Streptomyces albolongus ATCC 27414[J].Food Chemistry, 2019, 286:696-702.
