通过优化表达元件及培养基组分提高地衣芽胞杆菌中碱性丝氨酸蛋白酶产量
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摘要
【背景】碱性丝氨酸蛋白酶(Subtilisin)是一种具有广泛用途的工业酶制剂。【目的】旨在通过优化启动子、信号肽及培养基组分来提高地衣芽胞杆菌中碱性丝氨酸蛋白酶产量。【方法】以地衣芽胞杆菌BL10为出发菌株,构建了含有4种不同类型启动子(PbacA、P43、PaprE和PsrfA)及4种不同类型信号肽(SPVpr、SPSacB、SPSacC和SPAprE)的碱性丝氨酸蛋白酶表达菌株,并在获得高产菌株的基础上进行培养基优化。【结果】4种启动子的表达水平为PbacA>PaprE>P43>PsrfA,4种信号肽的分泌效率为SPAprE>SPSacC>SPSacB>SPVpr。其中,菌株BL10/pPbacA-aprE产生最高的碱性丝氨酸蛋白酶酶活(275.21 U/mL),相比于出发菌株BL10/pHY-aprE (167.98 U/mL)提高了64%。随后,通过对发酵培养基成分进行优化并结合正交优化,获得了一种高产碱性丝氨酸蛋白酶的培养基(g/L):玉米淀粉40.0,豆粕50.0,(NH4)2SO4 4.0,K2HPO4 3.0,CaCO3 1.0。最后,碱性丝氨酸蛋白酶酶活提高到747.37 U/mL,是初始酶活的4.45倍。【结论】为工业化高产碱性丝氨酸蛋白酶提供了一种有效策略。
[Background] Subtilisin is an important industrial protease with many applications. [Objective]The aim of this study is to improve the subtilisin production by screening promoters and signal peptides, and optimization of fermentation medium. [Methods] Bacillus licheniformis BL10, constructed in our previous research, was served as the host strain for subtilisin production, and four promoters(PbacA, P43, PaprE and PsrfA) and four signal peptides(SPVpr, SPSacB, SPSacC and SPAprE) were screened to improve subtilisin production, and the medium for subtilisin production was further optimized. [Results] Based on our results,the expression ratios among these four promoters were PbacA>Papr E>P43>PsrfA, and the secretion efficiencies were SPAprE>SPSacC>SPSacB>SPVpr, respectively. Among these strains, BL10/pPbacA-aprE displayed with the highest subtilisin activity(275.21 U/m L), which was increased by 64% compared with that of BL10/pHY-aprE(167.98 U/mL). Furthermore, the fermentation medium was optimized, and the orthogonal test was further applied to enhance subtilisin production. The maximum subtilisin activity(747.37 U/mL)was obtained in the optimized medium with 40.0 g/L corn starch, 50.0 g/L soybean meal, 4.0 g/L(NH4)2 SO4,3.0 g/L K2 HPO4, 1.0 g/L Ca CO3, increased by 4.45-fold compared with that of the initial condition.[Conclusion] Collectively, this study provided an effective strategy for enhanced production of subtilisin.
[Background] Subtilisin is an important industrial protease with many applications. [Objective]The aim of this study is to improve the subtilisin production by screening promoters and signal peptides, and optimization of fermentation medium. [Methods] Bacillus licheniformis BL10, constructed in our previous research, was served as the host strain for subtilisin production, and four promoters(PbacA, P43, PaprE and PsrfA) and four signal peptides(SPVpr, SPSacB, SPSacC and SPAprE) were screened to improve subtilisin production, and the medium for subtilisin production was further optimized. [Results] Based on our results,the expression ratios among these four promoters were PbacA>Papr E>P43>PsrfA, and the secretion efficiencies were SPAprE>SPSacC>SPSacB>SPVpr, respectively. Among these strains, BL10/pPbacA-aprE displayed with the highest subtilisin activity(275.21 U/m L), which was increased by 64% compared with that of BL10/pHY-aprE(167.98 U/mL). Furthermore, the fermentation medium was optimized, and the orthogonal test was further applied to enhance subtilisin production. The maximum subtilisin activity(747.37 U/mL)was obtained in the optimized medium with 40.0 g/L corn starch, 50.0 g/L soybean meal, 4.0 g/L(NH4)2 SO4,3.0 g/L K2 HPO4, 1.0 g/L Ca CO3, increased by 4.45-fold compared with that of the initial condition.[Conclusion] Collectively, this study provided an effective strategy for enhanced production of subtilisin.
引文
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[15]Wei XT,Zhou YH,Chen JB,et al.Efficient expression of nattokinase in Bacillus licheniformis:host strain construction and signal peptide optimization[J].Journal of Industrial Microbiology&Biotechnology,2015,42(2):287-295
[16]Cai DB,Wang H,He PH,et al.A novel strategy to improve protein secretion via overexpression of the SppA signal peptide peptidase in Bacillus licheniformis[J].Microbial Cell Factories,2017,16:70
[17]Suliburska J,Bogdański P,Chiniewicz B.The influence of selected cardiovascular and antidiabetic drugs on pepsin activity in vitro digestion[J].Acta Poloniae Pharmaceutica,2012,69(6):1049-1053
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[19]Li XY,Wang D,Cai DB,et al.Identification and high-level production of pulcherrimin in Bacillus licheniformis DW2[J].Applied Biochemistry and Biotechnology,2017,183(4):1323-1335
[20]Wang D,Wang Q,Qiu YM,et al.Untangling the transcription regulatory network of the bacitracin synthase operon in Bacillus licheniformis DW2[J].Research in Microbiology,2017,168(6):515-523
[21]He PH,Zhang ZY,Cai DB,et al.High-level production ofα-amylase by manipulating the expression of alanine racamase in Bacillus licheniformis[J].Biotechnology Letters,2017,39(9):1389-1394
[22]Bron S,Bolhuis A,Tjalsma H,et al.Protein secretion and possible roles for multiple signal peptidases for precursor processing in Bacilli[J].Journal of Biotechnology,1998,64(1):3-13
[23]Auclair SM,Bhanu MK,Kendall DA.Signal peptidase I:cleaving the way to mature proteins[J].Protein Science,2012,21(1):13-25
[24]Nam SE,Paetzel M.Structure of signal peptide peptidase A with C-termini bound in the active sites:insights into specificity,self-processing,and regulation[J].Biochemistry,2013,52(49):8811-8822
[25]Wang GQ,Chen HQ,Zhang H,et al.The secretion of an intrinsically disordered protein with different secretion signals in Bacillus subtilis[J].Current Microbiology,2013,66(6):566-572
[26]Qin XL,Liu CQ,Zheng LY.Efficiency of signal peptide sequence in yeast secretory expression system[J].Biotechnology,2010,20(3):95-97(in Chinese)覃晓琳,刘朝奇,郑兰英.信号肽对酵母外源蛋白质分泌效率的影响[J].生物技术,2010,20(3):95-97
[27]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
[2]Li CL,Chai XJ,Yang J,et al.Cloning,expression and sequence analysis of protease gene apr by Bacillus tequilensis[J].Chinese Agricultural Science Bulletin,2014,30(15):286-291(in Chinese)李曹龙,柴秀娟,杨晶,等.芽孢杆菌蛋白酶基因apr的克隆、表达及序列分析[J].中国农学通报,2014,30(15):286-291
[3]Espinosa-de-los-Monteros J,Martinez A,Valle F.Metabolic profiles and aprE expression in anaerobic cultures of Bacillus subtilis using nitrate as terminal electron acceptor[J].Applied Microbiology and Biotechnology,2001,57(3):379-384
[4]Kang Z,Yang S,Du GC,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
[5]Wang LF,Devenish RJ.Expression of Bacillus subtilis neutral protease gene(nprE)in Saccharomyces cerevisiae[J].Microbiology,1993,139(2):343-347
[6]Liu XY,Zhang PP,Li LK,et al.Cloning and expression of alkaline protease gene by Bacillus subtilis[J].Biotechnology,2007,17(2):13-16(in Chinese)刘新育,张品品,李林珂,等.枯草芽孢杆菌碱性蛋白酶基因的克隆和表达[J].生物技术,2007,17(2):13-16
[7]Wang YP,Liu YH,Wang ZX,et al.Influence of promoter and signal peptide on the expression of pullulanase in Bacillus subtilis[J].Biotechnology Letters,2014,36(9):1783-1789
[8]Strauch EM,Georgiou G.Escherichia coli tatC mutations that suppress defective twin-arginine transporter signal peptides[J].Journal of Molecular Biology,2007,374(2):283-291
[9]Yu XX,Xu JT,Liu XQ,et al.Identification of a highly efficient stationary phase promoter in Bacillus subtilis[J].Scientific Reports,2015,5:18405
[10]Cai D,Wei X,Qiu Y,et al.High-level expression of nattokinase in Bacillus licheniformis by manipulating signal peptide and signal peptidase[J].Journal of Applied Microbiology,2016,121(3):704-712
[11]Qiu YM,Xiao F,Wei XT,et al.Improvement of lichenysin production in Bacillus licheniformis by replacement of native promoter of lichenysin biosynthesis operon and medium optimization[J].Applied Microbiology and Biotechnology,2014,98(21):8895-8903
[12]Melicherova K,Krahulec J,?afránek M,et al.Optimization of the fermentation and downstream processes for human enterokinase production in Pichia pastoris[J].Applied Microbiology and Biotechnology,2017,101(5):1927-1934
[13]Qiu YM,Zhang JY,Li L,et al.Engineering Bacillus licheniformis for the production of meso-2,3-butanediol[J].Biotechnology for Biofuels,2016,9:117
[14]Wei XT,Tian GM,Ji ZX,et al.A new strategy for enhancement of poly-γ-glutamic acid production by multiple physicochemical stresses in Bacillus licheniformis[J].Journal of Chemical Technology and Biotechnology,2015,90(4):709-713
[15]Wei XT,Zhou YH,Chen JB,et al.Efficient expression of nattokinase in Bacillus licheniformis:host strain construction and signal peptide optimization[J].Journal of Industrial Microbiology&Biotechnology,2015,42(2):287-295
[16]Cai DB,Wang H,He PH,et al.A novel strategy to improve protein secretion via overexpression of the SppA signal peptide peptidase in Bacillus licheniformis[J].Microbial Cell Factories,2017,16:70
[17]Suliburska J,Bogdański P,Chiniewicz B.The influence of selected cardiovascular and antidiabetic drugs on pepsin activity in vitro digestion[J].Acta Poloniae Pharmaceutica,2012,69(6):1049-1053
[18]Song YF,Nikoloff JM,Fu G,et al.Promoter screening from Bacillus subtilis in various conditions hunting for synthetic biology and industrial applications[J].PLoS One,2016,11(7):e0158447
[19]Li XY,Wang D,Cai DB,et al.Identification and high-level production of pulcherrimin in Bacillus licheniformis DW2[J].Applied Biochemistry and Biotechnology,2017,183(4):1323-1335
[20]Wang D,Wang Q,Qiu YM,et al.Untangling the transcription regulatory network of the bacitracin synthase operon in Bacillus licheniformis DW2[J].Research in Microbiology,2017,168(6):515-523
[21]He PH,Zhang ZY,Cai DB,et al.High-level production ofα-amylase by manipulating the expression of alanine racamase in Bacillus licheniformis[J].Biotechnology Letters,2017,39(9):1389-1394
[22]Bron S,Bolhuis A,Tjalsma H,et al.Protein secretion and possible roles for multiple signal peptidases for precursor processing in Bacilli[J].Journal of Biotechnology,1998,64(1):3-13
[23]Auclair SM,Bhanu MK,Kendall DA.Signal peptidase I:cleaving the way to mature proteins[J].Protein Science,2012,21(1):13-25
[24]Nam SE,Paetzel M.Structure of signal peptide peptidase A with C-termini bound in the active sites:insights into specificity,self-processing,and regulation[J].Biochemistry,2013,52(49):8811-8822
[25]Wang GQ,Chen HQ,Zhang H,et al.The secretion of an intrinsically disordered protein with different secretion signals in Bacillus subtilis[J].Current Microbiology,2013,66(6):566-572
[26]Qin XL,Liu CQ,Zheng LY.Efficiency of signal peptide sequence in yeast secretory expression system[J].Biotechnology,2010,20(3):95-97(in Chinese)覃晓琳,刘朝奇,郑兰英.信号肽对酵母外源蛋白质分泌效率的影响[J].生物技术,2010,20(3):95-97
[27]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