基于信号肽策略提高外源蛋白在枯草芽孢杆菌中的表达

doi:10.13560/j.cnki.biotech.bull.1985.2020-1255

摘要/Abstract

摘要：

枯草芽孢杆菌(Bacillus subtilis)作为一种重要的原核表达宿主菌,具有蛋白分泌能力强、遗传背景清晰、无密码子偏好性、生长迅速和非致病性等优点,一直都是外源蛋白表达优选的模式菌株。信号肽(signal peptide)是一段存在于前体蛋白N-端的短肽链,其功能在于引导和调节前体蛋白的折叠,在蛋白转移和分泌过程中扮演着极其重要的角色。目前,利用枯草芽孢杆菌信号肽,针对不同外源蛋白的高效分泌暂无规律性可寻。为此,从枯草芽孢杆菌信号肽的结构特点、分类、转运途径和应用方式等方面进行综述,以期为进一步筛选外源蛋白在枯草芽孢杆菌表达系统中的最适信号肽提供一定的参考。

关键词: 枯草芽孢杆菌, 信号肽, 分泌途径, 信号肽文库

Abstract:

As an important prokaryotic expression host strain,Bacillus subtilis has always been regarded as preferred model strain for foreign protein expression,featuring strong protein secretion,clear genetic background,no codon preference,rapid growth and non-pathogenicity. Signal peptide is a short peptide chain located in the N-terminal of the precursor protein,with the function of guiding and regulating the folding of the precursor protein. Meanwhile,it plays a very important role in the process of protein transfer and secretion. At present,there is no regularity to find the efficient secretion of different foreign proteins by using the signal peptide of B. subtilis. For this reason,the structural characteristics,classification,transport pathway and application of signal peptides from B. subtilis are reviewed here,aiming to provide certain reference for further screening the optimal signal peptides of foreign proteins in B. subtilis expression system.

Key words: Bacillus subtilis, signal peptide, secretion pathway, signal peptide library

苗华彪, 曹艳, 杨梦瀚, 黄遵锡. 基于信号肽策略提高外源蛋白在枯草芽孢杆菌中的表达[J]. 生物技术通报, 2021, 37(6): 259-271.

MIAO Hua-biao, CAO Yan, YANG Meng-han, HUANG Zun-xi. The Strategy for Enhancing Foreign Proteins Expression by Signal Peptide in Bacillus subtilis[J]. Biotechnology Bulletin, 2021, 37(6): 259-271.

图/表 4

参考文献 87

[1]	Lee NK, Kim WS, Paik HD. Bacillus strains as human probiotics:characterization, safety, microbiome, and probiotic carrier[J]. Food Science and Biotechnology, 2019, 28(5):1297-1305. doi: 10.1007/s10068-019-00691-9 URL
[2]	Cui WJ, Han LH, Suo FY, et al. Exploitation of Bacillus subtilis as a robust workhorse for production of heterologous proteins and beyond[J]. World Journal of Microbiology & Biotechnology, 2018, 34(10):145-164. doi: 10.1007/s11274-018-2531-7 URL
[3]	Gu Y, Xu XH, Wu YK, et al. Advances and prospects of Bacillus subtilis cellular factories:from rational design to industrial applications[J]. Metabolic Engineering, 2018, 50:109-121. doi: 10.1016/j.ymben.2018.05.006 URL
[4]	Tjalsma H, Antelmann H, Jongbloed JD, et al. Proteomics of protein secretion by Bacillus subtilis:separating the “secrets” of the secretome[J]. Microbiology and Molecular Biology Reviews, 2004, 68(2):207-233. pmid: 15187182
[5]	Song Y, Nikoloff JM, Zhang D. Improving protein production on the level of regulation of both expression and secretion pathways in Bacillus subtilis[J]. World Journal of Microbiology & Biotechnology, 2015, 25(7):963-977.
[6]	熊海涛, 韦宇拓. 枯草芽孢杆菌表达系统及其启动子的研究进展[J]. 广西科学, 2018, 25(3):233-241.
	Xiong HT, Wei YT. Research progress of Bacillus subtilis expression on system and its promoter regulatory elements[J]. Guangxi Sciences, 2018, 25(3):233-241.
[7]	Blobel G. Transfer of proteins across membranes I. presence of proteolytically processed and unprocessed nascent immunoglobulin light chains on membrane-bound ribosomes of murine myeloma[J]. J Cell Biol, 1975, 67(3):835-851. pmid: 811671
[8]	Walter P, Blobel G. Purification of a membrane-associated protein complex required for protein translocation across the endoplasmic reticulum[J]. Proceedings of the National Academy of Sciences, 1980, 77(12):7112-7116. doi: 10.1073/pnas.77.12.7112 URL
[9]	Gilmore R, Walter P, Blobel G. Protein translocation across the endoplasmic reticulum II. isolation and characterization of the signal recognition particle receptor[J]. The Journal of Cell Biology, 1982, 95(2):470-477. doi: 10.1083/jcb.95.2.470 URL
[10]	Tjalsma H, Noback MA, Bron S, et al. Bacillus subtilis contains four closely related type I signal peptidases with overlapping substrate specificities:constitutive and temporally controlled expression of different sip genes[J]. Journal of Biological Chemistry, 1997, 272(41):25983-25992. doi: 10.1074/jbc.272.41.25983 URL
[11]	Tjalsma H, Bolhuis A, Jongbloed JDH, et al. Signal peptide-dependent protein transport in Bacillus subtilis:a genome-based survey of the secretome[J]. Microbiology and Molecular Biology Reviews, 2000, 64(3):515-547. pmid: 10974125
[12]	Brockmeier U, Caspers M, Freudl R, et al. Systematic screening of all signal peptides from Bacillus subtilis:a powerful strategy in optimizing heterologous protein secretion in Gram-positive bacteria[J]. J Mol Biol, 2006, 362(3):393-402. pmid: 16930615
[13]	Tjalsma H, Zanen G, Venema G, et al. The potential active site of the lipoprotein-specific(type II)signal peptidase of Bacillus subtilis[J]. Journal of Biological Chemistry, 1999, 242(40):28191-28197.
[14]	Georg K, Klemens W, Irmgard S. Structure of the complete bacterial SRP Alu domain[J]. Nucleic Acids Research, 2014, 42(19):12284-12294. doi: 10.1093/nar/gku883 URL
[15]	Struck JCR, Hartmann RK, Toschka HY, et al. Transcription and processing of Bacillus subtilis small cytoplasmic RNA[J]. Molecular & General Genetics, 1989, 215(3):478-482.
[16]	Carabetta VJ, Greco TM, Cristea IM, et al. YfmK is an N ε-lysine acetyltransferase that directly acetylates the histone-like protein HBsu in Bacillus subtilis[J]. Proceedings of the National Academy of Sciences, 2019, 116(9):1-6. doi: 10.1073/iti0119116 URL
[17]	Tomas F, Daniel W, Peter O, et al. Dynamic action of the Sec machinery during initiation, protein translocation and termination[J]. eLife, 2018, 7:35112-3538.
[18]	Adachi S, Murakawa Y, Hiraga S. Dynamic nature of SecA and its associated proteins in Escherichia coli[J]. Frontiers in Microbiology, 2015, 75(6):75-87.
[19]	Voros A, Simm R, Slamti L, et al. SecDF as part of the Sec-translocase facilitates efficient secretion of Bacillus cereus toxins and cell wall-associated proteins[J]. PLoS One, 2014, 8(9):e103326.
[20]	Chiba S, Ito K. MifM monitors total YidC activities of Bacillus subtilis, including that of YidC₂, the target of regulation[J]. Journal of Bacteriology, 2015, 197(1):99-107. doi: 10.1128/JB.02074-14 URL
[21]	Kudva R, Denks K, Kuhn P, et al. Protein translocation across the inner membrane of Gram-negative bacteria:the Sec and Tat dependent protein transport pathways[J]. Research in Microbiology, 2013, 164(6):505-534. doi: 10.1016/j.resmic.2013.03.016 URL
[22]	Wang H, Ma Y, Hsieh YH, et al. SecAAA trimer is fully functional as SecAA dimer in the membrane:existence of higher oligomers?[J]. Biochemical and Biophysical Research Communications, 2014, 447(2):250-254. doi: 10.1016/j.bbrc.2014.03.116 URL
[23]	Ma RJ, Wang YH, Liu L, et al. Production enhancement of the extracellular lipase LipA in Bacillus subtilis:effects of expression system and Sec pathway components[J]. Protein Expression and Purification, 2018, 142:81-87. doi: 10.1016/j.pep.2017.09.011 URL
[24]	Shapova YA, Paetzel M. Crystallographic analysis of Bacillus subtilis CsaA[J]. Acta Crystallographica Section D, Biological Crystallography, 2007, 63(4):478-485. doi: 10.1107/S0907444907005045 URL
[25]	Terra R, Stanley-Wall NR, Cao G, et al. Identification of Bacillus subtilis SipW as a bifunctional signal peptidase that controls surface-adhered biofilm formation[J]. Journal of Bacteriology, 2012, 194(11):2781-2790. doi: 10.1128/JB.06780-11 URL
[26]	Motojima F, Fujii K, Yoshida M. Chaperonin facilitates protein folding by avoiding initial polypeptide collapse[J]. Journal of Biochemistry, 2018, 164(5):369-379. doi: 10.1093/jb/mvy061 URL
[27]	Kang Z, Yang S, Du G, et al. Molecular engineering of secretory machinery components for high-level secretion of proteins in Bacillus species[J]. J Ind Microbiol Biotechnol, 2014, 41(11):1599-1607. doi: 10.1007/s10295-014-1506-4 URL
[28]	Seydlová G, Halada P, Fiser R, et al. DnaK and GroEL chaperones are recruited to the Bacillus subtilis membrane after short-term ethanol stress[J]. J Appl Microbiol, 2012, 112(4):765-742. doi: 10.1111/j.1365-2672.2012.05238.x pmid: 22268681
[29]	Anne J, Economou A, Bernaerts K. Protein secretion in Gram-positive acteria:from multiple pathways to biotechnology[J]. Curr Top Microbiol Immunol, 2017, 404:267-308.
[30]	Frain KM, Dijl JMV, Robinson C. The Twin-Arginine Pathway for Protein Secretion[M]// Protein Secretion in Bacteria. John Wiley & Sons, Ltd, 2019.
[31]	Frain KM, Robinson C, Dijl JMV. Transport of folded proteins by the Tat system[J]. Protein J, 2019, 38(4):377-388. doi: 10.1007/s10930-019-09859-y URL
[32]	Freudl R. Signal peptides for recombinant protein secretion in bacterial expression systems[J]. Microbial Cell Factories, 2018, 17(1):52-62. doi: 10.1186/s12934-018-0901-3 URL
[33]	Vivianne J, Goosens CG, Dijl JMV, et al. The Tat system of Gram-positive bacteria[J]. Biochimica et Biophysica Acta-Molecular Cell Research, 2014, 1843(8):1698-1706.
[34]	Van DPR, Mader U, Homuth G, et al. Environmental salinity determines the specificity and need for Tat-dependent secretion of the YwbN protein in Bacillus subtilis[J]. PLoS One, 2011, 6(3):e18140. doi: 10.1371/journal.pone.0018140 URL
[35]	Monteferrante CG, Miethke M, Vand PR, et al. Specific targeting of the metallophosphoesterase YkuE to the Bacillus cell wall requires the Twin-arginine translocation system[J]. Journal of Biological Chemistry, 2012, 287(35):29789-29800. doi: 10.1074/jbc.M112.378190 URL
[36]	Palmer T, Stansfeld PJ. Targeting of proteins to the twin-arginine translocation pathway[J]. Mol Microbiol, 2020, 113(5):861-871. doi: 10.1111/mmi.14461 pmid: 31971282
[37]	Goosens VJ, Van DJM. Twin-arginine protein translocation[J]. Curr Top Microbiol Immunol, 2017, 404:69-94.
[38]	Goosens VJ, De-San-Eustaquio-Campillo A, Carballido-López R, et al. A Tat menage a trois-the role of Bacillus subtilis TatAc in twin-arginine protein translocation[J]. Biochimica et Biophysica Acta-Molecular Cell Research, 2015, 1853(10):2425-2753.
[39]	Walther TH, Gottselig C, Grage SL, et al. Folding and self-assembly of the TatA translocation pore based on a charge zipper mechanism[J]. Cell, 2013, 152(1-2):316-326. doi: 10.1016/j.cell.2012.12.017 URL
[40]	Kang XM, Cai X, Huang ZX, et al. Construction of highly active secretory expression system in Bacillus subtilis of a recombinant amidase by promoter and signal peptide engineering[J]. Int J Biol Macromol, 2020, 143:833-841. doi: 10.1016/j.ijbiomac.2019.09.144 URL
[41]	Guan CR, Cui WJ, Cheng JT, et al. Construction of highly active secretory expression system via an engineered dual promoter and a highly efficient signal peptidein Bacillus subtilis[J]. New Biotechnology, 2016, 33(3):372-379. doi: 10.1016/j.nbt.2016.01.005 URL
[42]	Li YH, Shi CS, Li DK, et al. Engineering a highly effificient expression system to produce BcaPRO protease in Bacillus subtilis by an optimized promoter and signal peptide[J]. International Journal of Biological Macromolecules, 2019, 138:903-911. doi: 10.1016/j.ijbiomac.2019.07.175 URL
[43]	Tian JW, Long X, Tian YQ, et al. Enhanced extracellular recombinant keratinase activity in Bacillus subtilis SCK6 through signal peptide optimization and site-directed mutagenesis[J]. RSC Advances, 2019, 9(57):33337-33344. doi: 10.1039/C9RA07866E URL
[44]	Feng X, Liu S, Jiao Y, et al. Enhanced extracellular production of L-asparaginase from Bacillus subtilis 168 by B. subtilis WB600 through a combined strategy[J]. Applied Microbiology & Biotechnology, 2017, 101(4):1509-1520.
[45]	Song YF, Fu G, Dong HN, et al. High-efficiency Secretion of β-mannanase in Bacillus subtilis through protein synjournal and secretion optimization[J]. Journal of Agricultural and Food Chemistry, 2017, 65(12):2540-2548. doi: 10.1021/acs.jafc.6b05528 URL
[46]	Zhang JJ, Kang Z, Lin ZM, et al. High-level extracellular production of alkaline polygalacturonate lyase in Bacillus subtilis with optimized regulatory elements[J]. Bioresource Technology, 2013, 146:543-548. doi: 10.1016/j.biortech.2013.07.129 URL
[47]	Gu YY, Zheng JY, Feng J, et al. Improvement of levan production in Bacillus amyloliquefaciens through metabolic optimization of regulatory elements[J]. Applied Microbiology & Biotechnology, 2017, 101(10):4163-4142.
[48]	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. doi: 10.1007/s10529-014-1538-x URL
[49]	Huang X, Cao LC, Qin ZM, et al. Tat-independent secretion of polyethylene terephthalate hydrolase PETase in Bacillus subtilis 168 mediated by its native signal peptide[J]. Journal of Agricultural and Food Chemistry, 2018, 66(50):13217-13227. doi: 10.1021/acs.jafc.8b05038 URL
[50]	周勇. 嗜麦芽糖寡养单胞菌脂肪酶LipS在枯草芽孢杆菌中的高效分泌表达[D]. 杭州:浙江大学, 2015.
	Zhou Y. High level secretionexpression of a lipasee from Stenotrophompnas maltophilia in Bacillus subtilis[J]. Hangzhou:Zhejiang University, 2015.
[51]	Ren GH, Cao LC, Kong W, et al. Efficient secretion of the β-galactosidase Bgal1-3 via both Tat-dependent and Tat-independent pathways in Bacillus subtilis[J]. Journal of Agricultural and Food Chemistry, 2016, 64(28):5708-5726. doi: 10.1021/acs.jafc.6b01735 URL
[52]	Chen JQ, Gai YM, Fu G, et al. Enhanced extracellular production of α-amylase in Bacillus subtilis by optimization of regulatory elements and over-expression of PrsA lipoprotein[J]. Biotechnology Letters, 2015, 37(4):899-906. doi: 10.1007/s10529-014-1755-3 URL
[53]	马樱芳. 基于ARTP诱变、表达元件定向改造及发酵优化提高枯草芽孢杆菌生产重组碱性淀粉酶的水平[D]. 无锡:江南大学, 2016.
	Ma YF. Improving the yield of recombinant alkaline amylase in Bacillus subtillus by ARTP mutagenesis, directed modification of expression elements and fermentation optimization[J]. Wuxi:Jiangnan University, 2016.
[54]	刘金岚, 付刚, 董会娜, 等. 通过信号肽筛选优化耐高温α-淀粉酶在枯草芽孢杆菌中的分泌[J]. 工业微生物, 2017, 47(1):17-23.
	Liu JL, Fu G, Dong HN, et al. Optimization of thermostable α-amylase secretion by screening of optimal signal peptide in Bacillus subtilis[J]. Industrial Microbiology, 2017, 47(1):17-23.
[55]	袁林, 曾静, 郭建军, 等. 极端嗜热酸性α-淀粉酶PFA在枯草芽孢杆菌中的高效分泌表达[J]. 食品科学, 2018, 39(18):100-108.
	Yuan L, Zeng J, Guo JJ, et al. Effificient secretory expression of hyperthermoacidophilic α-Amylase PFA in Bacillus subtilis WB600[J]. Food Science, 2018, 39(18):100-108.
[56]	张士彬, 陈景奇, 崔艳艳, 等. 中温α-淀粉酶基因在枯草芽孢杆菌中的高效表达及其发酵条件优化[J]. 食品与发酵工业, 2016, 42(4):50-56.
	Zhang SB, Chen JQ, Cui YY, et al. High level expression of medium α-amylase in Bacillus subtilis and optimization of fermentation conditions[J]. Food and Fermentation Industries, 2016, 42(4):50-56.
[57]	Fu LL, Xu ZR, Shuai JB, et al. High-level secretion of a chimeric thermostable lichenase from Bacillus subtilis by screening of site-mutated signal peptides with structural alterations[J]. Current Microbiology, 2008, 56(3):287-292. doi: 10.1007/s00284-007-9077-5 URL
[58]	Caspers M, Brockmeier U, Degering C, et al. Improvement of Sec-dependent secretion of a heterologous model protein in Bacillus subtilis by saturation mutagenesis of the N-domain of the AmyE signal peptide[J]. Applied Microbiology & Biotechnology, 2010, 86(6):1877-1885.
[59]	祝发明, 刘辉, 曹要玲, 等. 枯草芽孢杆菌AmyX基因信号肽性能优化研究[J]. 西北农林科技大学学报:自然科学版, 2006, 36(9):11-16.
	Zhu FM, Liu H, Cao YL, et al. Studies on optimizing the signal peptide of AmyX protein from B. subtilis[J]. Journal of Northwest A&F University:Social Science Edition, 2006, 36(9):11-16.
[60]	Yao DB, Su LQ, Li N, et al. Enhanced extracellular expression of Bacillus stearothermophilus α-amylase in Bacillus subtilis through signal peptide optimization, chaperone overexpression and α-amylase mutant selection[J]. Microbial Cell Factories, 2019, 18(1):69-81. doi: 10.1186/s12934-019-1119-8 URL
[61]	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. doi: 10.1128/AEM.01146-10 URL
[62]	Tsuji S, Tanaka K, Takenaka S, et al. Enhanced secretion of natto phytase by Bacillus subtilis[J]. Journal of the Agricultural Chemical Society of Japan, 2015, 79(11):1906-1914.
[63]	蒋蕊. α-淀粉酶共表达系统的研究及在枯草芽孢杆菌中最优信号肽的筛选[D]. 昆明:云南师范大学, 2019.
	Jiang R. Research on co-expression of α-amylase and screening of optimal signal in Bacillus subtilis[J]. Kunming:Yunnan Normal University, 2019.
[64]	李雨桐. 嗜热脂肪芽孢杆菌麦芽糖淀粉酶在枯草芽孢杆菌中的重组表达及发酵优化[D]. 无锡:江南大学, 2018.
	Li YT. Recombinant expression and fermentation optimization of Bacillus stearothermophilus maltogenic amylase in Bacillus subtilis[J]. Wuxi:Jiangnan University, 2018.
[65]	Fu G, Liu JL, Li JS, et al. Systematic screening of optimal signal peptides for secretory production of heterologous proteins in Bacillus subtilis[J]. Journal of Agricultural and Food Chemistry, 2018, 66(50):13141-13151. doi: 10.1021/acs.jafc.8b04183 URL
[66]	Zhang WW, Yang MM, Yang YD, et al. Optimal secretion of alkali-tolerant xylanase in Bacillus subtilis by signal peptide screening[J]. Applied Microbiology & Biotechnology, 2016, 100(20):8425-8756.
[67]	Albiniak AM, Matos CFRO, Branston SD, et al. High-level secretion of a recombinant protein to the culture medium with a Bacillus subtilis twin-arginine translocation system in Escherichia coli[J]. FEBS Journal, 2013, 280(16):3810-3821. doi: 10.1111/febs.12376 URL
[68]	Low KO, Jonet MA, Ismail NF, et al. Optimization of a Bacillus sp signal peptide for improved recombinant protein secretion and cell viability in Escherichia coli[J]. Bioengineered Bugs, 2012, 3(6):334-338.
[69]	Zhang FY, He HH, Deng T, et al. N-terminal fused signal peptide prompted extracellular production of a Bacillus-derived alkaline and thermo stable xylanase in E. coli through cell autolysis[J]. Applied Biochemistry and Biotechnology, 2020, 192:339-352. doi: 10.1007/s12010-020-03323-9 URL
[70]	Hemmerich J, Rohe P, Kleine B, et al. Use of a Sec signal peptide library from Bacillus subtilis for the optimization of cutinase secretion in Corynebacterium glutamicum[J]. Microbial Cell Factories, 2016, 15(1):208-219. doi: 10.1186/s12934-016-0604-6 URL
[71]	刘慧玲. 分泌蛋白基因编码区5'端密码子使用偏好性研究[D]. 杨凌:西北农林科技大学, 2016.
	Liu HL. Codon usage bias in 5' terminal of coding sequences reveales an distinct enrichmant of gene functions[J]. Yangling:Northwest A&F University, 2016.
[72]	Zalucki YM, Power PM, Jennings MP. Selection for efficient translation initiation biases codon usage at second amino acid position in secretory proteins[J]. Nucleic Acids Research, 2007, 35(17):5748-5754. doi: 10.1093/nar/gkm577 URL
[73]	Power PM, Jones RA, Beacham IR, et al. Whole genome analysis reveals a high incidence of non-optimal codons in secretory signal sequences of Escherichia coli[J]. Biochemical & Biophysical Research Communications, 2004, 322(3):1038-1044.
[74]	Li YD, Li YQ, Chen JS, et al. Whole genome analysis of non-optimal codon usage in secretory signal sequences of Streptomyces coelicolor[J]. Biosystems, 2006, 85(3):225-230. doi: 10.1016/j.biosystems.2006.02.006 URL
[75]	Zalucki YM, Jennings MP. Experimental confirmation of a key role for non-optimal codons in protein export[J]. Biochemical and Biophysical Research Communications, 2007, 355(1):143-148. doi: 10.1016/j.bbrc.2007.01.126 URL
[76]	Zalucki YM, Gittins KL, Jennings MP. Secretory signal sequence non-optimal codons are required for expression and export of β-lactamase[J]. Biochemical & Biophysical Research Communications, 2008, 366(1):135-141.
[77]	Zalucki YM, Jones CE, Ng PSK, et al. Signal sequence non-optimal codons are required for the correct folding of mature maltose binding protein[J]. Biochimica Et Biophysica Acta, 2010, 1798(6):1244-1249.
[78]	Mahlab S, Linial M, Pilpel Y. Speed controls in translating secretory proteins in Eukaryotes-an evolutionary perspective[J]. Plos Computational Biology, 2014, 10(1):e1003294. doi: 10.1371/journal.pcbi.1003294 URL
[79]	Pechmann S, Chartron JW, Frydman J. Local slowdown of translation by nonoptimal codons promotes nascent-chain recognition by SRP in vivo[J]. Nature Structural & Molecular Biology, 2014, 21(12):1100-1105. doi: 10.1038/nsmb.2919 URL
[80]	陈景奇. α-淀粉酶的异源表达、调控元件优化和分泌瓶颈鉴定[D]. 天津:天津大学, 2015.
	Chen JQ. The heterologous expression of α-amylase regulatory element optimization and identification of secretion bottleneck[J]. Tianjin:Tianjin University, 2015.
[81]	Muller JP, Bron S, Venema G, et al. Chaperone-like activities of the CsaA protein of Bacillus subtilis[J]. Microbiology, 2000, 146(1):77-88. doi: 10.1099/00221287-146-1-77 URL
[82]	Muller JP, Ozegowski J, Vettermann S, et al. Interaction of Bacillus subtilis CsaA with SecA and precursor proteins[J]. Biochemical Journal, 2000, 348(2):367-373. doi: 10.1042/bj3480367 URL
[83]	Kakeshita H, Kageyama Y, Ara K, et al. Enhanced extracellular production of heterologous proteins in Bacillus subtilis by deleting the C-terminal region of the SecA secretory machinery[J]. Molecular Biotechnology, 2010, 46(3):250-257. doi: 10.1007/s12033-010-9295-0 URL
[84]	Mulder KCL, Bandola J, Schumann W. Construction of an artificial secYEG operon allowing high level secretion of α-amylase[J]. Protein Expression & Purification, 2013, 89(1):92-96.
[85]	Vitikainen M, Pummi T, Airaksinen U, et al. Quantitation of the capacity of the secretion apparatus and requirement for PrsA in growth and secretion of α-amylase in Bacillus subtilis[J]. Journal of Bacteriology, 2001, 183(6):1881-1890. doi: 10.1128/JB.183.6.1881-1890.2001 URL
[86]	Wu SC, Ye R, Wu XC, et al. Enhanced secretory production of a single-chain antibody fragment from Bacillus subtilis by coproduction of molecular chaperones[J]. Journal of Bacteriology, 1998, 180(11):2830-2835. doi: 10.1128/JB.180.11.2830-2835.1998 URL
[87]	Kontinen VP, Sarvas M. The PrsA lipoprotein is essential for protein secretion in Bacillus subtilis and sets a limit for high-level secretion[J]. Molecular Microbiology, 2010, 8(4):727-737. doi: 10.1111/mmi.1993.8.issue-4 URL

外源蛋白 Foreign protein	信号肽类型 Signal peptide type	最适信号肽 Optimal signal peptide	分泌途径 Secretory pathway	胞外活性 Extracellular activity	参考文献 Reference
酰胺酶 Amidase	20个Sec+1个Tat	Pac	Sec	+1.36倍	[40]
氨肽酶 Aminopeptidase	19个Sec	YncM	Sec	+1.20倍	[41]
碱性丝氨酸蛋白酶 Alkaline serine protease	73个Sec	DacB	Sec	953.00 U/mg	[42]
角蛋白酶 Keratinase	5个Sec+4个Tat	LipA	Tat	+2.00倍	[43]
L-天冬酰胺酶 L-asparaginase	5个Sec+3个Tat	WapA	Tat	+1.72倍	[44]
β-甘露聚糖酶 β-mannanase	3个Sec+3个Tat	LipA	Tat	+1.25倍	[45]
碱性果胶酶 Alkaline polygalacturonate lyase	11个Sec+3个Tat	Bpr	Sec	313.70 U/g	[46]
果聚糖蔗糖酶 Encoding levansucrase	7个Sec+1个Tat	YncM	Sec	+2.61倍	[47]
普鲁兰酶 Pullulanase	4个Sec	SacB	Sec	2.82 U/mL	[48]
聚对苯二甲酸乙二醇酯水解酶 PETase	3个Sec+3个Tat	SP_PETase	Tat	+3.80倍	[49]
脂肪酶 Lipase	6个Sec+2个Tat	PhoD	Tat	+2.33倍	[50]
β-半乳糖苷酶 β-galactosidase	3个Sec+2个Tat	PhoD	Tat	+10.10倍	[51]
α-淀粉酶 α-Amylase	5个Sec+1个Tat	NprE	Sec	260.00 U/mL	[52]
碱性淀粉酶 Alkaline amylase	9个Sec+4个Tat	YwbN	Tat	364.5 U/mL	[53]
α-淀粉酶 α-Amylase	44个Sec+2个Tat	BglS	Sec	1393.30 U/mL	[54]
α-淀粉酶 α-Amylase	8个Sec+1个Tat	YfkN	Sec	+10.00倍	[55]
α-淀粉酶 α-Amylase	6个Sec+2个Tat	NprE	Sec	242.00 U/mL	[56]

外源蛋白 Foreign protein	信号肽类型 Signal peptide type	最适信号肽 Optimal signal peptide	分泌途径 Secretory pathway	胞外活性 Extracellular activity	参考文献 Reference
酰胺酶 Amidase	20个Sec+1个Tat	Pac	Sec	+1.36倍	[40]
氨肽酶 Aminopeptidase	19个Sec	YncM	Sec	+1.20倍	[41]
碱性丝氨酸蛋白酶 Alkaline serine protease	73个Sec	DacB	Sec	953.00 U/mg	[42]
角蛋白酶 Keratinase	5个Sec+4个Tat	LipA	Tat	+2.00倍	[43]
L-天冬酰胺酶 L-asparaginase	5个Sec+3个Tat	WapA	Tat	+1.72倍	[44]
β-甘露聚糖酶 β-mannanase	3个Sec+3个Tat	LipA	Tat	+1.25倍	[45]
碱性果胶酶 Alkaline polygalacturonate lyase	11个Sec+3个Tat	Bpr	Sec	313.70 U/g	[46]
果聚糖蔗糖酶 Encoding levansucrase	7个Sec+1个Tat	YncM	Sec	+2.61倍	[47]
普鲁兰酶 Pullulanase	4个Sec	SacB	Sec	2.82 U/mL	[48]
聚对苯二甲酸乙二醇酯水解酶 PETase	3个Sec+3个Tat	SP_PETase	Tat	+3.80倍	[49]
脂肪酶 Lipase	6个Sec+2个Tat	PhoD	Tat	+2.33倍	[50]
β-半乳糖苷酶 β-galactosidase	3个Sec+2个Tat	PhoD	Tat	+10.10倍	[51]
α-淀粉酶 α-Amylase	5个Sec+1个Tat	NprE	Sec	260.00 U/mL	[52]
碱性淀粉酶 Alkaline amylase	9个Sec+4个Tat	YwbN	Tat	364.5 U/mL	[53]
α-淀粉酶 α-Amylase	44个Sec+2个Tat	BglS	Sec	1393.30 U/mL	[54]
α-淀粉酶 α-Amylase	8个Sec+1个Tat	YfkN	Sec	+10.00倍	[55]
α-淀粉酶 α-Amylase	6个Sec+2个Tat	NprE	Sec	242.00 U/mL	[56]

外源蛋白 Foreign protein	信号肽 Signal peptide	分泌途径 Secretory pathway	信号肽突变点 Signal peptide mutation site	胞外活性 Extracellular activity	参考文献 Reference
脂肪酶 Lipase	PhoD	Tat	N3K、N3K/L4K、D25R、D25R/F29R	+1.12-1.31倍	[50]
碱性淀粉酶 Alkaline amylase	YwbN	Tat	E4K/E9K、I16N/ L17S/W19S	+1.08-1.58倍	[53]
α-淀粉酶 α-Amylase	YfkN	Sec	I3G/Q4R	+13.00倍	[55]
地衣聚糖酶 Lichenase	H1	-	VNIAFMLF/RKIAGMAT	+4.60倍	[57]
角质酶 Cutinase	AmyE	Sec	F2D、F2E、F6W、K4L	+2-3倍	[58]
β-半乳糖苷酶 β-galactosidase	AmyX	Tat	N4I/ Y8F/N17I	+2.50倍	[59]

外源蛋白 Foreign protein	信号肽 Signal peptide	分泌途径 Secretory pathway	信号肽突变点 Signal peptide mutation site	胞外活性 Extracellular activity	参考文献 Reference
脂肪酶 Lipase	PhoD	Tat	N3K、N3K/L4K、D25R、D25R/F29R	+1.12-1.31倍	[50]
碱性淀粉酶 Alkaline amylase	YwbN	Tat	E4K/E9K、I16N/ L17S/W19S	+1.08-1.58倍	[53]
α-淀粉酶 α-Amylase	YfkN	Sec	I3G/Q4R	+13.00倍	[55]
地衣聚糖酶 Lichenase	H1	-	VNIAFMLF/RKIAGMAT	+4.60倍	[57]
角质酶 Cutinase	AmyE	Sec	F2D、F2E、F6W、K4L	+2-3倍	[58]
β-半乳糖苷酶 β-galactosidase	AmyX	Tat	N4I/ Y8F/N17I	+2.50倍	[59]

外源蛋白 Foreign protein	信号肽类型 Signal peptide type	分泌途径 Secretory pathway	最适信号肽 Optimal signal peptide	胞外活性 Extracellular activity	参考文献Reference
角质酶 Cutinase	173个Sec	Sec	Epr	4.67 U/mL	[12]
细胞质酯酶 Cytoplasmatic esterase	173个Sec	Sec	YwmC	1.50 U/mL	[12]
α-麦芽淀粉酶 Maltogenic α-amylase	173个Sec	Sec	YvcE	250.70 U/mL	[60]
蛋白酶 Protease	173个Sec	Sec	YbdN	+7.00倍	[61]
纳豆植酸酶 Natto phytase	173个Sec	Sec	Pbp	+2.00倍	[62]
α-淀粉酶 α-Amylase	173个Sec	Sec	YomL	113.25 U/mL	[63]
α-淀粉酶 α-Amylase	173个Sec	Sec	YojL	+3.5倍	[64]
α-淀粉酶 α-Amylase	173个Sec	Sec	PeI	+1.68倍	[65]
木聚糖酶 Xylanase	114个Sec+24个Tat	Sec	PhoB	327.20 U/mL	[66]