海藻糖合酶在枯草芽孢杆菌中的高效表达
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摘要
以枯草芽孢杆菌(Bacillus subtilis)为表达平台,海藻糖合酶为表达蛋白,研究了单启动子和多个启动子串联对外源蛋白表达的影响。分别选择6个启动子构建P_(srfA)、P_(43)单启动子和P_(abrB-spoVG-LytR-mmgA)四个时期特异性启动子串联表达系统进行验证。根据实验结果分析,组成型启动子P_(43)转录强度最高,摇瓶中酶活达到3 381 U/g,串联启动子P_(abrB-spoVG-LytR-mmgA)强度次之。针对验证酶活最高的重组菌pHT01-P_(43)-treS进行发酵优化,并在5 L发酵罐中进行放大实验,酶活达到7 215 U/g。实现了海藻糖合酶在枯草芽孢杆菌中自诱导高效表达,为获得高效率制备海藻糖合成酶的表达系统奠定了基础。
The effects of single promoter and multiple promoters on the expression of foreign proteins were studied using Bacillus subtilis as an expression platform and trehalose synthase as an expression protein. Six promoters were selected to construct single promoters P_(srfA) and P_(43). A four-stage specific promoter tandem expression system P_(abrB-spoVG-LytR-mmgA) was also constructed for validation. According to the experimental results, the constitutive promoter P_(43) had the highest transcriptional strength, the enzyme activity in the shake flask reached 3 381 U/g. The tandem promoter P_(abrB-spoVG-LytR-mmgA) had the second highest strength. The fermentation was optimized for recombinant enzyme pHT01-P_(43)-treS, which was verified to have the highest activity, and the scale-up experiment was carried out in a 5 L fermentor, and the enzyme activity reached 7 215 U/g. The self-induced and high-efficiency expression of trehalose synthase in B. subtilis was achieved, which laid a foundation for obtaining a highly efficient expression system of trehalose synthase.
The effects of single promoter and multiple promoters on the expression of foreign proteins were studied using Bacillus subtilis as an expression platform and trehalose synthase as an expression protein. Six promoters were selected to construct single promoters P_(srfA) and P_(43). A four-stage specific promoter tandem expression system P_(abrB-spoVG-LytR-mmgA) was also constructed for validation. According to the experimental results, the constitutive promoter P_(43) had the highest transcriptional strength, the enzyme activity in the shake flask reached 3 381 U/g. The tandem promoter P_(abrB-spoVG-LytR-mmgA) had the second highest strength. The fermentation was optimized for recombinant enzyme pHT01-P_(43)-treS, which was verified to have the highest activity, and the scale-up experiment was carried out in a 5 L fermentor, and the enzyme activity reached 7 215 U/g. The self-induced and high-efficiency expression of trehalose synthase in B. subtilis was achieved, which laid a foundation for obtaining a highly efficient expression system of trehalose synthase.
引文
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[25] ZWEERS J C,BARA K I,BECHER D,et al.Towards the development of Bacillus subtilis as a cell factory for membrane proteins and protein complexes[J].Microbial Cell Factories,2018,4:7-10.
[2] PAN Y T,CARROLL J D,ASANO N,et al.Trehalose synthase converts glycogen to trehalose[J].Febs Journal,2008,275(13):3 408-3 420
[3] ZUBER P,HEALY J M,LOSICK R.Effects of plasmid propagation of a sporulation promoter on promoter utilization and sporulation in Bacillus subtilis[J].Journal of Bacteriology,1987,169(2):461.
[4] PANAHI R,VASHEGHANI F E,SHOJAOSADATI SA,et al.Induction of Bacillus subtilis expression system using environmental stresses and glucose starvation[J].Annals of Microbiology,2014,64(2):879-882.
[5] GUAN C,CUI W,CHENG J,et al.Development of an efficient autoinducible expression system by promoter engineering in Bacillus subtilis[J].Microbial Cell Factories,2016,15(1):66.
[6] YANG S,DU G,CHEN J,et al.Characterization and application of endogenous phase-dependent promoters in Bacillus subtilis[J].Applied Microbiology and Biotechnology,2017,101(10):1-11.
[7] ZUBER P,LOSICK R.Role of AbrB in Spo0A- and Spo0B-dependent utilization of a sporulation promoter in Bacillus subtilis[J].Journal of Bacteriology,1987,169(5):23-2 230.
[8] LYCZAK J B,ZHANG K,SAXON A,et al.Roles of PucR,GlnR,and TnrA in regulating expression of the Bacillus subtilis ure P43 promoter[J].Journal of Bacteriology,2002,184(21):6 060.
[9] 刘强,王腾飞,汪俊卿,等.麦芽糖诱导海藻糖合成酶基因在B.subtilis WB800n中的表达[J].生物技术通报,2017,33(3):106-115.
[10] KENNEY T J,YORK K,YOUNGMAN P,et al.Genetic evidence that RNA polymerase associated with σA factor uses a sporulation-specific promoter in Bacillus subtilis[J].Proceedings of the National Academy of Sciences of the United States of America,1989,86(23):9 109.
[11] HAMOEN L W,KAUSCHE D,MARAHIEL M A,et al.The Bacillus subtilis transition state regulator AbrB binds to the -35 promoter region of comK[J].Fems Microbiology Letters,2003,218(2):299-304.
[12] 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):16-20.
[13] SONG Y,FU G,DONG H,et al.High-efficiency secretion of β-mannanase in Bacillus subtilis through protein synthesis and secretion optimization[J].Journal of Agricultural and Food Chemistry,2017,65 (12):2 540-2 548.
[14] 吕鑫,王腾飞,汪俊卿,等.Lys490饱和突变提高海藻糖合酶转化率的研究[J].食品与发酵工业,2018,44 (1):60-65.
[15] PHAN TTP,NGUYEN H D,SCHUMANN W.Development of a strong intracellular expression system for Bacillus subtilis by optimizing promoter elements[J].Journal of Biotechnology,2012,157(1):167-172.
[16] LIU H L,YANG S J,LIU Q,et al.A process for production of trehalose by recombinant trehalose synthase and its purification[J].Enzyme and Microbial Technology,2018,113:83
[17] SCHUMANN W.Production of recombinant proteins in Bacillus subtilis[J].Advance in Applied Microbiology,2007,27(3):137-189
[18] SCHUMANN W.Production of recombinant proteins in Bacillus subtilis Adv[J].Applied Microbiology and Biotechnology,2007,62:137e189.
[19] PANAHI R,VASHEGHANI-FARAHANI E,SHOJAOS-ADATI S A,et al.Induction of Bacillus subtilis,expression system using environmental stresses and glucose starvation[J].Annals of Microbiology,2014,64(2):879-882.
[20] SUN H,BIE X,LU F,et al.Enhancement of surfactin production of Bacillus subtilis fmbR by replacement of the native promoter with the pspac promoter[J].Canadian Journal of Microbiology,2009,55(8):1 003-1 006.
[21] JIAO S,LI X,YU H,et al.In situ enhancement of surfactin biosynthesis in Bacillus subtilis using novel artificial inducible promoters[J].Biotechnology and Bioengineering,2017,114(4):832.
[22] KIM J H,ROH H J,LEE J K,et al.Comparison of P_(aprE) P_(amyE),and P_(p43) promoter strength for ss-galactosidase and staphylokinase expression in Bacillus subtilis[J].Biotechnology and Bioprocess Engineering,2008,13(3):313-318.
[23] WENZEL M.Charakterisierung des regulators ManR und die anwendung des mannose expressionssystems in Bacillus subtilis[J].Applied and Environmental Microbiology,2013.
[24] YANG M M,ZHANG W W,CHEN Y L,et al.Development of a Bacillus subtilis expression system using the improved Pglv promoter[J].Microbial Cell Factories,2010,9(1):55.
[25] ZWEERS J C,BARA K I,BECHER D,et al.Towards the development of Bacillus subtilis as a cell factory for membrane proteins and protein complexes[J].Microbial Cell Factories,2018,4:7-10.