Self-induced Secretion Expression of Trehalose Synthase in Bacillus subtilis WB800n
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摘要
Bacillus subtilis is a non-pathogenic Gram-positive bacterium that has been widely used to produce industrially and pharmaceutically relevant proteins. Trehalose, a non reducing disaccharide used as protective agent and additive in foodstuffs and pharmaceutical products, can be prepared by trehalose synthase(TreS). The present work aims to construct a robust recombinant B. subtilis to achieve the secretory expression of TreS. In this study, the treS gene from Pseudomonas putida ATCC47054 was amplified by PCR and further cloned and expressed in B. subtilis WB800 N using pHT01 as expression vector. For avoiding the use of inducer, promoter P_(srfA) was used to replace the promoter P_(grac) in pHT01 and verify the activity of recombinant trehalose synthase. The TreS activity assay was employed to evaluate the performance of recombinant B. subtilis W800 N under different phosphate concentrations, carbon sources, carbon source concentrations, nitrogen sources and pH. The results showed that the P_(srfA) promoter had a good regulation effect under pH 8.0 condition, and the enzyme activity reached 6 000 U/L. Using the PhoD as the secretory signal peptide, TreS was effectively secreted, and the extracellular enzyme activity reached 2 100 U/L, accounting for 35% of the total enzyme activity. By optimizing the medium and fermentation conditions, the extracellular enzyme activity reached 6 900 U/L in 5 L of fermentor, and the proportion reached 48%. The pHT01-P_(srfA)-PhoD-treS secretory recombinant B. subtilis constructed in this study has great potential in trehalose synthase production.
Bacillus subtilis is a non-pathogenic Gram-positive bacterium that has been widely used to produce industrially and pharmaceutically relevant proteins. Trehalose, a non reducing disaccharide used as protective agent and additive in foodstuffs and pharmaceutical products, can be prepared by trehalose synthase(TreS). The present work aims to construct a robust recombinant B. subtilis to achieve the secretory expression of TreS. In this study, the treS gene from Pseudomonas putida ATCC47054 was amplified by PCR and further cloned and expressed in B. subtilis WB800 N using pHT01 as expression vector. For avoiding the use of inducer, promoter P_(srfA) was used to replace the promoter P_(grac) in pHT01 and verify the activity of recombinant trehalose synthase. The TreS activity assay was employed to evaluate the performance of recombinant B. subtilis W800 N under different phosphate concentrations, carbon sources, carbon source concentrations, nitrogen sources and pH. The results showed that the P_(srfA) promoter had a good regulation effect under pH 8.0 condition, and the enzyme activity reached 6 000 U/L. Using the PhoD as the secretory signal peptide, TreS was effectively secreted, and the extracellular enzyme activity reached 2 100 U/L, accounting for 35% of the total enzyme activity. By optimizing the medium and fermentation conditions, the extracellular enzyme activity reached 6 900 U/L in 5 L of fermentor, and the proportion reached 48%. The pHT01-P_(srfA)-PhoD-treS secretory recombinant B. subtilis constructed in this study has great potential in trehalose synthase production.
Bacillus subtilis is a non-pathogenic Gram-positive bacterium that has been widely used to produce industrially and pharmaceutically relevant proteins. Trehalose, a non reducing disaccharide used as protective agent and additive in foodstuffs and pharmaceutical products, can be prepared by trehalose synthase(TreS). The present work aims to construct a robust recombinant B. subtilis to achieve the secretory expression of TreS. In this study, the treS gene from Pseudomonas putida ATCC47054 was amplified by PCR and further cloned and expressed in B. subtilis WB800 N using pHT01 as expression vector. For avoiding the use of inducer, promoter P_(srfA) was used to replace the promoter P_(grac) in pHT01 and verify the activity of recombinant trehalose synthase. The TreS activity assay was employed to evaluate the performance of recombinant B. subtilis W800 N under different phosphate concentrations, carbon sources, carbon source concentrations, nitrogen sources and pH. The results showed that the P_(srfA) promoter had a good regulation effect under pH 8.0 condition, and the enzyme activity reached 6 000 U/L. Using the PhoD as the secretory signal peptide, TreS was effectively secreted, and the extracellular enzyme activity reached 2 100 U/L, accounting for 35% of the total enzyme activity. By optimizing the medium and fermentation conditions, the extracellular enzyme activity reached 6 900 U/L in 5 L of fermentor, and the proportion reached 48%. The pHT01-P_(srfA)-PhoD-treS secretory recombinant B. subtilis constructed in this study has great potential in trehalose synthase production.
引文
[1]OHTAKE S,WANG YJ.Trehalose:current use and future applications[J].Journal of Pharmaceutical Sciences,2011,100(6):2020-2053.
[2]PAN YT,CARROLL JD,ASANO N,et al.Trehalose synthase converts glycogen to trehalose[J].Febs journal,2008,275(13):3408-3420
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[4]PANAHI R,VASHEGHANI FE,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]KENNEY TJ,YORK K,YOUNGMAN P,et al.Genetic evidence that RNA polymerase associated withσA 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):9109.
[8]HAMOEN LW,KAUSCHE D,MARAHIEL MA,et al.The Bacillus subtilis transition state regulator Abr B binds to the-35 promoter region of com K[J].Fems Microbiology Letters,2003,218(2):299-304.
[9]LEE SJ,PAN JG,PARK SH,et al.Development of a stationary phasespecific autoinducible expression system in Bacillus subtilis[J].Journal of Biotechnology,2010,149(1):16-20.
[10]ROSHANI PATEL,SARAH M SMITH,COLIN ROBINSON.Protein transport by the bacterial Tat pathway[J].Biochimica et Biophysica Acta,1843(2014)1620-1628.
[11]GUAN CR,CUI WJ,CHENG JT,et al.Construction of a highly active secretory expression system via an engineered dual promoter and a highly efficient signal peptide in Bacillus subtilis[J].New Biotechnology,2016,33(3):372-379.
[2]PAN YT,CARROLL JD,ASANO N,et al.Trehalose synthase converts glycogen to trehalose[J].Febs journal,2008,275(13):3408-3420
[3]ZUBER P,HEALY JM,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 FE,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]KENNEY TJ,YORK K,YOUNGMAN P,et al.Genetic evidence that RNA polymerase associated withσA 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):9109.
[8]HAMOEN LW,KAUSCHE D,MARAHIEL MA,et al.The Bacillus subtilis transition state regulator Abr B binds to the-35 promoter region of com K[J].Fems Microbiology Letters,2003,218(2):299-304.
[9]LEE SJ,PAN JG,PARK SH,et al.Development of a stationary phasespecific autoinducible expression system in Bacillus subtilis[J].Journal of Biotechnology,2010,149(1):16-20.
[10]ROSHANI PATEL,SARAH M SMITH,COLIN ROBINSON.Protein transport by the bacterial Tat pathway[J].Biochimica et Biophysica Acta,1843(2014)1620-1628.
[11]GUAN CR,CUI WJ,CHENG JT,et al.Construction of a highly active secretory expression system via an engineered dual promoter and a highly efficient signal peptide in Bacillus subtilis[J].New Biotechnology,2016,33(3):372-379.