Self-induced Secretion Expression of Trehalose Synthase in Bacillus subtilis WB800n  

作　　者：Xihui WANG Hongling LIU Songsen SUI Shaojie YANG Tengfei WANG 

机构地区：[1]State Key Laboratory of Biobased Material and Green Papermaking(LBMP),Qilu University of Technology [2]Shandong Key Laboratory of Microbial Engineering,Qilu University of Technology [3]Key Laboratory of Industrial Fermentation Microbiology(Tianjin University of Science & Technology),Ministry of Education and Tianjin Key Lab of Industrial Microbiology,Tianjin University of Science & Technology [4]Zhucheng Dongxiao Biotechnology Co.,Ltd.

出　　处：《Agricultural Biotechnology》2019年第2期216-219,225,共5页农业生物技术（英文版）

基　　金：Supported by the National Nature Science Foundation of China(No.31501413);Shandong key project of Research&Development plan(No.2017GSF221019);Young doctorate Cooperation Fund Project,Qi Lu University of Technology(Shandong Academy of Sciences)(No.2017BSHZ021);Shandong Natural Science Foundation Project(ZR2017BC072)

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.

关 键 词：TREHALOSE SYNTHASE BACILLUS SUBTILIS W800N P srfA promoter Self-induction PhoD signal peptide 

分 类 号：S[农业科学]