碱性多聚半乳糖醛酸裂解酶在枯草芽孢杆菌中的高水平胞外生产与优化调控元件。
High-level extracellular production of alkaline polygalacturonate lyase in Bacillus subtilis with optimized regulatory elements.
机构信息
The Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, China; School of Biotechnology, Jiangnan University, Wuxi 214122, China.
The Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, China; School of Biotechnology, Jiangnan University, Wuxi 214122, China.
出版信息
Bioresour Technol. 2013 Oct;146:543-548. doi: 10.1016/j.biortech.2013.07.129. Epub 2013 Aug 3.
The present work aims to construct a robust recombinant Bacillus subtilis to achieve secretory production of alkaline polygalacturonate lyase (PGL). First, 6 signal peptides (amyX, bpr, vpr, yvgO, wapA and nprE) were screened with a semi-rational approach and comparatively investigated their effects on the production of PGL. The signal peptide bpr directed efficient PGL secretory expression and increased PGL titer to 313.7 U mL(-1). By optimizing and applying strong promoter P43 and Shine-Dalgarno sequence, higher titer of 446.3 U mL(-1) PGL was achieved. Finally, the capacity of the recombinant B. subtilis WB43CB was evaluated with a fed-batch strategy in 3 L fermentor. The PGL titer reached 632.6 U mL(-1) with a productivity of 17.6 U mL(-1) h(-1), which was the highest secretory production of PGL by the B. subtilis system. The recombinant B. subtilis strain WB43CB constructed in the present work has great potential in production of alkaline PGL.
本工作旨在构建一株稳健的重组枯草芽孢杆菌以实现碱性多聚半乳糖醛酸裂解酶（PGL）的分泌生产。首先，采用半理性方法筛选了 6 种信号肽（amyX、bpr、vpr、yvgO、wapA 和 nprE），并比较了它们对 PGL 生产的影响。信号肽 bpr 指导 PGL 的有效分泌表达，并将 PGL 效价提高到 313.7 U mL(-1)。通过优化和应用强启动子 P43 和 Shine-Dalgarno 序列，获得了更高的 PGL 效价 446.3 U mL(-1)。最后，在 3 L 发酵罐中采用分批补料策略评估了重组枯草芽孢杆菌 WB43CB 的能力。PGL 的效价达到 632.6 U mL(-1)，比生产率为 17.6 U mL(-1) h(-1)，这是枯草芽孢杆菌系统中 PGL 的最高分泌产量。本工作构建的重组枯草芽孢杆菌 WB43CB 在碱性 PGL 的生产中具有很大的潜力。
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