新型除草剂高粱素生物合成关键酶ARS的双控表达质粒构建及表达条件优化
Dualcontrolplasmidconstructionandexpressionoptimizationof
ARSforanovelsorgoleoneherbicidebiosynthesis
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摘要: 利用化感物质开发除草剂是目前新型除草剂发展的一个重要方向。高粱素是高粱中分泌的一种化感物质，可对多种杂草的生长表现出显著的抑制作用。高粱烷基间苯二酚合酶 (alkylresorcinol synthase，Sb_ARS) 是高粱素生物合成的关键酶。当以大肠杆菌Escherichia coli作为宿主进行Sb_ARS的胞内表达时，细胞破碎是其中最为费时且最昂贵的步骤之一。本研究通过构建新型双控表达质粒pBM123-ars，实现了Sb_ARS在大肠杆菌BL21 (DE3) 中的温控自裂解异源表达；在此基础上，通过优化诱导表达条件，可获得最高的Sb_ARS酶活力 (相对酶活力为160.04% ± 8.12%) 及最高的裂解率 (82.81% ± 3.14%)。研究结果可为新型除草剂高粱素的生物合成奠定理论基础。Abstract: Allelochemicals offer great potential for the development of future herbicides. Sorgoleone is a kind of allelochemicals secreted by Sorghum bicolor. It can significantly inhibit the growth of many weeds. S. bicolor alkylresorcinol synthase (Sb_ARS) is a key regulatory enzyme in the pathway of sorgoleone biosynthesis. When Sb_ARS is expressed intracellularly using Escherichia coli as the host, the disruption of cell membrane can be time-consuming and costly. In this study, Sb_ARS gene was optimized, chemically synthesized and expressed in a heat-inducible autolytic E. coli BL21(DE3) system by a novel dual control expression plasmid. After optimizing IPTG/heat-induced expression conditions, Sb_ARS activity was found to be 160.04% ± 8.12%, and the lysis efficiency was improved to 82.81% ± 3.14%. These results would lay a foundation for the biosynthesis of new herbicide sorghum.
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