Herbicides are often selective and kill only weeds without affecting crops. In rice, CYP81A6 is responsible for tolerance toward acetolactate synthase herbicides such as bensulfuron-methyl (BSM). CYP81A6 is thought to metabolize BSM into a nontoxic metabolite, that is, O-demethylated BSM. However, direct evidences for this reaction have not been reported, likely due to difficulties in the heterologous expression of plant P450s in bacterial expression systems. In this study, we established CYP81A6 expression in Escherichia coli via modification of its N-terminal basic region coding sequence, optimization of cultivation temperature, and selection of E. coli strains. Heterologous production of CYP81A6 was evaluated by the conversion of BSM in autoinduction medium after the culture, compared to that with an empty vector control. The optimized heterologous production condition of rice CYP81A6 in E. coli using 48-well plates involved replacement of the N-terminal basic region with that of CYP2C3, the use of E. coli BL21(DE3) as an expression host, and culture at 34°C. Further, co-expression of gene encoding glucose dehydrogenase enhanced the conversion of 0.5 mM BSM from 58.7 to 73.1% in the optimized condition. In the optimization process, we detected two metabolites, M1 and M2. Mass spectrometry and ¹H nuclear magnetic resonance analyses indicated that M1 was O-demethylated BSM. Although we could not isolate M2 because of its low abundance in the medium, mass spectrometry analysis suggested that it was the monooxygenated (+O) form of BSM. Overall, these results confirm that CYP81A6 catalyzes the formation of O-demethylated BSM from BSM.

中文翻译：

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水稻（Oryza sativa）CYP81A6 在大肠杆菌中的异源表达及苄嘧磺隆代谢物的结构分析

除草剂通常具有选择性，只杀死杂草而不影响作物。在水稻中，CYP81A6负责耐受乙酰乳酸合酶除草剂，如苯磺隆 (BSM)。CYP81A6 被认为将 BSM 代谢为无毒代谢物，即O-去甲基化 BSM。然而，尚未报道该反应的直接证据，可能是由于植物P450在细菌表达系统中异源表达的困难。在本研究中，我们通过修饰其 N 端基本区编码序列、优化培养温度和选择大肠杆菌来建立CYP81A6在大肠杆菌中的表达菌株。CYP81A6 的异源产生是通过 BSM 在培养后的自诱导培养基中的转化来评估的，与空载体对照相比。在稻CYP81A6的优化异源生产条件大肠杆菌使用48孔板参与替换与CYP2C3，使用的N末端碱性区域的大肠杆菌在34 BL21（DE3）作为表达宿主，并培养℃。此外，在优化条件下，编码葡萄糖脱氢酶的基因的共表达将 0.5 mM BSM 的转化率从 58.7% 提高到 73.1%。在优化过程中，我们检测到两种代谢物，M1 和 M2。质谱和¹ H 核磁共振分析表明 M1 是O-去甲基化的 BSM。虽然我们无法分离 M2，因为它在培养基中的丰度低，但质谱分析表明它是 BSM 的单氧化 (+O) 形式。总体而言，这些结果证实 CYP81A6 催化BSM形成O-去甲基化 BSM。