An indole-deficient Escherichia coli strain improves screening of cytochromes P450 for biotechnological applications
An indole-deficient Escherichia coli strain improves screening of cytochromes P450 for biotechnological applications
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DOI：
10.1002/bab.1488
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发表时间：
2017-05-01
影响因子：
2.8
通讯作者：
Bernhardt, Rita
中科院分区：
文献类型：
--
作者：
Brixius-Anderko, Simone;Hannemann, Frank;Bernhardt, Rita
大肠杆菌已发展成为异源细胞色素 P450 生产的有吸引力的生物体，但在某些情况下，由于色氨酸酶 (TnaA) 形成细胞色素 P450 抑制剂吲哚，因此在分子进化背景下筛选孤儿细胞色素 P450 或突变体文库时，大肠杆菌作为宿主受到限制。为了克服这种效应，我们破坏了大肠杆菌 C43(DE3) 的 tnaA 基因位点，并用三种吲哚敏感细胞色素 P450、粘细菌 CYP264A1 和 CYP109D1 以及牛类固醇生成 CYP21A2 评估了新菌株的全细胞底物转化能力。对于纯化的CYP264A1和CYP21A2，半数最大抑制吲哚浓度测定为140和500μM，其在复合培养基中培养大肠杆菌期间出现的生理浓度范围内。与 C43(DE3) 细胞相比，使用 C43(DE3) Delta tnaA 进行生物转化，CYP21A2 的产物形成量提高了 30%，而使用 CYP264A1 的产物形成量甚至增加了四倍。在基于 CYP109D1 的全细胞转化中，将吲哚转化为靛蓝，我们可以成功避免这种反应。微孔板形式的结果表明，我们新设计的菌株是在复杂培养基中快速有效筛选吲哚影响的细胞色素 P450 的合适宿主。 (C) 2016 国际生物化学与分子生物学联合会
Escherichia coli has developed into an attractive organism for heterologous cytochrome P450 production, but, in some cases, was restricted as a host in view of a screening of orphan cytochromes P450 or mutant libraries in the context of molecular evolution due to the formation of the cytochrome P450 inhibitor indole by the enzyme tryptophanase (TnaA). To overcome this effect, we disrupted the tnaA gene locus of E. coli C43(DE3) and evaluated the new strain for whole-cell substrate conversions with three indole-sensitive cytochromes P450, myxobacterial CYP264A1, and CYP109D1 as well as bovine steroidogenic CYP21A2. For purified CYP264A1 and CYP21A2, the half maximal inhibitory indole concentration was determined to be 140 and 500 mu M, which is within the physiological concentration range occurring during cultivation of E. coli in complex medium. Biotransformations with C43(DE3) Delta tnaA achieved a 30% higher product formation in the case of CYP21A2 and an even fourfold increase with CYP264A1 compared with C43(DE3) cells. In whole-cell conversion based on CYP109D1, which converts indole to indigo, we could successfully avoid this reaction. Results in microplate format indicate that our newly designed strain is a suitable host for a fast and efficient screening of indole-influenced cytochromes P450 in complex medium. (C) 2016 International Union of Biochemistry and Molecular Biology, Inc.