Kaderbhai M A, Ugochukwu C C, Kelly S L, Lamb D C
Institute of Biological Sciences, University of Wales Aberystwyth, Aberystwyth SY23 3DA, Wales, United Kingdom.
Appl Environ Microbiol. 2001 May;67(5):2136-8. doi: 10.1128/AEM.67.5.2136-2138.2001.
CYP105D1, a cytochrome P450 from Streptomyces griseus, was appended at its amino terminus to the secretory signal of Escherichia coli alkaline phosphatase and placed under the transcriptional control of the native phoA promoter. Heterologous expression in E. coli phosphate-limited medium resulted in abundant synthesis of recombinant CYP105D1 that was translocated across the bacterial inner membrane and processed to yield authentic, heme-incorporated P450 within the periplasmic space. Cell extract and whole-cell activity studies showed that the periplasmically located CYP105D1 competently catalyzed NADH-dependent oxidation of the xenobiotic compounds benzo[a]pyrene and erythromycin, further revealing the presence in the E. coli periplasm of endogenous functional redox partners. This system offers substantial advantages for the application of P450 enzymes to whole-cell biotransformation strategies, where the ability of cells to take up substrates or discard products may be limited.
来自灰色链霉菌的细胞色素P450 CYP105D1在其氨基末端附加了大肠杆菌碱性磷酸酶的分泌信号，并置于天然phoA启动子的转录控制之下。在大肠杆菌磷酸盐限制培养基中的异源表达导致重组CYP105D1大量合成，该重组蛋白穿过细菌内膜并进行加工，从而在周质空间内产生真正的、结合血红素的P450。细胞提取物和全细胞活性研究表明，位于周质的CYP105D1能够有效地催化异源生物化合物苯并[a]芘和红霉素的NADH依赖性氧化，进一步揭示了大肠杆菌周质中存在内源性功能性氧化还原伙伴。该系统为将P450酶应用于全细胞生物转化策略提供了显著优势，因为细胞摄取底物或丢弃产物的能力可能有限。
