Laboratory of Chemical Biotechnology, Department of Biochemical and Chemical Engineering, TU Dortmund University, Emil-Figge-Strasse 66, 44227 Dortmund, Germany.
J Ind Microbiol Biotechnol. 2012 Feb;39(2):275-87. doi: 10.1007/s10295-011-1026-4. Epub 2011 Aug 24.
Mammalian cytochrome P450 enzymes are of special interest as biocatalysts for fine chemical and drug metabolite synthesis. In this study, the potential of different recombinant microorganisms expressing rat and human cyp1a1 genes is evaluated for such applications. The maximum specific activity for 7-ethoxyresorufin O-deethylation and gene expression levels were used as parameters to judge biocatalyst performance. Under comparable conditions, E. coli is shown to be superior over the use of S. cerevisiae and P. putida as hosts for biocatalysis. Of all tested E. coli strains, E. coli DH5α and E. coli JM101 harboring rat CYP1A1 showed the highest activities (0.43 and 0.42 U g⁻¹(CDW), respectively). Detection of active CYP1A1 in cell-free E. coli extracts was found to be difficult and only for E. coli DH5α, expression levels could be determined (41 nmol g⁻¹(CDW)). The presented results show that efficient expression of mammalian cyp1a1 genes in recombinant microorganisms is troublesome and host-dependent and that enhancing expression levels is crucial in order to obtain more efficient biocatalysts. Specific activities currently obtained are not sufficient yet for fine chemical production, but are sufficient for preparative-scale drug metabolite synthesis.
哺乳动物细胞色素 P450 酶作为精细化学品和药物代谢物合成的生物催化剂具有特殊的意义。在这项研究中，评估了表达大鼠和人 CYP1A1 基因的不同重组微生物在这些应用中的潜力。7-乙氧基resorufin O-脱乙基化的最大比活性和基因表达水平被用作判断生物催化剂性能的参数。在可比条件下，与使用酿酒酵母和恶臭假单胞菌作为生物催化剂宿主相比，大肠杆菌表现出优越性。在所测试的所有大肠杆菌菌株中，携带大鼠 CYP1A1 的大肠杆菌 DH5α 和大肠杆菌 JM101 显示出最高的活性（分别为 0.43 和 0.42 U g⁻¹(CDW)）。发现细胞游离大肠杆菌提取物中活性 CYP1A1 的检测很困难，仅对大肠杆菌 DH5α 可以确定表达水平（41 nmol g⁻¹(CDW)）。所呈现的结果表明，哺乳动物 cyp1a1 基因在重组微生物中的高效表达是麻烦的且依赖于宿主，并且提高表达水平对于获得更有效的生物催化剂至关重要。目前获得的比活性还不足以用于精细化学品的生产，但足以用于药物代谢物的制备规模合成。
