Department of Chemical Engineering, The University of Melbourne, Parkville, VIC, 3010, Australia.
The Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, 30 Flemington Rd, Parkville, VIC, 3010, Australia.
J Ind Microbiol Biotechnol. 2020 Jul;47(6-7):449-464. doi: 10.1007/s10295-020-02283-7. Epub 2020 Jun 7.
Cytochrome P450 enzymes catalyse reactions of significant industrial interest but are underutilised in large-scale bioprocesses due to enzyme stability, cofactor requirements and the poor aqueous solubility and microbial toxicity of typical substrates and products. In this work, we investigate the potential for preparative-scale N-demethylation of the opium poppy alkaloid noscapine by a P450 (CYP102A1) mutant enzyme in a whole-cell biotransformation system. We identify and address several common limitations of whole-cell P450 biotransformations using this model N-demethylation process. Mass transfer into Escherichia coli cells was found to be a major limitation of biotransformation rate and an alternative Gram-positive expression host Bacillus megaterium provided a 25-fold improvement in specific initial rate. Two methods were investigated to address poor substrate solubility. First, a biphasic biotransformation system was developed by systematic selection of potentially biocompatible solvents and in silico solubility modelling using Hansen solubility parameters. The best-performing biphasic system gave a 2.3-fold improvement in final product titre compared to a single-phase system but had slower initial rates of biotransformation due to low substrate concentration in the aqueous phase. The second strategy aimed to improve aqueous substrate solubility using cyclodextrin and hydrophilic polymers. This approach provided a fivefold improvement in initial biotransformation rate and allowed a sixfold increase in final product concentration. Enzyme stability and cell viability were identified as the next parameters requiring optimisation to improve productivity. The approaches used are also applicable to the development of other pharmaceutical P450-mediated biotransformations.
细胞色素 P450 酶催化具有重要工业意义的反应，但由于酶稳定性、辅因子要求以及典型底物和产物的较差的水溶解度和微生物毒性，它们在大规模生物工艺中未得到充分利用。在这项工作中，我们研究了在全细胞生物转化系统中，通过 P450（CYP102A1）突变酶对罂粟碱生物碱北美黄连碱进行制备规模 N-去甲基化的潜力。我们使用这种模型 N-去甲基化过程来确定和解决全细胞 P450 生物转化的几个常见限制。质量传递到大肠杆菌细胞中被发现是生物转化速率的主要限制，替代的革兰氏阳性表达宿主巨大芽孢杆菌提供了 25 倍的比初始速率提高。研究了两种方法来解决较差的底物溶解度问题。首先，通过有针对性地选择潜在生物相容的溶剂和使用 Hansen 溶解度参数的计算溶解度建模，开发了两相生物转化系统。与单相系统相比，性能最佳的两相系统最终产物产率提高了 2.3 倍，但由于水相中的底物浓度低，初始生物转化速率较慢。第二种策略旨在使用环糊精和亲水性聚合物来提高水相中的底物溶解度。这种方法使初始生物转化速率提高了五倍，并允许最终产物浓度提高六倍。酶稳定性和细胞活力被确定为需要优化以提高生产力的下一个参数。所使用的方法也适用于其他药物 P450 介导的生物转化的开发。
