Integrative approach to promote hydroxylations with novel P450 enzymes for industrial processes

细胞色素p450是一个有趣的血红蛋白单加氧酶超家族，由于其巨大的反应能力，已经被研究了50多年。然而，先前对P450酶的研究表明，如何利用这种反应能力可能是一项具有挑战性的任务。在某些情况下，p450酶是膜结合的，它们需要复杂的电子传递系统。例如，分离酶需要高效的NAD(P)H再生系统。使用全细胞可以克服这些问题，但也可能出现其他生理问题，如底物摄取、产物/底物毒性、产物降解和底物溶解度差。哺乳动物细胞色素p450可以代谢药物、类固醇和除草剂，但其表达量有限。因此，可以克隆到大肠杆菌和其他宿主表达系统的新型p450的发现使它们具有工业应用的吸引力。由于上述原因，目前使用p450的工业过程数量有限。传统上，P450单加氧酶已被用于毒性测定或阐明体内外源化合物的作用。商业上的例子包括拜耳公司使用P450单加氧酶以100吨/年的速度生产类固醇氢化可的松的生物转化，辉瑞公司使用黄体酮生产可的松，百时美施贵宝公司通过微生物氧化压实蛋白销售普伐他汀。未来的应用可能包括抗生素合成、抗癌药物合成、生物修复、聚合物和香料生产。考虑使用该技术的工业过程需要旨在优化P450催化反应产率的策略。使用自动化微尺度技术快速生成整个生物过程数据的能力先前已得到证明。特别是，在一系列工艺条件下准确量化气液传质速率，为氧气限制的发生及其对酶表达、活性和工艺产量的影响提供了至关重要的见解。将这种方法应用于具有挑战性的P450催化生物转化，可以更快地确定优化的工艺条件，使其更适合大规模的开发研究和工业吸收。该提案的目标是建立自动化的微尺度方法，用于基于p450的新工艺的全过程评估。在这个项目中，以前在UCL进行的研究将通过引入能够并行评估大量变量的微尺度方法来扩展，这些方法有可能克服当前p450的局限性。提高产量被认为是确定具有适合商业应用的活性的p450的必要条件，同时也是在生物催化剂的选择和生产和应用过程中寻找协同改进的能力。

Cytochrome P450s are an interesting superfamily of haemoprotein monoooxygenases, which have been studied for over 50 years due to their vast reaction ability. However, previous studies on P450 enzymes have demonstrated how exploiting this reaction ability can be a challenging task. In some cases P450s enzymes are membrane bound and they require complex electron transport systems. For example isolated enzymes require efficient NAD(P)H regeneration systems. Using whole cells can overcome these issues but other physiological problems concerning substrate uptake, product/substrate toxicity, product degradation and poor substrate solubility may also arise. Mammalian cytochrome P450s can metabolise drugs, steroids and herbicides, however, their expression yields can be limited. Consequently, the discovery of novel P450s which can be cloned into E. coli and other host expression systems makes them attractive for industrial use. For the aforementioned reasons the number of industrial processes utilising P450s is currently limited. Traditionally, P450 monooxygenases have been utilised in toxicity determination or to elucidate the effects of xenobiotic compounds in vivo. Commercial examples include the biotransformation of the steroid hydrocortisone produced at 100 ton/yr by Bayer Pharmaceuticals using P450 monooxygenases to conduct hydroxylation, the production of cortisone from progesterone by Pfizer and Bristol-Myers Squibb Company markets Pravastatin by microbial oxidation of compactin. Future applications could include antibiotic synthesis, anticancer drug synthesis, bioremediation, polymer and flavour production. Strategies aimed at optimising product yields of P450 catalysed reactions are needed for industrial processes to consider using this technology. The ability to rapidly generate data on whole bioprocesses using automated microscale technologies has been previously demonstrated. In particular, accurate quantification of gas liquid mass transfer rates for a range of process conditions provided crucial insight into the occurrence of oxygen limitations and their effect on enzyme expression, activity and process yield. The application of this approach to challenging P450 catalysed bioconversions would allow quicker identification of optimised process conditions, making them more amenable to large scale development studies and industrial uptake. The proposal aim will be to establish automated microscale methodologies for the whole process evaluation of P450-based novel processes. In this project previous studies conducted at UCL will be extended by the introduction of microscale methods able to evaluate a large number of variables in parallel that have the potential to overcome the limitations of current P450s. An increase in throughput is considered as mandatory to identify P450s with activities suitable for commercial application as is the ability to look for synergistic improvements in both choice of biocatalyst and the processes by which it is produced and applied.