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

使用新型 P450 酶促进工业过程羟基化的综合方法

• 批准号: BB/L003546/1
• 负责人: Martina Micheletti
• 金额: $ 35.07万
• 依托单位: University College London
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2014
• 资助国家: 英国
• 起止时间: 2014 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FL003546%2F1
• 关键词: Integrative; approach; promote; hydroxylations; novel

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.

细胞色素P450是血蛋白单加氧酶的一个有趣的超家族，由于其巨大的反应能力，已经被研究了50多年。然而，以前对P450酶的研究已经证明了如何利用这种反应能力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，分离的酶需要有效的NAD（P）H再生系统。使用全细胞可以克服这些问题，但也可能出现关于底物摄取、产物/底物毒性、产物降解和底物溶解度差的其他生理问题。哺乳动物细胞色素P450可以代谢药物、类固醇和除草剂，然而，它们的表达产量可能有限。因此，新的P450基因的发现，可克隆到E。大肠杆菌和其它宿主表达系统使它们在工业应用上具有吸引力。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异生物质化合物在体内的作用。商业实例包括Bayer Pharmaceuticals使用P450单加氧酶进行羟基化以100吨/年生产的类固醇氢化可的松的生物转化，Pfizer和Bristol-Myers Squibb公司通过微生物氧化制羟甲纤维素生产孕酮的可的松。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。工业过程需要考虑使用这种技术，以优化P450催化反应的产物产率为目标的策略。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以前已经得到证明。特别是，气液传质速率的一系列工艺条件的准确定量提供了至关重要的洞察氧气的限制和它们对酶的表达，活性和工艺产率的影响的发生。将这种方法应用于挑战P450催化的生物转化将允许更快地确定优化的工艺条件，使其更适合大规模开发研究和工业应用。该提案的目的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价。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，这些变量有可能克服当前P450的局限性。生产量的增加被认为是鉴定具有适合商业应用的活性的P450的强制性因素，因为在生物催化剂的选择和生产和应用生物催化剂的过程中寻找协同改进的能力也是强制性的。