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P450s是血蛋白单氧酶的有趣超家族，由于其巨大的反应能力，已对其进行了50多年的研究。但是，先前对P450酶的研究证明了这种反应能力的利用如何成为一项具有挑战性的任务。在某些情况下，P450S酶是膜结合的，它们需要复杂的电子传输系统。例如，孤立的酶需要有效的NAD（P）H再生系统。使用全细胞可以克服这些问题，但是还可能出现有关底物摄取，产品/底物毒性，产品降解和底物溶解性差的其他生理问题。哺乳动物细胞色素P450可以代谢药物，类固醇和除草剂，但是，它们的表达产量可能受到限制。因此，可以将新颖的P450的发现可以克隆到大肠杆菌和其他宿主表达系统中，使其对工业使用有吸引力。由于上述原因，目前利用P450的工业流程数量有限。传统上，P450单加氧酶已用于确定毒性或阐明体内异生元化合物的作用。商业示例包括使用P450单加氧酶以100吨/年生产的类固醇氢化可的松的生物转化，从而进行羟基化，辉瑞公司和Bristol-Myers Marke Comparets Pravastatins Pravastatin pravastatin compactactin compactsication compactication of Compactication of Pfizer和Bristol-Myers Markets pfizer和Bristol-Myers Markets从孕激素中生产孕酮。未来的应用可能包括抗生素合成，抗癌药合成，生物修复，聚合物和风味产生。旨在优化P450催化反应的产品产量的策略是考虑使用该技术的策略。先前已经证明了使用自动微观技术快速生成整个生物过程的数据的能力。特别是，对一系列过程条件的气体液体传质速率的准确量化为氧气限制的发生及其对酶表达，活性和过程产量的影响提供了至关重要的见解。这种方法在挑战P450催化的生物转化中的应用将使更快地识别优化的过程条件，从而使它们更适合大规模开发研究和工业摄取。该提案的目的是建立自动化微观方法，用于基于P450的新过程的整个过程评估。在该项目中，将通过引入能够评估大量并联变量的微观方法来扩展在UCL进行的先前研究，该方法有可能克服当前P450的局限性。吞吐量的增加被认为是识别适合商业应用活动的P450的强制性，因为能够在生物催化剂的选择及其生产和应用过程中寻求协同改善。