Development of P450 and BVMO biocatalysts for the stereocontrolled production of the sulfoxide garlic components

该项目是国王学院和Almac之间新合作的结果，旨在开发新型生物催化剂P450和BVMO酶，用于大蒜及其衍生物的抗菌亚砜成分的可持续工业生产。该项目完全符合BBSRC关于“工业生物技术的新战略方法”和“对抗抗菌素耐药性”的战略重点。此外，英国化学工业热衷于确定新的战略研究，以支持创新的、可持续的制造路线的发展，以制备高价值的化学品和药物。蒜素、蒜素和蒜烯是在大蒜中发现的手性亚砜化合物。它们具有重要的饮食和治疗作用，包括对抗抗生素耐药细菌的潜在活性，这是英国医疗保健系统的主要威胁。从大蒜中提取对映纯亚砜可能存在问题，因为该过程的苛刻条件可能导致亚砜立体中心的外消旋化，从而导致抗菌活性的丧失。大蒜素及相关化合物可采用传统化学方法（硫化物前体氧化）合成；然而，这些没有选择性，导致立体异构体和副产物的混合物。为了解决这一问题，我们提出使用P450和BVMO酶催化硫化物立体选择性氧化成亚砜。选择性生物催化硫化物氧化在生物化学上是已知的，但尚未在工业上得到应用。与Almac公司合作，该公司是通过生物催化溶液生产药品的世界领先者，将开发一种更具选择性和“绿色”的方法，用于工业生产对对纯大蒜亚砜。利用Almac现有的设施和工业平台，将设计和开发新的P450/BVMO生物催化剂，并最终用于生产抗菌大蒜成分和新型衍生物。工作计划:Months1-12。在KCL的培训，文献回顾，基板设计和鉴定。Almac提供的selectAZymeTM筛选平台对选定硫化物前体进行生物催化氧化。铅生物催化剂的鉴定。13-18个月。在Almac实习和培训（6个月）。通过先进的基因组/宏基因组挖掘或蛋白质进化，优化和中试和实验室规模生产P450/BVMO（纯化或无细胞提取物CFE）。培训高通量筛选设备（即机器人菌落采摘机）和液体处理设备，用于生物催化剂的开发。Months19-30。实验室规模的大蒜亚砜及其衍生物的制造。工艺的改进（速率、收率、选择性、工艺稳健性）。确定最佳生物转化条件（共溶剂、辅助因子的回收、混合等）。访问Almac（3个月）进一步优化生物催化剂。然后，基材范围将扩大。Months31-40。替代硫氧化生物催化剂（如双加氧酶）的筛选和鉴定。在Almac进行鉴定和进一步优化（3个月）。大规模生产亚砜。Months41-48。与英国公共卫生部合作的项目中生产的亚砜衍生物的体外抗菌性能评价。数据收集，论文准备和提交。向BBSRC报告。

This project comes as a result of a new collaboration between King's College and Almac and it aims at the development of novel biocatalysts, P450 and BVMO enzymes, for the sustainable industrial production of the antibacterial sulfoxide components of garlic and their derivatives. The project fits firmly within the BBSRC strategic priorities on "New strategic approaches to industrial biotechnology" and "Combating antimicrobial resistance". Furthermore, the UK chemical industry is keen to identify new strategic research to underpin the development of innovative, sustainable manufacturing routes to prepare high-value chemicals and drugs. Allicin, aliin and ajoene are chiral sulfoxide compounds found in garlic. They have important dietary and therapeutic roles including poten activity against antibiotic resistant bacteria, a major threat in UK healthcare system. The industrial extraction of enantiopure sulfoxides from garlic can be problematic due to the harsh conditions of the process that can lead to the racemization of the sulfoxide stereocentres with consequent loss of antibacterial activity. Allicin and related comounds can be synthesied using traditional chemical methods (oxidation of the sulfide precursor); however these are not selective, resulting in mixtures of stereoisomers and byproducts. To solve this problem we propose to use P450 and BVMO enzymes to catalyse the stereoselective oxidation of sulfides into sulfoxides. Selective biocatalysed sulfide oxidation is known in biochemistry, but has not been exploited in industry. In collaboration with Almac, a world leader in the manufacture of pharmaceuticals through biocatalytic solutions, a more selective and "green" approach for the industrial production of enantiopure garlic sulfoxides will be developed. New P450/BVMO biocatalysts will be designed and developed taking advantage of the facilities and industrial platforms available at Almac and finally used for the manufacturing of the antibacterial garlic components and novel derivatives.Work plan:Months1-12. Training at KCL, literature review, substrate design and identification. Biocatalytic oxidation of selected sulfide precursors with selectAZymeTM screening platform provided by Almac. Identification of lead biocatalysts.Months13-18. Placement and training at Almac (6 months). Optimization and pilot- and lab scale production of P450/BVMO (purified or cell-free extract CFE) via advanced genome/metagenome mining or protein evolution. Training with high throughput screening equipment (i.e. robotic colony picker) and liquid handling equipment for biocatalysts development. Months19-30. Manufacturing of garlic sulfoxides and derivatives at lab scale. Improvements of the process (rate, yield, selectivity, process robustness). Identification of the best biotransformation conditions (co-solvents, recycling of the co-factors, mixing, etc.). Visit at Almac (3 months) for further biocatalysts optimization. The substrate scope will then be expanded. Months31-40. Screening and identification of alternative sulphur oxidizing biocatalysts (i.e. dioxygenase). Identification and further optimization at Almac (3 months). Large scale manufacturing of sulfoxides. Months41-48. Evaluation in-vitro of the antibacterial properties of the sulfoxide derivatives produced in the project in collaboration with Public Health England. Data collection, thesis preparation and submission. Report to BBSRC.