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抗菌亚砜组分的可持续工业生产。该项目完全符合BBSRC关于“工业生物技术新战略方法”和“对抗抗菌素耐药性”的战略优先事项。此外，英国化学工业热衷于确定新的战略研究，以支持开发创新的、可持续的制造路线，以制备高价值的化学品和药物。大蒜素、蒜氨酸和阿焦烯是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性细菌的潜在活性，这是英国医疗保健系统的主要威胁。从大蒜中工业提取对映体纯亚砜可能是有问题的，因为该方法的苛刻条件可能导致亚砜立构中心的外消旋化，从而丧失抗菌活性。大蒜素和相关化合物可以使用传统的化学方法（硫化物前体的氧化）合成;然而这些方法没有选择性，导致立体异构体和副产物的混合物。为了解决这个问题，我们建议使用P450和BVMO酶催化立体选择性氧化硫化物成亚砜。选择性生物催化的硫化物氧化在生物化学中是已知的，但尚未在工业中开发。将与通过生物催化溶液生产药品的世界领先企业Almac合作，开发一种更具选择性和“绿色”的对映体纯大蒜亚砜工业生产方法。新的P450/BVMO生物催化剂将利用Almac现有的设施和工业平台进行设计和开发，最终用于生产抗菌大蒜成分和新型衍生物。工作计划：1 -12月。在KCL培训，文献综述，基底设计和鉴定。利用Almac提供的selectAZymeTM筛选平台对选定的硫化物前体进行生物催化氧化。铅生物催化剂的鉴定。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及实验室规模生产P450/BVMO（纯化或无细胞提取物CFE）。高通量筛选设备（即机器人菌落采集器）和液体处理设备的生物催化剂开发培训。19 -30个月。大蒜亚砜及其衍生物的实验室规模生产。工艺改进（速率、收率、选择性、工艺耐用性）。确定最佳生物转化条件（共溶剂、辅助因子的再循环、混合等）。访问Almac（3个月），进一步优化生物催化剂。基板范围将扩大。31 -40个月。替代硫氧化生物催化剂（即双加氧酶）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。41 -48个月。与英国公共卫生部合作，对该项目中生产的亚砜衍生物的抗菌性能进行体外评价。资料收集、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.