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

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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.