Executive Summary : | Oxygen diffusion-enhancing compounds represent a class of potential therapeutic agents, particularly in ischemic conditions. These agents, represented by the most advanced and first-in-class molecule, trans sodium crocetinate (TSC), are the subject of intense clinical investigation, including Phase 1b/2b clinical trials for COVID-19. TSC is a successor of a natural product, crocetin, and is being investigated for various cancers as a radiosensitizer owing to its oxygen diffusion enhancement capability. Crocetin is a first-in-class, plasma oxygen-diffusion enhancing compound with potent antioxidant activity. Due to its unique ability, crocetin can be of potential use in hypoxia treatment, sleep disturbances, retinal damage, stroke, myocardial infarction, and as a radiosensitizer for cancer radiotherapy. The crocetin is a natural apocarotenoid dicarboxylic acid found majorly in the most expensive spice on earth, i.e., saffron, the Crocus sativus. Saffron cultivation is highly geographical specific and the collection of flowers and crocetin isolation from them is very labor-intensive and costly, whereas the multistep chemical synthesis is cumbersome with lower overall yield, uses hazardous chemicals, and leads to the formation of multiple structural isomers. We propose the bio-production of crocetin dialdehyde using metabolic engineering in Chlamydomonas reinharditii, one of the fastest-growing microalgae followed by a single-step, oxidative chemical conversion of crocetin dialdehyde into crocetin by green chemical methods. The hypothesis is based on the microalgae being an important source of carotenoids, plant counterparts having the same pathway that is used by the plant Crocus sativus to make crocetin and related compounds. Chlamydomonas reinharditii possess the carotenoid pathway making zeaxanthin, the immediate precursor of crocetin. Genetic insertion of crocetin pathway genes would lead to the diversion of zeaxanthin towards crocetin dialdehyde which can be followed by a single-step, oxidative chemical conversion of crocetin. Microalgae can be grown heterotrophically or mixotrophically in closed stirred tank reactors offering higher growth rate/biomass productivity, ease of harvesting, scalability, and require lesser cultivation area as compared to that of plants. The project will involve genetic engineering of Chlamydomonas reinharditii an industrial microalga by inserting a bifunctional rate-limiting gene of the IPP pathway to enhance the overall flux and crocetin pathway genes from Crocus sativus to channelize carotenoid pathway for making crocetin dialdehyde. This will be further converted to crocetin by a green chemical process. The successful process would be instrumental in the development of an alternative and less resource-intensive process for crocetin production thus reducing the import of this molecule for fulfilling the existing and prospective demand of the crocetin and derived compounds. |