CRISPR/Cas9-Mediated Metabolic Engineering of Endophytic Pseudomonas Loganensis Sp. Nov. for the Production of Nutritionally Valuable Carotenoids

Abstract

Carotenoids with significant nutritional and antioxidant properties have been widely utilized in the food, feed, pharmaceutical, and cosmetic industries. They improve the nutritional value of foodstuffs and have been used as natural food colorants. However, their current supply chain is mainly dependent on extraction from plants and chemical synthesis, both of which have bottlenecks, including environmental concerns, toxicity, and allergenicity. To address global demand for sustainable and environmentally friendly production of nutrients, we engineered the endophytic Pseudomonas loganensis sp. nov. as a niche microbial chassis for nutritionally valuable carotenoid production. Using CRISPR-Cas9, we knocked out key carotenogenic genes to construct strains capable of producing zeaxanthin, lycopene, and beta-carotene. Additionally, an overexpression plasmid was introduced to produce astaxanthin. HPLC analysis confirmed the successful production of four target carotenoids. The culture conditions and media compositions were optimized using response surface methodology, resulting in a similar to 5-fold increase in the titers of zeaxanthin (13.4 mg/L), lycopene (9.67 mg/L), and beta-carotene (23.53 mg/L), and a similar to 12-fold increase in astaxanthin titer (1 mg/L) compared to LB medium without optimization. Our results indicate the potential of endophytic bacteria as a microbial chassis for carotenoid bioproduction, underscoring the potential of synthetic biology to contribute to global efforts toward nutritional security and sustainable food systems.