Model-guided metabolic engineering of curcuminoid Production in Pseudomonas putida

Production of value-added, plant-derived compounds in microbes increasingly attracts commercially interest in food and pharmaceutical industries. However, plant metabolic pathways are complex, require a robust balance of enzymes, cofactors, ATP and other metabolites, and often result in low production when transplanted to bacteria. This is exemplified by the biosynthesis of curcuminoids from the Curcuma longa plant. Here, we combine dynamic pathway modeling, systematic testing of isoenzymes, and the optimization of gene expression levels and substrate concentrations for the biosynthesis of curcuminoids in Pseudomonas putida, leading to unprecedented conversion rates of caffeic acid and tyrosine to curcumin. The development of kinetic ensemble models guided the design of production strains, emphasizing the necessity of high relative expression of c3h, curs2 and dcs and, the low relative expression of tal, comt, ccoaomt, and 4cl4. This optimization resulted in a strain that achieved a 10.8 ± 1.8% of the maximum theoretical yield of curcumin from tyrosine. This represents a 4.1-fold increase in production efficiency and the highest yield reported to date, demonstrating the potential of P. putida as a promising platform for curcuminoid production. Our findings highlight the effectiveness of our strategy not only in the advances in the production of curcuminoids but also in setting a framework for the biosynthesis of other complex compounds. ### Competing Interest Statement Vitor A. P. Martins dos Santos (VAPMdS) has interests in LifeGlimmer GmbH and Richard van Kranenburg is employed by Corbion N.V.