Martin-Pascual, M. | bioRxiv Science

bioRxiv2024

Computer assisted multi-level optimization of malonyl-CoA availability in Pseudomonas putida

Malonyl-CoA is the major precursor for the biosynthesis of diverse industrially valuable products such as fatty acids/alcohols, flavonoids, and polyketides. However, its intracellular availability is limited in most microbial hosts, hampering the biological synthesis of such chemicals. To address this limitation, we present a multi-level optimization workflow using modern metabolic engineer-ing technologies to systematically increase the malonyl-CoA levels in Pseudomonas putida. The workflow involves the identification of gene downregulations, chassis selection, and optimization of the acetyl-CoA carboxylase complex through ribosome binding site engineering. Computa-tional tools and high-throughput screening with a malonyl-CoA biosensor enabled the rapid eval-uation of numerous genetic targets. Combining the most beneficial targets led to a 5.8-fold en-hancement in the production titer of the valuable polyketide phloroglucinol. This study demon-strates the effective integration of computational and genetic technologies for engineering P. putida, opening new avenues for the development of industrially relevant strains and the investi-gation of fundamental biological questions.

synthetic biology↗

bioRxiv2024

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.

synthetic biology↗