This proposal aims to change current paradigms of biotechnological production of complex plant natural compounds, specifically isoprenoids such as limonene, menthol, and p-cymene. MENTHOL main innovation lies in the synthetic decoupling of the complex biosynthetic pathways of these compounds into a distributed catalysis within monoclonal heterogeneous bacterial populations. Limonene and menthol are natural monoterpenes widely used as flavor and fragrance additives, while p-cymene is an important precursor in the bio-based production of terephthalic acid, a key precursor of Polyethylene Terephthalate (PET). Although they have been successfully produced by genetically engineered microorganisms, to reach industrial scale production there are still obstacles to overcome. On the one hand, the introduction of large biosynthetic pathways in a single bacterial strain may cause host unstable physiology and low performance; and on the other hand, the use of synthetic consortia to overcome this shortcoming can be limited by differences in the growth profile of species involved. This proposal is conceived as an alternative to avoid those obstacles, by decoupling the complex biosynthetic pathways of the target compounds into minimal functional modules, to be reassembled inside metabolically differentiated subpopulations of a single specie-based multifunctional bacterial culture. To achieve such ambitious goal, in this project we will applied cutting-edge systems and synthetic biology tools to engineer programmable metabolic heterogeneity inside a monoclonal population of Pseudomonas putida, to promote the division of labor during the production of limonene, menthol, and p-cymene using waste cooking oils as economic and reliable renewable feedstock.
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