Modular co-culture engineering of Yarrowia lipolytica for amorphadiene biosynthesis

Abstract

Amorphadiene is the precursor to synthesize the antimalarial drug artemisinin. The production of amorphadiene and artemisinin from metabolically engineered microbes may provide an alternate to plant secondary metabolite extraction. Microbial consortia can offer division of labor, and microbial co-culture system can be leveraged to achieve cost-efficient production of natural products. Using a co-culture system of Y. lipolytica Po1f and Po1g strains, subcellular localization of ADS gene (encoding amorphadiene synthase) into the endoplasmic reticulum, co-utilization of mixed carbon source, and enlargement of the endoplasmic reticulum (ER) surface area, we were able to significantly improve amorphadiene production in this work. Using Po1g/PPtM and Po1f/AaADSERx3/iGFMPDU strains and co-utilization of 5 µM sodium acetate with 20 g/L glucose in YPD media, amorphadiene titer were increased to 65.094 mg/L. The enlargement of the ER surface area caused by the deletion of the PAH1 gene provided more subcellular ER space for the action of the ADS-tagged gene. It further increased the amorphadiene production to 71.74 mg/L. The results demonstrated that the importance of the spatial localization of critical enzymes, and manipulating metabolic flux in the co-culture of Y. lipolytica can be efficient over a single culture for the bioproduction of isoprenoid-related secondary metabolites in a modular manner. Graphical Abstract

Modular Coculture to Reduce Substrate Competition and Off-Target Intermediates in Androstenedione Biosynthesis

“…Synthetic cocultures can facilitate bypassing undesirable reactions during the optimization of pathways with internal competition. More specifically, cell-level segregation, which involves the physical separation of specific pathway components into individual modules, (1) alleviates the loss of intermediates to competing pathways without reducing their activity, , (2) substantially reduces the metabolic burden on individual cells, (3) decreases the diffusion of certain intermediates, − and (4) enables the coordination of pathway modules to maximize biosynthetic capacity through modulating the ratio of different constituent strains. , Numerous previous studies involving microbial engineering have focused on the rational design of pathways that are limited by competing reactions catalyzed by enzymes belonging to other pathways. − Herein, we explored the potential of modular coculture engineering to resolve intrapathway competition via the rational rewiring of the pathway responsible for androstenedione (4AD) biosynthesis.…”