Modular Co-Culture Engineering of Yarrowia Lipolytica for Amorphadiene Biosynthesis

Amorphadiene is an intermediate product of the first dedicated step to artemisinin production. It has attracted wide research interest as an antimalaria drug precursor in recent years. The efficient conversion of renewable carbon sources and redirection of metabolic flux toward a metabolite of interest has become a fascinating strategy for plant secondary metabolite overproduction. A modular pathway that divides the hosts' labor, a co-culture system has shown great biosynthetic potential and can be leveraged to achieve cost-effective bioproduction of natural products. Using a co-culture system of Y. lipolytica Po1f and Po1g strains, subcellular localization of ADS gene into the endoplasmic reticulum, co-utilization of mixed carbon source, and enlargement of the endoplasmic reticulum size were investigated to improve amorphadiene producton in this work. Using Po1g/PPtM and Po1f/AaADSER x3 /iGFMPDU strains and co-utilization of 5 µM sodium acetate with 20 g/L glucose in YPD media, amorphadiene titer increased to 65.094 mg/L. The enlargement of the Endoplasmic reticulum membrane caused by the deletion of the PAH1 gene provided more subcellular space for the action of the ADS-tagged gene. It further increased the amorphadiene production to 71.74 mg/L. The results demonstrated that manipulating metabolic flux in the co-culture of Y. lipolytica can be efficient over a single culture for the bioproduction of many value-added metabolites in a whole or large biosynthetic pathway.