Metabolic Division in an Coculture System for Efficient Production of Kaempferide.

Kaempferide, a plant-derived natural flavonoid, exhibits excellent pharmacological activities with nutraceutical and medicinal applications in human healthcare. Efficient microbial production of complex flavonoids suffers from metabolic crosstalk and burden, which is a big challenge for synthetic biology. Herein, we identified 4'--methyltransferases and divided the artificial biosynthetic pathway of kaempferide into upstream, midstream, and downstream modules. By combining heterologous genes from different sources and fine-tuning the expression, we optimized each module for the production of kaempferide. Furthermore, we designed and evaluated four division patterns of synthetic labor in coculture systems by plug-and-play modularity. The linear division of three modules in a three-strain coculture showed higher productivity of kaempferide than that in two-strain cocultures. The U-shaped division by co-distributing the upstream and downstream modules in one strain led to the best performance of the coculture system, which produced 116.0 ± 3.9 mg/L kaempferide, which was 510, 140, and 50% higher than that produced by the monoculture, two-strain coculture, and three-strain coculture with the linear division, respectively. This is the first report of efficient production of kaempferide in a robust coculture. The strategy of U-shaped pathway division in the coculture provides a promising way for improving the productivity of valuable and complex natural products.