Evolution-guided high yield production of potent Gα_q/11-signalling inhibitors FR900359 and YM-254890

Heterotrimeric G proteins modulate a wide range of important intracellar effectors and play indispensable roles in diverse human physiological processes. The cyclic depsipeptides FR900359 and YM-254890 have emerged as potent and selective inhibitors for deconvoluting Gα_q/11-mediated signalling and targeted therapy of Gα_q/11-mutated tumors. Here we introduced an evolution-guided synthetic biology strategy to develop a set of robust heterologous microbial systems for high-yield producing FR900359 or YM-254890. First, following the uncultivable source (Candidatus Burkholderia crenata) of FR900359, Burkholderia gladioli Δgbn::attB was identified as an excellent, genetically tractable heterologous host. Second, inspired by high-GC-content feature of FR900359 or YM-254890 biosynthetic gene clusters (BGCs), the inter-genus, Streptomyces-derived superstrong promoter stnYp enabled FR900359 production to 101.7 mg/L in Δgbn::attB. Meanwhile, the stnYp-controlled YM-254890 BGC was de novo assembled due to the unavailability bottleneck of its native producer followed by efficiently heterologous production in Δgbn::attB. Finally, duplication of FR900359 or YM-254890 BGCs in our newly developed host Δgbn::2attB further increased their titers to 177.9 and 29.2 mg/L (the highest yields reported to date), respectively. Collectively, our study paved the way for the cost-efficient, sustainable microbial production of both FR900359 and YM-254890, significantly facilitating the biotechnological application and drug development of the two potent Gα_q/11-signalling inhibitors.