Refining genetic circuit design for enhanced type II polyketide biosynthesis in Escherichia coli

Polyketides represent a broad class of structurally diverse natural products with potent antibacterial and anticancer activities. Type II polyketide synthases (T2PKS) are multi-subunit complexes responsible for condensing acylated intermediates, including acetyl-CoA and malonyl-CoA, into complex fused-ring aromatic natural products. While type II polyketides have traditionally been associated with gram-positive actinomycetes, more recent discoveries have identified PKS systems in gram-negative bacteria. These findings have provided new genetic tools and opened new pathways for producing and manipulating polyketides in the versatile chassis organism Escherichia coli. In this study, we altered the genetic circuit of the Photorhabdus luminescens PKS for octaketide production in E. coli. Promoter engineering and ribosome binding site optimization of the antDEFBG minimal PKS (mPKS) resulted in 181 mg/L SEK4 and 392 mg/L SEK4b production titers. Further refinement of downstream ketoreductase, aromatase, and cyclase expression enabled the biosynthesis of native polyketide AQ-256 and naphthopyrone (S)-DNPA at titers exceeding 200 mg/L/d. Additional metabolic reprogramming strategies further increased yields by 25%. This work further illustrates the potential of E. coli as a versatile host for the biosynthesis of complex polyketides, paving the way for the scalable production of valuable natural products. By optimizing genetic circuits and pathway expression, this study provides a test case for engineering microbial systems to produce pharmaceutically relevant compounds more efficiently.