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A 61-kb biosynthetic gene cluster (BGC), which is accountable for the biosynthesis of hibarimicin (HBM) B from Microbispora rosea subsp. hibaria TP-A0121, was heterologously expressed in Streptomyces coelicolor M1154, which generated a trace of the target products but accumulated a large amount of shunt products. Based on rational analysis of the relevant secondary metabolism, directed engineering of the biosynthetic pathways resulted in the high production of HBM B, as well as new HBM derivates with improved antitumor activity. These results not only establish a biosynthetic system to effectively synthesize HBMs—a class of the largest and most complex Type-II polyketides, with a unique pseudo-dimeric structure—but also set the stage for further engineering and deep investigation of this complex biosynthetic pathway toward potent anticancer drugs.

Abstract Modulation of protein function through allosteric regulation is central in biology, but biomacromolecular systems involving multiple subunits and ligands may exhibit complex regulatory mechanisms at different levels, which remain poorly understood. Here, we discover an aldo-keto reductase termed AKRtyl and present its three-level regulatory mechanism. Specifically, by combining steady-state and transient kinetics, X-ray crystallography and molecular dynamics simulation, we demonstrate that AKRtyl exhibits a positive synergy mediated by an unusual Monod-Wyman-Changeux (MWC) paradigm of allosteric regulation at low concentrations of the cofactor NADPH, but an inhibitory effect at high concentrations is observed. While the substrate tylosin binds at a remote allosteric site with positive cooperativity. We further reveal that these regulatory mechanisms are conserved in AKR12D subfamily, and that substrate cooperativity is common in AKRs across three kingdoms of life. This work provides an intriguing example for understanding complex allosteric regulatory networks.