Enhancement and mechanism of in-cluster two-component regulatory factors on granaticin production in Streptomyces vilmorinianum YP1

Granaticins, a class of bioactive benzoisochromanequinones (BIQs), are natural blue pigments with biological activities, including antibacterial properties and promising clinical anticancer applications. However, their clinical and industrial applications are limited by low production yields and unclear biosynthetic regulation. In this study, we identified a rare in-cluster two-component system (TCS; orf10/orf11) within the granaticin biosynthesis-related gene cluster (BGC) of Streptomyces vilmorinianum YP1, a novel high-yielding producer. The orf10 encodes a MerR family transcription regulator, while the orf11 encodes a sensor histidine kinase. The roles of orf10 and orf11 were determined using single and double knockouts and complementation experiments. The yield of granaticins increased with the overexpression of the two genes in S. vilmorinianum YP1. Notably, orf10 overexpression resulted in a record high granaticin B titer of 716.27 mg/L, representing a substantial improvement over native production level. The metabolic map obtained using Ultra Performance Liquid Chromatography Tandem Mass Spectrometry (UPLC-MS/MS) analysis revealed that the metabolic processes of the combined overexpression mutant were significantly different from those of the single overexpression mutants. These findings not only establish the functional link between the in-cluster TCSs, and granaticin biosynthesis but also provide an effective engineering strategy to overcome production bottlenecks. Our work advances the mechanistic understanding of BIQ regulation and highlights S. vilmorinianum YP1 as a promising platform for industrial-scale granaticin production.