Integrated Transporter Engineering and Semi-Rational Enzyme Design in Corynebacterium glutamicum for High-Level Natural Blue Pigment Indigoidine Production

Abstract Indigoidine is a sustainable natural blue pigment with broad industrial prospects, while its microbial production is hampered by growth inhibition of the producing strain, enzyme-host incompatibility, and insufficient industrial-scale titer. In this study, de novo biosynthesis of indigoidine was achieved in Corynebacterium glutamicum. Efflux pump engineering was employed to alleviate growth inhibition of the production strain, and semi-rational design of the key synthetase BpsA generated the optimal mutant Y433Q with enhanced catalytic activity. Strain CGI23, which integrated the two aforementioned strategies, produced 29.8 g/L of indigoidine at a mass yield of 0.248 g/g glucose in shake flasks; when further scaled up in a 5 L fed-batch fermenter, the titer reached 60.7 g/L with a volumetric productivity of 0.723 g/L/h. This work establishes an efficient microbial platform for industrial indigoidine production, providing a reference for integrated engineering of high-performance microbial cell factories.