Toxin-Antitoxin System-Driven Metabolic Pathway Optimization for Antibiotic-Free Indigoidine Sustainable Production in Corynebacterium glutamicum

Abstract Indigoidine, a novel microbial pigment primarily applied in the textile dyeing industry, serves as a sustainable alternative to blue colorants, representing a bio-based pigment identified from natural and agricultural sources. Recently, a plasmid-based Corynebacterium glutamicum strain production system was constructed for scale-up indigoidine fermentation, however, it suffered from plasmid loss and reliance on antibiotic selection during cultivation. In this work, an antibiotic-free toxin-antitoxin (TA)-based plasmid system was employed to drive the systematic optimization of the green production of indigoidine in C. glutamicum. First, the key expression cassette bpsA-wlpl was separately integrated into various genomic loci of C. glutamicum to complete the indigoidine biosynthetic pathway. Furthermore, the glucose uptake system was modified from the PTS system to a non-PTS system to enhance carbon utilization. To fine-tune the overexpression levels of pathway genes, promoter and RBS engineering were employed to replace the corresponding elements in the plasmid for combinatorial optimization of precursor supply. Finally, the relE-relB TA system was identified from various microbial sources, which functions to prevent plasmid loss during cultivation. Engineered strain CGL49 exhibited sustainable indigoidine production in shake-flask and 5 L bioreactor, achieving titers of 3.83 g/L and 42.80 g/L, respectively, without antibiotics. This work provides a robust and sustainable plasmid-based bio-platform for antibiotic-free indigoidine production.