Systemic Metabolic Engineering of Plasmid-Free <i>Escherichia coli</i> for Sustainable High-Yield Indigoidine Production

Indigoidine, a novel blue microbial pigment with excellent dyeing performance, emerges as a sustainable alternative to synthetic dyes. However, prevailing plasmid-based production systems suffer from genetic instability and reliance on antibiotic selection, rendering them incompatible with green industrial manufacturing. In this study, we developed a genetically stable, plasmid-free Escherichia coli biocatalyst to drive the green synthesis of indigoidine utilizing glycerol, a renewable and abundant feedstock. First, CRISPR-based multicopy integration was employed to enhance expression of the key enzymes including indigoidine synthase and its phosphopantetheinyl transferase. Furthermore, to improve precursor supply, functionally analogous enzymes from different microbial sources were screened. Subsequently, to address the challenge of modifying the TCA cycle, a protein degradation tag system was introduced and the strength of specific genomic promoters was attenuated, two strategies that acted synergistically. Engineering of the NADPH regeneration pathway effectively elevated energy precursor pools, while knockout of acetate pathways alleviated metabolic stress. Ultimately, the optimal strain MIG41 achieved indigoidine titers of 6.09 g/L in shake flasks and 35.21 g/L in a 5 L fed-batch fermentation, with a productivity of 0.73 g/L/h. Moreover, the purified indigoidine exhibited excellent dyeing performance. Ultimately, this work provides a robust, scalable microbial cell factory platform for the sustainable, high-yield production of eco-friendly dyes.