Integrating Morphology and Transcriptional Regulation to Alleviate Intracellular Product Stress for Enhanced Indigo Biosynthesis in Escherichia coli.

Abstract

Indigo is an important biobased colorant used in textile dyeing and as a precursor for blue food colorants; however, its microbial production is often limited by cytotoxicity and intracellular accumulation of insoluble products. Here, we established a systems-level host engineering framework in Escherichia coli to improve indigo biosynthesis. CRISPRi screening of cell envelope-associated genes identified targets including sulA, ftsZ, fabF, and pgi. Mechanistic analyses showed that modulation of cell division and central carbon metabolism alleviates envelope stress and enhances reducing power for indigo formation, while electron microscopy confirmed reduced cellular perturbation caused by intracellular indigo accumulation. A second CRISPRi library targeting transcription factors revealed coordinated regulation of BolA, ArcA, and RpoN to balance metabolism and stress responses. Integration of targets enabled 9.38 g/L indigo production in a 5 L fed-batch bioreactor, a 2.55-fold improvement over the parental strain. This work provides a promising engineering strategy for microbial indigo production.