Development and optimization of a microbial co-culture system for heterologous indigo biosynthesis.

BACKGROUND: Indigo is a color molecule with a long history of being used as a textile dye. The conventional production methods are facing increasing economy, sustainability and environmental challenges. Therefore, developing a green synthesis method converting renewable feedstocks to indigo using engineered microbes is of great research and application interest. However, the efficiency of the indigo microbial biosynthesis is still low and needs to be improved by proper metabolic engineering strategies. RESULTS: In the present study, we adopted several metabolic engineering strategies to establish an efficient microbial biosynthesis system for converting renewable carbon substrates to indigo. First, a microbial co-culture was developed using two individually engineered E. coli strains to accommodate the indigo biosynthesis pathway, and the balancing of the overall pathway was achieved by manipulating the ratio of co-culture strains harboring different pathway modules. Through carbon source optimization and application of biosensor-assisted cell selection circuit, the indigo production was improved significantly. In addition, the global transcription machinery engineering (gTME) approach was utilized to establish a high-performance co-culture variant to further enhance the indigo production. Through the step-wise modification of the established system, the indigo bioproduction reached 104.3 mg/L, which was 11.4-fold higher than the parental indigo producing strain. CONCLUSION: This work combines modular co-culture engineering, biosensing, and gTME for addressing the challenges of the indigo biosynthesis, which has not been explored before. The findings of this study confirm the effectiveness of the developed approach and offer a new perspective for efficient indigo bioproduction. More broadly, this innovative approach has the potential for wider application in future studies of other valuable biochemicals' biosynthesis.