Insights into the Regulation of Indigo Production in an Engineered Escherichia coli Strain via Overexpression of Specific Transporter Genes and Proteomic Analyzes

Conventional extraction of indigo, a vital natural dye, provides low yields and has a negative environmental impact. However, microbial synthesis has emerged as a sustainable alternative. In this study, we describe the optimization of indigo biosynthesis in an engineered Escherichia coli strain called E216. This strain carries, on a replicative plasmid, the styAB genes originating from Pseudomonas putida that constitute the monooxygenase biosynthetic pathway of indigo, as well as mdh, encoding malate dehydrogenase, which plays a role in reducing power generation. In this strain, the overexpression of mtr (a gene encoding a transporter of tryptophan (Trp), the precursor of indigo biosynthesis) and acrA (a gene encoding a protein involved in indigo efflux) was found to substantially enhance indigo yields. Consistently, knocking out these two genes using CRISPR-Cas9 significantly reduced indigo production, whereas it was restored through the complementation of these mutants. This study thus revealed that stimulating tryptophan uptake and indigo efflux, the latter of which limits indigo’s toxic intracellular accumulation, has a positive impact on indigo yields. Furthermore, a comparative mass spectrometry-based proteomic analysis of E216 grown in fermentation medium with or without tryptophan supplementation, integrated with data-independent acquisition (DIA), revealed the global impact of tryptophan supplementation on cellular metabolism. This analysis identified upregulation of key proteins and enriched metabolic pathways under conditions of tryptophan supplementation. Integrating the results of the genetic engineering and proteomic analysis establishes a strong scientific and practical basis for developing a highly efficient method for the green industrial production of indigo using engineered E. coli strains.