Abstract
Indigo is a plant-based natural blue dye that can be produced via chemical synthesis and biological pathways. However, the toxic reduction processes and intracellular production of indigo through microbial metabolism are often limited by insolubility of indigo and complex downstream processing, causing environmental issues in the dyeing processes. Additionally, indican, a precursor of indigo with a glucose moiety, is highly soluble and can be easily converted into indoxyl by β-glucosidase, forming indigo under mild conditions. We constructed an indican-producing strain Escherichia coli BL21 HI201 by introducing a UDP-glycosyltransferase (ugt) into an indoxyl production system containing tryptophanse (tnaA) and flavin-containing monooxygenase (FMO) genes, enabling conversion of tryptophan into indican. Testing of the effect by various carbon sources suggested that glucose is one of the major factors affecting the ratio of indigo to indican, and increase in glucose concentration to more than 1.5% could produce sole indican without indigo. Under optimal conditions, E. coli BL21 HI201 biosynthesized 5.65 mM indican from tryptophan. Additionally, after deletion of various β-glucosidase genes, the bglA knockout strain E. coli BL21 HI204 produced more indican, achieving 6.79 mM after 24 hr of cultivation. This study demonstrated the strategic production of indican through the installation of a production system, deletion of a byproduct pathway, and control of glucose concentration. ONE-SENTENCE SUMMARY: This paper demonstrates the strategic enhancement of indican production in genetically engineered Escherichia coli BL21 by optimizing metabolic pathways and controlling glucose concentrations.