Enhanced production of bio-indigo in engineered Escherichia coli, reinforced by cyclopropane-fatty acid-acyl-phospholipid synthase from psychrophilic Pseudomonas sp. B14-6

Indigo dye is an organic compound with a distinctive blue color. Most of the indigo currently used in industry is produced via chemical synthesis, which generates a large amount of wastewater. Therefore, several studies have recently been conducted to find ways to produce indigo eco-friendly using microorganisms. Here, we produced indigo using recombinant Escherichia coli with both an indigo-producing plasmid and a cyclopropane fatty acid (CFA)-regulating plasmid. The CFA-regulating plasmid contains the cfa gene, and its expression increases the CFA composition of the phospholipid fatty acids of the cell membrane. Overexpression of cfa showed cytotoxicity resistance of indole, an intermediate product formed during the indigo production process. This had a positive effect on indigo production and cfa originated from Pseudomonas sp. B 14–6 was used. Optimal conditions for indigo production were determined by adjusting the expression strain, culture temperature, shaking speed, and isopropyl β-D-1-thiogalactopyranoside concentration. Treatment with Tween 80 at a particular concentration to increase the permeability of the cell membrane had a positive effect on indigo production. The strain with the CFA plasmid produced 4.1 mM of indigo after 24 h of culture and produced 1.5-fold higher indigo than the control strain without the CFA plasmid that produced 2.7 mM. Cfa expression increased cyclic fatty acid composition of phospholipid fatty acids.Overexpression of cfa had a positive effect on indigo production.Cfa from psychrophilic Pseudomonas sp. B14-6 showed the best indigo productionOverexpression of cfa showed increased robustness to indole.Indigo production was increased by1.52 fold (1.08 g/L) in CFA strain.