Eco-Friendly Indigo Biosynthesis in Escherichia coli: Functional Evaluation of a StyA/StyB and NDO Co-Expression Platform
Hayjun Kang
Seoul Scholars International Art and Design, Seoul, South Korea
Publication date: July 10, 2026
Seoul Scholars International Art and Design, Seoul, South Korea
Publication date: July 10, 2026
DOI: http://doi.org/10.34614/JIYRC2026I25
ABSTRACT
This study evaluated an eco-friendly microbial platform for indigo dye production in Escherichia coli using a dual-enzyme strategy based on two indole-oxidizing systems: the S-only condition, which overexpressed the styrene monooxygenase genes StyA/StyB; the N-only condition, which overexpressed the naphthalene dioxygenase (NDO) gene system; and the N+S condition, which co-expressed both enzyme systems. The aim was to determine whether dual-enzyme co-expression could improve indigo biosynthesis and downstream dyeing performance compared with single-enzyme expression alone. After plasmid transformation and selective colony confirmation, transformed cultures were grown in liquid medium and incubated at 17°C, 25°C, and 37°C. Indigo production was first evaluated by visible blue coloration and then quantified by indigo intensity analysis. The N-only condition showed minimal pigment formation at all temperatures, with very weak blue coloration and low signal intensity, indicating limited indigo production by NDO alone under the tested conditions. The S-only condition produced moderate pigment formation, suggesting that StyA/StyB had a stronger individual contribution to indigo biosynthesis. In contrast, the N+S condition consistently generated the highest indigo output, with quantified intensities of 0.80 ± 0.02 AU at 17°C, 0.90 ± 0.02 AU at 25°C, and 0.98 ± 0.03 AU at 37°C, demonstrating that co-expression was the most effective configuration and that 37°C was the optimal temperature among those tested. Indigo produced under the N+S condition also successfully stained cotton fabric and thread, although with lower intensity than commercial indigo. Overall, the results support StyA/StyB–NDO co-expression as a promising strategy for sustainable bio-based indigo production.
This study evaluated an eco-friendly microbial platform for indigo dye production in Escherichia coli using a dual-enzyme strategy based on two indole-oxidizing systems: the S-only condition, which overexpressed the styrene monooxygenase genes StyA/StyB; the N-only condition, which overexpressed the naphthalene dioxygenase (NDO) gene system; and the N+S condition, which co-expressed both enzyme systems. The aim was to determine whether dual-enzyme co-expression could improve indigo biosynthesis and downstream dyeing performance compared with single-enzyme expression alone. After plasmid transformation and selective colony confirmation, transformed cultures were grown in liquid medium and incubated at 17°C, 25°C, and 37°C. Indigo production was first evaluated by visible blue coloration and then quantified by indigo intensity analysis. The N-only condition showed minimal pigment formation at all temperatures, with very weak blue coloration and low signal intensity, indicating limited indigo production by NDO alone under the tested conditions. The S-only condition produced moderate pigment formation, suggesting that StyA/StyB had a stronger individual contribution to indigo biosynthesis. In contrast, the N+S condition consistently generated the highest indigo output, with quantified intensities of 0.80 ± 0.02 AU at 17°C, 0.90 ± 0.02 AU at 25°C, and 0.98 ± 0.03 AU at 37°C, demonstrating that co-expression was the most effective configuration and that 37°C was the optimal temperature among those tested. Indigo produced under the N+S condition also successfully stained cotton fabric and thread, although with lower intensity than commercial indigo. Overall, the results support StyA/StyB–NDO co-expression as a promising strategy for sustainable bio-based indigo production.
