Biotechnology and Bioprocess Engineering 2024; 29(5): 806-814  
Production of halogenated indigo by Escherichia coli whole-cell conversion system with novel halogenase derived from Pseudoalteromonas nigrifaciens
Byongson Yi1 · Byung Wook Lee1 · Kyungjae Yu1 · Hyun Gi Koh1 · Yung‑Hun Yang2 · Kwon‑Young Choi3 · Byung‑Gee Kim4 · Jung‑Oh Ahn5 · Kyungmoon Park1 · See‑Hyoung Park1
1 Department of Biological and Chemical Engineering, Hongik University, Sejong 30016, Korea
2 Department of Biological Engineering, Konkuk University, Seoul 05029, Korea
3 Department of Environmental Engineering, Ajou University, Suwon 16499, Korea
4 Interdisciplinary Program of Bioengineering, Seoul National University, Seoul 08826, Korea
5 Biotechnology Process Engineering Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Cheongju 28116, Korea
Correspondence to: ✉ Kyungmoon Park
pkm2510@hongik.ac.kr
✉ See‑Hyoung Park
shpark74@hongik.ac.kr
Received: April 2, 2024; Revised: May 22, 2024; Accepted: May 28, 2024; Published online: June 11, 2024.
© The Korean Society for Biotechnology and Bioengineering. All rights reserved.

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract
Indigo is a commercially significant dye extensively used in the textile industry for dyeing denim and other fabrics. The synthesis of various colored indigo derivatives necessitates the halogenation of the indole ring in indigo. However, the scarcity of halogenating enzymes, especially those with high positional specificity and commercial availability, limits the biological synthesis of various halogenated indigos. This study presents the discovery of a novel halogenase from Pseudoalteromonas nigrifaciens that is similar to sttH from Streptomyces toxytricini, an enzyme that specifically halogenates the 6th carbon of the indole in indigo. The cloned halogenase gene was validated for halogenation activity and regioselectivity using a recombinant Escherichia coli whole-cell conversion system. The addition of either NaCl or NaBr resulted in the production of 6-chloro indigo or 6-bromo indigo, respectively. Notably, 6-chloro indigo displayed a red coloration, while 6-bromo indigo appeared blue. To optimize the whole-cell conversion system, we evaluated the conversion rate of halogenated indigo production in response to varying concentrations of tryptophan and E. coli cells. The maximum conversion rate (32%) was achieved using 30 mM tryptophan and an E. coli cell density corresponding to an OD50 reading. In conclusion, we have designed a recombinant E. coli whole-cell conversion system capable of producing 6-halogenated indigo by introducing a novel sttH-like halogenase from P. nigrifaciens. This system holds promise for the production of various indigo derivatives.
Keywords: Halogenation · Indole · Indigo · sttH · Pseudoalteromonas nigrifaciens

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