Indigo is a unique blue dye that has been used in the textile industry for centuries and is currently mass-produced commercially through chemical synthesis. However, the use of toxic substrates and reducing agents for chemical synthesis is associated with environmental concerns, necessitating the development of eco-friendly alternatives based on microbial production. In this study, a robust industrial strategy for indigo production was developed using Pseudomonas putida KT2440 as the host strain, which is characterized by its excellent ability to degrade aromatic compounds and high resistance to environmental stress. By introducing the genes tryptophanase (tnaA) and Flavin-containing monooxygenase (FMO), a P. putida HI201 strain was constructed to produce indigo from tryptophan. To enhance the indigo yield, culture conditions, including medium composition, temperature, tryptophan concentration, and shaking speed, were optimized. Under optimal conditions such as TB medium, 15 mM tryptophan, 30°C, 200 rpm, P. putida HI201 biosynthesized 1.31 g/L indigo from tryptophan in a fed-batch fermentation system. The introduction of tnaA and FMO genes also enabled the production of indigo in various P. putida species, and the indigo-producing strain had a blue color, which served as a visual indicator. This study presents a strategy for using P. putida as a host for robust and sustainable microbial production of indigo, highlighting the strain's applicability and efficiency in environment friendly dye synthesis.
This work was supported by the National Research Foundation (NRF) of Korea, the Ministry of Science, ICT [grant numbers NRF-2022R1A2C2003138] and the R&D Program of MOTIE/KEIT [grant number 20014350, 20016324, 00467186] and the support of \u2018R&D Program for Forest Science Technology (Project No. \u201C2023473E10\u20132325- EE02)\u2019 provided by Korea Forest Service (KoreaForestry Promotion Institute). We wish to confirm that there are no known conflicts of interest associated with this publication. We confirm that the manuscript has been read and approved by all named authors and that there are no other persons who satisfied the criteria for authorship but are not listed. We further confirm that the order of authors listed in the manuscript has been approved by all of us. We understand that the Corresponding Author is the sole contact for the Editorial process (including Editorial Manager and direct communications with the office). He is responsible for communicating with the other authors about progress, submissions of revisions and final approval of proofs. We confirm that we have provided a current, correct email address which is accessible by the Corresponding Author.This work was supported by the National Research Foundation (NRF) of Korea, the Ministry of Science, ICT [grant numbers NRF-2022R1A2C2003138] and the R&D Program of MOTIE/KEIT [grant number 20014350, 20016324, 00467186] and the support of \u2018R&D Program for Forest Science Technology (Project No. \u201C2023473E10-2325- EE02)\u2019 provided by Korea Forest Service (KoreaForestry Promotion Institute).
