Development and optimization of a microbial co-culture system for heterologous indigo biosynthesis

Background Indigo is a color molecule with a long history of being used as a textile dye. The conventional production methods are facing increasing economy, sustainability and environmental challenges. Therefore, developing a green synthesis method converting renewable feedstocks to indigo using engineered microbes is of great research and application interest. However, the efficiency of the indigo microbial biosynthesis is still low and needs to be improved by proper metabolic engineering strategies. Results In the present study, we adopted several metabolic engineering strategies to establish an efficient microbial biosynthesis system for converting renewable carbon substrates to indigo. First, a microbial co-culture was developed using two individually engineered E. coli strains to accommodate the indigo biosynthesis pathway, and the balancing of the overall pathway was achieved by manipulating the ratio of co-culture strains harboring different pathway modules. Through carbon source optimization and application of biosensor-assisted cell selection circuit, the indigo production was improved significantly. In addition, the global transcription machinery engineering (gTME) approach was utilized to establish a high-performance co-culture variant to further enhance the indigo production. Through the step-wise modification of the established system, the indigo bioproduction reached 104.3 mg/L, which was 11.4-fold higher than the parental indigo producing strain. Conclusion This work combines modular co-culture engineering, biosensing, and gTME for addressing the challenges of the indigo biosynthesis, which has not been explored before. The findings of this study confirm the effectiveness of the developed approach and offer a new perspective for efficient indigo bioproduction. More broadly, this innovative approach has the potential for wider application in future studies of other valuable biochemicals’ biosynthesis.

Indigo in Precolonial South Asia

South Asia is the home of natural blue dye extracted from the indigo plant species indigofera tinctoria. Its production for commercial purposes began very early and peaked during the early modern period. Growing Asian and European demand for indigo in the 16th and early 17th centuries raised its status as a major commodity in Asian and Eurasian trade. Indigo production in South Asia increased, and Indian and other Asian merchants exported large quantities of it to West Asia from where some of it was re-exported to Europe via the Levantine trade of the eastern Mediterranean. From the mid-16th century, the Portuguese Estado da India exported large quantities of indigo to Lisbon. By the early 1600s, when the English and Dutch East India companies began trading with India, indigo had become a highly sought-after commodity in the markets of England and the Dutch Republic. Consequently, the English East India Company (EIC) and Verenigde Oost-indische Compagnie (Dutch East India Company or VOC) exported large quantities of it to Europe in the first half of the 17th century. With the rise of new indigo commodity chains in Europe’s transatlantic colonies, such as Guatemala, Jamaica, South Carolina, and Saint-Domingue, exports from South Asia declined. However, there was a substantial local demand, which kept the industry going well up to the end of the 18th century when indigo production would expand on an unprecedented scale in Bengal and some other parts of colonial India.

Light Intensity and Biofertilizers Effect on Natural Indigo Production and Nutrient Uptake of Indigofera tinctoria L.

This research investigated the effect of light intensity and biofertilizer on the yield, which includes the production of indigo compounds and plant nutrient uptake. The study used a randomized complete block design with a split plot design with 4 levels of light intensity as the main plots and 4 levels of biofertilizer as a sub plots with 3 replications. The combination of light intensity and biofertilizer affects fresh weight, biomass and tissue nitrogen. The highest fresh weight and biomass was found at 100% light intensity with double inoculation of mycorrhizae and rhizobium. Whereas the highest tissue nitrogen was at 10% light intensity with double inoculation of mycorrhizae and rhizobium. The production of indigo affected by light intensity, ie at 10% light intensity indicates the highest indigo. Mycorrhizae and rhizobium have a synergistic relationship as biofertilizer in increasing plant yields and nutrient uptakes in 100% light intensity.

An overview of microbial indigo-forming enzymes

Indigo is one of the oldest textile dyes and was originally prepared from plant material. Nowadays, indigo is chemically synthesized at a large scale to satisfy the demand for dyeing jeans. The current indigo production processes are based on fossil feedstocks; therefore, it is highly attractive to develop a more sustainable and environmentally friendly biotechnological process for the production of this popular dye. In the past decades, a number of natural and engineered enzymes have been identified that can be used for the synthesis of indigo. This mini-review provides an overview of the various microbial enzymes which are able to produce indigo and discusses the advantages and disadvantages of each biocatalytic system.

Structure-Based Redesign of a Self-Sufficient Flavin-Containing Monooxygenase towards Indigo Production

Indigo is currently produced by a century-old petrochemical-based process, therefore it is highly attractive to develop a more environmentally benign and efficient biotechnological process to produce this timeless dye. Flavin-containing monooxygenases (FMOs) are able to oxidize a wide variety of substrates. In this paper we show that the bacterial mFMO can be adapted to improve its ability to convert indole into indigo. The improvement was achieved by a combination of computational and structure-inspired enzyme redesign. We showed that the thermostability and the kcat for indole could be improved 1.5-fold by screening a relatively small number of enzyme mutants. This project not only resulted in an improved biocatalyst but also provided an improved understanding of the structural elements that determine the activity of mFMO and provides hints for further improvement of the monooxygenase as biocatalyst.