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

Abstract

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.

A modular and synthetic biosynthesis platform for de novo production of diverse halogenated tryptophan-derived molecules

“…Moreover, we hypothesized that this co-culture approach was necessary as a consolidated bioprocessing approach (wherein halogenase and downstream enzyme are co-localized) would lead to high competition for the intracellular tryptophan pool by both competing pathways and result in more un-halogenated products. Similar approaches have been used to reduce metabolic burden and generate a variety of products downstream of tryptophan, such as tryptamine and indigo 88,89 . Thus, one cell can act to primarily produce halogenated tryptophan whereas the other cell can specialize on the downstream conversion of halogenated tryptophan.…”

Synthetic mutualism in engineered E. coli mutant strains as functional basis for microbial production consortia

“…For example, recent developments focused on the formation of derivatives of aromatic amino acids with recombinant Escherichia coli cultures. For instance, indigo, a pigment conventionally produced chemically for the textile industry, could be bio‐synthesized from simple carbon substrates applying an engineered E. coli – E. coli system [ 14 ]. Additionally, tryptamine synthesis was established in subsequent studies [ 15 ] and the biosynthesis of rosmarinic acid was greatly improved compared to mono‐culture performance [ 16 ] by using a consortium of three fine‐tuned E. coli submodules.…”

Engineering microbial consortia of Elizabethkingia meningoseptica and Escherichia coli strains for the biosynthesis of vitamin K2

“…Compared with other expression systems, E. coli remains the preferred host for producing recombinant proteins because of several advantages, including rapid, inexpensive, and high-yield protein production, due to the well-characterised genetics and variety of available molecular tools [ 21 ]. Several studies have explored the benefits of developing microbial consortia based on the engineering of microbial chassis [ 22 – 25 ]. Two E. coli strains or E. coli strains co-cultured with other strains were constructed individually to accommodate different pathway modules, which helped reduce the overwhelming metabolic stress on each strain [ 26 , 27 ].…”