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Review
. 2023;10(1):20.
doi: 10.1186/s40643-023-00626-7. Epub 2023 Mar 13.

Production of indigo by recombinant bacteria

Julia A Linke  1   2 , Andrea Rayat  3 , John M Ward  3
Affiliations
Review

Production of indigo by recombinant bacteria

Julia A Linke et al. Bioresour Bioprocess. 2023.

Abstract

Indigo is an economically important dye, especially for the textile industry and the dyeing of denim fabrics for jeans and garments. Around 80,000 tonnes of indigo are chemically produced each year with the use of non-renewable petrochemicals and the use and generation of toxic compounds. As many microorganisms and their enzymes are able to synthesise indigo after the expression of specific oxygenases and hydroxylases, microbial fermentation could offer a more sustainable and environmentally friendly manufacturing platform. Although multiple small-scale studies have been performed, several existing research gaps still hinder the effective translation of these biochemical approaches. No article has evaluated the feasibility and relevance of the current understanding and development of indigo biocatalysis for real-life industrial applications. There is no record of either established or practically tested large-scale bioprocess for the biosynthesis of indigo. To address this, upstream and downstream processing considerations were carried out for indigo biosynthesis. 5 classes of potential biocatalysts were identified, and 2 possible bioprocess flowsheets were designed that facilitate generating either a pre-reduced dye solution or a dry powder product. Furthermore, considering the publicly available data on the development of relevant technology and common bioprocess facilities, possible platform and process values were estimated, including titre, DSP yield, potential plant capacities, fermenter size and batch schedule. This allowed us to project the realistic annual output of a potential indigo biosynthesis platform as 540 tonnes. This was interpreted as an industrially relevant quantity, sufficient to provide an annual dye supply to a single industrial-size denim dyeing plant. The conducted sensitivity analysis showed that this anticipated output is most sensitive to changes in the reaction titer, which can bring a 27.8% increase or a 94.4% drop. Thus, although such a biological platform would require careful consideration, fine-tuning and optimization before real-life implementation, the recombinant indigo biosynthesis was found as already attractive for business exploitation for both, luxury segment customers and mass-producers of denim garments.

Supplementary information: The online version contains supplementary material available at 10.1186/s40643-023-00626-7.

Keywords: Biocatalysis; Bioprocess; Commercialization; Heterologous DNA expression; Recombinant biology; Scale up; Sustainability; Vat dye.

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Conflict of interest statement

Competing interestsThe authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
Structural formula of indigo and leucoindigo with corresponding compound colours
Fig. 2
Fig. 2
Structural formula of indole, indoxyl, indican and isatan B
Fig. 3
Fig. 3
Reaction mechanism of indole production from tryptophan by enzyme tryptophanase
Fig. 4
Fig. 4
Natural pathway: from plant precursors, indican and isatan B, through intermediate indoxyl to indigo
Fig. 5
Fig. 5
Representative chemical pathways toward synthetic indigo. The main route is highlighted with purple lines and an arrow
Fig. 6
Fig. 6
Generic metabolic pathway from tryptophan to indigo
Fig. 7
Fig. 7
General flow diagram of the supply chain of the chemical manufacturing of 2 main indigo products, a powdered dye, or a liquid pre-reduced solution. The light blue area marks the steps that would be different if a biological manufacturing platform would be employed, and these alternative operations may be found in the light green area. The final processing steps of the formulation are anticipated as similar to the chemical and biological platforms; therefore, they have not been included in the blue or green shading. ‘High T’ and a thermometer denote high temperature. The splitting arrows, numbered 2. (light blue) and 3., have been used to visualize the formulation of liquid indigo, which usually commences after the drying process has been performed to a certain extent. The numbered steps denote: 1.—the main chemical reaction chain toward the synthesis of synthetic indigo; nowadays in the majority of industrial factories, the core reaction relies on aniline or its precursors as the raw materials. 2.—the formulation toward powdered indigo; usually performed via drying. 3.—the formulation toward pre-reduced liquid indigo; usually performed via catalytic hydrogenation and the addition of strong reducing agents. 4.—the fermentation reaction that leads to the synthesis of biological indigo. The grey centrifuge represents the downstream processing sequence for product purification, for which the individual unit operations have been described in detail in “Downstream processing considerations” section. The dark blue writing represents the main substrates that are required to perform a certain manufacturing step, while the violet writing has been used to underline the potentially most expensive compounds. The figure has been created with the help of BioRender
Fig. 8
Fig. 8
Representation of the supply chain extension of processing of the anticipated most expensive compounds required for the synthesis of biological indigo
Fig. 9
Fig. 9
Different enzymatic indole pathways toward indigo: dioxygenation (I), direct hydroxylation (II), and epoxidation (III)
Fig. 10
Fig. 10
Proposed process flowsheets, that are relevant for the production of commercial indigo dye products. Flowsheet A–C. represent flowsheets toward indigo derived via a biological platform, while for Flowsheet D. a chemical platform is assumed. Flowsheet A The flowsheet that is predicted for the considering platform. It has been assumed that indigo is in the medium after the fermentation, and no cell disruption has to take place. The process relies on the precipitation of indigo after the upstream chemical reaction. Depending on the chosen final step, this bioprocess could produce 2 different formulations of the product; either a powder indigo, or a pre-reduced dye solution. The normal writing represents the name of the considered step, while the cursive—the aim of the single flowsheet step or information about discarded or saved feed content. Flowsheet B The flowsheet is for the preparation of indigo by solubilization after the upstream chemical reaction. The process output is a pre-reduced solution only. It has been assumed that the indigo diffuses into the medium after production by cells. Flowsheet C This scenario represents in situ dyeing—the indigo is secreted onto the fabric. The final product is the dyed fabric. Flowsheet D is a comparative diagram of current steps for the production of synthetic indigo by a conventional chemical manufacturing platform, that has been sourced from Paul et al. (2021)
Fig. 11
Fig. 11
Different indigo forms: I.—insoluble parent pigment form; II.—acid leucoindigo; III.—monophenolate ionic form of alkaline leucoindigo; IV—biphenolate ionic form of alkaline leucoindigo. The drawn rectangles mark the expected colour of the compound
Fig. 12
Fig. 12
Sensitivity analysis chart, that presents percentage change in the annual output of indigo from a biological platform for scenarios. As the baseline (0%), the figure considers the anticipated product quantity (540 tonnes per annum), which has been calculated with the assumptions listed in Table 3., and based on the bioprocess sequence from Flowsheet A (Fig. 10). The light blue marks the best-case scenario, that could be achieved with short-to-medium-term process optimization, and the dark blue marks the predicted worst-case scenarios, where Table 3. values would not be achieved. For the value of upstream reaction titer, the worst-case scenario is 1 g/L, while the best-case scenario is 23 g/L. For the cumulative step yield, the former case is based on a sequence of 4 process steps, each of 80% yield, while the latter is based on the same number of steps, where each enables achieving 95% yield
Fig. 13
Fig. 13
A graph that represents the projected annual output for a recombinant biological indigo platform depending on the alterations of 2 main values, that have been dictated by the Sensitivity Analysis presented in Fig. 12. The assumption is that platform relies on the bioprocess sequence from Flowsheet A (Fig. 10). In Fig. 12, the basic scenario has been fully based on Table 3. values, and equals 540 tonnes. Each graph represents a scenario, where a singular factor from Fig. 11 fluctuates—the reaction titer or the DSP step yields—or for their simultaneous alteration. The equation used for output calculations is the same as the equation that was used for Table 3. output estimation, which has been Output = (fermenter number) × (output from single fermenter per batch) × (batch runs/year) (Table 3.). The values used for calculations have been taken from Table 3. assumptions, unless stated otherwise. For example, the 2nd bar from the left was calculated with Table 3. inputs, except for DSP yield, which for calculation purposes has changed from 0.75% (top-down approach) total to 4 × 95% (bottom-up approach)
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References

    1. Abduh MY, van Ulden W, Kalpoe V, van de Bovenkamp HH, Manurung R, Heeres HJ. Biodiesel synthesis from Jatropha curcas L. oil and ethanol in a continuous centrifugal contactor separator. Eur J Lipid Sci Technol. 2013;115:123–131. doi: 10.1002/ejlt.201200173. - DOI
    1. Agerkvist I, Enfors SO. Characterization of E. coli cell disintegrates from a bead mill and high pressure homogenizers. Biotechnol Bioeng. 1990;36:1083–1089. doi: 10.1002/bit.260361102. - DOI - PubMed
    1. Ajioka J, Yarkoni ORR (2017) Investigating the adoption of a new way of dyeing fabric in India [Online]. https://iteamsonline.org/investigating-the-adoption-of-a-new-way-of-dyei.... Accessed 03 Nov 2022.
    1. Alexandri M, Vlysidis A, Papapostolou H, Tverezovskaya O, Tverezovskiy V, Kookos IK, Koutinas A. Downstream separation and purification of succinic acid from fermentation broths using spent sulphite liquor as feedstock. Sep Purif Technol. 2019;209:666–675. doi: 10.1016/j.seppur.2018.08.061. - DOI
    1. Ameria SP, Jung HS, Kim HS, Han SS, Kim HS, Lee JH. Characterization of a flavin-containing monooxygenase from Corynebacterium glutamicum and its application to production of indigo and indirubin. Biotechnol Lett. 2015;37:1637–1644. doi: 10.1007/s10529-015-1824-2. - DOI - PubMed