Indigo & Indirubin - blue pill or red pill?

Indigo, the colorant that gives our jeans their characteristic appearance, is an industrially relevant compound indispensable in our everyday lives. In a survey of 196 participants, we asked the citizen of Freiburg, how many pairs of jeans they do have in their wardrobe. With an average of six pairs of jeans, we estimate that several 10,000 tons of indigo must be produced each year to cover the needs of the global fashion industry.

Indigo is a deep blue, crystalline organic chemical compound. It is an organic pigment with high color strength, has a rather high melting point of around 390–392°C and is poorly soluble in water, ethanol, and diethyl ether.
Historically, Indigo was extracted from certain plants through a tedious, time-consuming, and inconsistent process [60]. Nowadays, Indigo is produced by chemical synthesis. Although this method is very effective, it is not sustainable, and we think it is necessary to find alternatives for that! The biggest problems that emerge from chemical synthesis are its dependency on fossil fuels, such as oil as a source material, high energy consumption and the production of high quantities toxic wastewater [61]. Considering the environmental crisis, together with the declining availability of fossil fuels, one can quickly see that it is not bearable to continue the production of Indigo by chemical means for a much longer time. A better solution is needed. And where could this solution be harboured? In synthetic Biology of course! Already in the early 1960s, the first Indigo-producing bacteria were discovered and in the 1980s efforts were made to use bacteria to produce Indigo on a large scale [62]. However, until now, microbial production of Indigo hardly reached levels that compete with existing ways of synthesis on a global market.

How to improve indigo production with the use of genetic modified bacteria?
Microbial production of indigo requires four genes (TnaB, TnaA, Fre and XiaI) and the amino acids Tryptophan (Figure 1). L-Tryptophan is imported into bacteria with the help of a membrane protein called low affinity tryptophan permease, emerging from the gene TnaB [63]. Afterwards, L-tryptophan is split up into Indole, NH4+ and Pyruvate by the Tryptophanase that stems from the gene TnaA [64]. The reactions continue by the hydroxylation of Indole through XiaI. To enhance the effectivity of this enzyme, the NAD(P)H-flavin reductase is expressed by the gene Fre, as it provides XiaI with FADH2 by adding Hydrogen to FAD (which in turn emerges from the activity of XiaI) [65]. Finally, after going through two enzymatic reactions, L-Tryptophan is transformed to either 3-Hydroxyindole or 2-Hydroxyindole. These two substances spontaneously react to 3-Oxindole and 2-Oxindole through the secession of a Hydrogen from the OH-group. Through spontaneous dimerization Indigo and Indirubin are formed. Two molecules of 3-Oxindole form one molecule of Indigo, while one molecule of 3-Oxindole combined with one molecule of 2-Oxindole form one molecule of Indirubin (Figure 1) [65].

Schematic representation of the used Indigo/Indirubin pathway.

Figure 1: Schematic representation of the used Indigo/Indirubin pathway. L-Tryptophan is imported by the membrane protein TnaB (low affinity tryptophan permease). L-tryptophan is cleaved into Indole, NH4+ and Pyruvate by a Tryptophanase (TnaA). The reactions continues by the hydroxylation of Indole through XiaI. To enhance the effectivity of this enzyme, the NAD(P)H-flavin reductase provides XiaI with FADH2 by adding Hydrogen to FAD. Finally, Indole is transformed to either 3-Hydroxyindole or 2-Hydroxyindole. These two substances spontaneously react to 3-Oxindole and 2-Oxindole through the secession of a hydrogen from the OH-group. Through spontaneous dimerization Indigo and Indirubin are formed. Graphic was adapted from [65].

To improve production, we target TnaA and XiaI inside microcompartments, by adding the SnoopCatcher and SpyCatcher, respectively to the genes. By adding L-Cysteine to the mix, we can shift the production to Indirubin, an isomer of Indigo [65]. This is especially interesting as this compound is currently being tested as a supportive drug to treat leukaemia: current trials show the beneficial effects of indirubin while being scarce in side effects. A reason why indirubin might be of significant importance in the medical sector in the near future [66]. However, as of now, Indirubin is very costly even in small quantities. In case of being used as a drug, its production is likely to be the threshold limiting how many people will have the financial means to be treated with the help of Indirubin. Improvement of synthesis to reduce cost and enhance accessibility to this product are of high priority. Therefore, our compartmentalisation toolbox could be one way to achieve a higher yield of Indirubin, providing a solution to this scarceness of indirubin and possibly improving the life of many leukaemia patients by allowing for a fair distribution of this upcoming drug.


Trehalose is a non-reducing disaccharide consisting of two glucose molecules with an α (1-1) linkage. It has a wide range of applications, for example, as a sweetener, preservative, or as additive in pharmaceutical products [67] [68] [69] [70].

Trehalose occurs naturally in various plants, fungi and in the haemolymph of many insects. At first it was believed to be a rare sugar, but later it was found that many bacteria, fungi, plants, and invertebrates produce it. It is difficult to detect in most organisms because it is usually rapidly metabolized by an enzyme called trehalase [71]. In bacteria, it is expressed under osmotic stress [72]. It acts as an osmoprotectant in bacteria. One possible way in which trehalose preserves and protects macromolecules is by replacing the binding of water molecules with the binding of the sugar, thereby maintaining the structural integrity of these biological structures [73]. Two enzymes are involved in trehalose synthesis. The trehalose-6-phosphate synthetase (TPS) is encoded by the gene otsA. This enzyme converts glucose-6-phosphate (G-6-P) and UDP-glucose to trehalose-6-phosphate (T-6-P). The gene otsB expresses trehalose-6-phosphatase (TPP), which converts T-6-P to trehalose (Figure 2) [68].

Due to the wide range of applications and the need for only two enzymes, we decided to use trehalose as another pathway to encapsulate in our microcompartments.

Schematic representation of the used Trehalose pathway.

Figure 2: Schematic representation of the used Trehalose pathway. The trehalose-6-phosphate synthetase (TPS) is encoded by the gene otsA. This enzyme converts glucose-6-phosphate (G-6-P) and UDP-glucose to trehalose-6-phosphate (T-6-P). The gene otsB expresses trehalose-6-phosphatase (TPP), which converts T-6-P to trehalose.

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