A. Abstract

The world consumes over 40 million kilograms of indigo annually, primarily for dyeing denim. Indigo is currently derived from petroleum using a high energy process, and commercial dyeing involves the use of reducing agents to solubilize the dye. The development of biosynthetic and bioprocessing methodologies for indigo dyeing could have environmental and economic advantages. By combining the biosynthesis of indigo and the use of the natural indigo precursor indican, we propose a more sustainable dyeing method as an alternative to chemically-reduced indigo in the large scale production of indigo textiles. We achieved in vivo indigo production in high titers, and efficient cleavage of indican using a non-native glucosidase. Inspired by natural systems, we isolated and characterized several plant and bacterial glucosyl transferases hypothesized to produce indican. Lastly, we compare the cost and environmental impact of our alternative with the present chemical process.

B. Overview

The world produces over 40,000 tons of indigo per year to be able to dye 3 billion pairs of jeans. Unfortunately, the indigo production and dyeing industries utilize a variety of harmful chemicals (link to the image of harmful chemicals). Given previous iGEM interest in indigo and the need for a greener alternative to denim dyeing, we started our project – Blue Genes.

This summer we have taken inspiration from plant metabolic pathways to devise a biosynthetic approach to dyeing jeans with indigo. In the process, we have characterized main components of the metabolic pathway. In addition we have analyzed the scale up involved in taking our project from the bench to industry highlighting steps that need improvement as well as potential cost-energy savings.

One of our project goals this year is to improve the characterization of our indigo producing part so that other iGEM teams in the future can have access to this reliable and well characterized part. In order to characterize this enzyme, we looked to verify whether we were actually producing indigo, checked for factors affecting bio-indigo synthesis, and calculated kinetic data for our enzyme.

A. Indigo Titer

An important aspect in characterizing our FMO enzyme is to look into factors affecting indigo synthesis in order to be able to titrate the amount of indigo being produced. To do this, we ran a series of experiments with varying levels of tryptophan, pH buffering, and differing salts.

After culturing E. coli cells in plain LB media with resistance for over 24 hours, we quantified indigo concentration to be approximately 2.06 mg/L. Once we increased the tryptophan concentration in the media, we got an appreciable increase in bio-indigo production. We also tried two different salts, KCl and NaCl due to previous research suggesting potassium and sodium ions have an effect on the enzyme tryptophanase (tnaA), one of the enzymes involved in converting tryptophan to indole(superscript). However, there was little to no difference in indigo production from the different salts. Additionally, after buffering the pH level with sodium phosphate at pH 7, we observed little to no difference in indigo production.

From this, we concluded that the main factor affecting indigo production was the level of tryptophan present in the media. This makes sense because this is the raw material for cells in order to produce indole, the substrate the FMO enzyme acts on. The maximum amount of indigo we were able to produce was approximately 224 mg/L in buffered conditions and 2.4 g/L tryptophan concentration in the media. The results of these titrations are summarized below.

Experiment Scheme/Limitations

Cells were cultured in 5ml glass tubes in the appropriate media at 30o C for over 24 hours. YE stands for yeast extract. Buffering was done by the addition of sodium phosphate. Refreshment refers to the replacement of media after approximately 13 hours of growth.

Indigo Quantification

All indigo quantifications were done through absorbance measurements at 620 nm with TECAN instrument. In order to quantify our indigo production using this method, we created a standard indigo calibration curve with a stock solution of pure indigo in dimethyl sulfoxide (DMSO). The results of the calibration curve are below with a R2 value of 0.994. The calibration curve used in the experiment is shown below.

B. FMO Kinetics

Additionally, we wanted to measure how quickly FMO generated indigo from its substrate, indole. We used Michaelis-Menten kinetics to model the behavior of FMO with various concentrations of indole substrate to determine the Km and Vmax of the enzyme.

To generate kinetic data for FMO, we first needed to obtain purified enzyme. We used the T7 expression system and chemically competent BL21 cells to express a His6 tagged FMO and purify it using a nickel column. With this purified enzyme, we ran an assay with different concentrations of indole in a mixture containing 0.1 mM NADPH, 0.1 mM EDTA, and Bicine/KOH buffer (pH 8.5).



From this data, we generated a Michaelis-Menten kinetics graph, and found the Vmax and Km to be 1.166*10^(-5) mM/s and 0.8698 mM respectively. At the 95% confidence level, Vmax is in the range of [0.00000935 mM/s, 0.00001398 mM/s], while Km is in the range of [0.12 mM, 1.62 mM].

Experiment Scheme/Limitations

The kinetics assay was adopted from previous work with FMO on substrates such as trimethylamine (2 ref). To our knowledge, this is the first kinetic assay done with indole. All kinetic assays were carried out measuring absorbance at 620 nm with TECAN. A limitation in our kinetic assay is that we assumed that the oxidation step from indoxyl to indigo was nearly instantaneous, and subsequently measured only the indigo concentration. However, we believe that because of sufficient aeration and oxygen content during our assay, the error coming from this assumption would be very limited.

C. Indigo Toxicity

We were also interested in whether over-accumulation of indigo would be toxic to E. coli cells. This was of particular interest to us because large scale production of bio-indigo if toxicity occurred. To test the toxicity of indigo, we ran a time course experiment in which we measured the number of bacteria over a 24 hour period. Cells with the FMO plasmid and mutant FMO plasmid were cultured in 5ml tubes with 0.74 g/L tryptophan at 30 C. The mutant FMO, which contains mutagenized catalytic sites of the FMO, served as a control for this experiment. From this experiment, we observed no noticeable toxicity with indigo.

Limitations of Growth Assay

Soluble Indigo – Indican

Indigo is so insoluble in water that it is not capable of dyeing clothes by itself. Industrial dyeing gets around this problem by reducing indigo to leuco-indigo, a white soluble version of the dye. In our project, we have found a way to use indican (a natural occurring compound in indigo plants) to dye clothing (Link). Indican is a soluble precursor to indigo, and serves as a biosynthetic alternative to leuco-indigo.

Biosynthetic production of indican

Indigo producing plants have a naturally encoded pathway to produce indican. They utilize a glucosyl transferase (GT) to add a glucose molecule to the hydroxyl group of indoxyl. We intend to produce indican by co-expressing FMO and a GT in E. coli. Unfortunately, no sequence data is available for the glucosyl transferases that have activity on indoxyl. As part of our summer project, we embarked in a quest to find an indican producing GT.

Testing Homologus Enzymes

Glucosyl transferases(GTs) are ubiquitous and have been shown to act on a vast variety of substrates. Some known GTs have activity on substrates that resemble indoxyl which makes them attractive targets in our project.

This summer we have cloned a variety of GTs that have activity on compounds like benzoate, and jasmonate (See below for accession numbers and native substrates). We first attempted to express them in E.coli, but found most of them to form inclusion bodies.

As part of our future work, we intend on expressing them in eukaryotic systems (such as yeast and tobacco plants).

Active site modifications

This summer we have paid close attention to a bacterial glucosyl transferase (OleD).

OleD is known for its broad substrate specificity, and we have cloned it to co-express with the indoxyl producing enzyme FMO. In addition, we have purified OleD to conduct in-vitro testing for the production of indican as well as other glucosides.

OleD has been shown in the literature to further broaden its substrate specificity after introducing a set of active site mutations known as ASP mutations (A242V-S132F-P67T). Thorson et.al, 2010. We have recently cloned OleD-ASP and will be testing its activity on indoxyl as well as other substrates.

Indigo Plant cDNA

A very exciting approach at finding an indican producing GT involves going after those already encoded in Indigo Plants. This summer we have acquired 4 different indigo plants (Indigofera suffruticosa, Indigofera tinctoria, Polygonum tinctorium, and Isatis tinctoria), and used them to extract RNA from the leafs. RNA was reverse transcribed to generate cDNA libraries of all four plants. These cDNA libraries are being screened for indican producing GTs.

In order to screen cDNA, we have generated a multiple sequence alignment of Glucosyl Transferases (See image below).

Foot Note: Cropped version of a Multiple Sequence Alignment (MSA) of 122 B- UDP Glucosyl transferases found in the taxonomic group Core Eudicotyledons.

This alignment revealed a well conserved region which we have used to make degenerate primers for PCR. Our screening efforts have started to give good results! We have extracted 3 glucosyl transferases that have never been studied before from indigo producing plants. These new GTs will be submitted to NCBI and characterized.

Check out our first submission to NCBI!

A major goal of our project was to test whether indican could be used to dye fabric. Indican is a good candidate for dyeing because, like its industrial counterpart leuco-indigo, it is water soluble. The idea is that an enzyme called a glucosidase would cleave the sugar group from the indican leaving indoxyl. The freed indoxyl naturally oxidizes to form indigo, which would then, hopefully, be incorporated into the fabric.

First we wanted to see whether or not indican could be cleaved by a glucosidase. We suspected that a beta-glucosidase (GLU) found naturally in B. circulans would be able to recognize and cleave the beta-linked glucose moiety of indican. To test this hypothesis we cloned the gene into e. coli and expressed it with an attached 6XHis tag to allow for purification. Once we had obtained purified GLU, we set up reactions with the purified GLU and various levels of indican and visually tested for the production of a blue color - proof that indigo was being formed. The results can be seen below:

So, having confirmed we could cleave indican to make indigo, our next step was to test whether or not this process could be carried out on fabric itself. Would GLU continue to function in the presence of cotton polymers? Would the soluble indican be converted to insoluble indigo in such a way that it would strongly adhere to the cotton polymers? To test these ideas, we dunked cotton cloth into varying levels of indican dissolved in water. The cloth was then submerged in purified GLU. Over the course of 30 minutes, we could see the cloth change hues from white to varying levels of blue depending on the initial indican concentrations. See results below:

Just because the fabric turned blue however doesn’t necessarily mean that it would stay that way upon washing. The indican was converted into indigo, but was the indigo actually interacting with the cotton polymers in the same way as in a normal pair of blue jeans? To test the adherence of the indigo, we subjected the cloth to a number of different washes using water, SDS (like a laundry detergent), ethanol, and acetone. We did the same thing with a piece of actual blue jeans and compared our results. The results are below:

Remarkably, the indican dyed cloth proved to retain its color just as well as the blue jeans control. Neither showed any sign of color loss except for in the wash with acetone (an amphiphilic solvent) which is to be expected because of indigo’s higher solubility in acetone.