Team:Berkeley/Project/GT

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. Indican is a soluble precursor to indigo, and serves as a biosynthetic alternative to leuco-indigo.

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. We followed two main tactics in our search; Testing homologous enzymes and screening plant cDNA libraries. Hard work resulted in discovery of three new GTs, and the demonstration of indican biosynthesis! This represents the first time that a protein sequence has been linked to the production of indican.

Glucosyl transferases(GTs) are ubiquitous and have been shown to act on a vast variety of substrates. As part of our search for an indican producing GT we have tested enzymes shown in the literature to have activity on substrates that resemble indoxyl.

This summer we have cloned a variety of GTs that have activity on compounds like benzoate, and jasmonate. A table with accession numbers and native substrates of our GTs is shown below.

As a first experiment, we attempted to express all of our GTs in E.coli to check for solubility. We decided to test solubility by generating a C-terminus fusion of GTs with a yellow fluorescent protein (Venus). A sample parts plasmid is shown below. Imaging under a fluorescent microscope showed that most of our GTs formed inclusion bodies (Represented by punctate fluorescence as opposed to diffuse fluorescence).

OleD is a bacterial glucosyltransferase that natively glucosylates oleandomycin. Most interestingly, OleD is known to have a broad substrate specificity which includes compound that are similar to indoxyl. We cloned and purified OleD to test it invitro for indicant production. The vector is shown below.

To test indican production, we set up the following experiment.

Reaction mixes were set up following the conditions found in Thorson et. al. 2010

Unfortunately, no indican production was observed. Why was oleD not functional on indoxyl?
We came up with two possible solutions:
1. We can modify OleD’s substrate specificity to accommodate other compounds that are similar to indoxyl.
2. We can run the experiment under anaerobic conditions, thus preventing indoxyl from oxidizing into indigo.
A set of mutations identified by Thorson et al can impart a much broader substrate specificity to oleD than it already possesses. This set of mutations, called oleD-ASP, changes the A242V-S132F-P67T amino acid positions. We cloned and purified oleD-ASP.

Here we present the first instance of indican production using a recombinant enzyme. This is a major accomplishment in the development of our new biological dyeing process. The results from our anaerobic in-vitro experimentation with OleD is shown below. (HPLC DATA FOR OLED). (Mass Spectrometry data)

Absorbance data obtained from HPLC at 270nm. Reaction containing indoxyl, OleD, and donnor molecule UDP-glucosePeaks for indican and sodium complexed indican are observed along with the precursor indoxyl.

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).

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 NCBI submission
Accession number will be available soon!KF696704