Team:Berkeley/Project/GT

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.

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. For a table with accession numbers and native substrate of our GTs Click here. We have attempted to express all of our GTs 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). Once we express them in a functional form, we intend will test their activity on indoxyl.

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.

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).

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

Gantt , Richard W. "Probing the Aglycon Promiscuity of an Engineered Glycosyltransferase." 47.46 (2008): 8889–8892. Print. .
Warzecha, Heribert. "Formation of the indigo precursor indican in genetically engineered tobacco plants and cell cultures." 1467.7652 (2007): 185–191. Print. .
Links to Glucosyl Trasnferases. See table for more accession numbers: Accession Number Table
http://www.brenda-enzymes.org/literature/lit.php4?e=2.4.1.220&r=488950| BRENDA]
http://www.ncbi.nlm.nih.gov/protein/295854835?report=genbank&log$=prottop&blast_rank=3&RID=UA9TK3KH015
http://www.ncbi.nlm.nih.gov/nuccore/295854834?from=32&to=1462&sat=4&sat_key=63128784
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC168234/pdf/624121.pdf