Discovery and Directed Evolution of a Glyphosate Tolerance Gene

Abstract

The herbicide glyphosate is effectively detoxified by N-acetylation. We screened a collection of microbial isolates and discovered enzymes exhibiting glyphosate N-acetyltransferase (GAT) activity. Kinetic properties of the discovered enzymes were insufficient to confer glyphosate tolerance to transgenic organisms. Eleven iterations of DNA shuffling improved enzyme efficiency by nearly four orders of magnitude from 0.87 mM-1 min-1 to 8320 mM-1 min-1. From the fifth iteration and beyond, GAT enzymes conferred increasing glyphosate tolerance to Escherichia coli, Arabidopsis, tobacco, and maize. Glyphosate acetylation provides an alternative strategy for supporting glyphosate use on crops.

Molecular Plant Breeding as the Foundation for 21st Century Crop Improvement

“…The first glyphosate-tolerant maize hybrids used a modified version of the endogenous maize gene encoding 5-enol-pyruvylshikimate-3-P synthase (Spencer et al, 2000), which was followed later by events produced with a 5-enol-pyruvylshikimate-3-P synthase gene isolated from Agrobacterium (Behr et al, 2004). More recently, a synthetic gene with enhanced glyphosate acetyltransferase activity was created via gene shuffling and selection in a microbial system (Castle et al, 2004). Each of these glyphosate-tolerant maize events also illustrates another benefit of biotechnology, where new combinations of regulatory sequences (e.g.…”

PCR-Based Detection and Quantification of a Transgenic Glyphosate-Tolerant Canola Using a Novel Reference Gene System

“…Glyphosate tolerance, despite the occurrence of sporadic resistance in weeds (Powles 2008), remains a desirable trait, especially in combination with tolerance to herbicides with a different mode of action. A novel mode of glyphosatetolerant canola DP-Ø73496-4 has been developed by DuPont Pioneer, resulting from the incorporation of a glyphosate N-acetyltransferase (gat) transgene expressing an engineered acetylase capable of detoxifying the glyphosate herbicide (Castle et al 2004;Siehl et al 2005;Siehl et al 2007).…”

Glyphosate-based phospho-organic herbicides – an outline of action, metabolism and the selected effects on humans and other organisms

“…Recently, the possible use of class II EPSPS found in Ochrobactrum anthropi has also been considered. [9][10][11][12][13]. At present, after almost 20 years of their presence in agronomy, transgenic varieties constitute the essential profile of continent-wide cultivation.…”