Evolution of a microbial acetyltransferase for modification of glyphosate: a novel tolerance strategy

Siehl, Daniel L.; Castle, Linda A.; Gorton, Rebecca; Chen, Yong Hong; Bertain, Sean; Cho, Hyeon-Je; Keenan, Robert J.; Liu, Donglong; Lassner, Michael

doi:10.1002/ps.1014

Cited by 58 publications

(32 citation statements)

References 9 publications

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“…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).…”

Section: Introductionmentioning

confidence: 99%

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

2015

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Development and commercialization of a transgenic crop depends upon the availability of a reliable quantitative assay sensitive enough to detect low level presence of transgenic material in mixed seed or in a commercial product. An appropriate species-specific endogenous control also is necessary for accurate quantification. A novel mode of transgenic tolerance to glyphosate, whereupon an engineered acetylase detoxifies glyphosate, was recently developed and incorporated into transgenic canola (Brassica napus). Here, we describe two highly specific and sensitive PCR-based assays for the transgenic event, DP-Ø73496-4. We also developed and evaluated an appropriate endogenous control to be used in conjunction with the event-specific assays. This endogenous control is specific to the A genome of cultivated canola-quality oilseed rape based on our analysis of the diversity of FatA gene sequences.

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Section: Introductionmentioning

confidence: 99%

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

2015

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“…Glyphosate (N-phosphonomethylglycine) is a widely used herbicide that acts nonselectively through inhibition of 5-enolpyruvylshikimate-3-phosphate (EPSP) 4 synthase in the aromatic biosynthesis pathway (2). Its efficacy against all plant species, low cost, low mammalian toxicity, and benign environmental impact favor its use in crops that have a transgenic tolerance mechanism (3).…”

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confidence: 99%

“…1). Despite extensive screening of biological amines, including amino acids, nucleotides and antibiotics, the physiological substrates for the native enzymes are unknown (4).…”

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confidence: 99%

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The Molecular Basis of Glyphosate Resistance by an Optimized Microbial Acetyltransferase

Siehl

Castle

Gorton

et al. 2007

Journal of Biological Chemistry

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GAT is an N-acetyltransferase from Bacillus licheniformis that was optimized by gene shuffling for acetylation of the broad spectrum herbicide, glyphosate, forming the basis of a novel mechanism of glyphosate tolerance in transgenic plants ( Glyphosate (N-phosphonomethylglycine) is a widely used herbicide that acts nonselectively through inhibition of 5-enolpyruvylshikimate-3-phosphate (EPSP) 4 synthase in the aromatic biosynthesis pathway (2). Its efficacy against all plant species, low cost, low mammalian toxicity, and benign environmental impact favor its use in crops that have a transgenic tolerance mechanism (3). The predominant transgene currently in use encodes a bacterial EPSP synthase that is nearly insensitive to inhibition by glyphosate (2). We reported an alternative strategy for glyphosate resistance involving enzymatic conversion of glyphosate to N-acetylglyphosate (NAG) (1). Because the acetylated form of glyphosate has low affinity for the active site of EPSP synthase, it is nonherbicidal. Three variants of an enzyme that catalyzes N-acetylation of the secondary amine of glyphosate were discovered from Bacillus licheniformis. These GAT 5 enzymes are 17 kDa in size, are most active at pH 6.8, and have a K m for acetyl coenzyme A (AcCoA) of 1-2 M. The native enzymes are very poor catalysts for acetylation of glyphosate, with a k cat of 5.3 min Ϫ1 and K m,GPJ of 1.3 mM (for ST401 GAT, referred to hereafter as native GAT). 6 The B. licheniformis enzymes belong to a diverse family of largely uncharacterized bacterial N-acetyltransferases sharing between 30 and 64% amino acid identity to native GAT (Fig. 1). Despite extensive screening of biological amines, including amino acids, nucleotides and antibiotics, the physiological substrates for the native enzymes are unknown (4).To develop gat as a transgene for glyphosate resistance in crops, we subjected the three B. licheniformis enzymes to 11 rounds of gene shuffling and obtained optimized variants with up to a 4,500-fold increase in catalytic efficiency (k cat /K m ) relative to the native enzyme. This was achieved through a combination of improvements in k cat (190-fold) and K m,GPJ (24-fold). When introduced into plants, optimized gat genes confer robust tolerance to glyphosate (1).To gain insight into the molecular mechanism of glyphosate N-acetylation by GAT, we determined the structure of a 7th round GAT variant (termed R7 GAT) in ternary complex with AcCoA and the competitive inhibitor 3-phosphoglycerate (3PG) and in binary complex with oxidized CoA. We also car-* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. The atomic coordinates and structure factors (code 2JDC, 2JDD) have been deposited in the Protein

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“…Glyphosate acetylation activity was increased more than 2,000-fold by gene shuffling and site-directed mutagenesis. 7,8) In addition to CP4 and GAT, a gene from Ochrobactrum anthropi encoding glyphosate oxidoreductase (GOX) was employed to deactivate glyphosate and contributed to glyphosate tolerance in canola. 5) Transgenic glufosinate-tolerant crops have also been commercialized.…”

Section: Commercialized Herbicide-tolerant Crops Created By Thementioning

confidence: 99%

Creation of herbicide-tolerant crops by gene targeting

Endo

Toki

2013

J. Pestic. Sci.

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Herbicide-tolerant crops including glyphosate-tolerant crops, generated via transgenic approaches are now cultivated widely. However, only a few varieties of transgenic herbicide-tolerant crops have been commercialized due to the time and cost required to obtain regulatory clearance. In contrast, herbicide-tolerant crops resulting from mutagenesis approaches are less problematic. However, the low occurrence of spontaneous mutations at the required sequence in the target locus continues to limit this approach. A promising alternative for inducing desirable mutations in endogenous plant genomes is homologous recombination (HR)-dependent gene targeting (GT). Here, we review three different approaches to create herbicide-tolerant plants, i.e., transgenic approaches, mutagenesis and HR-dependent GT. Among these techniques, HR-dependent GT is relatively new. We introduce the basic mechanism of HR-dependent GT, present an example of how to construct herbicide-tolerant rice plants by this method, and suggest candidate genes that could be targeted to confer herbicide tolerance by HR-dependent GT.

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Evolution of a microbial acetyltransferase for modification of glyphosate: a novel tolerance strategy

Cited by 58 publications

References 9 publications

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

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

The Molecular Basis of Glyphosate Resistance by an Optimized Microbial Acetyltransferase

Creation of herbicide-tolerant crops by gene targeting

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