title: Evolution of a microbial acetyltransferase for modification of glyphosate: a novel tolerance strategy.
abstract: N-Acetylation is a modification of glyphosate that could potentially be used in transgenic crops, given a suitable acetyltransferase. Weak enzymatic activity (k(cat) = 5 min(-1), K(M) = 1 mM) for N-acetylation of glyphosate was discovered in several strains of Bacillus licheniformis (Weigmann) Chester by screening a microbial collection with a mass spectrometric assay. The parental enzyme conferred no tolerance to glyphosate in any host when expressed as a transgene. Eleven iterations of DNA shuffling resulted in a 7000-fold improvement in catalytic efficiency (k(cat)/K(M)), sufficient for conferring robust tolerance to field rates of glyphosate in transgenic tobacco and maize. In terms of k(cat)/K(M), the native enzyme exhibited weak activity (4-450% of that with glyphosate) with seven of the common amino acids. Evolution of the enzyme towards an improved k(cat)/K(M) for glyphosate resulted in increased activity toward aspartate (40-fold improved k(cat)), but activity with serine and phosphoserine almost completely vanished. No activity was observed among a broad sampling of nucleotides and antibiotics. Improved catalysis with glyphosate coincided with increased thermal stability.
authors.affiliation: Verdia, 200 Penobscot Dr, Redwood City, CA 94063, USA. dan.siehl@pioneer.com
authors.forename: Daniel L
authors.initials: DL
authors.lastname: Siehl
authors.forename: Linda A
authors.initials: LA
authors.lastname: Castle
authors.forename: Rebecca
authors.initials: R
authors.lastname: Gorton
authors.forename: Yong Hong
authors.initials: YH
authors.lastname: Chen
authors.forename: Sean
authors.initials: S
authors.lastname: Bertain
authors.forename: Hyeon-Je
authors.initials: HJ
authors.lastname: Cho
authors.forename: Robert
authors.initials: R
authors.lastname: Keenan
authors.forename: Donglong
authors.initials: D
authors.lastname: Liu
authors.forename: Michael W
authors.initials: MW
authors.lastname: Lassner
mesh_terms: D000123:Acetyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001407:Bacillus / Q000201:enzymology; D019020:Directed Molecular Evolution; D004795:Enzyme Stability; D005998:Glycine / Q000031:analogs & derivatives* / Q000378:metabolism*; D006540:Herbicides / Q000378:metabolism*; D030821:Plants, Genetically Modified / Q000187:drug effects; D013379:Substrate Specificity; D014026:Nicotiana / Q000235:genetics; D003313:Zea mays / Q000235:genetics; D000097797:Glyphosate
chemical_list: D000123:Acetyltransferases; D005998:Glycine; D006540:Herbicides; D000097797:Glyphosate; C486861:glyphosate N-acetyltransferase