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

作者：Daniel L. Siehl, Linda A. Castle, Rebecca Gorton, Yong Hong Chen, Sean Bertain, Hyeon‐Je Cho, Robert J. Keenan, Donglong Liu, Michael Lassner · 发表于：Pest Management Science · 年份：2005 · DOI：10.1002/ps.1014 · 被引用次数：60 · 研究领域：Advanced biosensing and bioanalysis techniques、Plant tissue culture and regeneration、CRISPR and Genetic Engineering

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.