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

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 m M ) 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. Copyright © 2005 Society of Chemical Industry