research2 - REPORTS Discovery and Directed Evolution of a...

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Discovery and Directed Evolution of a Glyphosate Tolerance Gene Linda A. Castle, 1 * Daniel L. Siehl, 1 Rebecca Gorton, 1 Phillip A. Patten, 2 Yong Hong Chen, 2 Sean Bertain, 1 Hyeon-Je Cho, 1 Nicholas Duck, 3 James Wong, 3 Donglong Liu, 3 Michael W. Lassner 1 The herbicide glyphosate is effectively detoxiFed by N -acetylation. We screened a collection of microbial isolates and discovered enzymes exhib- iting glyphosate N -acetyltransferase (GAT) activity. Kinetic properties of the discovered enzymes were insufFcient to confer glyphosate tolerance to transgenic organisms. Eleven iterations of DNA shuffling improved enzyme efFciency by nearly four orders of magnitude from 0.87 mM 2 1 min 2 1 to 8320 mM 2 1 min 2 1 . ±rom the Ffth 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. Herbicide tolerance is the most widely plant- ed transgenic crop trait. Globally, about 75% of genetically modified crops are engineered for herbicide tolerance ( 1 ). Glyphosate-toler- ant crops marketed as Roundup Ready occu- py the greatest acreage. The popularity of glyphosate stems from its effectiveness, low cost, and low environmental impact ( 2 ). Glyphosate inhibits the enzyme enolpyruvyl- shikimate-3-phosphate synthase (EPSPS) in the plant chloroplast-localized pathway that leads to the biosynthesis of aromatic amino acids ( 3 ). Some microbial EPSPS enzyme variants are not inhibited by glyphosate and provide the only mode of action for today’s commercial glyphosate-tolerant crops. How- ever, the glyphosate remains in the plant and accumulates in meristems ( 4 ), where it may interfere with reproductive development and may lower crop yield ( 5 ). Tolerance is more commonly achieved through metabolic de- toxification of herbicides by native plant or transgene-encoded enzymes. Commercial transgenic detoxification traits currently in use for other herbicides involve hydrolysis, acetylation, and oxidative cleavage ( 6 9 ). In each case, the relevant transgene is of micro- bial origin. The advantage of glyphosate detoxifica- tion is the removal of herbicidal residue, which may result in more robust tolerance and allow spraying during reproductive de- velopment. N -Acetylglyphosate is not herbi- cidal ( 10 ) and is not an effective inhibitor of EPSPS ( 11 ). We sought a soluble enzyme capable of carrying out N -acetylation of glyphosate (Scheme 1). Acetyl coenzyme A (AcCoA) acts as an acetyl donor to a wide variety of substrates. Typically, hydroxyl groups and primary amines are the acceptors. Chemical acetylation of secondary amines is feasible, but enzymatic acetylation of com- pounds such as glyphosate appears to be un- common although not unknown ( 12 , 13 ). We tested a variety of characterized and unchar- acterized N -acetyltransferases for their ability to accept glyphosate as a substrate ( 14 ), but none were capable of acetylating the second- ary amine.
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This note was uploaded on 12/04/2011 for the course CHEM 590A taught by Professor Staff during the Summer '10 term at University of Illinois, Urbana Champaign.

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research2 - REPORTS Discovery and Directed Evolution of a...

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