CHLOROPLAST-TARGETED ANTHRANILATE SYNTHASE VARIANTS LEAD TO INCREASED ACCUMULATION OF THE ESSENTIAL AMINO ACID TRYPTOPHAN IN SOYBEAN SEED

¹ Monsanto, 700 Chesterfield Parkway West, Chesterfield, MO, 63017, USA

² Renessen, 800 North Lindbergh Blvd, St. Louis, MO, 63167, USA

Soybeans represent a valuable source of protein for both human and animal nutrition although they do not contain a balanced proportion of dietary essential amino acids. One strategy to increase the tryptophan content in plants is to make the biosynthetic pathway less sensitive to end-product inhibition, which is controlled through anthranilate synthase (AS). Anthranilate synthase catalyzes the first committed step in the biosynthesis of tryptophan, and is typically found as a two-subunit enzyme. The alpha subunit catalyzes the conversion of chorismate to anthranilate with ammonia as a co-substrate. The beta subunit is an amidotransferase that provides ammonia, from glutamine, to the alpha subunit. A gene previously identified from the C28 mutant maize line encoding a tryptophan feedback resistant AS alpha subunit was transformed into soybean for expression in the seed, and the protein was directed to the chloroplast using a transit peptide. The resulting seed from positively transformed plants had enhanced tryptophan levels. The AS gene of Agrobacterium tumefaciens (agro) is unique for plant and most microbial anthranilate synthases in that it contains both the chorismate conversion and glutamine amidotransferase activities on a single polypeptide, but retains feedback inhibition by tryptophan. Variants showing less sensitivity to feedback inhibition were rationally designed and constructed. The wild type agro AS gene was expressed in soybean seed, directed to the chloroplast, and showed elevated tryptophan levels. Enzyme activities were 100-200 fold higher in transgenic seed than in controls. Transforming plants with one of the variant deregulated genes enhanced free tryptophan levels even further.