EFFECTS OF EXPRESSING ESCHERICHIA-COLI THREONINE SYNTHASE IN TOBACCO (NICOTIANA-TABACUM-L) SUSPENSION-CULTURE CELLS ON FREE AMINO-ACID LEVELS, ASPARTATE PATHWAY ENZYME-ACTIVITIES AND UPTAKE OF ASPARTATE INTO THE CELLS

The amino acids threonine and methionine are derived from aspartate in a multibranched biosynthetic pathway. In higher planes, the branchpoi nt enzymes, threonine synthase (TS;EC 4.2.99.2) and cystathionine gamm a-synthase (C gamma S; EC 4.2.99.9), which lead to threonine/isoleucin e and methionine synthesis, respectively, compete against each other f or pathway intermediates. In order to better understand the regulation and interplay between these competing pathways, the feedback-insensit ive E. roll TS was constitutively expressed in tobacco (Nicotiana taba cum L.) suspension cultured cells via Agrobacterium-mediated transform ation. Expression of E. coli TS in tobacco cells resulted in a 7-fold increase in total TS activity and a 5.7-fold increase in free threonin e levels. C gamma S activity increased 3.5 fold, apparently to compens ate for the heightened competition for the common pathway intermediate homoserine phosphate. Homoserine dehydrogenase and threonine-sensitiv e aspartate kinase activities were increased by almost 2 fold. Free as partate was decreased by 55 % in the transformed cells, while free lys ine and isoleucine levels were not significantly changed, indicating t hat threonine does not regulate its own synthesis by inhibiting an enz yme early in the pathway. Transformed cells had a markedly reduced abi lity to take up (14)[C] aspartate from the medium, suggesting that thr eonine may regulate its synthesis in vivo in part by limiting aspartat e availability after Met, Lys, and Ile pools are filled.