Gene Engineering and Protein Engineering of Secondaty Metabolism in Medicinal Plants.

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Cysteine synthase (CSase) (O-acetylserine(thiol)-lyase, EC 4.2.99.8) in higher plants catalyxes the formation of L-cysteine from Oacetyl-L-serine and hydrogen sulfide as the substrates. This enzyme is also reponsible for the biosynthesis of secondary non-protein heterocyclic beta-substituted alamines, e.g. beta-pyrazole alanine and mimosine. The final aims of uor long-term study are to reveal molecular basis for the biosynthesis of these amino acids and to engineer the producibility. We have obtained two cDNA clones encoding cytoplasmic CSaseA and chloroplast CSaseB from spinach by means of the microsequencing of peptide fragments followed by synthetic oligonucleotide. The cDNA for CSaseB contained the transit peptide sequence of52 amino acids. The experiments of site-directed mutagenesis teplacing 12 conserved Lysresidues into Ala indentified a functional Lys residue respansible for the binding of pyridoxal phosphate cofactor. Transgenic tobacco plants, integrated with the constructs for 35S-CSaseA cDNA, were obtained.analysis of transgenic plants showed the enhanced CSase activities in the plants with the sense constructs and the decreased activity in the antisense-construct plants. Theprokaryotic high expression system was estabkished with pET vector. beta-Purazole alanine, a specific metabolite of watermelon, was produced in bibo by feeding pyrazole to E.coli cuture expressing plant CSase.