Siritunga D and Sayre RT

Department of Plant Biology, The Ohio State University, Columbus OH 43210

Cassava is one of the most important food crops in tropical Africa, Asia and South America. Cassava, however, contains the cyanogenic glucoside, linamarin which when hydrolyzed yields cyanide. The initial dedicated step in linamarin synthesis is the conversion of valine to N-hydroxyvaline by a cytochrome P450. The source of electrons for the reductive oxigenation of valine is a non-specific NADPH: cytochrome P450 reductase (CPR). Our objective is to identify the gene encoding cassava Cyt-P450-Val so as to alter its pattern of expression in transgenic cassava. Our strategy is to identify the specific cassava cytochrome P450 that catalyzes the N-hydroxylation of valine by heterologous expression of this enzyme activity in transgenic yeast. Yeast strains WAT11 and WAT21, expressing a plant CPR from Arabidopsis, were transformed with a cassava cDNA library pre-screened for clones containing potential Cyt-P450 consensus regions. The transformed yeast then was incubated with ¹⁴C-Valine to determine which yeast transformants were capable of converting valine into intermediates of the linamarin biosynthetic pathway. Transformant I-11 has shown promising results with respect to the presence of altered forms of valine having a similar retention time on TLC-plates as the authentically modified valine obtained from cassava leaf microsomes. Significantly, synthesis of the valine product produced by I-11 is inhibited by both chlopromazine and O-methyl threonine, a known P450 inhibitor and a specific inhibitor of the cyanide synthesis pathway, respectively. The cDNA and the genomic clone of I11 has been sequenced and show high homology to known cytochrome P450s. Anti-sense I-11 was cloned to pKYLX plasmid and introduced into cassava via Agrobacterium mediated transformation. Analysis of the transformants is on-going.