Cytochromes P-450 from cassava (Manihot esculenta Crantz) catalyzing the first steps in the biosynthesis of the cyanogenic glucosides linamarin and lotaustralin - Cloning, functional expression in Pichia pastoris, and substrate specificity of the isolated recombinant enzymes

The first committed steps in the biosynthesis of the two cyanogenic glucosi des linamarin and lotaustralin in cassava are the conversion of L-valine an d L-isoleucine, respectively, to the corresponding oximes, Two full-length cDNA clones that encode cytochromes P-450 catalyzing these reactions have b een isolated, The two cassava cytochromes P-450 are 85% identical, share 54 % sequence identity to CYP79A1 from sorghum, and have been assigned CYP79D1 and CYP79D2. Functional expression has been achieved using the methylotrop hic yeast, Pichia pastoris, The amount of CYP79D1 isolated from 1 liter of P. pastoris culture exceeds the amounts that putatively could be isolated f rom 22,000 grown-up cassava plants. Each cytochrome P-450 metabolizes L-val ine as well as L-isoleucine consistent with the cooccurrence of linamarin a nd lotaustralin in cassava. CYP79D1 was isolated from P, pastoris, Reconsti tution in lipid micelles showed that CYP79D1 has a higher k(c) value with L -valine as substrate than with L-isoleucine, which is consistent with linam arin being the major cyanogenic glucoside in cassava, Both CYP79D1 and CYP7 9D2 are present in the genome of cassava cultivar MCol22 in agreement with cassava being allotetraploid, CYP79D1 and CYP79D2 are actively transcribed, and production of acyanogenic cassava plants would therefore require down- regulation of both genes.