The use of plant cell cultures for the production of podophyllotoxin and related lignans

Podophyllotoxin isolated from rhizomes of Podophyllum plants (Podophyllaceae) serves as precursor for the semi-synthesis of anticancer therapeutics. The plants are collected from the wild and are becoming threatened species due to overcollection. Suspension cultures of Linum album (Linaceae) accumulate podophyllotoxin as well and could therefore serve as an alternative source for this important aryltetralin lactone lignan. The culture characteristics of suspension cultures of Linum album are reported in the paper. The fast growth of the cell culture and a podophyllotoxin accumulation of about 0.2% of the cell dry weight would enable a podophyllotoxin production of ca. 28 mg in 11 days in one liter of suspension culture

Deoxypodophyllotoxin 6-hydroxylase, a cytochrome P450 monooxygenase from cell cultures of Linum flavum involved in the biosynthesis of cytotoxic lignans

Cell-suspension cultures of Linum flavum L. (Linaceae) synthesize and accumulate aryltetrallydronaphthalene lignans with 6-methoxypodophyllotoxin as the main component. The experimental data indicate that the biosynthesis of 6-methoxypodophyllotoxin occurs via deoxypodophyllotoxin, beta-peltatin, and beta-peltatin-A methyl ether. The enzyme catalyzing the introduction of the hydroxyl group in position 6 is deoxypodophyllotoxin 6-hydroxylase (DOP6H). The enzyme was shown to be a cytochrome P450-dependent monooxygenase by blue-light reversion of carbon monoxide inhibition and inhibition by cytochrome c. DOP6H is a membrane-bound microsomal enzyme with a pH optimum of 7.6 and a temperature optimum of 26 degreesC. Deoxypodophyllotoxin is specifically accepted with an apparent K-m of 20 mum and a saturation concentration of 200 muM; 4'-demethyldeoxypodophyllotoxin is the only other tested substrate accepted for hydroxylation. DOP6H predominantly accepts NADPH as electron donor; NADH can only sustain low hydroxylation activities. A synergistic effect of NADPH and NADH is not observed. The enzyme is saturated around 250 M NADPH; the apparent K-m for this substrate is 36 M.</p