Selected cell cultures and induction methods for cloning and assaying cytochromes P450 in alkaloid pathways

Abstract

This chapter focuses on selected cell cultures and induction methods for cloning and assaying cytochromes P450 in alkaloid pathways. The biosynthesis of selected members of alkaloid classes for which the enzymatic steps are elucidated in the chapter, such as the monoterpenoid indole alkaloid vindoline and the isoquinoline alkaloids morphine, macarpine, and berberine, involves highly substrate-specific cytochromes P450. Although in vitro enzyme assays have been successfully developed for most of these cytochromes P450, purification to apparent homogeneity of cytochromes P450 from plant tissues has, in many cases, proved elusive because of the low abundance and instability of these proteins. Because many alkaloid biosynthetic pathways can be induced in plant cell culture, the differential expression of cytochrome P450 encoding genes involved in these pathways can be exploited in the cloning of these genes. The protocols described in the chapter are used successfully in the laboratories to clone and functionally express cytochrome P450-encoding cDNAs of alkaloid biosynthesis.