Antisense RNA-mediated suppression of benzophenanthridine alkaloid biosynthesis in transgenic cell cultures of California poppy.

Abstract

California poppy (Eschscholzia californica Cham.) cell cultures produce several benzophenanthridine alkaloids, such as sanguinarine, chelirubine, and macarpine, with potent pharmacological activity. Antisense constructs of genes encoding two enzymes involved in benzophenanthridine alkaloid biosynthesis, the berberine bridge enzyme (BBE) and N-methylcoclaurine 3'-hydroxylase (CYP80B1), were introduced separately into California poppy cell cultures. Transformed cell lines expressing antisense BBE or antisense CYP80B1 constructs and displaying low levels of BBE or CYP80B1 mRNAs, respectively, showed reduced accumulation of benzophenanthridine alkaloids compared with control cultures transformed with a beta-glucuronidase gene. Pathway intermediates were not detected in any of the transformed cell lines. The suppression of benzophenanthridine alkaloid biosynthesis using BBE or CYP80B1 antisense RNA constructs also reduced the growth rate of the cultures. Two-dimensional (1)H-nuclear magnetic resonance and in vivo (15)N-nuclear magnetic resonance spectroscopy showed no difference in the abundance of carbohydrate metabolites in the various transgenic cell lines. However, transformed cells with reduced benzophenanthridine alkaloid levels contained larger cellular pools of several amino acids including alanine, leucine, phenylalanine, threonine, and valine compared with controls. The relative abundance of tyrosine, from which benzophenanthridine alkaloids are derived, was less than 2-fold higher in antisense-suppressed cells relative to controls. These results show that alterations in the metabolic flux through benzophenanthridine alkaloid biosynthesis can affect the regulation of amino acid pools. These data provide new insight into the metabolic engineering of benzophenanthridine alkaloid pathways.