Discovery of novel alkaloid biosynthetic genes using biochemical genomics.

摘要

The benzylisoquinoline alkaloids (BIAs) are a large and diverse group of plant specialized metabolites derived from the amino acid L-tyrosine via a complex array of biosynthetic enzymes. Many BIAs exhibit potent biological activities, such as the analgesic morphine and the antimicrobial sanguinarine, and are important therapeutic agents. However, plants remain the only commercial sources for many pharmaceutical alkaloids. Despite the occurrence of more than 2500 BIAs in plants, their biosynthesis is still poorly understood. This research aimed to isolate novel genes involved in BIA biosynthesis. Norcoclaurine synthase (NCS) catalyzes the first committed step in BIA biosynthesis in plants. NCS cDNAs were isolated from an opium poppy (Papaver somniferum) elicited cell culture expressed sequence tag (EST) collection and were functionally expressed in Escherichia coli. Recombinant forms of NCS homologs from other plants were shown to be devoid of NCS activity, and a virus-induced silencing of ncs expression significantly reduced the accumulation of morphinan alkaloids, suggesting that NCS is a unique enzyme responsible for norcoclaurine biosynthesis in plants. A cDNA encoding tetrahydroprotoberberine N-methyltransferase (TNMT), an enzyme in sanguinarine biosynthesis, was also isolated from the opium poppy EST collection. Recombinant TNMT was purified, and characterized with respect to substrate specificity and kinetic parameters. TNMT activity and gene expression were detected in all opium poppy organs, which along with its ability to utilize canadine as a substrate, suggests that TNMT might be involved in other branches of BIA biosynthesis. An integrated comparative genomics approach employing transcript and mass spectrometry-based alkaloid profiling of cell cultures of Eschscholzia californica, Thalictrum flavum, and Papaver bracteatum, was used to discover additional novel BIA N-methyltransferase (NMT) genes. Six novel NMT genes were identified and four were functionally characterized, including pavine N-methyltransferase from T. flavum, which represents the first characterized enzyme associated with pavine alkaloid biosynthesis. An integration of alkaloid profiling with the transcriptomic data generally facilitated the bidirectional prediction of both the substrate specificity of individual NMTs and the species-specific alkaloid content of different cellular systems, thereby demonstrating how this platform can be used to dissect the molecular basis of the chemical diversity generated in plant specialized metabolism.