Molecular and biochemical characterization of Catharanthus roseus perivine-Nß-methyltransferase.

The biosynthesis of monoterpenoid indole alkaloids in Catharanthus roseus has been most extensively studied, leading to the detailed characterization of the pathway for the formation of their well-known anticancer alkaloids. The present study describes the identification, molecular cloning, and functional expression of C. roseus perivine-Nß-methyltransferase (PeNMT) that converts perivine to Nß-methylperivine (vobasine). PeNMT is member of a recently discovered γ-tocopherol-like N-methyltransferase (γ-TLMT) gene family that displays high substrate specificity and that appears to have evolved in the Vinceae tribe of Apocynaceae family where most N-methylated MIAs have been identified in the phytochemical literature.

Canadian regulatory aspects of gene editing technologies.

The development of gene editing techniques, capable of producing plants and animals with new and improved traits, is revolutionizing the world of plant and animal breeding and rapidly advancing to commercial reality. However, from a regulatory standpoint the Government of Canada views gene editing as another tool that will join current methods used to develop desirable traits in plants and animals. This is because Canada focusses on the potential risk resulting from the novelty of the trait, or plant or animal product entering the Canadian environment or market place, rather than the process or method by which it was created. The Canadian Food Inspection Agency is responsible for the regulation of the environmental release of plants with novel traits, and novel livestock feeds, while Health Canada is responsible for the regulation of novel foods. Environment and Climate Change Canada, in partnership with Health Canada, regulates modified animals for entry into the environment. In all cases, these novel products may be the result of conventional breeding, mutagenesis, recombinant DNA techniques or other methods of plant or animal breeding such as gene editing. This novelty approach allows the Canadian regulatory system to efficiently adjust to any new developments in the science of plant and animal breeding and allows for risk-appropriate regulatory decisions. This approach encourages innovation while maintaining science-based regulatory expertise. Canadian regulators work cooperatively with proponents to determine if their gene editing-derived product meets the definition of a novel product, and whether it would be subject to a pre-market assessment. Therefore, Canada's existing regulatory system is well positioned to accommodate any new innovations or technologies in plant or animal breeding, including gene editing.

Rauvolfia serpentina N-methyltransferases involved in ajmaline and Nß -methylajmaline biosynthesis belong to a gene family derived from γ-tocopherol C-methyltransferase.

Ajmaline biosynthesis in Rauvolfia serpentina has been one of the most studied monoterpenoid indole alkaloid (MIA) pathways within the plant family Apocynaceae. Detailed molecular and biochemical information on most of the steps involved in the pathway has been generated over the last 30 years. Here we report the identification, molecular cloning and functional expression in Escherichia coli of two R. serpentinacDNAs that are part of a recently discovered γ-tocopherol-like N-methyltransferase (γ-TLMT) family and are involved in indole and side-chain N-methylation of ajmaline. Recombinant proteins showed remarkable substrate specificity for molecules with an ajmalan-type backbone and strict regiospecific N-methylation. Furthermore, N-methyltransferase gene transcripts and enzyme activity were enriched in R. serpentina roots which correlated with accumulation of ajmaline alkaloid. This study elucidates the final step in the ajmaline biosynthetic pathway and describes the enzyme responsible for the formation of Nß -methylajmaline, an unusual charged MIA found in R. serpentina.

A Picrinine N-Methyltransferase Belongs to a New Family of γ-Tocopherol-Like Methyltransferases Found in Medicinal Plants That Make Biologically Active Monoterpenoid Indole Alkaloids.

Members of the Apocynaceae plant family produce a large number of monoterpenoid indole alkaloids (MIAs) with different substitution patterns that are responsible for their various biological activities. A novel N-methyltransferase involved in the vindoline pathway in Catharanthus roseus showing distinct similarity to γ-tocopherol C-methyltransferases was used in a bioinformatic screen of transcriptomes from Vinca minor, Rauvolfia serpentina, and C. roseus to identify 10 γ-tocopherol-like N-methyltransferases from a large annotated transcriptome database of different MIA-producing plant species (www.phytometasyn.ca). The biochemical function of two members of this group cloned from V. minor (VmPiNMT) and R. serpentina (RsPiNMT) have been characterized by screening their biochemical activities against potential MIA substrates harvested from the leaf surfaces of MIA-accumulating plants. The approach was validated by identifying the MIA picrinine from leaf surfaces of Amsonia hubrichtii as a substrate of VmPiNMT and RsPiNMT. Recombinant proteins were shown to have high substrate specificity and affinity for picrinine, converting it to N-methylpicrinine (ervincine). Developmental studies with V. minor and R. serpentina showed that RsPiNMT and VmPiNMT gene expression and biochemical activities were highest in younger leaf tissues. The assembly of at least 150 known N-methylated MIAs within members of the Apocynaceae family may have occurred as a result of the evolution of the γ-tocopherol-like N-methyltransferase family from γ-tocopherol methyltransferases.

Discovery and functional analysis of monoterpenoid indole alkaloid pathways in plants.

Numerous difficulties have been associated with forward genetic approaches to identify, and functionally characterize genes involved in the biosynthesis, regulation, and transport of monoterpenoid indole alkaloids (MIAs). While the identification of certain classes of genes associated with MIA pathways has facilitated the use of homology-based approaches to clone other genes catalyzing similar reactions in other parts of the pathway, this has not greatly speeded up the pace of gene discovery for the diversity of reactions involved. Compounding this problem has been the lack of knowledge or even availability of certain MIA intermediates that would be required to establish a novel enzyme reaction to functionally identify a biosynthetic step or the candidate gene product involved. The advent of inexpensive sequencing technologies for transcriptome and genome sequencing, combined with proteomics and metabolomics, is now revolutionizing the pace of gene discovery associated with MIA pathways and their regulation. The discovery process uses large databases of genes, proteins, and metabolites from an ever-expanding list of nonmodel plant species competent to produce and accumulate MIAs. Comparative bioinformatics between species, together with gene expression analysis of particular tissue, cell, and developmental types, is helping to identify target genes that can then be investigated for their possible role in an MIA pathway by virus-induced gene silencing. Successful silencing not only confirms the involvement of the candidate gene but also allows identification of the pathway intermediate involved. In many circumstances, the pathway intermediate can be isolated for use as a substrate in order to confirm gene function in heterologous bacterial, yeast, or plant expression systems.

Application of carborundum abrasion for investigating the leaf epidermis: molecular cloning of Catharanthus roseus 16-hydroxytabersonine-16-O-methyltransferase.

The Madagascar periwinkle (Catharanthus roseus) produces the well-known and remarkably complex anti-cancer dimeric alkaloids vinblastine and vincristine that are derived from the coupling of vindoline and catharanthine monomers. This study describes the novel application of a carborundum abrasion (CA) technique for large-scale isolation of leaf epidermis-enriched proteins in order to purify to apparent homogeneity 16-hydroxytabersonine-16-O-methyltransferase (16OMT), which catalyses the second of six steps in the conversion of tabersonine into vindoline, and to clone the gene. Functional expression and biochemical characterization of recombinant 16OMT demonstrated its very narrow substrate specificity and high affinity for 16-hydroxytabersonine. In addition to allowing the cloning of this gene, the CA technique clearly showed that 16OMT is predominantly expressed in Catharanthus leaf epidermis. The results provide compelling evidence that most of the pathway for vindoline biosynthesis, including the O-methylation of 16-hydroxytabersonine, occurs exclusively in the leaf epidermis, with subsequent steps occurring in other leaf cell types.