How to make bioactive alkaloids: Vinblastine and vincristine are important, expensive anticancer agents that are produced by dimerization of the plant-derived alkaloids catharanthine and vindoline. The enzymes that transform tabersonine into vindoline are known; however, the mechanism by which the scaffolds of catharanthine and tabersonine are generated has been a mystery. Caputi et al. now describe the biosynthetic genes and corresponding enzymes responsible. This resolves a long-standing question of how plant alkaloid scaffolds are synthesized, which is important not only for vinblastine and vincristine biosynthesis, but also for understanding the many other biologically active alkaloids found throughout nature. Science , this issue p. 1235

Lochnericine is a major monoterpene indole alkaloid (MIA) in the roots of Madagascar periwinkle (Catharanthus roseus). Lochnericine is derived from stereoselective C6, C7 epoxidation of tabersonine and can be further metabolized to generate other complex MIAs. While the enzymes responsible for its downstream modifications have been characterized, those involved in lochnericine biosynthesis remain unknown. By combining gene correlation studies, functional assays, and transient gene inactivation, we identified two highly conserved P450s that efficiently catalyze the epoxidation of tabersonine: tabersonine 6,7-epoxidase isoforms 1 and 2 (TEX1 and TEX2). Both proteins are quite divergent from the previously characterized tabersonine 2, 3 epoxidase and are more closely related to tabersonine 16-hydroxylase, involved in vindoline biosynthesis in leaves. Biochemical characterization of TEX1/2 revealed their strict substrate specificity for tabersonine and their inability to epoxidize 19-hydroxytabersonine, indicating that they catalyze the first step in the pathway leading to hörhammericine production. TEX1 and TEX2 displayed complementary expression profiles, with TEX1 mainly expressed in roots and TEX2 in aerial organs. Our results suggest that TEX1 and TEX2 originated from a gene duplication event and later acquired divergent, organ-specific regulatory elements for lochnericine biosynthesis throughout the plant, as supported by the presence of lochnericine in flowers. Finally, through sequential expression of TEX1 and up to four other MIA biosynthetic genes in yeast, we reconstituted the 19-acetylhörhammericine biosynthetic pathway and produced tailor-made MIAs by mixing enzymatic modules that are naturally spatially separated in the plant. These results lay the groundwork for metabolic engineering of tabersonine/lochnericine derivatives of pharmaceutical interest.

A new method was developed to incorporate photocages site-specifically into plasmids. Plasmid photoregulation was demonstrated in both cell-free reactions and mammalian cells.

Plasmids are ubiquitous in biology, where they are used to study gene-function relationships and intricate molecular networks, and hold potential as therapeutic devices. Developing methods to control their function will advance their application in research and may also expedite their translation to clinical settings. Light is an attractive stimulus to conditionally regulate plasmid expression as it is non-invasive, and its properties such as wavelength, intensity, and duration can be adjusted to minimise cellular toxicity and increase penetration. Herein, we have developed a method to site-specifically introduce photocages into plasmids, by resynthesizing one strand in a manner similar to Kunkel mutagenesis. Unlike alternative approaches to chemically-modify plasmids, this method is sequence independent at the site of modification and uses commercially available phosphoramidites. To generate our light-activated (LA) plasmids, photocleavable biotinylated nucleobases were introduced at specific sites across the T7 and CMV promoters on plasmids and bound to streptavidin to sterically block access. These LA-plasmids were then successfully used to control expression in both cell-free systems (T7 promoter) and mammalian cells (CMV promoter). These light-activated plasmids might be used to remotely-control cellular activity and reduce off-target toxicity for future medical use. Our simple approach to plasmid modification might also be used to introduce novel chemical moieties for advanced function. Abstract Figure: