Molecular phylogeography and historical demography of a widespread herbaceous species from eastern North America, Podophyllum peltatum.

PREMISE: Glacial/interglacial cycles and topographic complexity are both considered to have shaped today's diverse phylogeographic patterns of taxa from unglaciated eastern North America (ENA). However, few studies have focused on the phylogeography and population dynamics of wide-ranging ENA herbaceous species occurring in forest understory habitat. We examined the phylogeographic pattern and evolutionary history of Podophyllum peltatum L., a widely distributed herb inhabiting deciduous forests of ENA. METHODS: Using chloroplast DNA (cpDNA) sequences and nuclear microsatellite loci, we investigated the population structure and genetic diversity of the species. Molecular dating, demographic history analyses, and ecological niche modeling were also performed to illustrate the phylogeographic patterns. RESULTS: Our cpDNA results identified three main groups that are largely congruent with boundaries along the Appalachian Mountains and the Mississippi River, two major geographic barriers in ENA. Populations located to the east of the Appalachians and along the central Appalachians exhibited relatively higher levels of genetic diversity. Extant lineages may have diverged during the late Miocene, and range expansions of different groups may have happened during the Pleistocene glacial/interglacial cycles. CONCLUSIONS: Our findings indicate that geographic barriers may have started to facilitate the population divergence in P. peltatum before the Pleistocene. Persistence in multiple refugia, including areas around the central Appalachians during the Quaternary glacial period, and subsequent expansions under hospitable climatic condition, especially westward expansion, are likely responsible for the species' contemporary genetic structure and phylogeographic pattern.

Total Biosynthesis for Milligram-Scale Production of Etoposide Intermediates in a Plant Chassis.

Etoposide is a plant-derived drug used clinically to treat several forms of cancer. Recent shortages of etoposide demonstrate the need for a more dependable production method to replace the semisynthetic method currently in place, which relies on extraction of a precursor natural product from Himalayan mayapple. Here we report milligram-scale production of (-)-deoxypodophyllotoxin, a late-stage biosynthetic precursor to the etoposide aglycone, using an engineered biosynthetic pathway in tobacco. Our strategy relies on engineering the supply of coniferyl alcohol, an endogenous tobacco metabolite and monolignol precursor to the etoposide aglycone. We show that transient expression of 16 genes, encoding both coniferyl alcohol and main etoposide aglycone pathway enzymes from mayapple, in tobacco leaves results in the accumulation of up to 4.3 mg/g dry plant weight (-)-deoxypodophyllotoxin, and enables isolation of high-purity (-)-deoxypodophyllotoxin after chromatography at levels up to 0.71 mg/g dry plant weight. Our work reveals that long (>10 step) pathways can be efficiently transferred from difficult-to-cultivate medicinal plants to a tobacco plant production chassis, and demonstrates mg-scale total biosynthesis for access to valuable precursors of the chemotherapeutic etoposide.

Six enzymes from mayapple that complete the biosynthetic pathway to the etoposide aglycone.

Podophyllotoxin is the natural product precursor of the chemotherapeutic etoposide, yet only part of its biosynthetic pathway is known. We used transcriptome mining in Podophyllum hexandrum (mayapple) to identify biosynthetic genes in the podophyllotoxin pathway. We selected 29 candidate genes to combinatorially express in Nicotiana benthamiana (tobacco) and identified six pathway enzymes, including an oxoglutarate-dependent dioxygenase that closes the core cyclohexane ring of the aryltetralin scaffold. By coexpressing 10 genes in tobacco-these 6 plus 4 previously discovered-we reconstitute the pathway to (-)-4'-desmethylepipodophyllotoxin (the etoposide aglycone), a naturally occurring lignan that is the immediate precursor of etoposide and, unlike podophyllotoxin, a potent topoisomerase inhibitor. Our results enable production of the etoposide aglycone in tobacco and circumvent the need for cultivation of mayapple and semisynthetic epimerization and demethylation of podophyllotoxin.