An Integrated Approach for Exploration of Plant Rhizomes’ Specialized Metabolism and Low-Cost Production of Related Valuable Compounds

Plants can synthesize an incredible myriad of specialized metabolites, providing one of the richest sources of bioactive molecules with applications in pharmaceuticals, agriculture, cosmetics, and even biofuels. Unfortunately, only a small percentage of these chemicals have been harnessed because extraction from natural sources and chemical synthesis of these metabolites is frequently challenging, low-yielding, and economically unviable. Engineering metabolic pathways into heterologous hosts, such as yeast and bacteria, has received increased attention as a cost-effective and environmentally sustainable alternative for generating large amounts of valuable compounds. However, heterologous production requires complete knowledge of a molecule’s biosynthetic pathway, which is missing for most plant specialized metabolites. Additionally, the RNA-seq and transcriptomic data needed to elucidate these pathways is sparse. Plant rhizomes are of particular interest because they are a specialized stem tissue that often contain unique metabolites with medicinal properties. Classic examples include ginseng (ginsenosides) and turmeric (curcumin), which have been heavily studied. Accordingly, extensive transcriptomic profiles of these species are available, and the corresponding biosynthetic pathways are broadly known. However, rhizomes are one of the most difficult tissues for nucleic acid extraction due to their high polyphenol and polysaccharide content, limiting RNA sequencing and thus transcriptome analysis. To overcome this challenge, additional extraction steps and optimization are required to obtain high-quality RNA. The rhizomes of Tacca sp. are particularly valuable but lack transcriptomic data. These modified stem tissues produce a novel class of anti-cancer compounds called taccalonolides that can circumvent resistance to current chemotherapeutic drugs, such as taxol. With an interest to engineer low-cost and sustainable production of taccalonolides in yeast, we set out to elucidate the biosynthetic pathway of these pentacyclic steroid-type compounds and reconstruct it in host cells. Considering the lack of transcriptomic data, we designed an integrated bioinformatic, metabolic and analytical workflow targeting rhizome specialized metabolism. This encompasses RNA extraction and sequencing, transcriptomic analysis combined with rhizome metabolomics, candidate library screening, and heterologous expression of biosynthetic enzymes in a microbial host. Our ongoing approach will facilitate exploration of the untapped diversity of rhizome-produced metabolites and can be readily expanded to other plant tissues for the discovery of novel bioactive compounds. From a chemical perspective, it will guide elucidation of unknown pathways for biosynthesis of other valued terpenoids, such as withanolides or pentacyclic triterpene saponins. Further engineering of dedicated microbial production platforms will enable scaled up precision fermentation for sustainable and low-cost biomanufacturing of important molecules.