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Diosgenin serves as an important precursor of most steroidal drugs in market. Cholesterol was previously deemed as a sterol origin leading to diosgenin biosynthesis. This study reports that cholesterol is not in parallel with diosgenin biosynthesis inTrigonella foenum-graecum. We first perturbed its sterol composition using inhibitors specific for the upstream isoprenoid pathway enzymes, HMGR (3-hydroxy-3-methylgutaryl-CoA reductase) on the mevalonate (MVA) and DXR (1-deoxy-D-xylulose-5-phosphate reductoisomerase) on the 2-C-methyl-D-erythritol-4-phophate (MEP) pathways, and have revealed that diosgenin and cholesterol reversely or differently accumulated in either the MVA or the MEP pathway-suppressed plants, challenging the previously proposed role of cholesterol in diosgenin biosynthesis. To further investigate this, we altered the sterol composition by suppressing and overexpressing the 24-sterol methyltransferase type 1 (SMT1) gene inT. foenum-graecum, as SMT1 acts in the first committed step of diverting the carbon flux of cholesterol toward biosynthesis of 24-alkyl sterols. Knockdown ofTfSMT1expression led to increased cholesterol level but caused a large reduction of diosgenin. Diosgenin was increased upon theTfSMT1-overexpressing, which, however, did not significantly affect cholesterol biosynthesis. These data consistently supported that diosgenin biosynthesis inT. foenum-graecumis not associated with cholesterol. Rather, campesterol, a 24-alkyl sterol, was indicative of being correlative to diosgenin biosynthesis inT. foenum-graecum.

Lupeol is a pentacyclic triterpene that shows a variety of pharmacological properties. Compared to engineering the production of sesquiterpenes and diterpenes, it is much more challenging to engineer the biosynthesis of triterpenes in microbial platforms. This study showed our efforts on engineering the triterpene pathway in Escherichia coli and Saccharomyces cerevisiae cells by recruiting the codon-optimized three lupeol pathway genes from different organisms. By comparing their activities with their respective counterparts, the squalene synthase from Thermosynechococcus elongates (tSQS), the squalene epoxidase from Rattus norvegicus (rSE) and the lupeol synthase from Olea europaea (OeLUP) were introduced into E. coli BL21(DE3), a break-through from zero was observed for lupeol biosynthesis in a prokaryotic host. We also assessed the lupeol pathway under two different yeast backgrounds-WAT11 and EPY300, and have found that the engineered strains based on EPY300, named ECHHOe, processed the best lupeol-producing ability with the maximum lupeol titer being 200.1 mg l−1 at 30 °C in a 72 h-flask culture, which so far was the highest amount of lupeol obtained by a microbial system and provides a basis for further industrial application of lupeol in the future.

(Iso)flavonoids are one of the largest groups of natural phenolic products conferring great value to the health of plants and humans. Pueraria lobata, a legume, has long been used in Chinese traditional medicine. (Iso)flavonoids mainly present as glycosyl-conjugates and accumulate in P. lobata roots. However, the molecular mechanism underlying the glycosylation processes in (iso)flavonoid biosynthesis are not fully understood. In the current study, three novel UDP-glycosyltransferases (PlUGT4, PlUGT15, and PlUGT57) were identified in P. lobata from RNA-seq data. Biochemical assays of these three recombinant PlUGTs showed all of them were able to glycosylate isoflavones (genistein and daidzein) at the 7-hydroxyl position in vitro. In comparison with the strict substrate specificity for PlUGT15 and PlUGT57, PlUGT4 displayed utilization of a broad range of sugar acceptors. Particularly, PlUGT15 exhibited a much higher catalytic efficiency toward isoflavones (genistein and daidzein) than any other identified 7-O-UGT from P. lobata. Moreover, the transcriptional expression patterns of these PlUGTs correlated with the accumulation of isoflavone glucosides in MeJA-treated P. lobata, suggesting their possible in vivo roles in the glycosylation process.

Lupeol is a pentacyclic triterpene which possesses many pharmacological activities. It has been found in many plants but generally with low content. Specifically, castor bean (Ricinus communis L.) contains high amounts of lupeol deposited on the outer surface, which is the major composition of epidermal wax. However, the biochemical pathways or regulatory genes responsible for lupeol accumulation in castor are largely unknown. In this study, a comparative transcriptome study was performed between the epidermis and stele of castor hypocotyl. Thirty-nine candidate genes involved in lupeol biosynthesis were identified and their expressions were analyzed. In particular, five selected genes associated with the mevalonate (MVA) pathway and triterpene downstream biosynthesis showed high correlation between their expression levels and the lupeol contents. Furthermore, several kinds of transcription factors were found to be specifically expressed in the epidermis. Especially, four transcription factors could activate the expression of Lupeol Synthase (RcLUS) in vivo, which is a key gene involved in lupeol synthesis. Taken together, these data will not only advance our understanding of triterpenoid synthesis in plants, but also provides potential candidate genes for enhancing lupeol biosynthesis by genetic engineering.

Trigonella foenum-graecum L. (fenugreek) is a valuable resource of producing diosgenin which serves as a substrate for synthesizing more than two hundred kinds of steroidal drugs. Phytochemical analysis indicated that methyl jasmonate (MeJA) efficiently induced diosgenin biosynthesis in fenugreek seedlings. Though early steps up to cholesterol have recently been elucidated in plants, cytochrome P450 (CYP)- and glycosyltransferase (GT)-encoding genes involved in the late steps from cholesterol to diosgenin remain unknown. This study established comparative fenugreek transcriptome datasets from the MeJA-treated seedlings and the corresponding control lines. Differential gene expression analysis identified a number of MeJA-induced CYP and GT candidate genes. Further gene expression pattern analysis across a different MeJA-treating time points, together with a phylogenetic analysis, suggested specific family members of CYPs and GTs that may participate in the late steps during diosgenin biosynthesis. MeJA-induced transcription factors (TFs) that may play regulatory roles in diosgenin biosynthesis were also discussed. This study provided a valuable genetic resource to functionally characterize the genes involved in diosgenin biosynthesis, which will push forward the production of diosgenin in microbial organisms using a promising synthetic biology strategy.