Lessons learned from metabolic engineering in hairy roots: Transcriptome and metabolic profile changes caused by Rhizobium-mediated plant transformation in Cucurbitaceae species.

Cucurbitaceae species are used in traditional medicine around the world. Cucurbitacins are highly oxygenated triterpenoids found in Cucurbitaceae species and exhibit potent anticancer activity alone and in combination with other existing chemotherapeutic drugs. Therefore, increasing production of these specialized metabolites is of great relevance. We recently showed that hairy roots of Cucurbita pepo can be used as a platform for metabolic engineering of cucurbitacins to modify their structure and increase their production. To study the changes in cucurbitacin accumulation upon formation of hairy roots, an empty vector (EV) control and Cucurbitacin inducing bHLH transcription factor 1 (CpCUCbH1)-overexpressing hairy roots of C. pepo were compared to untransformed (WT) roots. Whilst CpCUCbH1-overexpression increased production of cucurbitacins I and B by 5-fold, and cucurbitacin E by 3-fold when compared to EV lines, this increase was not significantly different when compared to WT roots. This indicated that Rhizobium rhizogenes transformation lowered the cucurbitacins levels in hairy roots, but that increasing expression of cucurbitacin biosynthetic genes by CpCUCbH1-overexpression restored cucurbitacin production to WT levels. Subsequent metabolomic and RNA-seq analysis indicated that the metabolic profile and transcriptome of hairy roots was significantly changed when compared to WT roots. Interestingly, it was observed that 11% of the differentially expressed genes were transcription factors. It was noteworthy that the majority of transcripts showing highest Pearson correlation coefficients to the Rhizobium rhizogenes genes rolB, rolC and ORF13a, were predicted to be transcription factors. In summary, hairy roots are an excellent platform for metabolic engineering of plant specialized metabolites, but these extensive transcriptome and metabolic profile changes should be considered in subsequent studies.

Comparative transcriptome analysis and identification of candidate genes involved in cucurbitacin IIa biosynthesis in Hemsleya macrosperma.

Hemsleya macrosperma (H. macrosperma) is widely used in southwestern China as folk medicine with various bioactivities. Cucurbitacin IIa is the main active component in H. macrosperma and draws increased attention for its potential pharmacological activities. In order to reveal the mechanism of cucurbitacin IIa biosynthesis and regulation in H. macrosperma, transcriptome analysis was performed to compare differentially expressed genes in three tissues (root tuber, stem and leaf). A total of 47 946 unigenes were generated from these tissues and 55 unigenes were identified as candidate genes involved in triterpenoid backbone biosynthesis. Three homologous genes encoding squalene epoxidase (HmSE) were discovered and successfully expressed in a prokaryotic system. HmSE1 was found to be responsible for oxidization of squalene. In addition, several cytochrome P450s and transcription factors were predicted as candidates associated to cucurbitacin IIa biosynthesis. Notably, the expression profiles of those putative genes showed a positive correlation with elevated curcurbitacin IIa production in methyl jasmonate-elicited suspension cells of H. macrosperma., suggesting probable functions of the candidates on curcurbitacin IIa biosynthesis. These findings provide insights on cucurbitacin IIa biosynthesis and regulation in H. macrosperma.

Metabolite Profiling and Quantitation of Cucurbitacins in Cucurbitaceae Plants by Liquid Chromatography coupled to Tandem Mass Spectrometry.

Cucurbitaceae is an important plant family because many of its species are consumed as food, and used in herbal medicines, cosmetics, etc. It comprises annual vines and is rich in various bioactive principles which include the cucurbitacins. These steroidal natural products, derived from the triterpene cucurbitane, are mainly the bitter principles of the family Cucurbitaceae. Their biological activities include anti-inflammatory, hepatoprotective, and anti-cancer activities. A total of 10 species belonging to 6 genera of the Cucurbitaceae family along with Cissampelos pareira (Menispermaceae) were included in this study. A comprehensive profiling of certain natural products was developed using HPLC-QTOF-MS/MS analysis and a distribution profile of several major natural products in this family was obtained. A total of 51 natural products were detected in both positive and negative ionization modes, based on accurate masses and fragmentation patterns. Along with this, quantitation of four bioactive cucurbitacins, found in various important plants of the Cucurbitaceae family, was carried out using multiple reaction monitoring (MRM) approach on an ion trap mass spectrometer. Cucurbitacin Q was found to be the most abundant in C. pareira, while Citrullus colocynthis contained all four cucurbitacins in abundant quantities. The developed quantitation method is simple, rapid, and reproducible.

The effect of red light and far-red light conditions on secondary metabolism in agarwood.

BACKGROUND: Agarwood, a heartwood derived from Aquilaria trees, is a valuable commodity that has seen prevalent use among many cultures. In particular, it is widely used in herbal medicine and many compounds in agarwood are known to exhibit medicinal properties. Although there exists much research into medicinal herbs and extraction of high value compounds, few have focused on increasing the quantity of target compounds through stimulation of its related pathways in this species. RESULTS: In this study, we observed that cucurbitacin yield can be increased through the use of different light conditions to stimulate related pathways and conducted three types of high-throughput sequencing experiments in order to study the effect of light conditions on secondary metabolism in agarwood. We constructed genome-wide profiles of RNA expression, small RNA, and DNA methylation under red light and far-red light conditions. With these profiles, we identified a set of small RNA which potentially regulates gene expression via the RNA-directed DNA methylation pathway. CONCLUSIONS: We demonstrate that light conditions can be used to stimulate pathways related to secondary metabolism, increasing the yield of cucurbitacins. The genome-wide expression and methylation profiles from our study provide insight into the effect of light on gene expression for secondary metabolism in agarwood and provide compelling new candidates towards the study of functional secondary metabolic components.

Identification of cucurbitacins and assembly of a draft genome for Aquilaria agallocha.

BACKGROUND: Agarwood is derived from Aquilaria trees, the trade of which has come under strict control with a listing in Appendix II of the Convention on International Trade in Endangered Species of Wild Fauna and Flora. Many secondary metabolites of agarwood are known to have medicinal value to humans, including compounds that have been shown to elicit sedative effects and exhibit anti-cancer properties. However, little is known about the genome, transcriptome, and the biosynthetic pathways responsible for producing such secondary metabolites in agarwood. RESULTS: In this study, we present a draft genome and a putative pathway for cucurbitacin E and I, compounds with known medicinal value, from in vitro Aquilaria agallocha agarwood. DNA and RNA data are utilized to annotate many genes and protein functions in the draft genome. The expression changes for cucurbitacin E and I are shown to be consistent with known responses of A. agallocha to biotic stress and a set of homologous genes in Arabidopsis thaliana related to cucurbitacin bio-synthesis is presented and validated through qRT-PCR. CONCLUSIONS: This study is the first attempt to identify cucurbitacin E and I from in vitro agarwood and the first draft genome for any species of Aquilaria. The results of this study will aid in future investigations of secondary metabolite pathways in Aquilaria and other non-model medicinal plants.