Biosynthesis, transport and combinatorial metabolic engineering of Tanacetum parthenium (feverfew) and Artemisia annua (sweet wormwood) sesquiterpene lactones

This thesis mainly focuses on sesquiterpene lactone biosynthesis, their (combinatorial) metabolic engineering and extracellular transport and accumulation. Terpenoids are the most diverse and largest class of natural products, many of which have important pharmaceutical, biological, nutraceutical and/or other beneficial properties. Chapter 1 of this thesis provides the background on the knowledge of biosynthesis of different terpenoid classes in plants at the onset of this thesis work. Also, a background is given of metabolic engineering approaches and different expression platforms (organisms) to overcome limitations of production of these attractive molecules. Finally the topic ‘terpenoid transport’ in plants is introduced, a research area which was largely underdeveloped at the start of my thesis. There are different classes of sesquiterpene lactones from which particularly the biosynthesis of germacranolides has been studied, mostly in plant species belonging to the Asteraceae. In contrast, how members of the guaianolide class of sesquiterpene lactones are synthesized has been a mystery for a very long time. In chapter 2, I report the first characterization of an enzyme from feverfew responsible for the first committed step branching the guaianolide pathway off from the germacranolides. This enzyme, which converts costunolide into kauniolide, is very special as it performs several sequential reactions, from hydroxylation, water elimination and cyclisation to regio-selective deprotonation. The mechanism of action of this enzyme was elucidated by testing different putative substrates and intermediates (obtained through chemical synthesis) and through in-silico substrate docking studies. Recent progress in metabolic engineering and pathway reconstruction in microbes and plants has resulted in the production of the bioactive costunolide and parthenolide from feverfew as well as dihydroartemisinic acid, the precursor of the antimalarial artemisinin, from Artemisia annua in heterologous expression platforms. In A. annua, a double bond reductase (AaDBR) enzyme catalyses a branch point in the pathway towards dihydroartemisinic acid. Because artemisinin biosynthesis in A. annua proceeds quite similar to costunolide biosynthesis in feverfew, we suspected that AaDBR may act on products from the feverfew pathway. This was confirmed in chapter 3 in a combinatorial metabolic engineering approach, in which I produced the novel dihydro-sesquiterpene lactones 3β-hydroxy-dihydroparthenolide and 3β-hydroxy-dihydrocostunolide. In Chapter 4 I switch from biosynthesis of sesquiterpene lactones to their transport. In this chapter I describe the involvement of Lipid Transfer Proteins (LTPs) and Pleiotropic Drug Resistance (PDR) membrane transporters in extracellular accumulation of (dihydro)artemisinic acid [(DH)AA], using transient expression assays in the heterologous host Nicotiana benthamiana. We show that AaLTP3 and AaPDR2 enhance (DH)AA accumulation in the N. benthamiana leaf apoplast as well as the overall flux through the (DH)AA pathway. For functional analysis of the LTPs I developed two novel assays: an in planta substrate export assay and an in planta substrate exclusion assay. Using these assays, we show that AaLTP3 is more effective than AaPDR2 in preventing influx of (DH)AA from the apoplast into the cells. This chapter also describes the first documented case of artemisinin and arteannuin B production in N. benthamiana. In chapter 5, I continue my research on sesquiterpene transport by functional characterization of Lipid Transfer Protein genes from feverfew trichomes. I demonstrate that some of these LTPs have acquired a specialized function in the transport of specific lipophilic products (sesquiterpene lactones) produced in feverfew trichomes. I show that TpLTP1 and TpLTP2 specifically improve costunolide export and that TpLTP3 highly specifically improves parthenolide export. Moreover, the substrate exclusion assays revealed TpLTP3 as the most effective in blocking influx of both costunolide and parthenolide. TpLTP3 has a GPI anchor domain and I show that this GPI-anchor domain is essential for the activity of TpLTP3, while addition of this domain to TpLTP1 resulted in loss of TpLTP1 activity. In chapter 6 I discuss the biosynthesis and transport results obtained during my thesis research. I address remaining questions related to biosynthesis of sesquiterpene lactones in yeast and discuss opportunities for combinatorial metabolic engineering, e.g. what are putative applications of the enzyme-substrate promiscuity concept for the double bond reductase (DBR)? In this chapter I also discuss the many remaining questions related to transport of sesquiterpene lactones, as many details about transport over the plasma membrane and over the cell wall are still missing. Finally, I provide a wider perspective on the role of LTPs, based on clues from the exclusion assays and the chemical properties of other biologically active molecules in plants. In summary, this thesis provides several novel discoveries in the field of sesquiterpene lactone metabolic engineering by identification of the biosynthetic branch point towards guaianolides, by combinatorial metabolic engineering to produce novel dihydro-sesquiterpene lactones and by revealing an important and very specific role of LTPs in extracellular transport of sesquiterpene lactones.</p

Transcriptomic data reveals the dynamics of terpenoids biosynthetic pathway of fenugreek

Abstract Medicinal plants are rich sources for treating various diseases due their bioactive secondary metabolites. Fenugreek (Trigonella foenum-graecum) is one of the medicinal plants traditionally used in human nutrition and medicine which contains an active substance, called diosgenin, with anticancer properties. Biosynthesis of this important anticancer compound in fenugreek can be enhanced using eliciting agents which involves in manipulation of metabolite and biochemical pathways stimulating defense responses. Methyl jasmonate elicitor was used to increase diosgenin biosynthesis in fenugreek plants. However, the molecular mechanism and gene expression profiles underlying diosgening accumulation remain unexplored. In the current study we performed an extensive analysis of publicly available RNA-sequencing datasets to elucidate the biosynthesis and expression profile of fenugreek plants treated with methyl jasmonate. For this purpose, seven read datasets of methyl jasmonate treated plants were obtained that were covering several post-treatment time points (6–120 h). Transcriptomics analysis revealed upregulation of several key genes involved in diosgenein biosynthetic pathway including Squalene synthase (SQS) as the first committed step in diosgenin biosynthesis as well as Squalene Epoxidase (SEP) and Cycloartenol Synthase (CAS) upon methyl jasmonate application. Bioinformatics analysis, including gene ontology enrichment and pathway analysis, further supported the involvement of these genes in diosgenin biosynthesis. The bioinformatics analysis led to a comprehensive validation, with expression profiling across three different fenugreek populations treated with the same methyl jasmonate application. Initially, key genes like SQS, SEP, and CAS showed upregulation, followed by later upregulation of Δ24, suggesting dynamic pathway regulation. Real-time PCR confirmed consistent upregulation of SQS and SEP, peaking at 72 h. Additionally, candidate genes Δ24 and SMT1 highlighted roles in directing metabolic flux towards diosgenin biosynthesis. This integrated approach validates the bioinformatics findings and elucidates fenugreek’s molecular response to methyl jasmonate elicitation, offering insights for enhancing diosgenin yield. The assembled transcripts and gene expression profiles are deposited in the Zenodo open repository at https://doi.org/10.5281/zenodo.8155183

Effect of Harvesting Time Variations on Essential Oil Yield and Composition of Sage (<i>Salvia officinalis</i>)

The objective of this study was to evaluate the production, contents, and essential oil (EO) components of sage as a function of the diurnal variation. The EOs from the aerial parts of the plant harvested at different day/night times were extracted by hydro-distillation. Plants were harvested in 2 h intervals (twelve harvesting times during each 24-h day). Harvesting between 4:00 and 6:00 p.m. revealed the highest EO percentage (1.14%), whereas harvesting between 04:00 and 06:00 a.m. indicated the minimum EO percentage (0.599%). The analysis of the EO identified 32 components. The major identified EO compounds were cis-thujone (34.38–46.18%), 1,8-cineol (8.70–11.07%), camphor (9.65–14.38%), and trans-thujone (9.43–14.19%). The highest value of cis-thujone (46.18%) was related to the harvest time of 04:00–06:00 a.m., and the lowest value (34.38%) was recorded at the harvest time of 00:00–02:00 a.m. The highest value of trans-thujone (14.19%) was obtained between 10:00–00:00 p.m., and the lowest value (9.43%) was obtained between 10:00–12:00 a.m. Camphor was another dominant compound where the highest (14.38%) was observed at 00:00–2:00 a.m. Our findings in sage, for the first time, may pave the route towards the optimization of sage EO quality and quantity by selecting the best harvesting time of the plants

Effect of Harvesting Time Variations on Essential Oil Yield and Composition of Sage (Salvia officinalis)

The objective of this study was to evaluate the production, contents, and essential oil (EO) components of sage as a function of the diurnal variation. The EOs from the aerial parts of the plant harvested at different day/night times were extracted by hydro-distillation. Plants were harvested in 2 h intervals (twelve harvesting times during each 24-h day). Harvesting between 4:00 and 6:00 p.m. revealed the highest EO percentage (1.14%), whereas harvesting between 04:00 and 06:00 a.m. indicated the minimum EO percentage (0.599%). The analysis of the EO identified 32 components. The major identified EO compounds were cis-thujone (34.38&ndash;46.18%), 1,8-cineol (8.70&ndash;11.07%), camphor (9.65&ndash;14.38%), and trans-thujone (9.43&ndash;14.19%). The highest value of cis-thujone (46.18%) was related to the harvest time of 04:00&ndash;06:00 a.m., and the lowest value (34.38%) was recorded at the harvest time of 00:00&ndash;02:00 a.m. The highest value of trans-thujone (14.19%) was obtained between 10:00&ndash;00:00 p.m., and the lowest value (9.43%) was obtained between 10:00&ndash;12:00 a.m. Camphor was another dominant compound where the highest (14.38%) was observed at 00:00&ndash;2:00 a.m. Our findings in sage, for the first time, may pave the route towards the optimization of sage EO quality and quantity by selecting the best harvesting time of the plants

Expression analyses of salinity stress- associated ESTs in Aeluropus littoralis

Salinity is among the most important abiotic stresses affecting crop production throughout the earth. Halophyte plants can sustain high salinity levels, therefore elucidating molecular mechanisms underlying their salinity resistance is beneficial for crop improvement. Aeluropus littoralis, a halophyte weed, is a great genetic resource for this purpose. Isolated expressed sequence taq (EST) sequences from A. littoralis under salinity stress, have given us the chance to find and analyze transcripts of genes involved in response to salinity. Transcriptome analyses indicated the expression levels of mRNAs corresponding to 10 of sequences were increased under treatments. All mRNAs were significantly induced under salt treatment with the highest peaks observed at different hours of treatments. Moreover, the full-length cDNA of vacuolar H+-pyrophosphatase (VP) was isolated utilizing 3′ and 5′ rapid amplification of cDNA ends polymerase chain reaction (RACE-PCR) and characterized (GenBank accession number of KT253223.1). The extracted full-length of VP was 2732 bp, which contained ORF of 2292 bp encoding 763 amino acids.</p

Individual lipid transfer proteins from Tanacetum parthenium show different specificity for extracellular accumulation of sesquiterpenes

Key message: A highly specialized function for individual LTPs for different products from the same terpenoid biosynthesis pathway is described and the function of an LTP GPI anchor is studied. Abstract: Sequiterpenes produced in glandular trichomes of the medicinal plant Tanacetum parthenium (feverfew) accumulate in the subcuticular extracellular space. Transport of these compounds over the plasma membrane is presumably by specialized membrane transporters, but it is still not clear how these hydrophobic compounds are subsequently transported over the hydrophilic cell wall. Here we identified eight so-called non-specific Lipid transfer proteins (nsLTPs) genes that are expressed in feverfew trichomes. A putative function of these eight nsLTPs in transport of the lipophilic sesquiterpene lactones produced in feverfew trichomes, was tested in an in-planta transport assay using transient expression in Nicotiana benthamiana. Of eight feverfew nsLTP candidate genes analyzed, two (TpLTP1 and TpLTP2) can specifically improve extracellular accumulation of the sesquiterpene costunolide, while one nsLTP (TpLTP3) shows high specificity towards export of parthenolide. The specificity of the nsLTPs was also tested in an assay that test for the exclusion capacity of the nsLTP for influx of extracellular substrates. In such assay, TpLTP3 was identified as most effective in blocking influx of both costunolide and parthenolide, when these substrates are infiltrated into the apoplast. The TpLTP3 is special in having a GPI-anchor domain, which is essential for the export activity of TpLTP3. However, addition of the TpLTP3 GPI-anchor domain to TpLTP1 resulted in loss of TpLTP1 export activity. These novel export and exclusion assays thus provide new means to test functionality of plant nsLTPs

Additional file 1 of Transcriptomic data reveals the dynamics of terpenoids biosynthetic pathway of fenugreek

Supplementary Material

Substrate promiscuity of enzymes from the sesquiterpene biosynthetic pathways from Artemisia annua and Tanacetum parthenium allows for novel combinatorial sesquiterpene production

The therapeutic properties of complex terpenes often depend on the stereochemistry of their functional groups. However, stereospecific chemical synthesis of terpenes is challenging. To overcome this challenge, metabolic engineering can be employed using enzymes with suitable stereospecific catalytic activity. Here we used a combinatorial metabolic engineering approach to explore the stereospecific modification activity of the Artemisia annua artemisinic aldehyde ∆11(13) double bond reductase2 (AaDBR2) on products of the feverfew sesquiterpene biosynthesis pathway (GAS, GAO, COS and PTS). This allowed us to produce dihydrocostunolide and dihydroparthenolide. For dihydroparthenolide we demonstrate that the preferred order of biosynthesis of dihydroparthenolide is by reduction of the exocyclic methylene of parthenolide, rather than through C4-C5 epoxidation of dihydrocostunolide. Moreover, we demonstrate a promiscuous activity of feverfew CYP71CB1 on dihydrocostunolide and dihydroparthenolide for the production of 3β-hydroxy-dihydrocostunolide and 3β-hydroxy-dihydroparthenolide, respectively. Combined, these results offer new opportunities for engineering novel sesquiterpene lactones with potentially improved medicinal value

Artemisinins in Combating Viral Infections Like SARS-CoV-2, Inflammation and Cancers and Options to Meet Increased Global Demand

Artemisinin is a natural bioactive sesquiterpene lactone containing an unusual endoperoxide 1, 2, 4-trioxane ring. It is derived from the herbal medicinal plant Artemisia annua and is best known for its use in treatment of malaria. However, recent studies also indicate the potential for artemisinin and related compounds, commonly referred to as artemisinins, in combating viral infections, inflammation and certain cancers. Moreover, the different potential modes of action of artemisinins make these compounds also potentially relevant to the challenges the world faces in the COVID-19 pandemic. Initial studies indicate positive effects of artemisinin or Artemisia spp. extracts to combat SARS-CoV-2 infection or COVID-19 related symptoms and WHO-supervised clinical studies on the potential of artemisinins to combat COVID-19 are now in progress. However, implementing multiple potential new uses of artemisinins will require effective solutions to boost production, either by enhancing synthesis in A. annua itself or through biotechnological engineering in alternative biosynthesis platforms. Because of this renewed interest in artemisinin and its derivatives, here we review its modes of action, its potential application in different diseases including COVID-19, its biosynthesis and future options to boost production