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Tropane alkaloids (TA) are valuable secondary plant metabolites which are mostly found in high concentrations in the Solanaceae and Erythroxylaceae families. The TAs, which are characterized by their unique bicyclic tropane ring system, can be divided into three major groups: hyoscyamine and scopolamine, cocaine and calystegines. Although all TAs have the same basic structure, they differ immensely in their biological, chemical and pharmacological properties. Scopolamine, also known as hyoscine, has the largest legitimate market as a pharmacological agent due to its treatment of nausea, vomiting, motion sickness, as well as smooth muscle spasms while cocaine is the 2nd most frequently consumed illicit drug globally. This review provides a comprehensive overview of TAs, highlighting their structural diversity, use in pharmaceutical therapy from both historical and modern perspectives, natural biosynthesis in planta and emerging production possibilities using tissue culture and microbial biosynthesis of these compounds.

The Arabian Peninsula is recognized as an arid area dominated by deserts and poor biodiversity. However, the Kingdom of Saudi Arabia (henceforth abbreviated into KSA) has a wide range of flora, consisting of different species of trees, herbs, and shrubs and containing numerous edible and medicinal plants. The KSA is characterized by its vast area of diverse geographical landscapes and climates. Consequently, there is enormous variation in the distribution of plants across the Kingdom. The traditional use of ethnomedical plants in the KSA represents a strong interconnection among familiar remedies, health, diet, and traditional healing practices characterized by specific cultures. The present paper reviews a collection of medicinal plants in KSA used in ethnomedicine. This review might be useful in developing strategies for the sustainable use of medicinal plants which are among the threatened important natural resources in folk medicine in the KSA. The present study reports 309 genera which cover 471 species from a total of 2253 known species belonging to 89 families. The most dominating families are Asteraceae, Fabaceae, Lamiaceae, Euphorbiaceae, Solanaceae, Apiaceae, Brassicaceae, Chenopodiaceae, Poaceae, Amaranthaceae, Boraginaceae, Apocynaceae, Convolvoulaceae, Asclepiadaceae, Capparaceae, Polygonaceae, and Zygophyllaceae.

Engineering of the central carbon metabolism of Saccharomyces cerevisiae to redirect metabolic flux towards cytosolic acetyl-CoA has become a central topic in yeast biotechnology. A cell factory with increased flux into acetyl-CoA can be used for heterologous production of terpenoids for pharmaceuticals, biofuels, fragrances, or other acetyl-CoA derived compounds. In a previous study, we identified promising metabolic engineering targets in S. cerevisiae using an in silico stoichiometric metabolic network analysis. Here, we validate selected in silico strategies in vivo. Patchoulol was produced by yeast via a heterologous patchoulol synthase of Pogostemon cablin. To increase the metabolic flux from acetyl-CoA towards patchoulol, a truncated HMG-CoA reductase was overexpressed and farnesyl diphosphate synthase was fused with patchoulol synthase. The highest increase in production could be achieved by modifying the carbon source; sesquiterpenoid titer increased from glucose to ethanol by a factor of 8.4. Two strategies predicted in silico were chosen for validation in this work. Disruption of α-ketoglutarate dehydrogenase gene (KGD1) was predicted to redirect the metabolic flux via the pyruvate dehydrogenase bypass towards acetyl-CoA. The metabolic flux was redirected as predicted, however, the effect was dependent on cultivation conditions and the flux was interrupted at the level of acetate. High amounts of acetate were produced. As an alternative pathway to synthesize cytosolic acetyl-CoA, ATP-citrate lyase was expressed as a polycistronic construct, however, in vivo performance of the enzyme needs to be optimized to increase terpenoid production. Stoichiometric metabolic network analysis can be used successfully as a metabolic prediction tool. However, this study highlights that kinetics, regulation and cultivation conditions may interfere, resulting in poor in vivo performance. Main sites of regulation need to be released and improved enzymes are essential to meet the required activities for an increased product formation in vivo.

Background Cannabis possesses a rich spectrum of phytochemicals i.e. cannabinoids, terpenes and phenolic compounds of industrial and medicinal interests. Most of these high-value plant products are synthesised in the disk cells and stored in the secretory cavity in glandular trichomes. Conventional trichome analysis was so far based on optical microscopy, electron microscopy or extraction based methods that are either limited to spatial or chemical information. Here we combine both information to obtain the spatial distribution of distinct secondary metabolites on a single-trichome level by applying Coherent anti-Stokes Raman scattering (CARS), a microspectroscopic technique, to trichomes derived from sepals of a drug- and a fibre-type. Results Hyperspectral CARS imaging in combination with a nonlinear unmixing method allows to identify and localise Δ 9 -tetrahydrocannabinolic acid (THCA) in the secretory cavity of drug-type trichomes and cannabidiolic acid (CBDA)/myrcene in the secretory cavity of fibre-type trichomes, thus enabling an easy discrimination between high-THCA and high-CBDA producers. A unique spectral fingerprint is found in the disk cells of drug-type trichomes, which is most similar to cannabigerolic acid (CBGA) and is not found in fibre-type trichomes. Furthermore, we differentiate between different cell types by a combination of CARS with simultaneously acquired two-photon fluorescence (TPF) of chlorophyll a from chloroplasts and organic fluorescence mainly arising from cell walls enabling 3D visualisation of the essential oil distribution and cellular structures. Conclusion Here we demonstrate a label-free and non-destructive method to analyse the distribution of secondary metabolites and distinguish between different cell and chemo-types with high spatial resolution on a single trichome. The record of chemical fingerprints of single trichomes offers the possibility to optimise growth conditions as well as guarantee a direct process control for industrially cultivated medicinal Cannabis plants. Moreover, this method is not limited to Cannabis related issues but can be widely implemented for optimising and monitoring all kinds of natural or biotechnological production processes with simultaneous spatial and chemical information.

Background The raised demand of cannabis as a medicinal plant in recent years led to an increased interest in understanding the biosynthetic routes of cannabis metabolites. Since there is no established protocol to generate stable gene knockouts in cannabis, the use of a virus-induced gene silencing (VIGS) method, resulting in a gene knockdown, to study gene functions is desirable. Results For this, a computational approach was employed to analyze the Cannabis sativa L. transcriptomic and genomic resources. Reporter genes expected to give rise to easily scorable phenotypes upon silencing, i.e. the phytoene desaturase ( PDS ) and magnesium chelatase subunit I ( ChlI ), were identified in C. sativa. Subsequently, the targets of specific small interfering RNAs (siRNAs) and silencing fragments were predicted and tested in a post-transcriptional gene silencing (PTGS) approach . Here we show for the first time a gene knockdown in C. sativa using the Cotton leaf crumple virus ( CLCrV ) in a silencing vector system. Plants transiently transformed with the Agrobacterium tumefaciens strain AGL1, carrying the VIGS-vectors, showed the desired phenotypes, spotted bleaching of the leaves. The successful knockdown of the genes was additionally validated by quantitative PCR resulting in reduced expression of transcripts from 70 to 73% for ChlI and PDS , respectively. This is accompanied with the reduction of the chlorophyll a and carotenoid content, respectively. In summary, the data clearly demonstrate the potential for functional gene studies in cannabis using the CLCrV -based vector system. Conclusions The applied VIGS-method can be used for reverse genetic studies in C. sativa to identify unknown gene functions. This will gain deeper inside into unknown biosynthetic routes and will help to close the gap between available genomic data and biochemical information of this important medicinal plant.

OBJECTIVE: The Δ9-tetrahydrocannabinolic acid synthase (THCAS) from Cannabis sativa was expressed intracellularly in different organisms to investigate the potential of a biotechnological production of Δ9-tetrahydrocannabinolic acid (THCA) using whole cells. RESULTS: Functional expression of THCAS was obtained in Saccharomyces cerevisiae and Pichia (Komagataella) pastoris using a signal peptide from the vacuolar protease, proteinase A. No functional expression was achieved in Escherichia coli. The highest volumetric activities obtained were 98 pkat ml⁻¹ (intracellular) and 44 pkat ml⁻¹ (extracellular) after 192 h of cultivation at 15 °C using P. pastoris cells. Low solubility of CBGA prevents the THCAS application in aqueous cell-free systems, thus whole cells were used for a bioconversion of cannabigerolic acid (CBGA) to THCA. Finally, 1 mM (0.36 g THCA l⁻¹) THCA could be produced by 10.5 gCDW l⁻¹ before enzyme activity was lost. CONCLUSION: Whole cells of P. pastoris offer the capability of synthesizing pharmaceutical THCA production.

The liverwort belongs to the bryophyte division of land plants and is a prospective alternate source of cannabinoid-like compounds. However, mechanistic insights into the molecular pathways directing the synthesis of these cannabinoid-like compounds have been hindered due to the lack of genetic information. This prompted us to do deep sequencing, assembly and annotation of transcriptome, which resulted in the identification and validation of the genes for cannabinoid biosynthetic pathway. In total, we have identified 11,421 putative genes encoding 1,554 enzymes from 145 biosynthetic pathways. Interestingly, we have identified all the upstream genes of the central precursor of cannabinoid biosynthesis, cannabigerolic acid (CBGA), including its two first intermediates, stilbene acid (SA) and geranyl diphosphate (GPP). Expression of all these genes was validated using quantitative real-time PCR. We have characterized the protein structure of stilbene synthase (STS), which is considered as a homolog of olivetolic acid in . Moreover, the metabolomics approach enabled us to identify CBGA-analogous compounds using electrospray ionization mass spectrometry (ESI-MS/MS) and gas chromatography mass spectrometry (GC-MS). Transcriptomic analysis revealed 1085 transcription factors (TF) from 39 families. Comparative analysis showed that six TF families have been uniquely predicted in . In addition, the bioinformatics analysis predicted a large number of simple sequence repeats (SSRs) and non-coding RNAs (ncRNAs). Our results collectively provide mechanistic insights into the putative precursor genes for the biosynthesis of cannabinoid-like compounds and a novel transcriptomic resource for . The large-scale transcriptomic resource generated in this study would further serve as a reference transcriptome to explore the family.

Even though maytansine was first discovered from Celastraceae plants, it was later proven to be an endophytic bacterial metabolite. However, a pure bacterial culture cannot synthesize maytansine. Therefore, an exclusive interaction between plant and endophytes is required for maytansine production. Unfortunately, our understanding of plant–endophyte interaction is minimal, and critical questions remain. For example: how do endophytes synthesize maytansine inside their plant host, and what is the impact of maytansine production in plant secondary metabolites? Our study aimed to address these questions. We selected Gymnosporia heterophylla as our model and used amino-hydroxybenzoic acid (AHBA) synthase and halogenase genes as biomarkers, as these two genes respond to biosynthesize maytansine. As a result, we found a consortium of seven endophytes involved in maytansine production in G. heterophylla, based on genome mining and gene expression experiments. Subsequently, we evaluated the friedelin synthase (FRS) gene’s expression level in response to biosynthesized 20-hydroxymaytenin in the plant. We found that the FRS expression level was elevated and linked with the expression of the maytansine biosynthetic genes. Thus, we achieved our goals and provided new evidence on endophyte–endophyte and plant–endophyte interactions, focusing on maytansine production and its impact on plant metabolite biosynthesis in G. heterophylla.

Medium chain fatty acids (MCFAs) are valuable platform compounds for the production of biotechnologically relevant chemicals such as biofuels and biochemicals. Two distinct pathways have been implemented in the yeast  Saccharomyces cerevisiae  for the biosynthetic production of MCFAs: (i) the mutant fatty acid biosynthesis (FAB) pathway in which the fatty acid synthase (FAS) complex is mutated and (ii) a heterologous multispecies-derived reverse β-oxidation (rBOX) pathway. Hexanoic acid has become of great interest as its acyl-CoA ester, hexanoyl-CoA, is required for the biosynthesis of olivetolic acid (OA), a cannabinoid precursor. Due to insufficient endogenous synthesis of hexanoyl-CoA, recombinant microbial systems to date require exogenous supplementation of cultures with hexanoate along with the overexpression of an acyl-CoA ligase to allow cannabinoid biosynthesis. Here, we engineer a recombinant  S. cerevisiae strain which was metabolically optimized for the production of hexanoic acid via the FAB and rBOX pathways and we combine both pathways in a single strain to achieve titers of up to 120 mg L −1 . Moreover, we demonstrate the biosynthesis of up to 15 mg L −1 OA from glucose using hexanoyl-CoA derived from the rBOX pathway.

Pantothenate kinase-associated neurodegeneration (PKAN), a progressive neurodegenerative disorder, is associated with impairment of pantothenate kinase function. Pantothenate kinase is the first enzyme required for de novo synthesis of CoA, an essential metabolic cofactor. The pathophysiology of PKAN is not understood, and there is no cure to halt or reverse the symptoms of this devastating disease. Recently, we and others presented a PKAN Drosophila model, and we demonstrated that impaired function of pantothenate kinase induces a neurodegenerative phenotype and a reduced lifespan. We have explored this Drosophila model further and have demonstrated that impairment of pantothenate kinase is associated with decreased levels of CoA, mitochondrial dysfunction, and increased protein oxidation. Furthermore, we searched for compounds that can rescue pertinent phenotypes of the Drosophila PKAN model and identified pantethine. Pantethine feeding restores CoA levels, improves mitochondrial function, rescues brain degeneration, enhances locomotor abilities, and increases lifespan. We show evidence for the presence of a de novo CoA biosynthesis pathway in which pantethine is used as a precursor compound. Importantly, this pathway is effective in the presence of disrupted pantothenate kinase function. Our data suggest that pantethine may serve as a starting point to develop a possible treatment for PKAN.