Steroidal scaffold decorations in Solanum alkaloid biosynthesis.

Steroidal glycoalkaloids (SGAs) are specialized metabolites produced by hundreds of Solanum species, including important vegetable crops such as tomato, potato, and eggplant. Although it has been known that SGAs play important roles in defense in plants and "anti-nutritional" effects (e.g., toxicity and bitterness) to humans, many of these molecules have documented anti-cancer, anti-microbial, anti-inflammatory, anti-viral, and anti-pyretic activities. Among these, α-solasonine and α-solamargine isolated from black nightshade (Solanum nigrum) are reported to have potent anti-tumor, anti-proliferative, and anti-inflammatory activities. Notably, α-solasonine and α-solamargine, along with the core steroidal aglycone solasodine, are the most widespread SGAs produced among the Solanum plants. However, it is still unknown how plants synthesize these bioactive steroidal molecules. Through comparative metabolomic-transcriptome-guided approach, biosynthetic logic, combinatorial expression in Nicotiana benthamiana, and functional recombinant enzyme assays, here we report the discovery of 12 enzymes from S. nigrum that converts the starting cholesterol precursor to solasodine aglycone, and the downstream α-solasonine, α-solamargine, and malonyl-solamargine SGA products. We further identified six enzymes from cultivated eggplant that catalyze the production of α-solasonine, α-solamargine, and malonyl-solamargine SGAs from solasodine aglycone via glycosylation and atypical malonylation decorations. Our work provides the gene tool box and platform for engineering the production of high-value, steroidal bioactive molecules in heterologous hosts using synthetic biology.

Development of Atropa belladonna L. Plants with High-Yield Hyoscyamine and without Its Derivatives Using the CRISPR/Cas9 System.

Atropa belladonna L. is one of the most important herbal plants that produces hyoscyamine or atropine, and it also produces anisodamine and scopolamine. However, the in planta hyoscyamine content is very low, and it is difficult and expensive to independently separate hyoscyamine from the tropane alkaloids in A. belladonna. Therefore, it is vital to develop A. belladonna plants with high yields of hyoscyamine, and without anisodamine and scopolamine. In this study, we generated A. belladonna plants without anisodamine and scopolamine, via the CRISPR/Cas9-based disruption of hyoscyamine 6ß-hydroxylase (AbH6H), for the first time. Hyoscyamine production was significantly elevated, while neither anisodamine nor scopolamine were produced, in the A. belladonna plants with homozygous mutations in AbH6H. In summary, new varieties of A. belladonna with high yields of hyoscyamine and without anisodamine and scopolamine have great potential applicability in producing hyoscyamine at a low cost.

Engineering Tropane Alkaloid Production Based on Metabolic Characterization of Ornithine Decarboxylase in Atropa belladonna.

Ornithine decarboxylase (ODC) plays an important role in various biological processes; however, its role in plant secondary metabolism, especially in the biosynthesis of tropane alkaloids (TAs) such as pharmaceutical hyoscyamine, anisodamine, and scopolamine, remains largely unknown. In this study, we characterized the physiological and metabolic functions of the ODC gene of Atropa belladonna (AbODC) and determined its role in TA production using metabolic engineering approaches. Feeding assays with enzyme inhibitors indicated that ODC, rather than arginine decarboxylase (ADC), plays a major role in TA biosynthesis. Tissue-specific AbODC expression analysis and ß-glucuronidase (GUS) staining assays showed that AbODC was highly expressed in secondary roots, especially in the cylinder tissue. Enzymatic assays indicated that AbODC was able to convert ornithine to putrescine, with the highest activity at pH 8.0 and 30 °C. Additionally, AbODC showed higher catalytic efficiency than other plant ODCs, as evident from the Km, Vmax, and Kcat values of AbODC using ornithine as the substrate. In A. belladonna root cultures, suppression of AbODC greatly reduced the production of putrescine, N-methylputrescine, and TAs, whereas overexpression of AbODC significantly increased the biosynthesis of putrescine, N-methylputrescine, hyoscyamine, and anisodamine. Moreover, transgenic A. belladonna plants overexpressing AbODC showed a significantly higher production of hyoscyamine and anisodamine compared with control plants. These findings indicate that AbODC plays a key role in TA biosynthesis and therefore is a valuable candidate for increasing TA production in A. belladonna.

Engineering a microbial biosynthesis platform for de novo production of tropane alkaloids.

Tropane alkaloids (TAs) are a class of phytochemicals produced by plants of the nightshade family used for treating diverse neurological disorders. Here, we demonstrate de novo production of tropine, a key intermediate in the biosynthetic pathway of medicinal TAs such as scopolamine, from simple carbon and nitrogen sources in yeast (Saccharomyces cerevisiae). Our engineered strain incorporates 15 additional genes, including 11 derived from diverse plants and bacteria, and 7 disruptions to yeast regulatory or biosynthetic proteins to produce tropine at titers of 6 mg/L. We also demonstrate the utility of our engineered yeast platform for the discovery of TA derivatives by combining biosynthetic modules from distant plant lineages to achieve de novo production of cinnamoyltropine, a non-canonical TA. Our engineered strain constitutes a starting point for future optimization efforts towards realizing industrial fermentation of medicinal TAs and a platform for the synthesis of TA derivatives with enhanced bioactivities.

Localization and Organization of Scopolamine Biosynthesis in Duboisia myoporoides R. Br.

Tropane alkaloids (TAs), especially hyoscyamine and scopolamine, are important precursors for anticholinergic and antispasmodic drugs. Hyoscyamine and scopolamine are currently obtained at commercial scale from hybrid crosses of Duboisia myoporoides × Duboisia leichhardtii plants. In this study, we present a global investigation of the localization and organization of TA biosynthesis in a Duboisia myoporoides R. Br. wild-type line. The tissue-specific spatial distribution of TAs within D. myoporoides is presented, including quantification of the TAs littorine, 6-hydroxy hyoscyamine, hyoscyamine, scopolamine and, additionally, hyoscyamine aldehyde as well as scopolamine glucoside. Scopolamine (14.77 ± 5.03 mg g-1), and to a lesser extent hyoscyamine (3.01 ± 1.54 mg g-1) as well as 6-hydroxy hyoscyamine (4.35 ± 1.18 mg g-1), are accumulated in leaves during plant development, with the highest concentration of total TAs detected in 6-month-old plants. Littorine, an early precursor in TA biosynthesis, was present only in the roots (0.46 ± 0.07 mg g-1). During development, the spatial distribution of all investigated alkaloids changed due to secondary growth in the roots. Transcripts of pmt, tr-I and cyp80f1 genes, involved in early stages of TA biosynthesis, were found to be most abundant in the roots. In contrast, the transcript encoding hyoscyamine 6ß-hydroxylase (h6h) was highest in the leaves of 3-month-old plants. This investigation presents the spatial distribution of biochemical components as well as gene expression profiles of genetic factors known to participate in TA biosynthesis in D. myoporoides. The results of this investigation may aid in future breeding or genetic enhancement strategies aimed at increasing the yields of TAs in these medicinally valuable plant species.

A Dioxygenase Catalyzes Steroid 16α-Hydroxylation in Steroidal Glycoalkaloid Biosynthesis.

Steroidal glycoalkaloids (SGAs) are toxic specialized metabolites that are found in the Solanaceae. Potato (Solanum tuberosum) contains the SGAs α-solanine and α-chaconine, while tomato (Solanum lycopersicum) contains α-tomatine, all of which are biosynthesized from cholesterol. However, although two cytochrome P450 monooxygenases that catalyze the 22- and 26-hydroxylation of cholesterol have been identified, the 16-hydroxylase remains unknown. Feeding with deuterium-labeled cholesterol indicated that the 16α- and 16ß-hydrogen atoms of cholesterol were eliminated to form α-solanine and α-chaconine in potato, while only the 16α-hydrogen atom was eliminated in α-tomatine biosynthesis, suggesting that a single oxidation at C-16 takes place during tomato SGA biosynthesis while a two-step oxidation occurs in potato. Here, we show that a 2-oxoglutarate-dependent dioxygenase, designated as 16DOX, is involved in SGA biosynthesis. We found that the transcript of potato 16DOX (St16DOX) was expressed at high levels in the tuber sprouts, where large amounts of SGAs are accumulated. Biochemical analysis of the recombinant St16DOX protein revealed that St16DOX catalyzes the 16α-hydroxylation of hydroxycholesterols and that (22S)-22,26-dihydroxycholesterol was the best substrate among the nine compounds tested. St16DOX-silenced potato plants contained significantly lower levels of SGAs, and a detailed metabolite analysis revealed that they accumulated the glycosides of (22S)-22,26-dihydroxycholesterol. Analysis of the tomato 16DOX (Sl16DOX) gene gave essentially the same results. These findings clearly indicate that 16DOX is a steroid 16α-hydroxylase that functions in the SGA biosynthetic pathway. Furthermore, St16DOX silencing did not affect potato tuber yield, indicating that 16DOX may be a suitable target for controlling toxic SGA levels in potato.

Mismatch repair deficiency increases the transfer of antibiosis and antixenosis properties against Colorado potato beetle in somatic hybrids of Solanum tuberosum + S. chacoense.

BACKGROUND: Colorado potato beetle (CPB) has become the biggest enemy of cultivated potato worldwide. One of the most effective sources of resistance to CPB is Solanum chacoense, an accession with a high leptine glycoalkaloid content. The aim of our study was to assay the repellence and toxicity of S. chacoense, its somatic hybrids (SHs) and their backcross progenies (BC1 ) with potato for CPB adults and larvae. Transgenic S. chacoense, deficient in DNA mismatch repair (MMR), was also used to produce SHs, in order to increase homeologous recombination and hence introgression of wild-species DNA into the potato gene pool. RESULTS: Wild-type SH was highly resistant to CPB. Resistance to CPB of BC1 progenies showed a 1:3 inheritance pattern. MMR-deficient SHs performed better in the resistance analysis. Most MMR-deficient SHs had a similar toxicity as S. chacoense and an intensely repellent effect on CPB adults. Resistance of SHs and BC1 clones may be attributed to leptine biosynthesis, which was confirmed using a RAPD marker. CONCLUSION: This is the first report of SHs and their progenies exhibiting both antibiosis and antixenosis against CPB. Resistant SHs are an important step forward in combating this voracious pest of potato. © 2016 Society of Chemical Industry.

Impact of light-exposure on the metabolite balance of transgenic potato tubers with modified glycoalkaloid biosynthesis.

Metabolite profiling (liquid chromatography-mass spectrometry (LC-MS) and gas chromatography (GC-MS)) was used to assess the impact of light on the composition of transgenic potato (Solanum tuberosum L. cv. Desirée) with reduced glycoalkaloid content via the down-regulation of the SGT1 gene. Transgenic tubers exhibited an almost complete knock-out of α-solanine production and light had little impact on its accumulation. Levels of α-chaconine increased significantly in the peel of both the control and transgenic lines when exposed to light, particularly in the transgenic line. Major differences in metabolite profiles existed between outer and inner tuber tissues, and between light and dark-treated tubers. Many of the light-induced changes are explicable in terms of pathways known to be affected by stress responses. The impact of transgenesis on profiles was much less than that of tissue type or light and most differences were explicable in terms of the modification to the glycoalkaloid pathway.

Solamargine production by a fungal endophyte of Solanum nigrum.

AIMS: The aim was to isolate, identify and characterize endophytes from Solanum nigrum L. as a new source of the cytotoxic steroidal alkaloid solamargine. METHODS AND RESULTS: Three endophytic fungi; SNFSt, SNFL and SNFF were isolated from S. nigrum and identified by molecular methods. Preliminary TLC screening showed a common metabolite between the plant and one of these fungi, SNFSt which was identified as Aspergillus flavus based on the phylogenetic analysis of its ITS sequence. Subsequent LC-HRESIMS analysis unambiguously established the identity of the compound based on its molecular formula and its characteristic MS(2) fragmentation pattern as solamargine. To ascertain its identity, fungal solamargine was isolated using preparative TLC and its structure was fully characterized using NMR spectroscopic techniques and high-resolution mass spectrometric analysis. Solamargine production could be followed and quantified for a total of 11 generations of this fungus with a titer of ~250-300 µg l(-1) . This study represents one of the first examples where host plant-derived compounds have been demonstrated to be steadily produced by an endophytic fungi in sizeable quantities. CONCLUSIONS: The production of solamargine (found in the host plant) by a cultivable fungal endophyte at a significant yield is a new observation. Further experiments such as media optimization, OSMAC (One Strain Many Compounds) or epigenetic modifiers could be applied to enhance the fungal solamargine production. SIGNIFICANCE AND IMPACT OF THE STUDY: The endophytic fungus SNFSt isolated from S. nigrum may be utilized for quantitative production of the potent cytotoxic metabolite solamargine.

Modifying glycoalkaloid content in transgenic potato--Metabolome impacts.

Metabolite profiling has been used to assess the potential for unintended composition changes in potato (Solanum tuberosum L. cv. Desirée) tubers, which have been genetically modified (GM) to reduce glycoalkaloid content, via the independent down-regulation of three genes SGT1, SGT2 and SGT3 known to be involved in glycoalkaloid biosynthesis. Differences between the three groups of antisense lines and control lines were assessed using liquid chromatography-mass spectrometry (LC-MS) and gas chromatography (GC)-MS, and data analysed using principal component analysis and analysis of variance. Compared with the wild-type (WT) control, LC-MS revealed not only the expected changes in specific glycoalkaloid levels in the GM lines, but also significant changes in several other metabolites, some of which were explicable in terms of known pathways. Analysis of polar and non-polar metabolites by GC-MS revealed other significant (unintended) differences between SGT lines and the WT, but also between the WT control and other control lines used.

Chemistry and anticarcinogenic mechanisms of glycoalkaloids produced by eggplants, potatoes, and tomatoes.

Inhibition of cancer can occur via apoptosis, a genetically directed process of cell self-destruction that involves numerous biomarkers and signaling pathways. Glycoalkaloids are nitrogen-containing secondary plant metabolites found in numerous Solanaceous plants including eggplants, potatoes, and tomatoes. Exposure of cancer cells to glycoalkaloids produced by eggplants (α-solamargine and α-solasonine), potatoes (α-chaconine and α-solanine), and tomatoes (α-tomatine) or their hydrolysis products (mono-, di-, and trisaccharide derivatives and the aglycones solasodine, solanidine, and tomatidine) inhibits the growth of the cells in culture (in vitro) as well as tumor growth in vivo. This overview comprehensively surveys and consolidates worldwide efforts to define the following aspects of these natural compounds: (a) their prevalence in the three foods; (b) their chemistry and structure-activity relationships; (c) the reported factors (biomarkers, signaling pathways) associated with apoptosis of bone, breast, cervical, colon, gastric, glioblastoma, leukemia, liver, lung, lymphoma, melanoma, pancreas, prostate, and squamous cell carcinoma cell lines in vitro and the in vivo inhibition of tumor formation and growth in fish and mice and in human skin cancers; and (d) future research needs. The described results may make it possible to better relate the structures of the active compounds to their health-promoting function, individually, in combination, and in food, and allow the consumer to select glycoalkaloid-containing food with the optimal content of nontoxic beneficial compounds. The described findings are expected to be a valuable record and resource for further investigation of the health benefits of food-related natural compounds.

Biosynthesis of antinutritional alkaloids in solanaceous crops is mediated by clustered genes.

Steroidal glycoalkaloids (SGAs) such as α-solanine found in solanaceous food plants--as, for example, potato--are antinutritional factors for humans. Comparative coexpression analysis between tomato and potato coupled with chemical profiling revealed an array of 10 genes that partake in SGA biosynthesis. We discovered that six of them exist as a cluster on chromosome 7, whereas an additional two are adjacent in a duplicated genomic region on chromosome 12. Following systematic functional analysis, we suggest a revised SGA biosynthetic pathway starting from cholesterol up to the tetrasaccharide moiety linked to the tomato SGA aglycone. Silencing GLYCOALKALOID METABOLISM 4 prevented accumulation of SGAs in potato tubers and tomato fruit. This may provide a means for removal of unsafe, antinutritional substances present in these widely used food crops.

Glycoalkaloid and calystegine levels in table potato cultivars subjected to wounding, light, and heat treatments.

Potato tubers naturally contain a number of defense substances, some of which are of major concern for food safety. Among these substances are the glycoalkaloids and calystegines. We have here analyzed levels of glycoalkaloids (α-chaconine and α-solanine) and calystegines (A3, B2, and B4) in potato tubers subjected to mechanical wounding, light exposure, or elevated temperature: stress treatments that are known or anticipated to induce glycoalkaloid levels. Basal glycoalkaloid levels in tubers varied between potato cultivars. Wounding and light exposure, but not heat, increased tuber glycoalkaloid levels, and the relative response differed among the cultivars. Also, calystegine levels varied between cultivars, with calystegine B4 showing the most marked variation. However, the total calystegine level was not affected by wounding or light exposure. The results demonstrate a strong variation among potato cultivars with regard to postharvest glycoalkaloid increases, and they suggest that the biosynthesis of glycoalkaloids and calystegines occurs independently of each other.

Cloning and expression analysis of squalene synthase, a key enzyme involved in antifungal steroidal glycoalkaloids biosynthesis from Solanum nigrum.

Steroidal glycoalkaloids (SGAs) are a family of nitrogenous secondary metabolites produced in solanaceous plants. In our present study, γ-solamargine and its aglycone solasodine from Solanum nigrum were found to inhibit hyphae formation of Fusarium oxysporum. As phytoalexins, the formation of SGAs was significantly increased in the plants when infected with the spore of F. oxysporum. In order to understand this inducible defense mechanism, the rate-limiting enzyme squalene synthase in the biosynthesis process of SGAs was investigated well. A full-length cDNA encoding squalene synthase was isolated from S. nigrum (the squalene synthase in S. nigrum was designated as SnSS). The full-length cDNA of SnSS was 1,765 bp and contained a 1,236 bp open reading frame (ORF) encoding a polypeptide of 411 amino acids. Bioinformatic analysis revealed that the deduced SnSS protein had a high similarity with other plant squalene synthases. Real-time RT-PCR analysis showed that SnSS was expressed constitutively in all tested tissues, with the highest expression in stems. After treatment with the spore of F. oxysporum, the mRNA level of SnSS was significantly increased in the infected plants in accordance with the change of SGAs.

Effects of auxins on the production of steroidal alkaloids in rapidly proliferating tissue and cell cultures of Solanum lyratum.

INTRODUCTION: Solanum lyratum, a rare species, is used to treat cancer, tumours and warts. Plant cell and tissue culture of S. lyratum, producing steroidal alkaloids, could be useful supplements to natural sources. OBJECTIVE: To study the production of solanine, solanidine and solasodine by adding auxin-type phytohormones including indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), naphthaleneacetic acid (NAA) and 2,4-dichlorophenoxyacetic acid (2,4-D) to cell and callus cultures of S. lyratum. METHODOLOGY: Methanolic extracts were made from callus and cell cultures of S. lyratumand and analysed using RP C18 HPLC with UV detection. RESULTS: 2,4-D-induced calli from roots led to a significant enhancement in solanine production with a value of 4.13 mg/g dry weight (DW). The maximal solanidine and solasodine levels of 6.26 and 7.69 mg/g DW were respectively obtained with IBA- and IAA-treated S. lyratum cells at concentrations of 1 and 5 mg/L. CONCLUSION: Auxins were found to be useful phytohormones for the production of steroidal alkaloids. The callus and cell culture system developed is simple and can hence be a method of production of steroidal alkaloids in S. lyratum and other Solanaceae species.

Anisodamine production from natural sources: seedlings and hairy root cultures of Argentinean and Colombian Brugmansia candida plants.

The tropane alkaloid anisodamine ( 2) is obtained by 6 beta-hydroxylation of hyoscyamine ( 1). The application of this alkaloid in medicine is gaining attention due to the wide range of therapeutic applications described in addition to its anticholinergic activity. In this work, the production of anisodamine ( 2) by IN VITRO cultures of BRUGMANSIA CANDIDA (Argentinean and Colombian samples) was studied. This alkaloid was estimated in different organs of IN VITRO-germinated seedlings as well as in hairy roots obtained from seedlings from both sources. Colombian roots exhibited the highest content of tropane alkaloids, with anisodamine ( 2) being the main alkaloid measured. In the leaves, the main alkaloid was scopolamine ( 3) and no significant differences were observed between Argentinean and Colombian leaves. The tropane alkaloid content in Argentinean hairy roots was significantly higher than in Colombian ones. Also, in the Argentinean samples the main alkaloid detected was anisodamine ( 2). Argentinean and Colombian B. CANDIDA seedlings and hairy roots appear to be a promising system for the production of anisodamine ( 2).

Salinity stress enhances production of solasodine in Solanum nigrum L.

Various in vitro grown tissues (non-regenerative callus, regenerative callus and microshoot derived leaves) of Solanum nigrum L. were cultured under salinity stress (0-150 mM NaCl) for enhanced production of solasodine, a steroidal alkaloid and an alternative to diosgenin, which is used as a precursor for the commercial production of steroidal drugs. The role of plant growth regulators and various concentrations of NaCl during in vitro production of solasodine was studied. The in vitro yield was compared with the yield from leaves of field grown plant. Solasodine content was maximum (2.39 mg/g dry wt.) in regenerative callus when grown on medium added with 150 mM NaCl; followed by in vitro raised leaf of microshoot. Quantitative estimation of solasodine was carried out using a new HPTLC method, which is validated for its recovery and precession. The proposed HPTLC method showed a good linear relationship (r(2)=0.994) in 50-2000 ng/spot concentration ranges. The data demonstrate that the solasodine production in cultures was growth dependent.

Growth and production optimization of tropane alkaloids in Datura stramonium cell suspension culture.

Abstract: A number of physicochemical conditions such different concentration of glucose, sucrose, potassium nitrate, ammonium nitrate, calcium chloride and temperatures were tested to optimize growth and production of tropane alkaloids from Datura stramonium (Solanaceae) plants. Cell suspension from semi-clear calli of leave explants developed in MS medium containing kinetin (0.5 mg L(-1)) and NAA (2 mg L(-1)) hormones was used to measure biomass and total alkaloids and comparison of treatments. The results showed that 30 and 40 g L(-1) glucose led to the highest level of alkaloids and biomass productions, respectively. 20 and 40 g L(-1) sucrose concentrations resulted in order the most rates of alkaloids and biomass productions. The results showed that increasing of nitrate concentration led to the reduction of the alkaloids. The best concentration of potassium nitrate for the production of tropane alkaloids and biomass were in order 9.4 and 3.76 mM. Also it was evinced that the optimized concentration of ammonium nitrate for alkaloids production was 10.3 mM and for the biomass was 41.22 mM. The best concentration of calcium chloride for growth and production of the alkaloids was 7.92 mM. Testing different temperature specified that the best condition for production of the alkaloids was 20 degrees C whereas it was 25 degrees C for biomass production. The results of this study could be recommended to farmers involved in production of D. stramonium for tropain alkaloids at industrial and semi-industrial scales.

Production of solasodine by Solanum laciniatum using plant tissue culture technique.

Leaf and hypocotyl explants of 15 days old aseptically grown seedlings of Solanum laciniatum were cultured on MS medium supplemented with NAA (2 mg/l) and kinetin (0.5 mg/l) for callus initiation. For maintenance and proliferation of callus MS medium supplemented with 2,4-D (1 mg/l) and kinetin (0.5 mg/l) was used. The growth of the calli derived from hypocotyls increased with time of incubation and remained almost constant after 45 days. The solasodine content in callus culture was maximum after 30 days of incubation. Addition of L-arginine in the medium (50-150 mg/l) increased growth as well as chlorophyll content in the callus culture. The solasodine content also increased up to 1.2 to 1.4 times in these cultures. High frequency shoot regeneration was obtained in MS medium having BA (4 mg/l) and IBA (0.25 mg/l). For shoot multiplication, MS medium having BA (4 mg/l) was used. Shoots rooted on the same medium. Organogenesis promoted solasodine accumulation in the cultures. Regenerated shoots yielded higher solasodine content than undifferentiated as well as organogenic callus. Solasodine contents in the regenerated shoots was found to be 10 times higher than the callus culture and approached towards the field grown plants. Thin layer chromatography revealed the presence of three compounds. The most predominant spot (Rf 0.789) corresponded to the reference solasodine.

Solasodine glycoside production by hairy root cultures of Physalis minima Linn.

Hairy root cultures of Physalis minima L. were developed using Agrobacterium rhizogenes, strain ATCC 15834 mediated transformation and grown in half strength of Murashige and Skoog medium containing 8% (w/v) sucrose. Media supplementation with 1 mg naphthalenacetic acid l(-1) and 1 mg benzyladenine increased solasodine glycoside up to 900 g dry wt, which was 20 times higher than that in the native root.