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The industrial value of various plants has been improved through the of plant cell culture systems with elicitation. In this study, the adventitious root of Abeliophyllum distichum (AdAR) was treated with gibberellic acid 3 (GA3) to improve its anticancer property. The hexane fraction of the GA3-treated A. distichum adventitious root exhibited a stronger cytotoxic activity against A549 cells than the hexane fraction of AdAR. Through GC/MS and principal component analysis, we identified ferruginol and sugiol as anticancer compounds, which were induced by GA3 treatment in AdAR. Gene expression analysis combined with functional characterisation suggests that the GA3 treatment increased the transcription of geranylgeranyl pyrophosphate synthases and copalyl diphosphate synthase, which led to the accumulation of diterpenoids, including ferruginol and sugiol. Overall, these findings can contribute to the advancement of metabolic engineering for enhancing the biosynthesis of active diterpenoids, and facilitate the large-scale production of bioactive compounds sourced from A. distichum . more...

Camptothecin (CPT), an indole alkaloid popular for its anticancer property, is considered the third most promising drug after taxol and famous alkaloids from Vinca for the treatment of cancer in humans. Camptothecin was first identified in Camptotheca acuminata followed by several other plant species and endophytic fungi. Increased harvesting driven by rising global demand is depleting the availability of elite plant genotypes, such as Camptotheca acuminata and Nothapodytes nimmoniana, crucial for producing alkaloids used in treating diseases like cancer. Conservation of these genotypes for the future is imperative. Therefore, research on different plant tissue culture techniques such as cell suspension culture, hairy roots, adventitious root culture, elicitation strategies, and endophytic fungi has been adopted for the production of CPT to meet the increasing demand without affecting the source plant's existence. Currently, another strategy to increase camptothecin yield by genetic manipulation is underway. The present review discusses the plants and endophytes that are employed for camptothecin production and throws light on the plant tissue culture techniques for the regeneration of plants, callus culture, and selection of cell lines for the highest camptothecin production. The review further explains the simple, accurate, and cost-effective extraction and quantification methods. There is enormous potential for the sustainable production of CPT which could be met by culturing of suitable endophytes or plant cell or organ culture in a bioreactor scale production. Also, different gene editing tools provide opportunities for engineering the biosynthetic pathway of CPT, and the overall CPT production can be improved . KEY POINTS: • Camptothecin is a naturally occurring alkaloid with potent anticancer properties, primarily known for its ability to inhibit DNA topoisomerase I. • Plants and endophytes offer a potential approach for camptothecin production. • Biotechnology approaches like plant tissue culture techniques enhanced camptothecin production., (© 2024. The Author(s).) more...

Paclitaxel, a rare diterpene extracted from the bark of Chinese yew ( Taxus chinensis ), is renowned for its anti-cancer activity and serves as a primary drug for treating cancers. Due to the exceptionally low content of paclitaxel in the bark, a semi-synthetic method that depletes Chinese yew resources is used in the production of paclitaxel, which, however, fails to meet the escalating clinical demand. In recent years, researchers have achieved significant progress in heterologous biosynthesis and metabolic engineering for the production of paclitaxel. This article comprehensively reviews the advancements in paclitaxel production, encompassing chemical synthesis, heterologous biosynthesis, and cell engineering. It provides an in-depth introduction to the biosynthetic pathway and transcriptional regulation mechanisms of paclitaxel, aiming to provide a valuable reference for further research on paclitaxel biosynthesis. more...

It is known that Senna obtusifolia has been used in medicine since ancient times due to the content of many valuable compounds with a pro-health effect. One of them is betulinic acid, which is a pentacyclic triterpene with antimalarial, antiviral, anti-inflammatory and anticancer properties. In this work, a continuation of our previous research, an attempt was made to increase the level of betulinic acid accumulation by the cultivation of transgenic hairy roots that overexpress the squalene synthase gene in a 10 L sprinkle bioreactor with methyl jasmonate elicitation. We present that the applied strategy allowed us to increase the content of betulinic acid in hairy root cultures to the level of 48 mg/g dry weight. The obtained plant extracts showed a stronger cytotoxic effect on the U87MG glioblastoma cell line than the roots grown without elicitors. Additionally, the induction of apoptosis, reduction of mitochondrial membrane potential, chromosomal DNA fragmentation and activation of caspase cascades are demonstrated. Moreover, the tested extract showed inhibition of topoisomerase I activity. more...

Seven phenolic compounds (ferulic acid, caffeic acid, 4-methoxycinnamic acid, 3,4-dimethoxycinnamic acid, 3-hydroxy-4-methoxybenzaldehyde, 3-methoxy-4-hydroxypropiophenone and 1-O,2-O-digalloyl-6-O-trans-p-coumaroyl-β-D-glucopyranoside), a flavanonol (7-O-methylaromadendrin), two lignans (pinoresinol and matairesinol) and six diterpenic acids/alcohol (19-acetoxy-13-hydroxyabda-8(17),14-diene, totarol, 7-oxodehydroabietic acid, dehydroabietic acid, communic acid and isopimaric acid) were isolated from the hydroalcoholic extract of a Brazilian Brown Propolis and characterized by NMR spectral data analysis. The volatile fraction of brown propolis was characterized by CG-MS, composed mainly of monoterpenes and sesquiterpenes, being the major α-pinene (18.4 %) and β-pinene (10.3 %). This propolis chemical profile indicates that Pinus spp., Eucalyptus spp. and Araucaria angustifolia might be its primary plants source. The brown propolis displayed significant activity against Plasmodium falciparum D6 and W2 strains with IC 50 of 5.3 and 9.7 μg/mL, respectively. The volatile fraction was also active with IC 50 of 22.5 and 41.8 μg/mL, respectively. Among the compounds, 1-O,2-O-digalloyl-6-O-trans-p-coumaroyl-β-D-glucopyranoside showed IC 50 of 3.1 and 1.0 μg/mL against D6 and W2 strains, respectively, while communic acid showed an IC 50 of 4.0 μg/mL against W2 strain. Cytotoxicity was determined on four tumor cell lines (SK-MEL, KB, BT-549, and SK-OV-3) and two normal renal cell lines (LLC-PK1 and VERO). Matairesinol, 7-O-methylaromadendrin, and isopimaric acid showed an IC 50 range of 1.8-0.78 μg/mL, 7.3-100 μg/mL, and 17-18 μg/mL, respectively, against the tumor cell lines but they were not cytotoxic against normal cell lines. The crude extract of brown propolis displayed antimicrobial activity against C. neoformans, methicillin-resistant Staphylococcus aureus, and P. aeruginosa at 29.9 μg/mL, 178.9 μg/mL, and 160.7 μg/mL, respectively. The volatile fraction inhibited the growth of C. neoformans at 53.0 μg/mL. The compounds 3-hydroxy-4-methoxybenzaldehyde, 3-methoxy-4-hydroxypropiophenone and 7-oxodehydroabietic acid were active against C. neoformans, and caffeic and communic acids were active against methicillin-resistant Staphylococcus aureus., (© 2021 Wiley-VHCA AG, Zurich, Switzerland.) more...

Chlorophyllides can be found in photosynthetic organisms. Generally, chlorophyllides have a -, b -, c -, d -, and f -type derivatives, and all chlorophyllides have a tetrapyrrole structure with a Mg ion at the center and a fifth isocyclic pentanone. Chlorophyllide a can be synthesized from protochlorophyllide a , divinyl chlorophyllide a , or chlorophyll. In addition, chlorophyllide a can be transformed into chlorophyllide b , chlorophyllide d , or chlorophyllide f . Chlorophyllide c can be synthesized from protochlorophyllide a or divinyl protochlorophyllide a . Chlorophyllides have been extensively used in food, medicine, and pharmaceutical applications. Furthermore, chlorophyllides exhibit many biological activities, such as anti-growth, antimicrobial, antiviral, antipathogenic, and antiproliferative activity. The photosensitivity of chlorophyllides that is applied in mercury electrodes and sensors were discussed. This article is the first detailed review dedicated specifically to chlorophyllides. Thus, this review aims to describe the definition of chlorophyllides, biosynthetic routes of chlorophyllides, purification of chlorophyllides, and applications of chlorophyllides. more...

Triptolide, an abietane-type diterpenoid isolated from Tripterygium wilfordii Hook. F., has significant pharmacological activity. Research results show that triptolide has obvious inhibitory effects on many solid tumors. Therefore, triptolide has become one of the lead compounds candidates for being the next "blockbuster" drug, and multiple triptolide derivatives have entered clinical research. An increasing number of researchers have developed triptolide synthesis methods to meet the clinical need. To provide new ideas for researchers in different disciplines and connect different disciplines with researchers aiming to solve scientific problems more efficiently, this article reviews the research progress made with analyzes of triptolide pharmacological activity, biosynthetic pathways, and chemical synthesis pathways and reported in toxicological and clinical studies of derivatives over the past 20 years, which have laid the foundation for subsequent researchers to study triptolide in many ways., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).) more...

Camptothecin (CPT), a well-known monoterpenoid indole alkaloid with broad-spectrum anti-cancer activity, is produced from plants and endophytes. In view of the limitations of plants as sources of camptothecin in productivity and efficiency, endophytes serve as the fast growth, high cost-effectiveness, good reproducibility, and feasible genetic manipulation, so they have the potential to meet the huge market demand of the pharmaceutical industry. In this review, we summarized the isolation, identification and fermentation of CPT-producing endophytes, as well as the biosynthesis, extraction and detection of camptothecin from endophytes. Among them, we put emphasis on increasing the production of camptothecin in endophytes through different strategies such as changing the proportion of carbon, nitrogen and phosphate source, adding the precursors, elicitors or adsorbent resin, utilizing co-culture fermentation or fermenter culture. However, cell subculture and metabolic reprogramming affect the expression of camptothecin biosynthetic genes in CPT-producing endophytes, which poses a challenge to the industrial production of camptothecin. Therefore, it will be useful to gain insights through the review of these researches and provide alternative approaches to develop economical, eco-friendly and reliable natural products., (Copyright © 2021 Elsevier Ltd. All rights reserved.) more...

Background: Taxus is a valuable woody species with important medicinal value. The bark of Taxus can produce taxol, a natural antineoplastic drug that is widely used in the treatment of breast, ovarian and lung cancers. However, the low content of taxol in the bark of Taxus can not meet the growing clinical demands, so the current research aims at finding ways to increase taxol production., Objective: In this review, the research progress of taxol including the factors affecting the taxol content, biosynthesis pathway of taxol, production of taxol in vitro and the application of multi-omics approaches in Taxus as well as future research prospects will be discussed., Results: The taxol content is not only dependent on the species, age and tissues but is also affected by light, moisture levels, temperature, soil fertility and microbes. Most of the enzymes in the taxol biosynthesis pathway have been identified and characterized. Total chemical synthesis, semi-synthesis, plant cell culture and biosynthesis in endophytic fungi have been explored to product taxol. Multi-omics have been used to study Taxus and taxol., Conclusion: Further efforts in the identification of unknown enzymes in the taxol biosynthesis pathway, establishment of the genetic transformation system in Taxus and the regulatory mechanism of taxol biosynthesis and Taxus cell growth will play a significant role in improving the yield of taxol in Taxus cells and plants., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.) more...

Paclitaxel, a tetracyclic diterpenoid compounds, was firstly isolated from the bark of the Pacific yew trees. Currently, as a low toxicity, high efficiency, and broad-spectrum natural anti-cancer drug, paclitaxel has been widely used against ovarian cancer, breast cancer, uterine cancer, and other cancers. As the matter of fact, natural paclitaxel from Taxus species has been proved to be environmentally unsustainable and economically unfeasible. For this reason, researchers from all over the world are devoted to searching for new ways of obtaining paclitaxel. At present, other methods, including artificial cultivation of Taxus plants, microbial fermentation, chemical synthesis, tissue and cell culture have been sought and developed subsequently. Meanwhile, the biosynthesis of paclitaxel is also an extremely attractive method. Unlike other anti-cancer drugs, paclitaxel has its unique anti-cancer mechanisms. Here, the source, production, and anti-cancer mechanisms of paclitaxel were summarized and reviewed, which can provide theoretical basis and reference for further research on the production, anti-cancer mechanisms and utilization of paclitaxel., (Copyright © 2020 China Pharmaceutical University. Published by Elsevier B.V. All rights reserved.) more...

In the study, endophytic fungi isolated from Ophiorrhiza mungos were screened for camptothecin (CPT) biosynthetic potential by high performance liquid chromatography (HPLC). Among the 16 fungi screened, OmF3, OmF4, and OmF6 were identified to synthesize CPT. Further LC-MS analysis also showed the presence of CPT specific m/z of 349 for the extracts from OmF3, OmF4, and OmF6. However, the fragmentation masses with m/z of 320, 305, 277 and 220 specific to the CPT could be identified only for the OmF3 and OmF4. These CPT producing fungi were further identified as Meyerozyma sp. OmF3 and Talaromyces sp. OmF4. The cultures of these two fungi were then supplemented with nanoparticles and analyzed for the quantitative enhancement of CPT production by LC-MS/MS. From the result, Meyerozyma sp. OmF3 was found to produce 947.3 ± 12.66 μg/L CPT, when supplemented with 1 μg/mL zinc oxide nanoparticles and the same for uninduced parental strain OmF3 was only 1.77 ± 0.13 μg/L. At the same time, Talaromyces sp. OmF4 showed the highest production of 28.97 ± 0.37 μg/L of CPT when cultured with 10 μg/mL silver nanoparticles and the same for uninduced strain was 1.19 ± 0.24 μg/L. The observed quantitative enhancement of fungal CPT production is highly interesting as it is a rapid and cost effective method. The study is remarkable due to the identification of novel fungal sources for CPT production and its enhancement by nanoparticle supplementation., (© 2020 American Institute of Chemical Engineers.) more...

Glucoraphanin is a methionine-derived glucosinolate that imparts numerous health-benefits with broad bioactivity. Low amounts in plant tissues and high cost of extraction have limited the production of glucoraphanin. Metabolic engineering in heterologous microorganisms is an attractive approach to achieve efficient production of valuable natural products. In this study, a microbial fermentation process for glucoraphanin production was demonstrated. The engineered bacterial strain stably expressed 10 allogeneic enzymes in E. coli chromosome, including nine heterologous genes from Arabidopsis and Brassica and one from fungus Neurospora crassa, which could produce the specialized glucosinolate compound glucoraphanin with a titer of 0.675 μg/L by fermentation from glucose. The cofactor supplements and individual gene overexpression for glucoraphanin production were also investigated. This work highlights the possibility of supplying specialized plant glucosinolates by microbial fermentation process, instead of chemical extraction. Additionally, the limiting step enzyme, UDP-glucose-thiohydroximate glucosyltransferase, identified in this study also laid a foundation for further optimizing the glucoraphanin-producing cell factory., Competing Interests: Declaration of Competing Interest The authors declare that they have no competing interests., (Copyright © 2020 Elsevier GmbH. All rights reserved.) more...

Advances in recombinant DNA technology have opened up new possibilities of exploiting toxic proteins for therapeutic purposes. Bringing forth these protein toxins from the bench to the bedside strongly depends on the availability of production methods that are reproducible, scalable and comply with good manufacturing practice (GMP). The type I ribosome-inhibiting protein, gelonin, has great potential as an anticancer drug, but is sequestrated in endosomes and lysosomes. This can be overcome by combination with photochemical internalization (PCI), a method for endosomal drug release. The combination of gelonin-based drugs and PCI represents a tumor-targeted therapy with high precision and efficiency. The aim of this study was to produce recombinant gelonin (rGel) at high purity and quantity using an automated liquid chromatography system. The expression and purification process was documented as highly efficient (4.4 mg gelonin per litre induced culture) and reproducible with minimal loss of target protein (~50% overall yield compared to after initial immobilized metal affinity chromatography (IMAC)). The endotoxin level of 0.05-0.09 EU/mg was compatible with current standards for parenteral drug administration. The automated system provided a consistent output with minimal human intervention and close monitoring of each purification step enabled optimization of both yield and purity of the product. rGel was shown to have equivalent biological activity and cytotoxicity, both with and without PCI-mediated delivery, as rGel ref produced without an automated system. This study presents a highly refined and automated manufacturing procedure for recombinant gelonin at a quantity and quality sufficient for preclinical evaluation. The methods established in this report are in compliance with high quality standards and compose a solid platform for preclinical development of gelonin-based drugs. more...

Aims: Paclitaxel is a type of broad-spectrum anticancer drug in short supply. The price of acetyl-CoA (17 709 677·4 USD mol -1 ), which is the acetyl group donor for the enzymatic synthesis of the intermediate, baccatin Ⅲ, is still the bottleneck of the mass production of paclitaxel. This study reports a novel acetyl group donor, which could substantially reduce the cost of production., Methods and Results: In this study, a substrate spectrum with 14 kinds of representative acetyl-donor substitutes predicted by computer-aided methods was tested in a 10-deacetylbaccatin Ⅲ-10-O-acetyltransferase (DBAT) heterogeneous-expressed open-whole-cell catalytic system. The results of computer prediction and experimental analysis revealed the rule of the acetyl-donor compounds based on this substrate spectrum. N-acetyl-d-glucosamine (30·95 USD mol -1 , about 572 202-fold cheaper than acetyl-CoA) is selected as a suitable substitute under the rule. The yield when using N-acetyl-d-glucosamine as acetyl donor in open-whole-cell catalytic system was 2·13-fold of that when using acetyl-CoA. In the in vivo system, the yield increased 24·17%, which may indicate its cooperation with acetyl-CoA., Conclusion: The success of open-whole-cell synthesis and in vivo synthesis of baccatin Ⅲ by adding N-acetyl-d-glucosamine as acetyl substrate demonstrates that it is a useful substrate to improve the yield of baccatin Ⅲ., Significance and Impact of the Study: All these findings provided a potential acetyl-donor substitute for acetyl-CoA, as well as a low cost and efficient method of preparing paclitaxel through baccatin Ⅲ semi-synthesis., (© 2020 The Society for Applied Microbiology.) more...

Many of the plant-derived compounds used in chemotherapies are currently produced by semisynthesis, which results in limited supplies at exorbitant market prices. However, the synthetic biology era, which began ca 15 years ago, has progressively yielded encouraging advances by using engineered microbes for the practical production of cheaper plant anticancer drugs., (Copyright © 2020 Elsevier Inc. All rights reserved.) more...

The graphene-based nanomaterials have been measured as a most promising nanomaterials in the various fields. Graphene oxide having attractive and effective attention in the modified of medicine. Also, graphene oxide is one of the distinct bio-chemical properties with minimum cytotoxicity compared to the other nanomaterials. Up to till date, gastric treatments with reduced graphene oxide not studied so far. In this report, 7-ethyl- 10-hydroxycamptothecin (SN-38) coated graphene oxide synthesized (SN-rGO) effectively and are characterized using various analytical methods. The hydroxyl and carbonyl gatherings of oxidized SN38 will in general ingest onto GO by means of hydrogen bond arrangement with their leftover oxygen functionalities and offers steadiness to SN38. The morphological analyses showed the foldable fields of SN38-rGO NPs with acquire transparency, thin sheets and the crumpled structures. Further the photothermal efficiency and cytotoxicity of the SN-rGO were examined by MTT assay using two NCI-N87 and SGC-791 gastric carcinoma cells. In addition, the morphological changes were examined through the live and dead cells and nuclear staining biochemical techiniques and apoptosis were evaluated through flow cytometry analysis. Our result's suggested that the SN-38 coated reduced graphene oxide can be used for the photothermal treatment of gastric carcinoma for the future nanotechnology cancer therapies without using functionalized polymeric nanoparticles., Competing Interests: Declaration of Competing Interest None., (Copyright © 2020 Elsevier B.V. All rights reserved.) more...

Triptolide is a trace natural product of Tripterygium wilfordii. It has antitumor activities, particularly against pancreatic cancer cells. Identification of genes and elucidation of the biosynthetic pathway leading to triptolide are the prerequisite for heterologous bioproduction. Here, we report a reference-grade genome of T. wilfordii with a contig N50 of 4.36 Mb. We show that copy numbers of triptolide biosynthetic pathway genes are impacted by a recent whole-genome triplication event. We further integrate genomic, transcriptomic, and metabolomic data to map a gene-to-metabolite network. This leads to the identification of a cytochrome P450 (CYP728B70) that can catalyze oxidation of a methyl to the acid moiety of dehydroabietic acid in triptolide biosynthesis. We think the genomic resource and the candidate genes reported here set the foundation to fully reveal triptolide biosynthetic pathway and consequently the heterologous bioproduction. more...

Taxol is one of the anticancer drugs synthesized naturally in the evergreen Taxus brevifolia forest tree belonging to the yew family ( Taxaceae ) growing on the Pacific. There are reportedly evidence for treating ovarian, breast and lung cancers through this drug given its unique structural and functional features. Extraction of this drug from yew trees bark is one of the most common ways of producing this drug, but 3000 trees are needed to obtain a kilogram of Taxol. Hence, further attention has recently been attracted to the metabolic engineering strategies, including, engineering cellular metabolism of microorganisms and their optimization. Accordingly, the present paper article was aimed to review recent advances in elevating the production and commercialization of Taxol through metabolic engineering techniques. more...

Background: Dracocephalum kotschyi Boiss. is a valuable source of rosmarinic acid (RA) and methoxylated hydroxyflavones (such as xanthomicrol and cirsimaritin) with antioxidative and antiplatelet effects and with antiproliferative potential against various cancer cells. The extensive application of nanotechnology in hairy root cultures is a new sustainable production platform for producing these active constituents. In the present study, hairy roots derived from 4-week-old leaves and Agrobacterium rhizogenes strain ATCC15834 were used to investigate the impact of various concentrations of iron oxide nanoparticles (Fe NPs) in two elicitation time exposures (24 and 48 h) on growth, antioxidant enzyme activity, total phenolic and flavonoid content (TPC and TFC), and some polyphenols. Gene expression levels of phenylalanine ammonia-lyase (pal) and rosmarinic acid synthase (ras) were also analyzed., Results: Iron nanoparticles enhanced biomass accumulation in hairy roots. The treatment time and Fe NP dosage largely improved the activity of antioxidant enzymes, TPC and TFC. The highest RA (1194 μg g -1 FW) content (9.7-fold), compared to controls, was detected with 24 h of exposure to 75 mg L -1 Fe NP, which was consistent with the expression of pal and ras genes under the influence of elicitation. The xanthomicrol, cirsimaritin, and isokaempferide content was increased 11.87, 3.85, and 2.27-fold, respectively., Conclusion: Stimulation of D. kotschyi hairy roots by Fe NPs led to a significant increase in the induction and production of important pharmaceutical compounds such as rosmarinic acid and xanthomicrol. © 2019 Society of Chemical Industry., (© 2019 Society of Chemical Industry.) more...

Sesquiterpene lactones (STL) are a subclass of isoprenoids with many known bioactivities frequently found in the Asteraceae family. In recent years, remarkable progress has been made regarding the biochemistry of STL, and today the biosynthetic pathway of the core backbones of many STLs has been elucidated. Consequently, the focus has shifted to the discovery of the decorating enzymes that can modify the core skeleton with functional hydroxy groups. Using in vivo pathway reconstruction assays in heterologous organisms such as Saccharomyces cerevisiae and Nicotiana benthamiana, we have analyzed several cytochrome P450 enzyme genes of the CYP71AX subfamily from Helianthus annuus clustered in close proximity to one another on the sunflower genome. We show that one member of this subfamily, CYP71AX36, can catalyze the conversion of costunolide to 14-hydroxycostunolide. The catalytic activity of CYP71AX36 may be of use for the chemoenzymatic production of antileukemic 14-hydroxycostunolide derivatives and other STLs of pharmaceutical interest. We also describe the full 2D-NMR assignment of 14-hydroxycostunolide and provide all 13C chemical shifts of the carbon skeleton for the first time. more...

Cancer is the leading cause of death in industrialized countries. Cancer therapy often involves monoclonal antibodies or small-molecule drugs, but carbohydrate-binding lectins such as mistletoe (Viscum album) viscumin offer a potential alternative treatment strategy. Viscumin is toxic in mammalian cells, ruling them out as an efficient production system, and it forms inclusion bodies in Escherichia coli such that purification requires complex and lengthy refolding steps. We therefore investigated the transient expression of viscumin in intact Nicotiana benthamiana plants and Nicotiana tabacum Bright Yellow 2 plant-cell packs (PCPs), comparing a full-length viscumin gene construct to separate constructs for the A and B chains. As determined by capillary electrophoresis the maximum yield of purified heterodimeric viscumin in N. benthamiana was ~7 mg/kg fresh biomass with the full-length construct. The yield was about 50% higher in PCPs but reduced 10-fold when coexpressing A and B chains as individual polypeptides. Using a single-step lactosyl-Sepharose affinity resin, we purified viscumin to ~54%. The absence of refolding steps resulted in estimated cost savings of more than 80% when transient expression in tobacco was compared with E. coli. Furthermore, the plant-derived product was ~3-fold more toxic than the bacterially produced counterpart. We conclude that plants offer a suitable alternative for the production of complex biopharmaceutical proteins that are toxic to mammalian cells and that form inclusion bodies in bacteria., (© 2019 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.) more...

(-)-Podophyllotoxin is one of the most potent microtubule depolymerizing agents and has served as an important lead compound in antineoplastic drug discovery. Reported here is a short chemoenzymatic total synthesis of (-)-podophyllotoxin and related aryltetralin lignans. Vital to this approach is the use of an enzymatic oxidative C-C coupling reaction to construct the tetracyclic core of the natural product in a diastereoselective fashion. This strategy allows gram-scale access to (-)-deoxypodophyllotoxin and is readily adaptable to the preparation of related aryltetralin lignans., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.) more...

Background: Nothapodytes nimmoniana, a plant of pivotal medicinal significance is a source of potent anticancer monoterpene indole alkaloid (MIA) camptothecin (CPT). This compound owes its potency due to topoisomerase-I inhibitory activity. However, biosynthetic and regulatory aspects of CPT biosynthesis so far remain elusive. Production of CPT is also constrained due to unavailability of suitable in vitro experimental system. Contextually, there are two routes for the biosynthesis of MIAs: the mevalonate (MVA) pathway operating in cytosol and the methylerythritol phosphate (MEP) pathway in the plastids. Determination of relative precursor flux through either of these pathways may provide a new vista for manipulating the enhanced CPT production., Results: In present study, specific enzyme inhibitors of MVA (lovastatin) and MEP pathways (fosmidomycin) were used to perturb the metabolic flux in N. nimmoniana. Interaction of both these pathways was investigated at transcriptional level by using qRT-PCR and at metabolite level by evaluating secologanin, tryptamine and CPT contents. In fosmidomycin treated plants, highly significant reduction was observed in both secologanin and CPT accumulation in the range 40-57% and 64-71.5% respectively, while 4.61-7.69% increase was observed in tryptamine content as compared to control. Lovastatin treatment showed reduction in CPT (7-11%) and secologanin (7.5%) accumulation while tryptamine registered slight increase (3.84%) in comparison to control. These inhibitor mediated changes were reflected at transcriptional level via altering expression levels of deoxy-xylulose-5-phosphate reductoisomerase (DXR) and hydroxymethylglutaryl-CoA reductase (HMG). Further, mRNA expression of four more genes downstream to DXR and HMG of MEP and MVA pathways respectively were also investigated. Expression analysis also included secologanin synthase (SLS) and strictosidine synthase (STR) of seco-iridoid pathway. Present investigation also entailed development of an efficient in vitro multiplication system as a precursor to pathway flux studies. Further, a robust Agrobacterium-mediated transformed hairy root protocol was also developed for its amenability for up-scaling as a future prospect., Conclusions: Metabolic and transcriptional changes reveal differential efficacy of cytosolic and plastidial inhibitors in context to pathway flux perturbations on seco-iridoid end-product camptothecin. MEP pathway plausibly is the major precursor contributor towards CPT production. These empirical findings allude towards developing suitable biotechnological interventions for enhanced CPT production. more...

Paclitaxel is a well-known anticancer agent with a unique mechanism of action. It is considered to be one of the most successful natural anticancer drugs available. This study summarizes the recent advances in our understanding of the sources, the anticancer mechanism, and the biosynthetic pathway of paclitaxel. With the advancement of biotechnology, improvements in endophytic fungal strains, and the use of recombination techniques and microbial fermentation engineering, the yield of extracted paclitaxel has increased significantly. Recently, paclitaxel has been found to play a large role in tumor immunity, and it has a great potential for use in many cancer treatments., Competing Interests: Competing interestsThe authors declare that they have no competing interests. more...

Covering: 1960s to end of August 2018 Plant glandular trichomes (GTs) are adaptive structures that are well known as "phytochemical factories" due to their impressive capacity to biosynthesize and store large quantities of specialized natural products. The natural products in GTs are chemically diverse and mostly function as defense chemicals, therefore GTs are frequently regarded as "the first defense line" of plants against biotic and abiotic stresses. More importantly, many GT natural products are commercially desirable, thanks to their significant biological activities, thus attracting extensive interest in their biosynthesis. Consequently, it is well known that plant GTs are not only important reservoirs of biologically active natural products but are also a valuable bank of novel biosynthetic genes and enzymes. The non-volatile or oxygenated natural products in plant GTs, which need longer biosynthetic pathways and more energy from the plants, are of particular interest due to their more extensive biological activities and high commercial value. This review mainly focuses on these non-volatile natural products in plant GTs, including their chemistry, biological activities and biosynthesis. The methods employed for investigating natural products and their biosynthesis in plant GTs are also comprehensively discussed. more...

The plant derived camptothecin (CPT) is a pentacyclic pyrroloquinoline alkaloid with unique antitumor activity. Successive discoveries of new CPT-producing plants occurred in recent years due to market demands. The scattered distribution among angiosperms drew researchers' attention. The aim of this review is to appraise the literature available to date for CPT distribution and the phytochemistry of these CPT-producing plants. Metabolite comparative analyses between the plants were also conducted for tracking of possible clues for CPT biosynthesis. Forty-three plant species in total were reported to possess CPT-producing capability, and one hundred twenty-five alkaloids classified into three major categories are summarized herein. Metabolite comparative analysis between these plants suggests the probability that the formation of the central intermediate for CPT biosynthesis has multiple origins. A more complete biogenetic reasoning for CPT and its structural homolog was delineated based on this fragmentary phytochemical evidence from a chemical point of view. Furthermore, an in-house compound database was constructed for further metabolomic analysis., (Copyright © 2019 Elsevier B.V. All rights reserved.) more...

To enhance production of Antrodia cinnamomea triterpenoids (ACTs) from mycelia in solid-state culture, α-terpineol was added to the medium as an elicitor at an optimal concentration of 0.05 mL L-1. Multi-stage solvent extraction and HPLC analysis were performed, and the compositions of ACTs-E (from culture with elicitor) and ACTs-NE (from culture without elicitor) were found to be quite different. In assays of in vitro antitumor activity, ACTs-E, in comparison with ACTs-NE, produced stronger viability reduction in several tumor cell lines and stronger apoptosis induction in HeLa in a dose-dependent manner. Several related proteins involved in the mitochondrial pathway of apoptosis (p53, Bax, caspase-3) did not show expression upregulation by ACTs-E, suggesting that apoptosis induction occurred through a p53-independent process. Further analysis revealed that ACTs-E strongly inhibited synthesis of topoisomerase I (TOP1) and tyrosyl-DNA phosphodiesterase I (TDP1), which are involved in DNA repair, at both transcriptional and protein levels. Our findings suggest that ACTs-E have potential for applications in the pharmaceutical, clinical, and functional food industries, as a novel antitumor agent and a dual TOP1/TDP1 inhibitor. more...

Viola odorata L. (Violaceae), an Indian medicinal plant, contains a plethora of cyclotides, which are a class of cyclic peptides derived from plants, possessing several applications. Somatic embryo culture of V. odorata was developed, via indirect somatic embryogenesis, to serve as an alternative to natural plant biomass for sustainable and continuous production of its bioactive ingredients, such as cyclotides. Among the various combinations of phytohormones tested, Murashige and Skoog medium supplemented with 1 mg/l thidiazuron gave rise to the maximum frequency of induction (86.7%) and a high number of somatic embryos (3) from an embryogenic callus. Identification and characterization of cyclotides in the somatic embryos were carried out using a Fourier transform mass spectrometer coupled with liquid chromatography (LC-FTMS). Among the cyclotides identified in the study, few were found to be exclusively present in the somatic embryo culture. Furthermore, the relative abundance of the cyclotides was higher in somatic embryo extract than in the natural plant extract. The biological activities (cytotoxic, haemolytic and antimicrobial) of the somatic embryos and the parent plant were compared. Unlike the natural plants, the somatic embryo extracts demonstrated specificity i.e. they were found to be potent against cancerous cells but not against non-cancerous cell line or red blood cells. In contrast to the plant extract, the somatic embryos extracts were found to be potent against Escherichia coli and Staphylococcus aureus. These results suggest that somatic embryos of V. odorata (rich in cyclotides) can be used as an alternative to plant biomass for its therapeutic applications and germplasm conservation., (Copyright © 2018 Elsevier Ltd. All rights reserved.) more...

Cancer stem cells (CSCs) as a subpopulation of cancer cells are drug-resistant and radiation-resistant cancer cells to be responsible for tumor progress, maintenance and recurrence of cancer, and metastasis. This study isolated and investigated a new cancer stem cell (CSC) inhibitor derived from lactic acid fermentation products using culture broth with 2% aronia juice. The anti-CSC activity of aronia-cultured broth was significantly higher than that of the control. Activity-guided fractionation and repeated chromatographic preparation led to the isolation of one compound. Using nuclear magnetic resonance and ESI mass spectrometry, we identified the isolated compound as catechol. In this study, we report that aronia-fermented catechol has a novel inhibitory effect on human breast CSCs. Catechol inhibited breast cancer cell proliferation and mammosphere formation in a dose-dependent manner. This compound reduced the CD44 high /CD24 low subpopulation, ALDH-expressing cell population and the self-renewal-related genes nanog, sox2, and oct4. Catechol preferentially reduced mRNA transcripts and protein levels of Stat3 and did not induce c-Myc degradation. These findings support the novel utilization of catechol for breast cancer therapy via the Stat3/IL-6 signaling pathway. Our results suggest that catechol can be used for breast cancer therapy and that Stat3 expression is a marker of CSCs. Catechol inhibited Stat3 signaling by reducing Stat3 expression and secreted IL-6, a CSC survival factor. These findings support the novel utilization of catechol for breast cancer therapy via Stat3/IL-6 signaling., (© 2018 Wiley Periodicals, Inc.) more...

Rosmarinic acid (RA) is a highly valued natural phenolic compound that is very commonly found in plants of the families Lamiaceae and Boraginaceae, including Coleus blumei, Heliotropium foertherianum, Rosmarinus officinalis, Perilla frutescens, and Salvia officinalis. RA is also found in other members of higher plant families and in some fern and horned liverwort species. The biosynthesis of RA is catalyzed by the enzymes phenylalanine ammonia lyase and cytochrome P450-dependent hydroxylase using the amino acids tyrosine and phenylalanine. Chemically, RA can be produced via methods involving the esterification of 3,4-dihydroxyphenyllactic acid and caffeic acid. Some of the derivatives of RA include melitric acid, salvianolic acid, lithospermic acid, and yunnaneic acid. In plants, RA is known to have growth-promoting and defensive roles. Studies have elucidated the varied pharmacological potential of RA and its derived molecules, including anticancer, antiangiogenic, anti-inflammatory, antioxidant, and antimicrobial activities. The demand for RA is therefore, very high in the pharmaceutical industry, but this demand cannot be met by plants alone because RA content in plant organs is very low. Further, many plants that synthesize RA are under threat and near extinction owing to biodiversity loss caused by unscientific harvesting, over-collection, environmental changes, and other inherent features. Moreover, the chemical synthesis of RA is complicated and expensive. Alternative approaches using biotechnological methodologies could overcome these problems. This review provides the state of the art information on the chemistry, sources, and biosynthetic pathways of RA, as well as its anticancer properties against different cancer types. Biotechnological methods are also discussed for producing RA using plant cell, tissue, and organ cultures and hairy-root cultures using flasks and bioreactors. The recent developments and applications of the functional genomics approach and heterologous production of RA in microbes are also highlighted. This chapter will be of benefit to readers aiming to design studies on RA and its applicability as an anticancer agent. more...

Natural production of anti-cancer drug taxol from Taxus has proved to be environmentally unsustainable and economically unfeasible. Currently, bioengineering the biosynthetic pathway of taxol is an attractive alternative production approach. 10-deacetylbaccatin III-10-O-acetyl transferase (DBAT) was previously characterized as an acyltransferase, using 10-deacetylbaccatin III (10-DAB) and acetyl CoA as natural substrates, to form baccatin III in the taxol biosynthesis. Here, we report that other than the natural acetyl CoA (Ac-CoA) substrate, DBAT can also utilize vinyl acetate (VA), which is commercially available at very low cost, acylate quickly and irreversibly, as acetyl donor in the acyl transfer reaction to produce baccatin III. Furthermore, mutants were prepared via a semi-rational design in this work. A double mutant, I43S/D390R was constructed to combine the positive effects of the different single mutations on catalytic activity, and its catalytic efficiency towards 10-DAB and VA was successfully improved by 3.30-fold, compared to that of wild-type DBAT, while 2.99-fold higher than the catalytic efficiency of WT DBAT towards 10-DAB and Ac-CoA. These findings can provide a promising economically and environmentally friendly method for exploring novel acyl donors to engineer natural product pathways. more...

Vinblastine, a potent anticancer drug, is produced by Catharanthus roseus (Madagascar periwinkle) in small quantities, and heterologous reconstitution of vinblastine biosynthesis could provide an additional source of this drug. However, the chemistry underlying vinblastine synthesis makes identification of the biosynthetic genes challenging. Here we identify the two missing enzymes necessary for vinblastine biosynthesis in this plant: an oxidase and a reductase that isomerize stemmadenine acetate into dihydroprecondylocarpine acetate, which is then deacetoxylated and cyclized to either catharanthine or tabersonine via two hydrolases characterized herein. The pathways show how plants create chemical diversity and also enable development of heterologous platforms for generation of stemmadenine-derived bioactive compounds., (Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.) more...

Epidemiological data confirmed a strong correlation between regular consumption of cruciferous vegetables and lower cancer risk. This cancer preventive property is mainly attributed to the glucosinolate products, such as glucoraphanin found in broccoli that is derived from methionine. Here we report the first successful reconstruction of the complete biosynthetic pathway of glucoraphanin from methionine in Escherichia coli via gene selection, pathway design, and protein engineering. We used branched-chain amino transferase 3 to catalyze two transamination steps to ensure the purity of precursor molecules and used cysteine as a sulfur donor to simplify the synthesis pathway. Two chimeric cytochrome P450 enzymes were engineered and expressed in E. coli functionally. The original plant C-S lyase was replaced by the Neurospora crassa hercynylcysteine sulfoxide lyase. Other pathway enzymes were successfully mined from Arabidopsis thaliana, Brassica rapa, and Brassica oleracea. Biosynthesis of glucoraphanin upon coexpression of the optimized enzymes in vivo was confirmed by liquid chromatography-tandem mass spectrometry analysis. No other glucosinolate analogues (except for glucoiberin) were identified that could facilitate the downstream purification processes. Production of glucoraphanin in this study laid the foundation for microbial production of such health-beneficial glucosinolates in a large-scale. more...

Betulin (B) and betulinic acid (BA) are two triterpenes with diverse pharmacological and physiological actions. Elicitation of Betula pendula Roth cell cultures by elicitors is an excellent strategy to increase B and BA levels. Six abiotic and biotic elicitors were studied to improve accumulation of B and BA in the cell culture of B. pendula. The B and BA production in treated cells was verified by HPLC. The results showed the maximum growth index (7) on day 3 in cells treated with 0.5 mg L -1 chlorocholine chloride (CCC). The increased accumulation of BA in the cells treated with 200 mg L -1 of chitosan was found to be 5.9 × (6.5 mg g -1 DW) higher over control cells. Treating the cells with 2 mg L -1 of CCC, after 7 days, led to 149.3× enhancement of B content (19.4 mg g -1 DW) over the controls. Production of this triterpenoid at a much shorter time with a much higher growth rate can be economic and lead to producing large amounts of B and BA for anti-cancer and HIV drugs preparation. more...

Angelica gigas Nakai (AGN) is a crucial oriental medicinal herb that grows especially in Korea and the Far-East countries. It contains chemically active compounds like pyranocoumarins, polyacetylenes and essential oils, which might be useful for treatment of several chronic diseases. It has been used for centuries as a traditional medicine in Southeast Asia, but in Western countries is used as a functional food and a major ingredient of several herbal products. The genus Angelica is also known as 'female ginseng' due to its critical therapeutic role in female afflictions, such as gynecological problems. However, it is well-documented that the AGN pyranocoumarins may play vital beneficial roles against cancer, neurodisorders, inflammation, osteoporosis, amnesia, allergies, depression, fungi, diabetes, ischemia, dermatitis, reactive oxygen species (ROS) and androgen. Though numerous studies revealed the role of AGN pyranocoumarins as therapeutic agents, none of the reviews have published their molecular mechanism of action. To the best of our knowledge, this would be the first review that aims to appraise the biosynthesis of AGN's major active pyranocoumarins, discuss effective extraction and formulation methods, and detail the molecular action mechanism of decursin (D), decursinol angelate (DA) and decursinol (DOH) in chronic diseases, which would further help extension of research in this area., Competing Interests: The authors declare that they have no competing interests. more...

Among 60 fungal endophytes isolated from twigs, bark, and mature leaves of different plant species, two fungal isolates named TXD105 and TER995 were capable of producing paclitaxel in amounts of up to 84.41 and 37.92 μg L -1 , respectively. Based on macroscopic and microscopic characteristics, ITS1-5.8S-ITS2 rDNA sequence, and phylogenetic characteristic analysis, the two respective isolates were identified as Aspergillus fumigatus and Alternaria tenuissima. In the effort to increase paclitaxel magnitude by the two fungal strains, several fermentation conditions including selection of the proper fermentation medium, agitation rate, incubation temperature, fermentation period, medium pH, medium volume, and inoculum nature (size and age of inoculum) were tried. Fermentation process carried out in M1D medium (pH 6.0) and maintained at 120 rpm for 10 days and at 25 °C using 4% (v/v) inoculum of 5-day-old culture stimulated the highest paclitaxel production to attain 307.03 μg L -1 by the A. fumigatus strain. In the case of the A. tenuissima strain, fermentation conditions conducted in flask basal medium (pH 6.0) and maintained at 120 rpm for 14 days and at 25 °C using 8% (v/v) inoculum of 7-day-old culture were found the most favorable to attain the highest paclitaxel production of 124.32 μg L -1 . Using the MTT-based assay, fungal paclitaxel significantly inhibited the proliferation of five different cancer cell lines with 50% inhibitory concentration values varied from 3.04 to 14.8 μg mL -1 . Hence, these findings offer new and alternate sources with excellent biotechnological potential for paclitaxel production by fungal fermentation. more...

Grammothele lineata, an endophyte isolated in our laboratory from jute (Corchorus olitorius acc. 2015) was found to be a substantial paclitaxel producer. Taxol and its related compounds, produced by this endophyte were extracted by growing the fungus in simple nutrient media (potato dextrose broth, PDB). Taxol was identified and characterized by different analytical techniques (TLC, HPLC, FTIR, LC-ESI-MS/MS) following its extraction by ethyl acetate. In PDB media, this fungus was found to produce 382.2 μgL-1 of taxol which is about 7.6 x103 fold higher than the first reported endophytic fungi, Taxomyces andreanae. The extracted taxol exhibited cytotoxic activity in an in vitro culture of HeLa cancer cell line. The fungal extract also exhibited antifungal and antibacterial activities against different pathogenic strains. This is the first report of a jute endophytic fungus harboring the capacity to produce taxol and also the first reported taxol producing species that belongs to the Basidiomycota phylum, so far unknown to be a taxol producer. These findings suggest that the fungal endophyte, Grammothele lineata can be an excellent source of taxol and can also serve as a potential species for chemical and genetic engineering to enhance further the production of taxol. more...

The Camptotheca acuminata cell suspension cultures were established to produce the well-known antitumor monoterpene indole alkaloid camptothecin (CAM). Most CAM was present in the broth of the C. acuminata cell suspension cultures. The CAM production was evidenced to be attenuated when the C. acuminata cell suspension cultures were continuously subcultured and grown under identical axenic conditions. A practical cryopreservation and recovery procedure was established to maintain the C. acuminata cell suspension cultures. Biotic and abiotic elicitors were administrated to the C. acuminata cell suspension cultures to restore and enhance CAM production. Of them, sorbitol, a well-known hyperosmotic stressor, was proven to be the most effective elicitor that stimulates a ∼500-fold increase of CAM production. The committed biosynthetic precursors of CAM, tryptamine and secologanin, were feed to the C. acuminata cell suspension cultures and the CAM production is not remarkably increased. However, N 1 -acetylkynuramine (NAK), an important metabolite of kynuramine pathway, was isolated and identified from the cell suspension cultures feeding with tryptamine. The present work provides an efficient method to produce CAM and NAK using the C. acuminata cell suspension cultures. The biotransformation of tryptamine to NAK sheds lights on the biosynthetic formation of the pyrroloquinoline moiety of CAM. more...

In the present study, resveratrol and various oligomeric derivatives were obtained from a 14 L bioreactor culture of elicited grapevine cell suspensions (Vitis labrusca L.). The crude ethyl acetate stilbene extract obtained from the culture medium was fractionated by centrifugal partition chromatography (CPC) using a gradient elution method and the major stilbenes contained in the fractions were subsequently identified by using a 13 C-NMR-based dereplication procedure and further 2D NMR analyses including HSQC, HMBC, and COSY. Beside δ-viniferin (2), leachianol F (4) and G (4'), four stilbenes (resveratrol (1), ε-viniferin (5), pallidol (3) and a newly characterized dimer (6)) were recovered as pure compounds in sufficient amounts to allow assessment of their biological activity on the cell growth of three different cell lines, including two human skin malignant melanoma cancer cell lines (HT-144 and SKMEL-28) and a healthy human dermal fibroblast HDF line. Among the dimers obtained in this study, the newly characterized resveratrol dimer (6) has never been described in nature and its biological potential was evaluated here for the first time. ε-viniferin as well as dimer (6) showed IC 50 values on the three tested cell lines lower than the ones exerted by resveratrol and pallidol. However, activities of the first two compounds were significantly decreased in the presence of fetal bovine serum although that of resveratrol and pallidol was not. The differential tumor activity exerted by resveratrol on healthy and cancer lines was also discussed. more...

Structure-activity relationship studies show that the phenylisoserinyl moiety of paclitaxel (Taxol) is largely necessary for the effective anticancer activity. Several paclitaxel analogues with a variant isoserinyl side chain have improved pharmaceutical properties versus those of the parent drug. To produce the isoserinyl CoAs as intermediates needed for enzyme catalysis on a semibiosynthetic pathway to paclitaxel analogues, we repurposed the adenylation and thiolation domains (Phe-AT) of a nonribosomal peptide synthetase (TycA) so that they would function as a CoA ligase. Twenty-eight isoserine analogue racemates were synthesized by an established procedure based on the Staudinger [2+2] cycloaddition reaction. Phe-AT converted 16 substituted phenylisoserines, one β-(heteroaryl)isoserine, and one β-(cyclohexyl)isoserine to their corresponding isoserinyl CoAs. We imagine that these CoA thioesters can likely serve as linchpin biosynthetic acyl donors transferred by a 13-O-acyltransferase to a paclitaxel precursor baccatin III to make drug analogues with better efficacy. It was also interesting to find that an active site mutant [Phe-AT (W227S)] turned over 2-pyridylisoserine and the sterically demanding p-methoxyphenylisoserine substrates to their CoA thioesters, while Phe-AT did not. This mutant is promising for further development to make 3-fluoro-2-pyridylisoserinyl CoA, a biosynthetic precursor of the oral pharmaceutical tesetaxel used for gastric cancers. more...

N-hydroxysuccinimide ester of monomethoxy polyethylene glycol hemisuccinate was synthesized. It acylated amino groups in a molecule of recombinant L-asparaginase from Erwinia carotovora. A method of L-asparaginase modification by the obtained activated polyethylene glycol derivative was developed. The best results were produced by modification of the enzyme with a 25-fold excess of reagent relative to the enzyme tetramer. The modified L-asparaginase was isolated from the reaction mixture by gel filtration on Sepharose CL-6B. The purified bioconjugate did not contain PEG unbound to the protein, demonstrated high catalytic activity, and exhibited antiproliferative action on cell cultures. more...

Biotransformation of the soy isoflavone genistein by sequential 3'-hydroxylation using recombinant Escherichia coli expressing tyrosinase from Bacillus megaterium and then methylation using another recombinant E. coli expressing O -methyltransferase from Streptomyces peucetius was conducted. The results showed that two metabolites were produced from the biotransformation, identified as 5,7,4'-trihydroxy-3'-methoxyisoflavone and 5,7,3'-trihydroxy-4'-methoxyisoflavone, respectively, based on their mass and nuclear magnetic resonance spectral data. 5,7,4'-Trihydroxy-3'-methoxyisoflavone showed potent antiproliferative activity toward mouse B16 melanoma cells with an IC 50 value of 68.8 μM. In contrast, the compound did not show any cytotoxicity toward mouse normal fibroblast cells, even at 350 μM concentration. The results of the present study offer insight on the production of both 5,7,4'-trihydroxy-3'-methoxyisoflavone and 5,7,3'-trihydroxy-4'-methoxyisoflavone by two recombinant E. coli strains and the potential anti-melanoma applications of 5,7,4'-trihydroxy-3'-methoxyisoflavone. more...

The Euphorbiaceae produce a wide variety of bioactive diterpenoids. These include the lathyranes, which have received much interest due to their ability to inhibit the ABC transporters responsible for the loss of efficacy of many chemotherapy drugs. The lathyranes are also intermediates in the biosynthesis of range of other bioactive diterpenoids with potential applications in the treatment of pain, HIV and cancer. We report here a gene cluster from Jatropha curcas that contains the genes required to convert geranylgeranyl pyrophosphate into a number of diterpenoids, including the lathyranes jolkinol C and epi-jolkinol C. The conversion of casbene to the lathyranes involves an intramolecular carbon-carbon ring closure. This requires the activity of two cytochrome P450s that we propose form a 6-hydroxy-5,9-diketocasbene intermediate, which then undergoes an aldol reaction. The discovery of the P450 genes required to convert casbene to lathyranes will allow the scalable heterologous production of these potential anticancer drugs, which can often only be sourced in limited quantities from their native plant., (© 2016 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.) more...

The seed oil of Euphorbia lathyris L. contains a series of macrocyclic diterpenoids known as Euphorbia factors. They are the current industrial source of ingenol mebutate, which is approved for the treatment of actinic keratosis, a precancerous skin condition. Here, we report an alcohol dehydrogenase-mediated cyclization step in the biosynthetic pathway of Euphorbia factors, illustrating the origin of the intramolecular carbon-carbon bonds present in lathyrane and ingenane diterpenoids. This unconventional cyclization describes the ring closure of the macrocyclic diterpene casbene. Through transcriptomic analysis of E. lathyris L. mature seeds and in planta functional characterization, we identified three enzymes involved in the cyclization route from casbene to jolkinol C, a lathyrane diterpene. These enzymes include two cytochromes P450 from the CYP71 clan and an alcohol dehydrogenase (ADH). CYP71D445 and CYP726A27 catalyze regio-specific 9-oxidation and 5-oxidation of casbene, respectively. When coupled with these P450-catalyzed monooxygenations, E. lathyris ADH1 catalyzes dehydrogenation of the hydroxyl groups, leading to the subsequent rearrangement and cyclization. The discovery of this nonconventional cyclization may provide the key link to complete elucidation of the biosynthetic pathways of ingenol mebutate and other bioactive macrocyclic diterpenoids., Competing Interests: D.L., M.T.N., R.C., and Björn Hamberger have filed a patent application (PA 2015 70069) covering “Methods and materials for producing oxidized macrocyclic diterpenes” related to the manufacture of jolkinol C and precursors thereof. more...

Plant lectins are proteins that possess at least one non-catalytic domain, which could reversibly bind to specific monosaccharides or oligosaccharides. The important roles played by plant lectins in immune regulation, signaling pathways, and plant defense could be attributed to their specific binding activities with carbohydrates. In this study, a Pinellia integrifolia lectin gene, designated pia, was cloned using rapid amplification of cDNA ends. The open reading frame (ORF) of pia was constructed into the pET-28a vector, and a 33-kDa recombinant protein was induced in Escherichia coli BL21. The hemagglutination and anticancer properties of the purified recombinant protein were assayed in vitro. The results indicated that the full-length cDNA of pia was 1210 bp long, containing an 807-bp ORF encoding a 268-amino acid peptide. The putative P. integrifolia lectin protein (PIA) contained three mannose-binding sites. The agglutinating activity exhibited by PIA was inhibited by D-mannose. PIA was also shown to exert an anti-proliferative activity against nasopharyngeal carcinoma, human cervical carcinoma, and human breast cancer cell lines in vitro. These results could be applied to determine the function of PIA in the future., Competing Interests: The authors declare no conflict of interest. more...

This study examined the effect of different elicitors (seven, different concentrations) and environmental factors (water activity (aw), pH) on taxol production by strains of two endophytic fungi, Paraconiothyrium variabile and Epicoccum nigrum, isolated from temperate yew trees. A defined liquid broth medium was modified with elicitors, solute aw depressors at different pH values. For P. variabile, the best elicitor was salicylic acid at 50mg/l which gave a taxol yield of 14.7±4.8μg/l. The study of synergistic effects between elicitor, aw and pH on taxol production showed that the highest yield of taxol (68.9±11.9μg/l) was produced under modified ionic stress of 0.98aw (KCl) at pH 5 when supplemented with 20mg/l of salicylic acid. For E. nigrum, serine was the best elicitor which increased yield significantly (29.6 fold) when KCL was used as the aw depressor (0.98aw) at pH 5.0 with 30mg/l of serine. The maximum taxol yield produced by E. nigrum was 57.1±11.8μg/l. Surface response models were used to build contour maps to determine the conditions for maximum and marginal conditions for taxol yield in relation to the best elicitor and aw, and the best pH for the first time. This will be beneficial for identifying key parameters for improvement of taxol yields by endophytic fungi., (Copyright © 2016 Elsevier Inc. All rights reserved.) more...

Andrographolide and neoandrographolide are major bioactive molecules of Andrographis paniculata, a well-known medicinal plant. These molecules exhibited varying degrees of anti-inflammatory and anticancer activities in-vitro and in-vivo. Role of begomovirus protein C2/TrAP in biosynthesis of andrographolide was identified through molecular modeling, docking and predicted results were substantiated by in vitro studies. Homology molecular modeling and molecular docking were performed to study the binding conformations and different bonding behaviors, in order to reveal the possible mechanism of action behind higher accumulation of andrographolide. It was concluded that C2/TrAP inhibit the activation of SNF1-Related Protein Kinase-1 (SnRK1) in terpenoid pathway and removes the negative regulation of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) by SnRK1, leading to higher accumulation of andrographolide and neoandrographolide in begomovirus infected plants. The binding site residues of SnRK1 docked with C2/TrAP were found to be associated with ATP binding site, substrate binding site and activation loop. Predicted results were also validated by HPTLC. This study provides important insights into understanding the role of viral protein in altering the regulation of biosynthesis of andrographolide and could be used in future research to develop biomimetic methods for increasing the production of such phytometabolites having anti-cancerous and anti-inflammatory properties., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.) more...

Three new diterpenoids, mannolides A-C (1-3), and two new Cephalotaxus troponoids, 4 and 5, were isolated from Cephalotaxus mannii and structurally characterized by spectroscopic data and X-ray crystallography. The discovery of compounds 1-3 featuring a new intact carbon skeleton, proposed as cephalotane, sheds new light on the biogenesis of Cephalotaxus troponoids, a rare class of antitumor C19 norditerpenoids. Antitumor tests showed that the tropone motif is essential for the activity. more...