Production of Active Compounds in Medicinal Plants: From Plant Tissue Culture to Biosynthesis
TL;DR: In order to improve the contents of active compounds in medicinal plants, following aspects could be carried out gene interference or gene silencing, gene overexpression, combination with chemical synthesis, application of elicitors, and site-directed mutagenesis of the key enzymes.
About: This article is published in Chinese Herbal Medicines.The article was published on 2017-04-01. It has received 36 citations till now. The article focuses on the topics: Plant tissue culture.
Citations
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TL;DR: Given the growing clinical importance of anti-M deliriant hallucinogens, the use and abuse, clinical importance, and the growing value in preclinical (experimental) animal models relevant to modeling CNS functions and dysfunctions are discussed.
Abstract: Anticholinergic drugs based on tropane alkaloids, including atropine, scopolamine, and hyoscyamine, have been used for various medicinal and toxic purposes for millennia. These drugs are competitive antagonists of acetylcholine muscarinic (M-) receptors that potently modulate the central nervous system (CNS). Currently used clinically to treat vomiting, nausea, and bradycardia, as well as alongside other anesthetics to avoid vagal inhibition, these drugs also evoke potent psychotropic effects, including characteristic delirium-like states with hallucinations, altered mood, and cognitive deficits. Given the growing clinical importance of anti-M deliriant hallucinogens, here we discuss their use and abuse, clinical importance, and the growing value in preclinical (experimental) animal models relevant to modeling CNS functions and dysfunctions.
41 citations
TL;DR: A systematic analysis on the effects of growth years and post-harvest processing on the contents of medicinal active ingredients of S. baicalensis showed that the best harvesting period should be determined as 2–3 years based on comprehensive evaluation of active ingredient content, annual yield increment, and land use efficiency.
32 citations
TL;DR: In this paper, MeJA was used as an elicitor to treat Oplopanax elatus Nakai root culturing, which significantly increased the contents of quercetin, aloe-emodin, rhein and emodin.
Abstract: Adventitious root (AR) culturing is an effective approach for obtaining bioactive compounds from the endangered plant species of Oplopanax elatus Nakai. In the present study, flavonoids and anthraquinones, both important bioactive compounds were identified via HPLC-ESI–MS/MS, and flavonoid monomers of quercetin and kaempferide and anthraquinone monomers of aloe-emodin, rhein, and emodin were detected in O. elatus AR cultures. To enhance synthesis of both compounds, methyl jasmonate (MeJA) was used as the elicitor to treat ARs. At 200 µM, MeJA significantly increased the contents of quercetin, aloe-emodin, rhein, and emodin, whereas 225 µM was the optimal concentration for kaempferide accumulation. The antioxidant property in ARs was evaluated by determining activities of 1,1-diphenyl-2-picrylhydrazyl (DPPH) and alkyl radical scavenging using the an electron spin resonance spectrometer. The IC50 of DPPH (6.2 µg/mL) and alkyl (20.2 µg/mL) radical scavenging activities with MeJA treatment were lower than that of the control, thereby indicating that ARs treated with MeJA possess stronger antioxidant activity. Hence, AR cultures can be deployed as raw materials or additives in the production of antioxidant-related O. elatus products using the MeJA as elicitor.
26 citations
TL;DR: It is shown that MeJA and SA could effectively be used as potent elicitors to enhance the production of camphor and phenolic compounds, along with cell growth, in cell suspension cultures of the endemic Turkish yarrow species Achillea gypsicola.
Abstract: Elicitors have been widely used as biotic and abiotic stimulants in triggering the production of secondary metabolites in plant cell culture. The present study aimed to enhance the production of camphor and phenolic compounds and cell growth using methyl jasmonate (MeJA) and salicylic acid (SA) in the cell suspension culture of Turkish endemic species Achillea gypsicola. Various concentrations (0, 10, 50, and 100 μM) of MeJA and SA were applied to 8-day-old cell cultures. The camphor and phenolic compound contents were determined using a headspace gas chromatographic-mass spectrometer device and spectrophotometer. Increasing doses of MeJA and SA significantly enhanced the accumulation of the camphor and phenolic compounds in general. The highest amount of camphor accumulation occurred in cells treated with 100 μM MeJA (0.3449 μg/g) and 50 μM SA (0.3816 μg/g). Increasing concentrations of MeJA resulted in a significant decrease in the total anthocyanin when compared to the initial culture. The present study showed that MeJA and SA could effectively be used as potent elicitors to enhance the production of camphor and phenolic compounds, along with cell growth, in cell suspension cultures of the endemic Turkish yarrow species Achillea gypsicola.
24 citations
Cites background from "Production of Active Compounds in M..."
...…duration of elicitor exposure, age of culture, cell line, growth regulation, and nutrient composition, should be considered in the correct evaluation of the elicitation process regarding cell growth and product yield for plant cell suspension cultures (Smetanska, 2008; Wang et al., 2017)....
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...Plant cell culture, in this respect, is of considerable importance and is widely used as an attractive alternative for producing valuable secondary metabolites (Georgiev et al., 2009; Wang et al., 2017)....
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...A number of factors, including the elicitor concentration and selectivity, duration of elicitor exposure, age of culture, cell line, growth regulation, and nutrient composition, should be considered in the correct evaluation of the elicitation process regarding cell growth and product yield for plant cell suspension cultures (Smetanska, 2008; Wang et al., 2017)....
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TL;DR: 5 new cytochromes P450 and 5 berberine enzymes are proposed as candidates to be involved into tanshinone biosynthesis, a novel finding that opens new avenues to improve tansinone production through biotechnological approaches.
Abstract: Salvia miltiorrhiza is a medicinal plant highly appreciated by its content of tanshinones and salvianolic acids. Tanshinones are of particular relevance for their anti-oxidant, anti-tumoral and anti-inflammatory properties. Abiotic and biotic agents as silver nitrate and yeast extract have shown efficiently to stimulate tanshinone accumulation, but the underlying molecular mechanism remains essentially unknown. By using hairy roots as experimental material and the elicitors mentioned, were obtained up to 22 mg of tanshinones per gram of dry weight. Differential label-free quantitative proteomic analysis was applied to study the proteins involved in tanshinone biosynthesis. A total of 2650 proteins were identified in roots extracts, of which 893 showed statistically (p < 0.05) significant change in relative abundance compared to control roots, 251 proteins were upregulated and 642 downregulated. Among the upregulated proteins the predominant functional categories were metabolism (47%), stress defense (18%) and redox homeostasis (10%). Within the metabolism category, isoprenoid metabolism enzymes, cytochromes P450 and FAD-binding berberine proteins showed abundance profile linked to tanshinone concentration. The results presented here allowed to propose 5 new cytochromes P450 and 5 berberine enzymes as candidates to be involved into tanshinone biosynthesis, a novel finding that opens new avenues to improve tanshinone production through biotechnological approaches.
20 citations
References
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TL;DR: The strains and processes described here form the basis of a viable industrial process for the production of semi-synthetic artemisinin to stabilize the supply of art Artemisinin for derivatization into active pharmaceutical ingredients (for example, artesunate) for incorporation into ACTs.
Abstract: Saccharomyces cerevisiae is engineered to produce high concentrations of artemisinic acid, a precursor of the artemisinin used in combination therapies for malaria treatment; an efficient and practical chemical process to convert artemisinic acid to artemisinin is also developed. Artemisinin-based combination therapies are the treatment of choice for uncomplicated Plasmodium falciparum malaria, but the supply of plant-derived artemisinin can sometimes be unreliable, causing shortages and high prices. This manuscript describes a viable industrial process for the production of semisynthetic artemisinin, with the potential to help stabilize artemisinin supply. The process uses Saccharomyces cerevisiae yeast engineered to produce high yields of artemisinic acid, a precursor of artemisinin. The authors have also developed an efficient and scalable chemical process to convert artemisinic acid to artemisinin. In 2010 there were more than 200 million cases of malaria, and at least 655,000 deaths1. The World Health Organization has recommended artemisinin-based combination therapies (ACTs) for the treatment of uncomplicated malaria caused by the parasite Plasmodium falciparum. Artemisinin is a sesquiterpene endoperoxide with potent antimalarial properties, produced by the plant Artemisia annua. However, the supply of plant-derived artemisinin is unstable, resulting in shortages and price fluctuations, complicating production planning by ACT manufacturers2. A stable source of affordable artemisinin is required. Here we use synthetic biology to develop strains of Saccharomyces cerevisiae (baker’s yeast) for high-yielding biological production of artemisinic acid, a precursor of artemisinin. Previous attempts to produce commercially relevant concentrations of artemisinic acid were unsuccessful, allowing production of only 1.6 grams per litre of artemisinic acid3. Here we demonstrate the complete biosynthetic pathway, including the discovery of a plant dehydrogenase and a second cytochrome that provide an efficient biosynthetic route to artemisinic acid, with fermentation titres of 25 grams per litre of artemisinic acid. Furthermore, we have developed a practical, efficient and scalable chemical process for the conversion of artemisinic acid to artemisinin using a chemical source of singlet oxygen, thus avoiding the need for specialized photochemical equipment. The strains and processes described here form the basis of a viable industrial process for the production of semi-synthetic artemisinin to stabilize the supply of artemisinin for derivatization into active pharmaceutical ingredients (for example, artesunate) for incorporation into ACTs. Because all intellectual property rights have been provided free of charge, this technology has the potential to increase provision of first-line antimalarial treatments to the developing world at a reduced average annual price.
1,588 citations
"Production of Active Compounds in M..." refers background in this paper
...Paddon et al (2013) used synthetic biology to develop strains of S. cerevisiae for high-yielding biological production of artemisinic acid, a precursor of artemisinin....
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Journal Article•
TL;DR: In this paper, a multivariate-modular approach to metabolic-pathway engineering that succeeded in increasing titers of taxadiene, the first committed Taxol intermediate, was reported.
Abstract: Taxol (paclitaxel) is a potent anticancer drug first isolated from the Taxus brevifolia Pacific yew tree. Currently, cost-efficient production of Taxol and its analogs remains limited. Here, we report a multivariate-modular approach to metabolic-pathway engineering that succeeded in increasing titers of taxadiene—the first committed Taxol intermediate—approximately 1 gram per liter (~15,000-fold) in an engineered Escherichia coli strain. Our approach partitioned the taxadiene metabolic pathway into two modules: a native upstream methylerythritol-phosphate (MEP) pathway forming isopentenyl pyrophosphate and a heterologous downstream terpenoid–forming pathway. Systematic multivariate search identified conditions that optimally balance the two pathway modules so as to maximize the taxadiene production with minimal accumulation of indole, which is an inhibitory compound found here. We also engineered the next step in Taxol biosynthesis, a P450-mediated 5α-oxidation of taxadiene to taxadien-5α-ol. More broadly, the modular pathway engineering approach helped to unlock the potential of the MEP pathway for the engineered production of terpenoid natural products.
1,362 citations
TL;DR: Stable co-culture in the same bioreactor was achieved by designing a mutualistic relationship between the two species in which a metabolic intermediate produced by E. coli was used and functionalized by yeast.
Abstract: Metabolic engineering of microorganisms such as Escherichia coli and Saccharomyces cerevisiae to produce high-value natural metabolites is often done through functional reconstitution of long metabolic pathways. Problems arise when parts of pathways require specialized environments or compartments for optimal function. Here we solve this problem through co-culture of engineered organisms, each of which contains the part of the pathway that it is best suited to hosting. In one example, we divided the synthetic pathway for the acetylated diol paclitaxel precursor into two modules, expressed in either S. cerevisiae or E. coli, neither of which can produce the paclitaxel precursor on their own. Stable co-culture in the same bioreactor was achieved by designing a mutualistic relationship between the two species in which a metabolic intermediate produced by E. coli was used and functionalized by yeast. This synthetic consortium produced 33 mg/L oxygenated taxanes, including a monoacetylated dioxygenated taxane. The same method was also used to produce tanshinone precursors and functionalized sesquiterpenes.
477 citations
"Production of Active Compounds in M..." refers background in this paper
...Recently, Zhou et al (2015) reported the co-culture of engineered bacteria strains, each of which contains the part of the pathway that it is best suited to host....
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TL;DR: A multivariate-modular approach to metabolic-pathway engineering that succeeded in increasing titers of taxadiene—the first committed Taxol intermediate—approximately 1 gram per liter in an engineered Escherichia coli strain helped to unlock the potential of the MEP pathway for the engineered production of terpenoid natural products.
Abstract: Taxol (paclitaxel) is a potent anticancer drug first isolated from the Taxus brevifolia Pacific yew tree. Currently, cost-efficient production of Taxol and its analogs remains limited. Here, we report a multivariate-modular approach to metabolic-pathway engineering that succeeded in increasing titers of taxadiene—the first committed Taxol intermediate—approximately 1 gram per liter (~15,000-fold) in an engineered Escherichia coli strain. Our approach partitioned the taxadiene metabolic pathway into two modules: a native upstream methylerythritol-phosphate (MEP) pathway forming isopentenyl pyrophosphate and a heterologous downstream terpenoid–forming pathway. Systematic multivariate search identified conditions that optimally balance the two pathway modules so as to maximize the taxadiene production with minimal accumulation of indole, which is an inhibitory compound found here. We also engineered the next step in Taxol biosynthesis, a P450-mediated 5α-oxidation of taxadiene to taxadien-5α-ol. More broadly, the modular pathway engineering approach helped to unlock the potential of the MEP pathway for the engineered production of terpenoid natural products.
434 citations
TL;DR: The results and the approaches that were described here provide a solid foundation to further elucidate the biosynthesis of tanshinones and related diterpenoids and should facilitate the construction of microbial cell factories for the production of phytoterpenoids.
Abstract: Cytochrome P450 enzymes (CYPs) play major roles in generating highly functionalized terpenoids, but identifying the exact biotransformation step(s) catalyzed by plant CYP in terpenoid biosynthesis is extremely challenging. Tanshinones are abietane-type norditerpenoid naphthoquinones that are the main lipophilic bioactive components of the Chinese medicinal herb danshen (Salvia miltiorrhiza). Whereas the diterpene synthases responsible for the conversion of (E,E,E)-geranylgeranyl diphosphate into the abietane miltiradiene, a potential precursor to tanshinones, have been recently described, molecular characterization of further transformation of miltiradiene remains unavailable. Here we report stable-isotope labeling results that demonstrate the intermediacy of miltiradiene in tanshinone biosynthesis. We further use a next-generation sequencing approach to identify six candidate CYP genes being coregulated with the diterpene synthase genes in both the rhizome and danshen hairy roots, and demonstrate that one of these, CYP76AH1, catalyzes a unique four-electron oxidation cascade on miltiradiene to produce ferruginol both in vitro and in vivo. We then build upon the previous establishment of miltiradiene production in Saccharomyces cerevisiae, with incorporation of CYP76AH1 and phyto-CYP reductase genes leading to heterologous production of ferruginol at 10.5 mg/L. As ferruginol has been found in many plants including danshen, the results and the approaches that were described here provide a solid foundation to further elucidate the biosynthesis of tanshinones and related diterpenoids. Moreover, these results should facilitate the construction of microbial cell factories for the production of phytoterpenoids.
283 citations
"Production of Active Compounds in M..." refers methods in this paper
...Guo et al (2013) used a next-generation sequencing approach to identify six candidate CYP genes being coregulated with the diterpene synthase genes in both the rhizome and danshen hairy roots, and demonstrated that one of these, CYP76AH1, catalyzes a unique four-electron oxidation cascade on…...
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