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      Production of Plant Secondary Metabolites: Examples, Tips and Suggestions for Biotechnologists

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          Abstract

          Plants are sessile organisms and, in order to defend themselves against exogenous (a)biotic constraints, they synthesize an array of secondary metabolites which have important physiological and ecological effects. Plant secondary metabolites can be classified into four major classes: terpenoids, phenolic compounds, alkaloids and sulphur-containing compounds. These phytochemicals can be antimicrobial, act as attractants/repellents, or as deterrents against herbivores. The synthesis of such a rich variety of phytochemicals is also observed in undifferentiated plant cells under laboratory conditions and can be further induced with elicitors or by feeding precursors. In this review, we discuss the recent literature on the production of representatives of three plant secondary metabolite classes: artemisinin (a sesquiterpene), lignans (phenolic compounds) and caffeine (an alkaloid). Their respective production in well-known plants, i.e., Artemisia, Coffea arabica L., as well as neglected species, like the fibre-producing plant Urtica dioica L., will be surveyed. The production of artemisinin and caffeine in heterologous hosts will also be discussed. Additionally, metabolic engineering strategies to increase the bioactivity and stability of plant secondary metabolites will be surveyed, by focusing on glycosyltransferases (GTs). We end our review by proposing strategies to enhance the production of plant secondary metabolites in cell cultures by inducing cell wall modifications with chemicals/drugs, or with altered concentrations of the micronutrient boron and the quasi-essential element silicon.

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          Production of the antimalarial drug precursor artemisinic acid in engineered yeast.

          Malaria is a global health problem that threatens 300-500 million people and kills more than one million people annually. Disease control is hampered by the occurrence of multi-drug-resistant strains of the malaria parasite Plasmodium falciparum. Synthetic antimalarial drugs and malarial vaccines are currently being developed, but their efficacy against malaria awaits rigorous clinical testing. Artemisinin, a sesquiterpene lactone endoperoxide extracted from Artemisia annua L (family Asteraceae; commonly known as sweet wormwood), is highly effective against multi-drug-resistant Plasmodium spp., but is in short supply and unaffordable to most malaria sufferers. Although total synthesis of artemisinin is difficult and costly, the semi-synthesis of artemisinin or any derivative from microbially sourced artemisinic acid, its immediate precursor, could be a cost-effective, environmentally friendly, high-quality and reliable source of artemisinin. Here we report the engineering of Saccharomyces cerevisiae to produce high titres (up to 100 mg l(-1)) of artemisinic acid using an engineered mevalonate pathway, amorphadiene synthase, and a novel cytochrome P450 monooxygenase (CYP71AV1) from A. annua that performs a three-step oxidation of amorpha-4,11-diene to artemisinic acid. The synthesized artemisinic acid is transported out and retained on the outside of the engineered yeast, meaning that a simple and inexpensive purification process can be used to obtain the desired product. Although the engineered yeast is already capable of producing artemisinic acid at a significantly higher specific productivity than A. annua, yield optimization and industrial scale-up will be required to raise artemisinic acid production to a level high enough to reduce artemisinin combination therapies to significantly below their current prices.
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            Elicitor signal transduction leading to production of plant secondary metabolites.

            Plant secondary metabolites are unique sources for pharmaceuticals, food additives, flavors, and other industrial materials. Accumulation of such metabolites often occurs in plants subjected to stresses including various elicitors or signal molecules. Understanding signal transduction paths underlying elicitor-induced production of secondary metabolites is important for optimizing their commercial production. This paper summarizes progress made on several aspects of elicitor signal transduction leading to production of plant secondary metabolites, including: elicitor signal perception by various receptors of plants; avirulence determinants and corresponding plant R proteins; heterotrimeric and small GTP binding proteins; ion fluxes, especially Ca2+ influx, and Ca2+ signaling; medium alkalinization and cytoplasmic acidification; oxidative burst and reactive oxygen species; inositol trisphosphates and cyclic nucleotides (cAMP and cGMP); salicylic acid and nitric oxide; jasmonate, ethylene, and abscisic acid signaling; oxylipin signals such as allene oxide synthase-dependent jasmonate and hydroperoxide lyase-dependent C12 and C6 volatiles; as well as other lipid messengers such as lysophosphatidylcholine, phosphatidic acid, and diacylglycerol. All these signal components are employed directly or indirectly by elicitors for induction of plant secondary metabolite accumulation. Cross-talk between different signaling pathways is very common in plant defense response, thus the cross-talk amongst these signaling pathways, such as elicitor and jasmonate, jasmonate and ethylene, and each of these with reactive oxygen species, is discussed separately. This review also highlights the integration of multiple signaling pathways into or by transcription factors, as well as the linkage of the above signal components in elicitor signaling network through protein phosphorylation and dephosphorylation. Some perspectives on elicitor signal transduction and plant secondary metabolism at the transcriptome and metabolome levels are also presented.
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              Cannabis sativa: The Plant of the Thousand and One Molecules

              Cannabis sativa L. is an important herbaceous species originating from Central Asia, which has been used in folk medicine and as a source of textile fiber since the dawn of times. This fast-growing plant has recently seen a resurgence of interest because of its multi-purpose applications: it is indeed a treasure trove of phytochemicals and a rich source of both cellulosic and woody fibers. Equally highly interested in this plant are the pharmaceutical and construction sectors, since its metabolites show potent bioactivities on human health and its outer and inner stem tissues can be used to make bioplastics and concrete-like material, respectively. In this review, the rich spectrum of hemp phytochemicals is discussed by putting a special emphasis on molecules of industrial interest, including cannabinoids, terpenes and phenolic compounds, and their biosynthetic routes. Cannabinoids represent the most studied group of compounds, mainly due to their wide range of pharmaceutical effects in humans, including psychotropic activities. The therapeutic and commercial interests of some terpenes and phenolic compounds, and in particular stilbenoids and lignans, are also highlighted in view of the most recent literature data. Biotechnological avenues to enhance the production and bioactivity of hemp secondary metabolites are proposed by discussing the power of plant genetic engineering and tissue culture. In particular two systems are reviewed, i.e., cell suspension and hairy root cultures. Additionally, an entire section is devoted to hemp trichomes, in the light of their importance as phytochemical factories. Ultimately, prospects on the benefits linked to the use of the -omics technologies, such as metabolomics and transcriptomics to speed up the identification and the large-scale production of lead agents from bioengineered Cannabis cell culture, are presented.
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                Author and article information

                Journal
                Genes (Basel)
                Genes (Basel)
                genes
                Genes
                MDPI
                2073-4425
                20 June 2018
                June 2018
                : 9
                : 6
                : 309
                Affiliations
                [1 ]Research and Innovation Department, Luxembourg Institute of Science and Technology, 5 avenue des Hauts-Fourneaux, L-4362 Esch/Alzette, Luxembourg; jean-francois.hausman@ 123456list.lu
                [2 ]Department of Life Sciences, University of Siena, via P.A. Mattioli 4, 53100 Siena, Italy; berni10@ 123456student.unisi.it (R.B.); giampiero.cai@ 123456unisi.it (G.C.)
                [3 ]Trees and timber institute-National research council of Italy (CNR-IVALSA), via Aurelia 49, 58022 Follonica (GR), Italy; cantini@ 123456ivalsa.cnr.it
                [4 ]Unidad de Bioquímica y Biología Molecular de Plantas, Centro de Investigación Científica de Yucatán, Calle 43 # 130 X 32 y 34, Col. Chuburná de Hidalgo, Mérida, Yucatán 97205, Mexico; arms@ 123456cicy.mx (J.A.M.-S.); ths@ 123456cicy.mx (S.M.T.H.-S.)
                [5 ]Arterra Biosciences srl/Vitalab srl, via B. Brin 69, 80142 Naples, Italy; fapone@ 123456arterrabio.it
                [6 ]Department of Botany & Microbiology, College of Science, King Saud University, P.O. Box 2455, Riyadh 11451, Saudi Arabia; eabdelsalam@ 123456ksu.edu.sa (E.M.A.-S.); ahmadaqq@ 123456gmail.com (A.A.Q.); aalatar@ 123456ksu.edu.sa (A.A.A.)
                [7 ]Life Sciences Department, King Fahd University of Petroleum and Minerals (KFUPM), 31261 Dhahran, Saudi Arabia; ksiddiqui@ 123456kfupm.edu.sa
                Author notes
                [* ]Correspondence: gea.guerriero@ 123456list.lu (G.G.); mofaisal@ 123456ksu.edu.sa or faisalm15@ 123456yahoo.com (M.F.); Tel.: +352-275-888-5023 (G.G.); +966-11-4675-877 (M.F.)
                Author information
                https://orcid.org/0000-0001-6586-6997
                https://orcid.org/0000-0001-6103-7817
                Article
                genes-09-00309
                10.3390/genes9060309
                6027220
                29925808
                51bd1b70-c59e-4191-9b48-3c0322d7c10b
                © 2018 by the authors.

                Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ( http://creativecommons.org/licenses/by/4.0/).

                History
                : 30 May 2018
                : 20 June 2018
                Categories
                Review

                secondary metabolites,artemisia,artemisinin,urtica dioica l.,lignans,coffea arabica l.,caffeine,bioactivity,heterologous hosts,uridine diphosphate glycosyltransferases,cell wall

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