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      Anchoring a Plant Cytochrome P450 via PsaM to the Thylakoids in Synechococcus sp. PCC 7002: Evidence for Light-Driven Biosynthesis

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          Abstract

          Plants produce an immense variety of specialized metabolites, many of which are of high value as their bioactive properties make them useful as for instance pharmaceuticals. The compounds are often produced at low levels in the plant, and due to their complex structures, chemical synthesis may not be feasible. Here, we take advantage of the reducing equivalents generated in photosynthesis in developing an approach for producing plant bioactive natural compounds in a photosynthetic microorganism by functionally coupling a biosynthetic enzyme to photosystem I. This enables driving of the enzymatic reactions with electrons extracted from the photosynthetic electron transport chain. As a proof of concept, we have genetically fused the soluble catalytic domain of the cytochrome P450 CYP79A1, originating from the endoplasmic reticulum membranes of Sorghum bicolor, to a photosystem I subunit in the cyanobacterium Synechococcus sp. PCC 7002, thereby targeting it to the thylakoids. The engineered enzyme showed light-driven activity both in vivo and in vitro, demonstrating the possibility to achieve light-driven biosynthesis of high-value plant specialized metabolites in cyanobacteria.

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          Most cited references 38

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          High-level semi-synthetic production of the potent antimalarial artemisinin.

          In 2010 there were more than 200 million cases of malaria, and at least 655,000 deaths. 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 manufacturers. 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 acid. 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.
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            Plants as a source of anti-cancer agents.

            Plant-derived compounds have been an important source of several clinically useful anti-cancer agents. These include vinblastine, vincristine, the camptothecin derivatives, topotecan and irinotecan, etoposide, derived from epipodophyllotoxin, and paclitaxel (taxol A number of promising new agents are in clinical development based on selective activity against cancer-related molecular targets, including flavopiridol and combretastin A4 phosphate, while some agents which failed in earlier clinical studies are stimulating renewed interest.
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              Plant cell cultures: Chemical factories of secondary metabolites.

              This review deals with the production of high-value secondary metabolites including pharmaceuticals and food additives through plant cell cultures, shoot cultures, root cultures and transgenic roots obtained through biotechnological means. Plant cell and transgenic hairy root cultures are promising potential alternative sources for the production of high-value secondary metabolites of industrial importance. Recent developments in transgenic research have opened up the possibility of the metabolic engineering of biosynthetic pathways to produce high-value secondary metabolites. The production of the pungent food additive capsaicin, the natural colour anthocyanin and the natural flavour vanillin is described in detail.
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                Author and article information

                Affiliations
                [1 ]Center for Synthetic Biology “bioSYNergy”, the VILLUM Research Center “Plant Plasticity”, Copenhagen Plant Science Center, Department of Plant and Environmental Sciences, University of Copenhagen, Frederiksberg C, Copenhagen, Denmark
                [2 ]Department of Biophysics, Faculty of Biotechnology, University of Wroclaw, Wroclaw, Poland
                University of Hyderabad, India
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                Conceived and designed the experiments: LML AZN KJ BLM PEJ. Performed the experiments: LML CEO. Analyzed the data: LML AZN PEJ. Contributed reagents/materials/analysis tools: WB. Contributed to the writing of the manuscript: LML AZN PEJ.

                [¤a]

                Current address: Nixon Group, Sir Ernst Chain Building - Wolfson Laboratories, Imperial College London, London, United Kingdom

                [¤b]

                Current address: Novozymes A/S, Bagsvaerd, Denmark

                Contributors
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, USA )
                1932-6203
                2014
                15 July 2014
                : 9
                : 7
                PONE-D-14-21317
                10.1371/journal.pone.0102184
                4099078
                25025215

                This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                Counts
                Pages: 10
                Funding
                The authors gratefully acknowledge financial support from 1) the VILLUM Center of Excellence “Plant Plasticity”, 2) Center of Synthetic Biology “bioSYNergy” funded by the UCPH Excellence Program for Interdisciplinary Research, 3) “Plant Power: Light-driven synthesis of complex terpenoids using cytochrome P450s” (12-131834) funded by the Danish Council for Strategic Research, Programme Commission on Strategic Growth Technologies and 4) ERC Advanced Research Grant No. 323034 “Light driven P450s”. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology and Life Sciences
                Biochemistry
                Metabolism
                Metabolites
                Plant Biochemistry
                Photosynthesis
                Biotechnology
                Bioengineering
                Organisms
                Bacteria
                Cyanobacteria
                Synthetic Biology
                Custom metadata
                The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and its Supporting Information files.

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