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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 references32

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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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            Natural plant chemicals: sources of industrial and medicinal materials.

            Many higher plants produce economically important organic compounds such as oils, resins, tannins, natural rubber, gums, waxes, dyes, flavors and fragrances, pharmaceuticals, and pesticides. However, most species of higher plants have never been described, much less surveyed for chemical or biologically active constituents, and new sources of commercially valuable materials remain to be discovered. Advances in biotechnology, particularly methods for culturing plant cells and tissues, should provide new means for the commercial processing of even rare plants and the chemicals they produce. These new technologies will extend and enhance the usefulness of plants as renewable resources of valuable chemicals. In the future, biologically active plant-derived chemicals can be expected to play an increasingly significant role in the commercial development of new products for regulating plant growth and for insect and weed control.
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              The many roles of cytochrome b5.

              Four distinct suggestions have been made to explain the mechanism of the cytochrome b(5)-imposed positive modifier action of the cytochrome P450 monooxygenase reaction. The first mechanism involves a direct input of an electron into the monooxygenase cycle. This is the second of the two electrons necessary for activation of molecular oxygen, and appears to be a rate-limiting step in the monooxygenase reaction. P450 monooxygenases all appear to be uncoupled to varying extents, releasing superoxide and hydrogen peroxide instead of oxidized substrate. A second mechanism suggests that cytochrome b(5) acts as a positive modifier of the monooxygenase by decreasing the extent of uncoupling of the monooxygenase reaction. The implication is that a slow input of the second electron allows uncoupling of a superoxide anion instead of formation of two-electron reduced oxygen. Faster input of the second electron via cytochrome b(5) would result in formation of more of the activated oxygen that reacts with substrate to form product. A third suggestion involves formation of a two-hemoprotein complex between cytochrome b(5) and cytochrome P450 that allows acceptance of two electrons from NADPH-cytochrome P450 reductase. Uncomplexed cytochrome P450 accepts an electron from the reductase, dissociates from it, binds oxygen, and re-associates with the reductase to accept another electron. Complexation with cytochrome b(5) enhances the rate of formation of the active oxygen by obviating the need for two interactions with reductase. The fourth mechanism has cytochrome b(5) serving as an effector without a reduction-oxidation role in the monooxygenation reaction. This effector function may be to enhance the breakdown of the oxygenated hemoprotein to products or to facilitate flow of electrons through the system. Copyright 2002 Elsevier Science Inc.
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                Author and article information

                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
                : e102184
                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

                Article
                PONE-D-14-21317
                10.1371/journal.pone.0102184
                4099078
                25025215
                1d97e019-abdf-40c5-a673-5ab2b66c1ba3
                Copyright @ 2014

                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.

                History
                : 13 May 2014
                : 16 June 2014
                Page count
                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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