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Fusion of Ferredoxin and Cytochrome P450 Enables Direct Light-Driven Biosynthesis

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      Abstract

      Cytochrome P450s (P450s) are key enzymes in the synthesis of bioactive natural products in plants. Efforts to harness these enzymes for in vitro and whole-cell production of natural products have been hampered by difficulties in expressing them heterologously in their active form, and their requirement for NADPH as a source of reducing power. We recently demonstrated targeting and insertion of plant P450s into the photosynthetic membrane and photosynthesis-driven, NADPH-independent P450 catalytic activity mediated by the electron carrier protein ferredoxin. Here, we report the fusion of ferredoxin with P450 CYP79A1 from the model plant Sorghum bicolor, which catalyzes the initial step in the pathway leading to biosynthesis of the cyanogenic glucoside dhurrin. Fusion with ferredoxin allows CYP79A1 to obtain electrons for catalysis by interacting directly with photosystem I. Furthermore, electrons captured by the fused ferredoxin moiety are directed more effectively toward P450 catalytic activity, making the fusion better able to compete with endogenous electron sinks coupled to metabolic pathways. The P450-ferredoxin fusion enzyme obtains reducing power solely from its fused ferredoxin and outperforms unfused CYP79A1 in vivo. This demonstrates greatly enhanced electron transfer from photosystem I to CYP79A1 as a consequence of the fusion. The fusion strategy reported here therefore forms the basis for enhanced partitioning of photosynthetic reducing power toward P450-dependent biosynthesis of important natural products.

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      Most cited references 55

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      Engineering a platform for photosynthetic isoprene production in cyanobacteria, using Synechocystis as the model organism.

      The concept of "photosynthetic biofuels" envisions application of a single organism, acting both as photo-catalyst and producer of ready-made fuel. This concept was applied upon genetic engineering of the cyanobacterium Synechocystis, conferring the ability to generate volatile isoprene hydrocarbons from CO(2) and H(2)O. Heterologous expression of the Pueraria montana (kudzu) isoprene synthase (IspS) gene in Synechocystis enabled photosynthetic isoprene generation in these cyanobacteria. Codon-use optimization of the kudzu IspS gene improved expression of the isoprene synthase in Synechocystis. Use of the photosynthesis psbA2 promoter, to drive the expression of the IspS gene, resulted in a light-intensity-dependent isoprene synthase expression. Results showed that oxygenic photosynthesis can be re-directed to generate useful small volatile hydrocarbons, while consuming CO(2), without a prior requirement for the harvesting, dewatering and processing of the respective biomass.
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        Supramolecular organization of thylakoid membrane proteins in green plants.

        The light reactions of photosynthesis in green plants are mediated by four large protein complexes, embedded in the thylakoid membrane of the chloroplast. Photosystem I (PSI) and Photosystem II (PSII) are both organized into large supercomplexes with variable amounts of membrane-bound peripheral antenna complexes. PSI consists of a monomeric core complex with single copies of four different LHCI proteins and has binding sites for additional LHCI and/or LHCII complexes. PSII supercomplexes are dimeric and contain usually two to four copies of trimeric LHCII complexes. These supercomplexes have a further tendency to associate into megacomplexes or into crystalline domains, of which several types have been characterized. Together with the specific lipid composition, the structural features of the main protein complexes of the thylakoid membranes form the main trigger for the segregation of PSII and LHCII from PSI and ATPase into stacked grana membranes. We suggest that the margins, the strongly folded regions of the membranes that connect the grana, are essentially protein-free, and that protein-protein interactions in the lumen also determine the shape of the grana. We also discuss which mechanisms determine the stacking of the thylakoid membranes and how the supramolecular organization of the pigment-protein complexes in the thylakoid membrane and their flexibility may play roles in various regulatory mechanisms of green plant photosynthesis.
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          Cyanogenic glycosides: synthesis, physiology, and phenotypic plasticity.

          Cyanogenic glycosides (CNglcs) are bioactive plant products derived from amino acids. Structurally, these specialized plant compounds are characterized as α-hydroxynitriles (cyanohydrins) that are stabilized by glucosylation. In recent years, improved tools within analytical chemistry have greatly increased the number of known CNglcs by enabling the discovery of less abundant CNglcs formed by additional hydroxylation, glycosylation, and acylation reactions. Cyanogenesis--the release of toxic hydrogen cyanide from endogenous CNglcs--is an effective defense against generalist herbivores but less effective against fungal pathogens. In the course of evolution, CNglcs have acquired additional roles to improve plant plasticity, i.e., establishment, robustness, and viability in response to environmental challenges. CNglc concentration is usually higher in young plants, when nitrogen is in ready supply, or when growth is constrained by nonoptimal growth conditions. Efforts are under way to engineer CNglcs into some crops as a pest control measure, whereas in other crops efforts are directed toward their removal to improve food safety. Given that many food crops are cyanogenic, it is important to understand the molecular mechanisms regulating cyanogenesis so that the impact of future environmental challenges can be anticipated.
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            Author and article information

            Affiliations
            []Copenhagen Plant Science Center, Department of Plant and Environmental Sciences, University of Copenhagen , Thorvaldsensvej 40, 1871 Frederiksberg C, Denmark
            []DynaMo Center of Excellence, Department of Plant and Environmental Sciences, University of Copenhagen , Thorvaldsensvej 40, 1871 Frederiksberg C, Denmark
            [§ ]Center for Synthetic Biology “bioSYNergy” , Thorvaldsensvej 40, 1871 Frederiksberg, Denmark
            []Villum Research Center of Excellence ”Plant Plasticity”, University of Copenhagen , Thorvaldsensvej 40, 1871 Frederiksberg C, Denmark
            Author notes
            [* ]Tel.: +45 3533 3340. E-mail: peje@ 123456plen.ku.dk .
            Journal
            ACS Chem Biol
            ACS Chem. Biol
            cb
            acbcct
            ACS Chemical Biology
            American Chemical Society
            1554-8929
            1554-8937
            27 April 2016
            15 July 2016
            : 11
            : 7
            : 1862-1869
            27119279
            4949584
            10.1021/acschembio.6b00190
            Copyright © 2016 American Chemical Society

            This is an open access article published under an ACS AuthorChoice License, which permits copying and redistribution of the article or any adaptations for non-commercial purposes.

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            Biochemistry

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