51
views
0
recommends
+1 Recommend
0 collections
    0
    shares
      • Record: found
      • Abstract: found
      • Article: found
      Is Open Access

      Heterologous expression of the isopimaric acid pathway in Nicotiana benthamiana and the effect of N-terminal modifications of the involved cytochrome P450 enzyme

      research-article

      Read this article at

      Bookmark
          There is no author summary for this article yet. Authors can add summaries to their articles on ScienceOpen to make them more accessible to a non-specialist audience.

          Abstract

          Background

          Plant terpenoids are known for their diversity, stereochemical complexity, and their commercial interest as pharmaceuticals, food additives, and cosmetics. Developing biotechnology approaches for the production of these compounds in heterologous hosts can increase their market availability, reduce their cost, and provide sustainable production platforms. In this context, we aimed at producing the antimicrobial diterpenoid isopimaric acid from Sitka spruce. Isopimaric acid is synthesized using geranylgeranyl diphosphate as a precursor molecule that is cyclized by a diterpene synthase in the chloroplast and subsequently oxidized by a cytochrome P450, CYP720B4.

          Results

          We transiently expressed the isopimaric acid pathway in Nicotiana benthamiana leaves and enhanced its productivity by the expression of two rate-limiting steps in the pathway (providing the general precursor of diterpenes). This co-expression resulted in 3-fold increase in the accumulation of both isopimaradiene and isopimaric acid detected using GC-MS and LC-MS methodology. We also showed that modifying or deleting the transmembrane helix of CYP720B4 does not alter the enzyme activity and led to successful accumulation of isopimaric acid in the infiltrated leaves. Furthermore, we demonstrated that a modified membrane anchor is a prerequisite for a functional CYP720B4 enzyme when the chloroplast targeting peptide is added. We report the accumulation of 45–55 μg/g plant dry weight of isopimaric acid four days after the infiltration with the modified enzymes.

          Conclusions

          It is possible to localize a diterpenoid pathway from spruce fully within the chloroplast of N. benthamiana and a few modifications of the N-terminal sequences of the CYP720B4 can facilitate the expression of plant P450s in the plastids. The coupling of terpene biosynthesis closer to photosynthesis paves the way for light-driven biosynthesis of valuable terpenoids.

          Related collections

          Most cited references49

          • Record: found
          • Abstract: found
          • Article: not found

          An enhanced transient expression system in plants based on suppression of gene silencing by the p19 protein of tomato bushy stunt virus.

          Transient gene expression is a fast, flexible and reproducible approach to high-level expression of useful proteins. In plants, recombinant strains of Agrobacterium tumefaciens can be used for transient expression of genes that have been inserted into the T-DNA region of the bacterial Ti plasmid. A bacterial culture is vacuum-infiltrated into leaves, and upon T-DNA transfer, there is ectopic expression of the gene of interest in the plant cells. However, the utility of the system is limited because the ectopic protein expression ceases after 2-3 days. Here, we show that post-transcriptional gene silencing (PTGS) is a major cause for this lack of efficiency. We describe a system based on co-expression of a viral-encoded suppressor of gene silencing, the p19 protein of tomato bushy stunt virus (TBSV), that prevents the onset of PTGS in the infiltrated tissues and allows high level of transient expression. Expression of a range of proteins was enhanced 50-folds or more in the presence of p19 so that protein purification could be achieved from as little as 100 mg of infiltrated leaf material. The effect of p19 was not saturated in cells that had received up to four individual T-DNAs and persisted until leaf senescence. Because of its simplicity and rapidity, we anticipate that the p19-enhanced expression system will have value in industrial production as well as a research tool for isolation and biochemical characterisation of a broad range of proteins without the need for the time-consuming regeneration of stably transformed plants.
            Bookmark
            • Record: found
            • Abstract: found
            • Article: not found

            Network analysis of the MVA and MEP pathways for isoprenoid synthesis.

            Isoprenoid biosynthesis is essential for all living organisms, and isoprenoids are also of industrial and agricultural interest. All isoprenoids are derived from prenyl diphosphate (prenyl-PP) precursors. Unlike isoprenoid biosynthesis in other living organisms, prenyl-PP, as the precursor of all isoprenoids in plants, is synthesized by two independent pathways: the mevalonate (MVA) pathway in the cytoplasm and the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway in plastids. This review focuses on progress in our understanding of how the precursors for isoprenoid biosynthesis are synthesized in the two subcellular compartments, how the underlying pathway gene networks are organized and regulated, and how network perturbations impact each pathway and plant development. Because of the wealth of data on isoprenoid biosynthesis, we emphasize research in Arabidopsis thaliana and compare the synthesis of isoprenoid precursor molecules in this model plant with their synthesis in other prokaryotic and eukaryotic organisms.
              Bookmark
              • Record: found
              • Abstract: found
              • Article: not found

              Biogenesis and homeostasis of chloroplasts and other plastids.

              Chloroplasts are the organelles that define plants, and they are responsible for photosynthesis as well as numerous other functions. They are the ancestral members of a family of organelles known as plastids. Plastids are remarkably dynamic, existing in strikingly different forms that interconvert in response to developmental or environmental cues. The genetic system of this organelle and its coordination with the nucleocytosolic system, the import and routing of nucleus-encoded proteins, as well as organellar division all contribute to the biogenesis and homeostasis of plastids. They are controlled by the ubiquitin-proteasome system, which is part of a network of regulatory mechanisms that integrate plastid development into broader programmes of cellular and organismal development.
                Bookmark

                Author and article information

                Contributors
                thgn@plen.ku.dk
                kva@plen.ku.dk
                joar@berkeley.edu
                az@plen.ku.dk
                ceo@plen.ku.dk
                bjoernh@plen.ku.dk
                peje@plen.ku.dk
                Journal
                J Biol Eng
                J Biol Eng
                Journal of Biological Engineering
                BioMed Central (London )
                1754-1611
                22 December 2015
                22 December 2015
                2015
                : 9
                : 24
                Affiliations
                [ ]Department of Plant and Environmental Sciences, Copenhagen Plant Science Centre, UNIK Center for Synthetic Biology, Villum Research Center “Plant Plasticity”, University of Copenhagen, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Copenhagen, Denmark
                [ ]Present address: Plant and Microbial Biology, University of California, 371 Koshland Hall, Berkeley, CA 94720 USA
                Article
                22
                10.1186/s13036-015-0022-z
                4688937
                26702299
                9fa26360-ceb6-49da-83d9-79215805144d
                © Gnanasekaran et al. 2015

                Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License ( http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

                History
                : 10 November 2015
                : 7 December 2015
                Funding
                Funded by: Innovation Fund Denmark
                Award ID: 12-131834
                Award Recipient :
                Categories
                Research
                Custom metadata
                © The Author(s) 2015

                Biotechnology
                chloroplasts,cytochrome p450,diterpenoids,isopimaric acid,protein engineering
                Biotechnology
                chloroplasts, cytochrome p450, diterpenoids, isopimaric acid, protein engineering

                Comments

                Comment on this article