DOCSLIB.ORG

Production of Muconic Acid in Plants T ⁎ Aymerick Eudesa,B, , Roland Berthomieua,C, Zhangying Haoa,B, Nanxia Zhaoa,D, ⁎ Veronica Teixeira Benitesa,E, Edward E.K

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Production of Muconic Acid in Plants T ⁎ Aymerick Eudesa,B, , Roland Berthomieua,C, Zhangying Haoa,B, Nanxia Zhaoa,D, ⁎ Veronica Teixeira Benitesa,E, Edward E.K Metabolic Engineering 46 (2018) 13–19 Contents lists available at ScienceDirect Metabolic Engineering journal homepage: www.elsevier.com/locate/meteng Production of muconic acid in plants T ⁎ Aymerick Eudesa,b, , Roland Berthomieua,c, Zhangying Haoa,b, Nanxia Zhaoa,d, ⁎ Veronica Teixeira Benitesa,e, Edward E.K. Baidooa,e, Dominique Loquéa,b,f,g, a Joint BioEnergy Institute, EmeryStation East, 5885 Hollis St, 4th Floor, Emeryville, CA 94608, USA b Environmental Genomics and Systems Biology Division, Lawrence Berkeley National Laboratory, 1 Cyclotron Road, Berkeley, CA 94720, USA c Ecole Polytechnique, Université Paris-Saclay, Palaiseau 91120, France d Department of Bioengineering, Department of Chemical & Biomolecular Engineering, University of California, Berkeley, CA 94720, USA e Biological Systems and Engineering Division, Lawrence Berkeley National Laboratory, 1 Cyclotron Road, Berkeley, CA 94720, USA f Department of Plant and Microbial Biology, University of California, Berkeley, CA 94720, USA g Université Lyon 1, INSA de Lyon, CNRS, UMR5240, Microbiologie, Adaptation et Pathogénie, 10 rue Raphaël Dubois, F-69622, Villeurbanne, France ARTICLE INFO ABSTRACT Keywords: Muconic acid (MA) is a dicarboxylic acid used for the production of industrially relevant chemicals such as Muconic acid adipic acid, terephthalic acid, and caprolactam. Because the synthesis of these polymer precursors generates Salicylic acid toxic intermediates by utilizing petroleum-derived chemicals and corrosive catalysts, the development of al- Catechol ternative strategies for the bio-based production of MA has garnered significant interest. Plants produce organic Shikimate carbon skeletons by harvesting carbon dioxide and energy from the sun, and therefore represent advantageous Plastid hosts for engineered metabolic pathways towards the manufacturing of chemicals. In this work, we engineered Arabidopsis Arabidopsis to demonstrate that plants can serve as green factories for the bio-manufacturing of MA. In parti- cular, dual expression of plastid-targeted bacterial salicylate hydroxylase (NahG) and catechol 1,2-dioxygenase (CatA) resulted in the conversion of the endogenous salicylic acid (SA) pool into MA via catechol. Sequential increase of SA derived from the shikimate pathway was achieved by expressing plastid-targeted versions of bacterial salicylate synthase (Irp9) and feedback-resistant 3-deoxy-D-arabino-heptulosonate synthase (AroG). Introducing this SA over-producing strategy into engineered plants that co-express NahG and CatA resulted in a 50-fold increase in MA titers. Considering that MA was easily recovered from senesced plant biomass after harvest, we envision the phytoproduction of MA as a beneficial option to add value to bioenergy crops. 1. Introduction shikimate pathway for the biosynthesis of catechol precursors such as protocatechuate, anthranilate, salicylic acid (SA), and 2,3-dihydrox- Muconic acid (MA) is a platform chemical that serves as a precursor ybenzoic acid (Kruyer and Peralta-Yahya, 2017). Recently, MA bio- for the synthesis of products such as adipic acid, terephthalic acid, and synthetic pathways have been implemented in various microbial strains caprolactam which are widely used in the nylon and thermoplastic capable of growing in the presence of aromatics derived from lig- polymer industries. Current processes for the manufacturing of MA or nocellulosic biomass. These include engineered strains of Escherichia its derivatives mainly rely on non-renewable petroleum-based chemi- coli (Sonoki et al., 2014; Wu et al., 2017), Amycolatopsis sp. (Barton cals. Such processes are not sustainable and eco-friendly since they et al., 2017), Pseudomonas sp. (Vardon et al., 2015; Johnson et al., require a high energy input and yield large quantities of toxic by-pro- 2016, 2017; Sonoki et al., 2017), and Sphingobium sp. (Sonoki et al., ducts (Xie et al., 2014). 2017). As an alternative, the biological production of MA using engineered In addition to microbial synthesis, the metabolic engineering of microorganisms and inexpensive carbohydrate feedstocks has received photosynthetic organisms like plants also provides a sustainable ap- increasing attention over the past 20 years (Xie et al., 2014). Most of the proach for the production of valuable metabolites and materials established biological routes consist in the production catechol and its (Börnke and Broer, 2010; Farré et al., 2014). These chemicals, when subsequent conversion into MA by ring-cleaving catechol 1,2-dioxy- produced in engineered bioenergy and oilseed crops, represent value- genase (Vaillancourt et al., 2006). All these routes exploit the intrinsic added renewable co-products on top of the lignocellulose and seed oil Abbreviations: DAHP, 3-deoxy-D-arabino-heptulosonate; MA, muconic acid; SA, salicylic acid ⁎ Corresponding authors at: Environmental Genomics and Systems Biology Division, Lawrence Berkeley National Laboratory, 1 Cyclotron Road, Berkeley, CA 94720, USA. E-mail addresses: [email protected] (A. Eudes), [email protected] (D. Loqué). https://doi.org/10.1016/j.ymben.2018.02.002 Received 1 October 2017; Received in revised form 6 January 2018; Accepted 13 February 2018 Available online 21 February 2018 1096-7176/ © 2018 The Authors. Published by Elsevier Inc. on behalf of International Metabolic Engineering Society. This is an open access article under the CC BY license (http://creativecommons.org/licenses/BY/4.0/). A. Eudes et al. Metabolic Engineering 46 (2018) 13–19 1 A Glycolysis Fig. 1. Strategy used for the production of muconic PEP AroG* 234567Irp9 Irp9 NahG CatA acid in plants. (A) De novo biosynthetic pathway for DAHP DHQ DHS SHIK S3P EPSP CHA ISOCHA SA CAT MA muconic acid synthesis. Bacterial enzymes AroG*, Pentose phosphate Irp9, NahG, and CatA were expressed in Arabidopsis E4P Shikimate pathway pathway for the production of muconic acid from salicylic acid derived from the shikimate pathway. B pAtIRX5 pAtCCoAOMT pAtIRX8 pAtC4H Abbreviations are: CAT, catechol; CHA, chorismate; DAHP, 3-deoxy-D-arabino-heptulosonate; DHQ, de- pA6 (hyg ) schl1 irp9 schl2 aroG* pTKan (kan ) schl3 catA schl3 nahG hydroquinate; DHS, dehydroshikimate; E4P, ery- throse 4-phosphate; EPSP, 5-enolpyruvoylshikimate 3-phosphate; F6P, fructose 6-phosphate; G3P, glyceraldehyde 3-phosphate; ISOCHA, isochorismate; MA, muconic acid; PEP, phosphoenolpyruvate; PYR, pyruvate; S3P, shikimate 3- phosphate; SA, salicylic acid; SHIK, shikimate. The enzymes are as follows: 1, enolase; AroG*, feedback-resistant DAHP synthase (L175Q); 2, DHQ synthase; 3, DHQ dehydratase; 4, shikimate dehydrogenase; 5, shikimate kinase; 6, EPSP synthase; 7, chorismate synthase; Irp9, bifunctional ISOCHA synthase / ISOCHA pyruvate lyase; NahG, salicylate hydroxylase; CatA, catechol 1,2-dioxygenase. (B) Binary vectors used in this study. Boxes labelled “schl” denote plastid transit peptides. pA6 and pTKan denote the two binary vector backbones conferring hygromycin (hygR) and kanamycin (kanR) resistance in plants. Abbreviations are: schl1, plastid transit peptide from Arabidopsis ferredoxin2 (At1g60950); schl2, plastid transit peptide from pea (Pisum sativum) ribulose-1,5-bisphosphate carboxylase small subunit (GenBank: AAG45569.1); schl3, plastid transit peptide sunflower (Helianthus annuus) ribulose-1,5- bisphosphate carboxylase small subunit (UniProtKB/Swiss-Prot: P08705.1). pAtIRX5, pAtCCoAOMT, pAtIRX8, and pAtC4H designate the promoters of Arabidopsis cellulose synthase 4 (At5G44030), caffeoyl coenzyme A O-methyltransferase 1 (At4G34050), galacturonosyltransferase 12 (At5G54690), and cinnamate 4-hydroxylase (At2G30490) genes, respectively. used to generate energy (Snell et al., 2015). Because plants are auto- construct (Fig. 1B), the attB1-schl1-irp9-attB2 sequence was amplified trophs able to capture solar energy, they represent an attractive chassis by PCR to replace the Gateway attB2 recombination site (3’-end) by an for implementing de novo metabolic pathways for cost-effective pro- attB4 recombination site, and cloned into the Gateway pDONR221- duction of important chemicals (Yuan and Grotewold, 2015). P1P4 entry vector by BP recombination (Life technologies, Foster City, In plants, the shikimate pathway is confined to plastids and provides CA) to produce a pDONR221-L1-schl1-irp9-L4 construct. A chimeric the precursors for the synthesis of aromatic amino acids and derived DNA construct was synthesized (GenScript, Piscatway, NJ): it was metabolites, vitamins K1 and B9, and SA (Maeda and Dudareva, 2012). flanked by the gateway sequences attB4r (5’-end) and attB3r (3’-end), We therefore used Arabidopsis as a model system to investigate a novel and contained the tG7 terminator and a 2.2-Kb sequence corresponding bio-based approach for the phytoproduction of MA. In particular, the to the Arabidopsis CCoAOMT1 (At4g34050) promoter (pCCoAOMT). SA pool derived from chorismate via the shikimate pathway was con- This attB4r-tG7-pCCoAOMT-attB3r construct (Supplementary Data S1) verted to catechol and MA by dual expression and plastid-targeting of was then subcloned into the Gateway pDONR221-P4rP3r entry vector bacterial salicylate hydroxylase (NahG) and catechol 1,2-dioxygenase by BP recombination (Life technologies, Foster City, CA) to produce (CatA) (Fig. 1A). Additional supply of SA to the MA pathway was pDONR221-L4R-tG7-pCCoAOMT-L3R. A gene sequence encoding feed- achieved via the expression of bacterial salicylate synthase Irp9 and back-insensitive AroG (L175Q) from E. coli (NCBI Reference Sequence: feedback-resistant
Load more

Recommended publications
  • Part One Amino Acids As Building Blocks
    Part One Amino Acids as Building Blocks Amino Acids, Peptides and Proteins in Organic Chemistry. Vol.3 – Building Blocks, Catalysis and Coupling Chemistry. Edited by Andrew B. Hughes Copyright Ó 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN: 978-3-527-32102-5 j3 1 Amino Acid Biosynthesis Emily J. Parker and Andrew J. Pratt 1.1 Introduction The ribosomal synthesis of proteins utilizes a family of 20 a-amino acids that are universally coded by the translation machinery; in addition, two further a-amino acids, selenocysteine and pyrrolysine, are now believed to be incorporated into proteins via ribosomal synthesis in some organisms. More than 300 other amino acid residues have been identified in proteins, but most are of restricted distribution and produced via post-translational modification of the ubiquitous protein amino acids [1]. The ribosomally encoded a-amino acids described here ultimately derive from a-keto acids by a process corresponding to reductive amination. The most important biosynthetic distinction relates to whether appropriate carbon skeletons are pre-existing in basic metabolism or whether they have to be synthesized de novo and this division underpins the structure of this chapter. There are a small number of a-keto acids ubiquitously found in core metabolism, notably pyruvate (and a related 3-phosphoglycerate derivative from glycolysis), together with two components of the tricarboxylic acid cycle (TCA), oxaloacetate and a-ketoglutarate (a-KG). These building blocks ultimately provide the carbon skeletons for unbranched a-amino acids of three, four, and five carbons, respectively. a-Amino acids with shorter (glycine) or longer (lysine and pyrrolysine) straight chains are made by alternative pathways depending on the available raw materials.
    [Show full text]
  • Yeast Genome Gazetteer P35-65
    gazetteer Metabolism 35 tRNA modification mitochondrial transport amino-acid metabolism other tRNA-transcription activities vesicular transport (Golgi network, etc.) nitrogen and sulphur metabolism mRNA synthesis peroxisomal transport nucleotide metabolism mRNA processing (splicing) vacuolar transport phosphate metabolism mRNA processing (5’-end, 3’-end processing extracellular transport carbohydrate metabolism and mRNA degradation) cellular import lipid, fatty-acid and sterol metabolism other mRNA-transcription activities other intracellular-transport activities biosynthesis of vitamins, cofactors and RNA transport prosthetic groups other transcription activities Cellular organization and biogenesis 54 ionic homeostasis organization and biogenesis of cell wall and Protein synthesis 48 plasma membrane Energy 40 ribosomal proteins organization and biogenesis of glycolysis translation (initiation,elongation and cytoskeleton gluconeogenesis termination) organization and biogenesis of endoplasmic pentose-phosphate pathway translational control reticulum and Golgi tricarboxylic-acid pathway tRNA synthetases organization and biogenesis of chromosome respiration other protein-synthesis activities structure fermentation mitochondrial organization and biogenesis metabolism of energy reserves (glycogen Protein destination 49 peroxisomal organization and biogenesis and trehalose) protein folding and stabilization endosomal organization and biogenesis other energy-generation activities protein targeting, sorting and translocation vacuolar and lysosomal
    [Show full text]
  • Genetic Characterisaton of Rhodococcus Rhodochrous ATCC
    The copyright of this thesis vests in the author. No quotation from it or information derived from it is to be published without full acknowledgement of the source. The thesis is to be used for private study or non- commercial research purposes only. Published by the University of Cape Town (UCT) in terms of the non-exclusive license granted to UCT by the author. University of Cape Town Genetic characterization of Rhodococcus rhodochrous ATCC BAA-870 with emphasis on nitrile hydrolysing enzymes n ow Joni Frederick A thesis submitted in fulfilment of the requirements for the degree of Doctor of Philosophy in the Departmentty of of MolecularCape and T Cell Biology, Universitysi of Cape Town er UnivSupervisor: Professor B. T. Sewell Co-supervisor: Professor D. Brady February 2013 Keywords Nitrile hydrolysis Biocatalysis Rhodococcus rhodochrous ATCC BAA-870 Genome sequencing Nitrilase Nitrile hydratase n ow ty of Cape T si er Univ ii Keywords Abstract Rhodococcus rhodochrous ATCC BAA-870 (BAA-870) had previously been isolated on selective media for enrichment of nitrile hydrolysing bacteria. The organism was found to have a wide substrate range, with activity against aliphatics, aromatics, and aryl aliphatics, and enantioselectivity towards beta substituted nitriles and beta amino nitriles, compounds that have potential applications in the pharmaceutical industry. This makes R. rhodochrous ATCC BAA-870 potentially a versatile biocatalyst for the synthesis of a broad range of compounds with amide and carboxylic acid groups that can be derived from structurally related nitrile precursors. The selectivity of biocatalysts allows for high product yields and better atom economyn than non- selective chemical methods of performing this reaction, suchow as acid or base hydrolysis.
    [Show full text]
  • Crystal Structure of Chorismate Synthase: a Novel FMN-Binding Protein Fold and Functional Insights
    doi:10.1016/j.jmb.2003.12.072 J. Mol. Biol. (2004) 336, 903–915 Crystal Structure of Chorismate Synthase: A Novel FMN-binding Protein Fold and Functional Insights Hyung Jun Ahn, Hye-Jin Yoon, Byung Il Lee and Se Won Suh* Department of Chemistry Chorismate synthase catalyzes the conversion of 5-enolpyruvylshikimate College of Natural Sciences 3-phosphate to chorismate in the shikimate pathway, which represents Seoul National University an attractive target for discovering antimicrobial agents and herbicides. Seoul 151-0742, South Korea Chorismate serves as a common precursor for the synthesis of aromatic amino acids and many aromatic compounds in microorganisms and plants. Chorismate synthase requires reduced FMN as a cofactor but the catalyzed reaction involves no net redox change. Here, we have deter- mined the crystal structure of chorismate synthase from Helicobacter pylori in both FMN-bound and FMN-free forms. It is a tetrameric enzyme, with each monomer possessing a novel “b-a-b sandwich fold”. Highly con- served regions, including several flexible loops, cluster together around the bound FMN to form the active site. The unique FMN-binding site is formed largely by a single subunit, with a small contribution from a neighboring subunit. The isoalloxazine ring of the bound FMN is signifi- cantly non-planar. Our structure illuminates the essential functional roles played by the cofactor. q 2004 Elsevier Ltd. All rights reserved. Keywords: aroC; chorismate synthase; FMN-binding protein; Helicobacter *Corresponding author pylori; shikimate
    [Show full text]
  • Molecular Targets for Antifungals in Amino Acid and Protein Biosynthetic Pathways
    Amino Acids https://doi.org/10.1007/s00726-021-03007-6 REVIEW ARTICLE Molecular targets for antifungals in amino acid and protein biosynthetic pathways Aleksandra Kuplińska1 · Kamila Rząd1 Received: 1 March 2021 / Accepted: 17 May 2021 © The Author(s) 2021 Abstract Fungi cause death of over 1.5 million people every year, while cutaneous mycoses are among the most common infections in the world. Mycoses vary greatly in severity, there are long-term skin (ringworm), nail or hair infections (tinea capitis), recurrent like vaginal candidiasis or severe, life-threatening systemic, multiorgan infections. In the last few years, increas- ing importance is attached to the health and economic problems caused by fungal pathogens. There is a growing need for improvement of the availability of antifungal drugs, decreasing their prices and reducing side efects. Searching for novel approaches in this respect, amino acid and protein biosynthesis pathways appear to be competitive. The route that leads from amino acid biosynthesis to protein folding and its activation is rich in enzymes that are descriptive of fungi. Blocking the action of those enzymes often leads to avirulence or growth inhibition. In this review, we want to trace the principal processes of fungi vitality. We present the data of genes encoding enzymes involved in amino acid and protein biosynthesis, potential molecular targets in antifungal chemotherapy, and describe the impact of inhibitors on fungal organisms. Keywords Antifungal targets · Amino acid biosynthesis · Protein biosynthesis · Candida · Aspergillus · Plant pathogens Introduction is approximately 30–55% and 50–100% for aspergillosis (Brown et al. 2012; Verweij et al. 2016; Haidar and Singh Invasive fungal mycoses may afect over 300 million peo- 2018).
    [Show full text]
  • Analysis of Allosteric Communication Pathways in Class I Glutamine Amidotransferases
    Analysis of Allosteric Communication Pathways in Class I Glutamine Amidotransferases Dissertation zur Erlangung des Doktorgrades der Naturwissenschaften (Dr. rer. nat.) der Fakultät für Biologie und vorklinische Medizin der Universität Regensburg vorgelegt von Florian Semmelmann aus Deggendorf Juli 2019 Das Promotionsgesuch wurde eingereicht am: 12.07.2019 Die Arbeit wurde angeleitet von: Prof. Dr. Reinhard Sterner Unterschrift: .............................................. Florian Semmelmann Die vorliegende Arbeit wurde im Zeitraum von Oktober 2015 bis Juli 2019 am Lehrstuhl Bio- chemie II des Institutes für Biophysik und Physikalische Biochemie der Fakultät für Biologie und Vorklinische Medizin der Universität Regensburg unter Leitung von Prof. Dr. Reinhard Sterner angefertigt. Diese Arbeit wurde vom 01.01.2016 bis zum 31.12.2018 durch ein Promotionsstipendium der Konrad-Adenauer-Stiftung gefördert. Abstract Class I glutamine amidotransferases (GATases) represent a ubiquitous family of enzymes that catalyze the incorporation of ammonia within various metabolic pathways. GATases are het- eromeric enzyme complexes consisting of at least one pair of glutaminase and synthase subunits in various oligomeric associations. The activities of the glutaminase and synthase subunits are allosterically coupled in most class I GATases in a way that the presence of substrate at the synthase subunit stimulates glutamine hydrolysis at the glutaminase active site. The structural basis of allosteric communication between both subunits is, however, not well understood. The first part of this thesis focuses on the molecular basis of allosteric coupling in 4-amino-4- deoxychorismate synthase (ADCS), an important but hitherto sparsely characterized member of class I GATases. Using ancestral sequence reconstruction, we generated a thermostable glu- taminase subunit, anc2PabA, solved its crystal structure by molecular replacement and used it to compute for the first time a reliable model of the E.
    [Show full text]
  • Supplementary Material Toxicological Impacts and Likely Protein Targets Of
    Supplementary Material Toxicological impacts and likely protein targets of bisphenol A in Paramecium caudatum Marcus V. X. Senra† & Ana Lúcia Fonseca Instituto de Recursos Naturais, Universidade Federal de Itajubá, 37500-903, Itajubá, Minas Gerais – Brazil †To whom correspondence should be addressed – [email protected]; Orcid - 0000-0002-3866- 8837 Table S1. Annotation data on the P. caudatum 3D modelled proteins and their binding energies to BPA. BINDING ID DESCRIPTION CHROMOSOME NT_START NT_END ENERGIES (kcal/mol) PCAU.43c3d.1.P00010012 Tryptophan synthase beta subunit-like PLP-dependent enzyme scaffold_0001 23197 24853 -7.4 PCAU.43c3d.1.P00010015 Ribosomal protein L32e scaffold_0001 26373 26859 -6.2 PCAU.43c3d.1.P00010044 Catalase, mono-functional, haem-containing scaffold_0001 71821 73367 -6.5 PCAU.43c3d.1.P00010050 Dihydroorotate dehydrogenase, class 1/ 2 scaffold_0001 76614 79650 -6.6 PCAU.43c3d.1.P00010054 Serine/threonine/dual specificity protein kinase, catalytic domain scaffold_0001 83399 84653 -6.7 PCAU.43c3d.1.P00010070 Peptidyl-prolyl cis-trans isomerase, FKBP-type scaffold_0001 104909 105387 -5.9 PCAU.43c3d.1.P00010103 V-ATPase proteolipid subunit C-like domain scaffold_0001 168736 169346 -5.6 PCAU.43c3d.1.P00010112 DNA-directed RNA polymerase, RBP11-like dimerisation domain scaffold_0001 180310 181372 -6.0 PCAU.43c3d.1.P00010165 Vacuolar (H+)-ATPase G subunit scaffold_0001 252653 253112 -5.6 PCAU.43c3d.1.P00010176 Coproporphyrinogen III oxidase, aerobic scaffold_0001 262051 263168 -6.7 PCAU.43c3d.1.P00010205 Metalloenzyme,
    [Show full text]
  • Targeting Mycobacterium Tuberculosis Proteins: Structure and Function Studies of Five Essential Proteins
    Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 411 Targeting Mycobacterium tuberculosis Proteins: Structure and Function Studies of Five Essential Proteins ADRIAN SUAREZ COVARRUBIAS ACTA UNIVERSITATIS UPSALIENSIS ISSN 1651-6214 UPPSALA ISBN 978-91-554-7134-7 2008 urn:nbn:se:uu:diva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ist of publications This thesis consists of a comprehensive summary based on the following papers. In the text, the papers will be referred to by their roman numerals. I Covarrubias, A. S., Larsson, A. M., Högbom, M., Lindberg, J., Bergfors, T., Björkelid, C., Mowbray, S. L., Unge, T., and Jones, T. A. (2005). Structure and
    [Show full text]
  • Suppression of Chorismate Synthase, Which Is Localized in Chloroplasts
    www.nature.com/scientificreports OPEN Suppression of chorismate synthase, which is localized in chloroplasts and peroxisomes, results in abnormal fower development and anthocyanin reduction in petunia Shiwei Zhong1,2, Zeyu Chen1, Jinyi Han1, Huina Zhao3, Juanxu Liu1 & Yixun Yu 1,2* In plants, the shikimate pathway generally occurs in plastids and leads to the biosynthesis of aromatic amino acids. Chorismate synthase (CS) catalyses the last step of the conversion of 5-enolpyruvylshikimate 3-phosphate (EPSP) to chorismate, but the role of CS in the metabolism of higher plants has not been reported. In this study, we found that PhCS, which is encoded by a single- copy gene in petunia (Petunia hybrida), contains N-terminal plastidic transit peptides and peroxisomal targeting signals. Green fuorescent protein (GFP) fusion protein assays revealed that PhCS was localized in chloroplasts and, unexpectedly, in peroxisomes. Petunia plants with reduced PhCS activity were generated through virus-induced gene silencing and further characterized. PhCS silencing resulted in reduced CS activity, severe growth retardation, abnormal fower and leaf development and reduced levels of folate and pigments, including chlorophylls, carotenoids and anthocyanins. A widely targeted metabolomics analysis showed that most primary and secondary metabolites were signifcantly changed in pTRV2-PhCS-treated corollas. Overall, the results revealed a clear connection between primary and specialized metabolism related to the shikimate pathway in petunia. Te shikimate pathway
    [Show full text]
  • Modeling and Computational Prediction of Metabolic Channelling
    MODELING AND COMPUTATIONAL PREDICTION OF METABOLIC CHANNELLING by Christopher Morran Sanford A thesis submitted in conformity with the requirements for the degree of Master of Science Graduate Department of Molecular Genetics University of Toronto © Copyright by Christopher Morran Sanford 2009 Abstract MODELING AND COMPUTATIONAL PREDICTION OF METABOLIC CHANNELLING Master of Science 2009 Christopher Morran Sanford Graduate Department of Molecular Genetics University of Toronto Metabolic channelling occurs when two enzymes that act on a common substrate pass that intermediate directly from one active site to the next without allowing it to diffuse into the surrounding aqueous medium. In this study, properties of channelling are investigated through the use of computational models and cell simulation tools. The effects of enzyme kinetics and thermodynamics on channelling are explored with the emphasis on validating the hypothesized roles of metabolic channelling in living cells. These simulations identify situations in which channelling can induce acceleration of reaction velocities and reduction in the free concentration of intermediate metabolites. Databases of biological information, including metabolic, thermodynamic, toxicity, inhibitory, gene fusion and physical protein interaction data are used to predict examples of potentially channelled enzyme pairs. The predictions are used both to support the hypothesized evolutionary motivations for channelling, and to propose potential enzyme interactions that may be worthy of future investigation. ii Acknowledgements I wish to thank my supervisor Dr. John Parkinson for the guidance he has provided during my time spent in his lab, as well as for his extensive help in the writing of this thesis. I am grateful for the advice of my committee members, Prof.
    [Show full text]
  • Supplemental Table S1: Comparison of the Deleted Genes in the Genome-Reduced Strains
    Supplemental Table S1: Comparison of the deleted genes in the genome-reduced strains Legend 1 Locus tag according to the reference genome sequence of B. subtilis 168 (NC_000964) Genes highlighted in blue have been deleted from the respective strains Genes highlighted in green have been inserted into the indicated strain, they are present in all following strains Regions highlighted in red could not be deleted as a unit Regions highlighted in orange were not deleted in the genome-reduced strains since their deletion resulted in severe growth defects Gene BSU_number 1 Function ∆6 IIG-Bs27-47-24 PG10 PS38 dnaA BSU00010 replication initiation protein dnaN BSU00020 DNA polymerase III (beta subunit), beta clamp yaaA BSU00030 unknown recF BSU00040 repair, recombination remB BSU00050 involved in the activation of biofilm matrix biosynthetic operons gyrB BSU00060 DNA-Gyrase (subunit B) gyrA BSU00070 DNA-Gyrase (subunit A) rrnO-16S- trnO-Ala- trnO-Ile- rrnO-23S- rrnO-5S yaaC BSU00080 unknown guaB BSU00090 IMP dehydrogenase dacA BSU00100 penicillin-binding protein 5*, D-alanyl-D-alanine carboxypeptidase pdxS BSU00110 pyridoxal-5'-phosphate synthase (synthase domain) pdxT BSU00120 pyridoxal-5'-phosphate synthase (glutaminase domain) serS BSU00130 seryl-tRNA-synthetase trnSL-Ser1 dck BSU00140 deoxyadenosin/deoxycytidine kinase dgk BSU00150 deoxyguanosine kinase yaaH BSU00160 general stress protein, survival of ethanol stress, SafA-dependent spore coat yaaI BSU00170 general stress protein, similar to isochorismatase yaaJ BSU00180 tRNA specific adenosine
    [Show full text]
  • Proteomic Analysis of SUMO1-Sumoylome Changes During Defense Elicitation in Arabidopsis
    Supporting Information Kishor D. Ingole, Shraddha K. Dahale and Saikat Bhattacharjee* Laboratory of Signal Transduction and Plant Resistance, UNESCO-Regional Centre for Biotechnology (RCB), NCR Biotech Science Cluster, 3rd Milestone, Faridabad-Gurgaon Expressway, Faridabad- 121 001, Haryana, India. Proteomic analysis of SUMO1-SUMOylome changes during defense elicitation in Arabidopsis Supplementary Figures Figure S1 Figure S1. EDS1 mutation (eds1-2) restores enhanced SUMOylome in srfr1-4 to Col-0 level. Total protein extracts from indicated plants grown for 3-weeks were immunoblotted with anti- SUMO1/2-antibodies. Migration position of molecular weight standards (in kDa), SUMO1/2- conjugates and free-SUMO1/2 are shown. Actin immunoblot and PonceauS staining of Rubisco demonstrate comparable loading across samples. Figure S2 Figure S2. Validation of His-SUM1 and srfr1-4:His-SUM1 systems. (A) Growth phenotypes of indicated plants 4-weeks post-germination. (B-C) SUMOylation enhancements are comparable between srfr1-4 and srfr1-4::His-SUM1 (B), or between Col-0 and His-SUM1 that are mock or PstDC3000 infected (C). (D-F) Transcriptional changes in PR1, PR2, or ICS1 (D), or SUMOylation-associated genes (E-G) are also comparable between srfr1-4 and srfr1-4::His-SUM1. Figure S3 Figure S3. Chromosome-wise distribution of genes corresponding to the enriched SUMO1- substrate proteins in Arabidopsis genome analysed by ShinyGo (v0.61; http://bioinformatics.sdstate.edu/go/). Figure S4 Figure S4. STRING-based protein-protein interaction (PPI) networks (https://string-db.org) of SUMO1-substrates identified in this study. (Red- Defense response; Blue-response to hormones, Green-response to reactive oxygen species, Pale yellow-response to stress).
    [Show full text]