DOCSLIB.ORG

Global Cardiac Phosphoproteome Analysis in Nitric Oxide-Induced Heart Failure

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Global Cardiac Phosphoproteome Analysis in Nitric Oxide-Induced Heart Failure Global Cardiac Phosphoproteome Analysis In Nitric Oxide-Induced Heart Failure Inaugural-Dissertation to obtain the scientific degree Dr. rer. nat. of the Department of Mathematics and Natural Sciences of the Heinrich-Heine-University Düsseldorf submitted by Annamária Simon from Keszthely (Hungary) Düsseldorf, June 2009 From the Institute of Cardiovascular Physiology of the Heinrich-Heine-University Düsseldorf Printed with the authorization of the Department of Mathematics and Natural Sciences of the Heinrich-Heine-University Düsseldorf First Supervisor: Prof. MD Jürgen Schrader Second Supervisor: Prof. Dr. Rainer Weinkauf Day of the oral examination: 16. July 2009 This work is dedicated to my family ACKNOWLEDGEMETNS ACKNOWLEDGEMENTS First and foremost I would like to thank Prof. Dr. med. Jürgen Schrader for giving me the opportunity to work at his laboratory. I am deeply grateful for his excellent supervision at every stage of this work. Furthermore, I would like to thank Prof. Dr. Rainer Weinkauf for the fast preparation of the expert opinion. I would also like to thank the Graduate College 1089 “Protein interactions and modifications in the heart“ for the interesting courses and for the financial support. Additionally I would like to thank Prof. Dr. Albert Sickmann for giving me some practical tips and tricks in proteomics. I am grateful to have the possibility of performing some test measurements on LTQ Orbitrap XL at Thermo Scientific in Bremen. Furthermore, I would like to thank Dr. Michael Reinartz and Dr. Urlich Flögel for performing the heart perfusion experiments, additional thanks to Dr. Michael Reinartz also for mentoring me during my PhD thesis and for the critical reading of this manuscript. I would also like to thank Dr. Christoph Jacobi for helping me in computer problems and for programming MSanalysis. Hereby I would like to thank Dr. Peter Schneider for taking care of the MASCOT server cluster and introducing me into the basics of intranet to reach analyzed results and used databases. Additionally, I would like to thank Dr. Peter Mortensen for providing dimethyl labeling compatible version of MSQuant and Dr. Bas van Breukelen for some bioinformatics programs enabling easier and quantitative analysis of measured data. I am especially grateful for Jan Heye Buß for helping me in the MATLAB program based proteomic data management. Furthermore, I would like to thank all my colleagues and PhD students from the Institute of Cardiovascular Physiology and from the Graduate School 1089 for fruitful scientifical discussions and for a nice atmosphere at the laboratory. At last but not at least I thank my family and all my friends, who supported and motivated me during the last years. TABLE OF CONTENTS TABLE OF CONTENTS ABSTRACT.................................................................................................8 ABSTRAKT (IN GERMAN)...........................................................................9 ABBREVIATIONS.......................................................................................10 I.INTRODUCTION ...............................................................................17 PHYSICAL AND CHEMICAL PROPERTIES OF NO ............................................................17 NITRIC OXIDE AS REGULATOR MOLECULE IN THE HEART .........................................18 NO SYNTHESIS – THE NOSS .........................................................................................................................................18 NO DEPENDENT SIGNALING PATHWAYS ......................................................................................................................20 HEART FAILURE AND NO ...............................................................................................................................................22 HEART FAILURE MODEL (INOS+/MYO-/-) MICE ...........................................................................................................23 MASS SPECTROMETRY IN PROTEOMICS ...........................................................................25 PRINCIPLE ........................................................................................................................................................................25 ION SOURCES ..................................................................................................................................................................25 MASS ANALYZER ..............................................................................................................................................................26 ANALYSIS OF PROTEIN PHOSPHORYLATION USING MASS SPECTROMETRY .................................................................28 OVERVIEW OF MS BASED QUANTIFICATION STRATEGIES ............................................................................................29 II. AIM OF THE STUDY ..........................................................................32 III. MATERIALS AND METHODS ..........................................................33 USED MATERIALS ....................................................................................................................33 CHEMICALS AND MATERIALS .........................................................................................................................................33 MS STANDARDS ...............................................................................................................................................................34 ANIMALS ..........................................................................................................................................................................34 PRIMARY AND SECONDARY ANTIBODIES .......................................................................................................................34 LABORATORY INSTRUMENTS ..........................................................................................................................................35 SOFTWARE .......................................................................................................................................................................36 LANGENDORFF-PERFUSION OF ISOLATED MOUSE HEARTS ......................................37 BIOANALYTICAL METHODS .................................................................................................37 PROTEIN ASSAY USING BICINCHONINIC ACID (BCA) .................................................................................................37 TWO DIMENSIONAL GEL ELECTROPHORESIS (2-D PAGE) .........................................................................................38 ISOELECTRICAL FOCUSING (1. DIMENSION) ..........................................................................................................38 GRADIENT SDS-PAGE (2. DIMENSION) ...............................................................................................................39 PROQ DIAMOND PHOSPHOPROTEIN STAIN ................................................................................................................40 MS COMPATIBLE SILVER STAIN ........................................................................................................................................40 COLLOIDAL COOMASSIE STAIN .....................................................................................................................................40 WESTERN BLOT ...............................................................................................................................................................41 IN GEL DIGESTION WITH TRYPSIN ..................................................................................................................................42 PREPARATION OF HEART SAMPLES FOR COMPARATIVE PHOSPHOPROTEOME ANALYSIS USING STABLE ISOTOPE DIMETHYL LABELING .........................................................................................................................................................................42 PROTEIN EXTRACTION AND FRACTIONATION FOR IN SOLUTION DIGEST ..........................................................42 INTERNAL STANDARD – A REFERENCE FOR RELATIVE QUANTITATIVE ANALYSIS ..................................................42 REDUCTION AND ALKYLATION OF CYSTEINE RESIDUES ........................................................................................43 METHANOL-CHLOROFORM PRECIPITATION OF PROTEINS ....................................................................................43 5 TABLE OF CONTENTS ETHANOL PRECIPITATION ........................................................................................................................................43 PROTEIN DIGESTION .................................................................................................................................................43 STABLE ISOTOPE DIMETHYL LABELING .....................................................................................................................43 SAMPLE CLEAN UP USING SOLID PHASE EXTRACTION ...........................................................................................44 PHOSPHOPEPTIDE ENRICHMENT USING TITANIUM DIOXIDE MICRO PARTICLES FILLED TOPTIP .........................44 PHOSPHOPEPTIDE ENRICHMENT USING CALCIUM PHOSPHATE PRECIPITATION ..................................................45 SAMPLE FRACTIONATION USING SCX FILLED TOPTIP .........................................................................................45
Load more

Recommended publications
  • Structural Insights Into the Mechanism of Inhibition of AHAS by Herbicides
    Structural insights into the mechanism of inhibition of PNAS PLUS AHAS by herbicides Thierry Lonhiennea,1,2, Mario D. Garciaa,1, Gregory Pierensb, Mehdi Moblia,b, Amanda Nouwensa, and Luke W. Guddata,2 aSchool of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, QLD 4072, Australia; and bCenter for Advanced Imaging, The University of Queensland, Brisbane, QLD 4072, Australia Edited by María-Jazmin Abraham-Juarez, University of California, Berkeley, CA, and accepted by Editorial Board Member Gregory A. Petsko January 22, 2018 (received for review August 16, 2017) Acetohydroxyacid synthase (AHAS), the first enzyme in the FAD cofactor is required for the enzyme to be active (14). The branched amino acid biosynthesis pathway, is present only in reversible accumulative inhibition described here is not equivalent plants and microorganisms, and it is the target of >50 commercial to the “reversible time-dependent inhibition” described by herbicides. Penoxsulam (PS), which is a highly effective broad- Morrison and Walsh (15). Reversible time-dependent inhibition spectrum AHAS-inhibiting herbicide, is used extensively to control generally describes a process in which the inhibitor–enzyme weed growth in rice crops. However, the molecular basis for its complex requires time to acquire a conformation in which the inhibition of AHAS is poorly understood. This is despite the avail- inhibitor is fully effective. In that mode of inhibition, reversibility ability of structural data for all other classes of AHAS-inhibiting herbicides. Here, crystallographic data for Saccharomyces cerevisiae relates to the binding alone, with the inhibitor being able to AHAS (2.3 Å) and Arabidopsis thaliana AHAS (2.5 Å) in complex dissociate from the complex.
    [Show full text]
  • An Escherichiacoli Mutant Deficient in Pyruvate Oxidase Activity Due To
    Proc. Natl. Acad. Sci. USA Vol. 81, pp. 4348-4352, July 1984 Biochemistry An Escherichia coli mutant deficient in pyruvate oxidase activity due to altered phospholipid activation of the enzyme (lipid activation/lipid binding/hydrophobic site/conformational change/proteolytic activation) YING-YING CHANG AND JOHN E. CRONAN, JR. Department of Microbiology, University of Illinois, 131 Burrill Hall, 407 South Goodwin, Urbana, IL 61801 Communicated by Ralph S. Wolfe, April 4, 1984 ABSTRACT The pyruvate oxidase (pyruvate:ferricyto- lipid activation of an enzyme occurs in vivo and is physiolog- chrome b, oxidoreductase, EC 1.2.2.2) of Escherichia coli is ically significant. markedly activated by phospholipids in vitro. To test the phys- iological relevance of this activation, we isolated an E. coil mu- MATERIALS AND METHODS tant producing an oxidase that is deficient in activation by (and binding to) phospholipids. The mutant oxidase could be Bacterial Strains, Media, and Extract Preparation. Strains fully activated by a specific proteolytic cleavage, indicating CY265 (pox') and YYC124 (poxB3) are isogenic derivatives that the catalytic site is normal. The mutant enzyme functions (4) of E. coli K-12 that carry a deletion of the aceEF (pyru- poorly in vivo, indicating that activation of the oxidase by vate dehydrogenase) genes. Strain YYC124 is a pyruvate ox- phospholipids plays an important physiological role. idase mutant derived from strain CY265 (4). The strains were grown in broth containing 10 mM sodium acetate at 330C, Enzyme activation by membrane phospholipids in vitro has and cell extracts were prepared as described (3, 4). In some often been observed (1, 2) and is generally ascribed a physio- cases, the cell-free extract was heated to 600C for 5 min and logical role.
    [Show full text]
  • Recent Progress in the Microbial Production of Pyruvic Acid
    fermentation Review Recent Progress in the Microbial Production of Pyruvic Acid Neda Maleki 1 and Mark A. Eiteman 2,* 1 Department of Food Science, Engineering and Technology, University of Tehran, Karaj 31587-77871, Iran; [email protected] 2 School of Chemical, Materials and Biomedical Engineering, University of Georgia, Athens, GA 30602, USA * Correspondence: [email protected]; Tel.: +1-706-542-0833 Academic Editor: Gunnar Lidén Received: 10 January 2017; Accepted: 6 February 2017; Published: 13 February 2017 Abstract: Pyruvic acid (pyruvate) is a cellular metabolite found at the biochemical junction of glycolysis and the tricarboxylic acid cycle. Pyruvate is used in food, cosmetics, pharmaceutical and agricultural applications. Microbial production of pyruvate from either yeast or bacteria relies on restricting the natural catabolism of pyruvate, while also limiting the accumulation of the numerous potential by-products. In this review we describe research to improve pyruvate formation which has targeted both strain development and process development. Strain development requires an understanding of carbohydrate metabolism and the many competing enzymes which use pyruvate as a substrate, and it often combines classical mutation/isolation approaches with modern metabolic engineering strategies. Process development requires an understanding of operational modes and their differing effects on microbial growth and product formation. Keywords: auxotrophy; Candida glabrata; Escherichia coli; fed-batch; metabolic engineering; pyruvate; pyruvate dehydrogenase 1. Introduction Pyruvic acid (pyruvate at neutral pH) is a three carbon oxo-monocarboxylic acid, also known as 2-oxopropanoic acid, 2-ketopropionic acid or acetylformic acid. Pyruvate is biochemically located at the end of glycolysis and entry into the tricarboxylic acid (TCA) cycle (Figure1).
    [Show full text]
  • United States Patent (19) 11 Patent Number: 4,458,686 Clark, Jr
    United States Patent (19) 11 Patent Number: 4,458,686 Clark, Jr. 45) Date of Patent: Jul. 10, 1984 (54), CUTANEOUS METHODS OF MEASURING 4,269,516 5/1981 Lubbers et al. ..................... 356/427 BODY SUBSTANCES 4,306,877 12/1981 Lubbers .............................. 128/633 Primary Examiner-Benjamin R. Padgett 75) Inventor: Leland C. Clark, Jr., Cincinnati, Ohio Assistant Examiner-T. J. Wallen 73. Assignee: Children's Hospital Medical Center, Attorney, Agent, or Firm-Wood, Herron & Evans Cincinnati, Ohio 57 ABSTRACT (21) Appl. No.: 491,402 Cutaneous methods for measurement of substrates in 22 Filed: May 4, 1983 mammalian subjects are disclosed. A condition of the skin is used to measure a number of important sub - Related U.S. Application Data stances which diffuse through the skin or are present (62) Division of Ser. No. 63,159, Aug. 2, 1979, Pat. No. underneath the skin in the blood or tissue. According to 4,401,122. the technique, an enzyme whose activity is specific for a particular substance or substrate is placed on, in or 51). Int. Cl. ................................................ A61B5/00 under the skin for reaction. The condition of the skin is 52). U.S. Cl. .................................... 128/635; 128/636; then detected by suitable means as a measure of the 436/11 amount of the substrate in the body. For instance, the (58), Field of Search ............... 128/632, 635, 636, 637, enzymatic reaction or by-product of the reaction is 128/633; 356/41, 417, 427; 422/68; 23/230 B; detected directly through the skin as a measure of the 436/11 amount of substrate.
    [Show full text]
  • Rapid Expression and Purification of 100 Nmol Quantities of Active Protein Using Cell-Free Protein Synthesis
    102 Biotechnol. Prog. 2004, 20, 102−109 Rapid Expression and Purification of 100 nmol Quantities of Active Protein Using Cell-Free Protein Synthesis Michael C. Jewett and James R. Swartz* Department of Chemical Engineering, Stanford University, Stanford, California 94305-5025 Two strategies for ATP regeneration during cell-free protein synthesis were applied to the large-scale production and single-column purification of active chloramphenicol acetyl transferase (CAT). Fed-batch reactions were performed on a 5-10 mL scale, approximately 2 orders of magnitude greater than the typical reaction volume. The pyruvate oxidase system produced 104 nmol of active CAT ina5mLreaction over the course of 5 h. The PANOx system produced 261 ( 42 nmol, about 7 mg, of active CAT in a 10 mL reaction over the course of 4 h. The reaction product was purified to apparent homogeneity with approximately 70% yield by a simple affinity chromatog- raphy adsorption and elution. To our knowledge, this is the largest amount of actively expressed protein to be reported in a simple, fed-batch cell-free protein synthesis reaction. Introduction enous acetate kinase present in the cell extract prepared from E. coli. The most notable feature of this system is Cell-free protein synthesis offers a method to exploit that phosphate released during protein synthesis is biological processes without intact cells. It is a convenient recycled into acetyl-phosphate (Figure 1A). This attribute tool for the production of active recombinant DNA (rDNA) allows for numerous additions of the energy source proteins and has several advantages over in vivo protein without inhibition of the protein synthesis reaction via expression systems (1-4).
    [Show full text]
  • Oxoglutarate Dehydrogenase Multienzyme Complexes Reconstituted from Recombinant
    *RevisedView metadata, Manuscript citation and (text similar UNmarked) papers at core.ac.uk brought to you by CORE Click here to view linked References provided by Repository of the Academy's Library Formation of reactive oxygen species by human and bacterial pyruvate and 2- oxoglutarate dehydrogenase multienzyme complexes reconstituted from recombinant components Attila Ambrusa, Natalia S. Nemeriab, Beata Torocsika, Laszlo Trettera, Mattias Nilssona, Frank Jordanb, and Vera Adam-Vizia,* aDepartment of Medical Biochemistry, MTA-SE Laboratory for Neurobiochemistry, Semmelweis University, Budapest, 1094, Hungary bDepartment of Chemistry, Rutgers, the State University, Newark, NJ 07102, USA *To whom correspondence should be addressed at: Department of Medical Biochemistry, Semmelweis University, 37-47 Tuzolto Street, Budapest, 1094, Hungary. Tel.: +361 266 2773, Fax.: +361 267 0031, E-mail: [email protected] 1 Abstract Individual recombinant components of pyruvate and 2-oxoglutarate dehydrogenase multienzyme complexes (PDHc, OGDHc) of human and Escherichia coli (E. coli) origin were expressed and purified from E. coli with optimized protocols. The four multienzyme complexes were each reconstituted under optimal conditions at different stoichiometric ratios. Binding stoichiometries for the highest catalytic efficiency were determined from the rate of NADH generation by the complexes at physiological pH. Since some of these complexes were shown to possess ‘moonlighting’ activities under pathological conditions often accompanied by acidosis, activities were also determined at pH 6.3. As reactive oxygen species (ROS) generation by the E3 component of hOGDHc is a pathologically relevant feature, superoxide generation by the complexes with optimal stoichiometry was measured by the acetylated cytochrome c reduction method in both the forward and the reverse catalytic directions.
    [Show full text]
  • Pyruvate Oxidase, Its Preparation and Use
    ft-: .pr Europaisches Patentamt J European Patent Office ® Publication number: 0 274 4SS Office europeen des brevets A2 EUROPEAN PATENT APPLICATION @ Application number: 68300073.9 Int.CI.*: C 12 N 15/00 C 12 N 9/02, 0 12 Q 1/50, @ Date of filing: 06.01.88 C 12 Q 1/48, C 12 Q 1/26 Priority: 06.01.87 JP 903/87 Imamura, Shlgeyuki 15.05.87 JP 118161/87 696, Mifuku Ohito-cho Tagata-gun Shizuoka-ken (JP) Date of publication of application: 13.07.88 Bulletin 88/28 Sagai, Hitoshi 1 28-51 , Naka MiShima-shi Shizuoka-ken (JP) Designated Contracting States: DE FR IT Misaki, Hidfeo Applicant: TOYO JOZO KABUSHIKI KAISHA 774,Yosh!daOhlto-cho 632-1 Mifuku Ohito-cho Tagata-gun Shizuoka-ken (JP) tagata-gun Shizuoka-ken (JP) Nogata, Keiko 855, Yoka-machi Nirayama-cho Inventor: Matsumura, Eiji Tagata-gun Shizuoka-ken (JP) 530-1, Yoka-machi Nirayama-cho Tagate-gun Shizuoka-ken (JP) Representative : Woods, Geoffrey Corlett et at J.A. KEMP & CO. 14 South Square Gray's Inn London WC1R5EU (GB) @ Pyruvate oxidase, its preparation and use. @ A pyruvate oxidase having the ability to catalyse a reaction from pyruvate, phosphate and oxygen with thS formation of acetylphosphate, carbon dioxide and hydrogen peroxide, an ATP-ass content of below 0.0005% and substantially no lactate oxidase activity is prepared by genetic engineering techniques. A pyruvate oxidase gene is isolated from a known enzymatic source of the enzyme and used to transform a microorganism and provide a transformant which can be cultured to provide substantially pure pyruvate oxidase.
    [Show full text]
  • Protein Engineering of Pyruvate Oxidase from Lactobacillus Plantarum for Application in Biosensors
    Research Collection Doctoral Thesis Protein engineering of pyruvate oxidase from Lactobacillus plantarum for application in biosensors Author(s): Kramer, Matthias Publication Date: 2008 Permanent Link: https://doi.org/10.3929/ethz-a-005708619 Rights / License: In Copyright - Non-Commercial Use Permitted This page was generated automatically upon download from the ETH Zurich Research Collection. For more information please consult the Terms of use. ETH Library Doctoral Thesis ETH No. 17765 PROTEIN ENGINEERING OF PYRUVATE OXIDASE FROM LACTOBACILLUS PLANTARUM FOR APPLICATION IN BIOSENSORS A dissertation submitted to the SWISS FEDERAL INSTITUTE OF TECHNOLOGY ZURICH for the degree of Doctor of Natural Sciences presented by MATTHIAS KRAMER Eidg. dipl. Apotheker, ETH Zürich born 25.05.1975 citizen of Full-Reuenthal AG deposited to examination Prof. Dr. G. Folkers, examiner Prof. Dr. L. Scapozza, co-examiner Prof. Dr. U. Spichiger-Keller, co-examiner Prof. Dr. A. Schubiger, co-examiner 2008 Für meine Familie “Nicht im Wissen liegt das Glück, sondern im Erwerben von Wissen.“ Edgar Allen Poe Table of Contents Table of Contents I Abbreviations V Summary IX Zusammenfassung XI 1 Introduction 1 1.1 The Aim of the Work...................................................................................................... 3 1.1.1 Working Hypothesis ...................................................................................... 3 1.2 Phosphate Analysis ........................................................................................................
    [Show full text]
  • Introduction to Enzymes What Is Enzymes?
    Introduction to Enzymes Enzyme Engineering What is enzymes? Life depends on well-orchestrated series of chemical reactions : E. coli has 4288 proteins, 2656 of which are characterized, and 64% (1701) of the characterized ones code enzymes Chemical reactions are far slow to maintain life Living system has designed catalysts to fasten the specific reactions 1 1.2 History of enzyme study Rock and key model Fig. 1.1 Demonstrated that enzymes do not require a cell Enzyme is proved to be a protein 1957, Myoglobin structure was deduced by X-ray crystallography Kendrew 1963, The first aa sequence of enzyme, ribonuclease was reported 1965, The first enzyme structure of lysozyme was reported 2 1.2 History of enzyme study 1958, “Induced fit” model was proposed, Koshland 1965, “Allosteric model” of enzyme was porposed, Monod 1969, the first chemical synthesis of an enzyme was reported, proving an enzyme is a protein Mechanisms of thousands enzymes have been studied by X-ray crystallography and NMR DNA recombinant methods were used to overproduce enzymes and to pinpoint the important amino acids 2004, the first computer designed enzyme was reported Kaplan, J. and DeGrado, W. F. (2004) Proc. Natl. Acad. Sci. USA 101, 11566-11570 3 1.2 History of enzyme study Catalytic biological molecules other than conventional enzymes Antibody RNA (Ribozyme) : Usually involved in RNA processing (phosphate ester hydrolysis)-Cech, 1986 As short as 30 nucleotide (hammerhead ribozyme)-Fig. 1.3 1.3 Properties of enzymes I. Catalytic power 17 It increases the rate as much as 10 fold It operates in moderate temperature and neutral pH (Enzymes from archeabacteria are exceptions) Extreme example is Nitrogen fixation (N2 to ammonia) 700 ~ 900K, 100 ~ 900atm with iron catalysts vs.
    [Show full text]
  • Understanding Physiology of Diseases and Cell Lines Using Omics Based Approaches
    UNDERSTANDING PHYSIOLOGY OF DISEASES AND CELL LINES USING OMICS BASED APPROACHES by Amit Kumar A dissertation submitted to Johns Hopkins University in conformity with the requirements for the degree of Doctor of Philosophy Baltimore, Maryland May, 2015 © 2015 Amit Kumar All Rights Reserved Abstract This thesis focuses on understanding physiology of diseases and cell lines using OMICS based approaches such as microarrays based gene expression analysis and mass spectrometry based proteins analysis. It includes extensive work on functionally characterizing mass spectrometry based proteomics data for identifying secreted proteins using bioinformatics tools. This dissertation also includes work on using omics based techniques coupled with bioinformatics tools to elucidate pathophysiology of diseases such as Type 2 Diabetes (T2D). Although the well-known characteristic of T2D is hyperglycemia, there are multiple other metabolic abnormalities that occur in T2D, including insulin resistance and dyslipidemia. In order to attain a greater understanding of the alterations in metabolic tissues associated with T2D, microarray analysis of gene expression in metabolic tissues from a mouse model of pre-diabetes and T2D to understand the metabolic abnormalities that may contribute to T2D was performed. This study also uncovered the novel genes and pathways regulated by the insulin sensitizing agent (CL-316,243) to identify key pathways and target genes in metabolic tissues that can reverse the diabetic phenotype. Specifically, he found significant decreases in the expression of mitochondrial and peroxisomal fatty acid oxidation genes in the skeletal muscle and adipose tissue of adult MKR mice, and in the liver of pre-diabetic MKR mice, compared to healthy mice. In addition, this study also explained the lower free fatty acid levels in MKR mice after treatment with CL-316,243 and provided biomarker genes such as ACAA1 and HSD17b4.
    [Show full text]
  • Eliminating Acetate Formation Improves Citramalate Production by Metabolically Engineered Escherichia Coli Naga Sirisha Parimi, Ian A
    Parimi et al. Microb Cell Fact (2017) 16:114 DOI 10.1186/s12934-017-0729-2 Microbial Cell Factories RESEARCH Open Access Eliminating acetate formation improves citramalate production by metabolically engineered Escherichia coli Naga Sirisha Parimi, Ian A. Durie, Xianghao Wu, Afaq M. M. Niyas and Mark A. Eiteman* Abstract Background: Citramalate, a chemical precursor to the industrially important methacrylic acid (MAA), can be syn- thesized using Escherichia coli overexpressing citramalate synthase (cimA gene). Deletion of gltA encoding citrate synthase and leuC encoding 3-isopropylmalate dehydratase were critical to achieving high citramalate yields. Acetate is an undesirable by-product potentially formed from pyruvate and acetyl-CoA, the precursors of citramalate during aerobic growth of E. coli. This study investigated strategies to minimize acetate and maximize citramalate production in E. coli mutants expressing the cimA gene. Results: Key knockouts that minimized acetate formation included acetate kinase (ackA), phosphotransacetylase (pta), and in particular pyruvate oxidase (poxB). Deletion of glucose 6-phosphate dehydrogenase (zwf) and ATP synthase (atpFH) aimed at improving glycolytic fux negatively impacted cell growth and citramalate accumulation in shake fasks. In a repetitive fed-batch process, E. coli gltA leuC ackA-pta poxB overexpressing cimA generated 54.1 g/L citramalate with a yield of 0.64 g/g glucose (78% of theoretical maximum yield), and only 1.4 g/L acetate in 87 h. Conclusions: This study identifed the gene deletions critical to reducing acetate accumulation during aerobic growth and citramalate production in metabolically engineered E. coli strains. The citramalate yield and fnal titer relative to acetate at the end of the fed-batch process are the highest reported to date (a mass ratio of citramalate to acetate of nearly 40) without being detrimental to citramalate productivity, signifcantly improving a potential process for the production of this fve-carbon chemical.
    [Show full text]
  • Pyruvate Oxidase As a Key Determinant of Pneumococcal Viability During Transcytosis
    bioRxiv preprint doi: https://doi.org/10.1101/2020.12.31.424967; this version posted January 2, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 Pyruvate oxidase as a key determinant of pneumococcal viability during transcytosis 2 across the blood-brain barrier endothelium. 3 4 AUTHORS 5 Anjali Anil1, Akhila Parthasarathy1, Shilpa Madhavan1, Kwang Sik Kim2, Anirban Banerjee1* 6 7 AFFILIATIONS 8 1Department of Biosciences and Bioengineering, Indian Institute of Technology Bombay, 9 Powai, Mumbai-400076, Maharashtra, INDIA. 10 2Division of Pediatric Infectious Diseases, School of Medicine, Johns Hopkins University, 11 Baltimore, MD, United States of America. 12 13 *CORRESPONDING AUTHOR: 14 Anirban Banerjee; E-mail: [email protected], Phone: +91-22-25767794 15 16 KEYWORDS: Streptococcus pneumoniae, blood-brain barrier, transcytosis, endocytic 17 pathway, acid tolerance response, two component system, pyruvate oxidase. 18 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.12.31.424967; this version posted January 2, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 19 ABSTRACT 20 Streptococcus pneumoniae (SPN / pneumococcus), invades myriad of host tissues following 21 efficient breaching of cellular barriers. However, strategies adopted by pneumococcus for 22 evasion of host intracellular defences governing successful transcytosis across host cellular 23 barriers remain elusive. In this study, using brain endothelium as a model host barrier, we 24 observed that pneumococcus containing endocytic vacuoles (PCVs) formed following SPN 25 internalization into brain microvascular endothelial cells (BMECs), undergo early maturation 26 and acidification, with a major subset acquiring lysosome-like characteristics.
    [Show full text]