Enhancing Polyhydroxyalkanoate Biosynthesis in Escherichia Coli

Total Page:16

File Type:pdf, Size:1020Kb

Enhancing Polyhydroxyalkanoate Biosynthesis in Escherichia Coli SUNY College of Environmental Science and Forestry Digital Commons @ ESF Dissertations and Theses Spring 4-15-2020 Enhancing polyhydroxyalkanoate biosynthesis in Escherichia coli: A genetic engineering and process optimization approach towards functionalized polymeric nanomedicine Ryan Scheel SUNY College of Environmental Science and Forestry, [email protected] Follow this and additional works at: https://digitalcommons.esf.edu/etds Part of the Environmental Engineering Commons Recommended Citation Scheel, Ryan, "Enhancing polyhydroxyalkanoate biosynthesis in Escherichia coli: A genetic engineering and process optimization approach towards functionalized polymeric nanomedicine" (2020). Dissertations and Theses. 161. https://digitalcommons.esf.edu/etds/161 This Open Access Dissertation is brought to you for free and open access by Digital Commons @ ESF. It has been accepted for inclusion in Dissertations and Theses by an authorized administrator of Digital Commons @ ESF. For more information, please contact [email protected], [email protected]. ENHANCING POLYHYDROXYALKANOATE BIOSYNTHESIS IN ESCHERICHIA COLI: A GENETIC ENGINEERING AND PROCESS OPTIMIZATION APPROACH TOWARDS FUNCTIONALIZED POLYMERIC NANOMEDICINE By Ryan A. Scheel A dissertation submitted in partial fulfillment of the requirements for the Doctor of Philosophy Degree State University of New York College of Environmental Science and Forestry Syracuse, New York April 2020 Department of Chemistry Approved by: Christopher T. Nomura, Major Professor John C. Stella, Chair, Examining Committee Avik P. Chatterjee, Department Chair S. Scott Shannon, Dean, The Graduate School Acknowledgements The ESF community has played a pivotal role in shaping me into the person I am today; I have discovered new passions, formed many incredible friendships, and learned so much about the environment from many different angles. There are too many people here who have touched my life to properly thank you all, so to the ESF community at large: thank you for helping me become the person I want to be. May you continue to inspire future generations! The completion of this dissertation would not have been possible without the support of my committee members, Dr. Chris Nomura, Dr. Art Stipanovic, and Dr. Lee Newman. Thank you for your insight and guidance, as well as your patience throughout the research process. I am especially grateful to my advisor Dr. Chris Nomura. You have taught me what it means to be a good scientist. I would also like to extend my sincere thanks to Dr. Erica Majumder. Although our time together at ESF was brief, I truly value the scientific, professional, and personal insight you offered me in the final year of my research. I look forward to seeing the great things you will accomplish here at ESF. To my colleagues in the Nomura Research Group, thank you for both your friendship and your endless assistance in the lab. I’m extremely grateful to Dr. Ben Lundgren, Dr. Zaara Sarwar, and Dr. Ata Pinto; thank you for devoting your time to training me in all manner of lab techniques, and for the research opportunities within your own projects that you offered me. I would also like to thank all the undergraduates who worked with me on my research. You allowed me to become a better mentor, and your work in the lab was instrumental to this research. I am particularly indebted to Sarah Alam el Din and Seamus McKenney, whose work laid the foundations for the research on nanoparticles and high-density fermentation, respectively. And finally, to my family. To my mother, Carla: for always teaching and supporting me, especially when I didn’t appreciate it as you deserved. To my grandmother, Marcia: for instilling scientific curiosity in me for as long as I can remember. To my partner, Megan Gallagher: for your encouragement and your inspiring work ethic, which impresses me to this day. And to my best friend, Ryan Crandall: for your enduring friendship, your endless enthusiasm, and for teaching me nearly everything I know about bugs. ii Table of Contents List of Tables ................................................................................................................................. vi List of Figures ............................................................................................................................... vii List of Appendices ......................................................................................................................... ix Abstract ......................................................................................................................................... xii Chapter 1: Introduction and literature review for polyhydroxyalkanoates ..................................... 1 1.1 Introduction to polyhydroxyalkanoates ............................................................................ 1 1.2 PHA composition and physical properties ....................................................................... 2 1.3 PHA biosynthesis in native producers ............................................................................. 7 1.4 PHA biosynthesis in Escherichia coli LSBJ .................................................................... 9 1.4.1 Engineering of E. coli LS5218.................................................................................. 9 1.4.2 SCL and MCL biosynthesis pathways .................................................................... 10 1.4.3 Transcriptional regulation of MCL PHA biosynthesis pathway............................. 13 1.5 Recent developments in the biosynthesis of chemically modifiable PHAs ................... 28 1.5.1 Fluorination of the PHA α-carbon .......................................................................... 29 1.5.2 Development and biosynthesis of azido-PHAs....................................................... 35 1.5.3 Biosynthesis and modifications of unsaturated PHAs ............................................ 40 1.6 Objectives of this dissertation ........................................................................................ 42 1.7 References ...................................................................................................................... 44 Chapter 2: Enhancing poly(3-hydroxyalkanoate) production in Escherichia coli by the removal of the regulatory gene arcA........................................................................................................... 70 2.1 Abstract ............................................................................................................................... 70 2.2 Introduction ......................................................................................................................... 71 2.3 Methods ............................................................................................................................... 75 2.3.1 Materials ....................................................................................................................... 75 2.3.2 Gene deletions .............................................................................................................. 76 2.3.3 PHA production ............................................................................................................ 77 2.3.4 GC analysis ................................................................................................................... 78 2.3.5 Polymer purification and molecular weight determination .......................................... 78 2.3.6 Growth profiles of LSBJ and RSC02 ........................................................................... 79 2.3.7 RNA extraction and qPCR analysis .............................................................................. 80 2.4 Results ................................................................................................................................. 81 2.4.1 PHA homopolymer production .................................................................................... 81 iii 2.4.2 Molecular weight comparison ...................................................................................... 84 2.4.3 Growth profile of LSBJ and RSC02 ............................................................................. 85 2.4.4 PHA yield at 24 vs 48 hours ......................................................................................... 86 2.4.5 Relative gene expression of RSC02 ............................................................................. 87 2.5 Discussion ........................................................................................................................... 88 2.6 References ........................................................................................................................... 94 Chapter 3: Increased production of the value-added biopolymers poly(R-3-hydroxyalkanoate) and poly(γ-glutamic acid) from hydrolyzed paper recycling waste fines ................................... 104 3.1 Abstract ............................................................................................................................. 104 3.2 Introduction ....................................................................................................................... 105 3.3 Methods ............................................................................................................................. 106 3.3.1 Hydrolysate from waste fines ....................................................................................
Recommended publications
  • Inactivation Mechanisms of Alternative Food Processes on Escherichia Coli O157:H7
    INACTIVATION MECHANISMS OF ALTERNATIVE FOOD PROCESSES ON ESCHERICHIA COLI O157:H7 DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Aaron S. Malone, M.S. ***** The Ohio State University 2009 Dissertation Committee: Approved by Professor Ahmed E. Yousef, Adviser Professor Polly D. Courtney ___________________________________ Professor Tina M. Henkin Adviser Food Science and Nutrition Professor Robert Munson ABSTRACT Application of high pressure (HP) in food processing results in a high quality and safe product with minimal impact on its nutritional and organoleptic attributes. This novel technology is currently being utilized within the food industry and much research is being conducted to optimize the technology while confirming its efficacy. Escherichia coli O157:H7 is a well studied foodborne pathogen capable of causing diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome. The importance of eliminating E. coli O157:H7 from food systems, especially considering its high degree of virulence and resistance to environmental stresses, substantiates the need to understand the physiological resistance of this foodborne pathogen to emerging food preservation methods. The purpose of this study is to elucidate the physiological mechanisms of processing resistance of E. coli O157:H7. Therefore, resistance of E. coli to HP and other alternative food processing technologies, such as pulsed electric field, gamma radiation, ultraviolet radiation, antibiotics, and combination treatments involving food- grade additives, were studied. Inactivation mechanisms were investigated using molecular biology techniques including DNA microarrays and knockout mutants, and quantitative viability assessment methods. The results of this research highlighted the importance of one of the most speculated concepts in microbial inactivation mechanisms, the disruption of intracellular ii redox homeostasis.
    [Show full text]
  • Supplementary Material
    Lacoux et al. Dynamics of transcription initiation and RNA processing Supplementary Material Dynamic insights on transcription initiation and RNA processing during bacterial adaptation Caroline Lacoux1,#, Aymeric Fouquier d’Hérouël2, Françoise Wessner- Le Bohec1, Nicolas Innocenti1,3,#, Chantal Bohn4, Sean P. Kennedy5,#, Tatiana Rochat6, Rémy A. Bonnin4,#, Pascale Serror1, Erik Aurell3,7, Philippe Bouloc4 and Francis Repoila1,* Email adresses: CL: [email protected], AFD: [email protected], FW: [email protected], NI: [email protected], CB: [email protected], SPK: [email protected], TR: [email protected], RB: [email protected], PS: [email protected], EA: [email protected], PB: [email protected], FR: [email protected] Corresponding author : [email protected] Running head: Dynamics of transcription initiation and RNA processing This file contains: Section S1: Dynamics of RNA levels in response to σE induction (Contains Figures S1 and S2). Section S2: Assignment of 5’ RNA ends. Section S3: Figure S3. Patterns of changes in TIF at σE-TSSs assigned as UNDs or PSSs in this study. Figure S4. Examples of changes in TIF at σE-independent TSSs Section S4: RNA dynamics at the bepA-yfgD operon (Contains Figures S5) References 1 Lacoux et al. Dynamics of transcription initiation and RNA processing Section S1: Dynamics of RNA levels in response to σE induction Global view of the evolving transcriptome Our experimental system consisted of a plasmid (pZE21-rpoE) that harbor the σE encoding sequence under control of the inducible promoter PLtetO-1 in E. coli K12 strain MG1655 (Fig.
    [Show full text]
  • Influence of Glucose Availability and CRP Acetylation on The
    ORIGINAL RESEARCH published: 23 May 2018 doi: 10.3389/fmicb.2018.00941 Influence of Glucose Availability and CRP Acetylation on the Genome-Wide Transcriptional Response of Escherichia coli: Assessment by an Optimized Factorial Microarray Analysis Daniel V. Guebel 1 and Néstor V. Torres 2* 1 Biotechnology Counselling Services, Buenos Aires, Argentina, 2 Systems Biology and Mathematical Modelling Group, Department of Biochemistry, Microbiology, Cellular Biology and Genetics, Institute of Biomedical Technologies, Center for Biomedical Research of the Canary Islands, University of La Laguna, San Cristóbal de La Laguna, Spain Background: While in eukaryotes acetylation/deacetylation regulation exerts multiple Edited by: pleiotropic effects, in Escherichia coli it seems to be more limited and less known. Hence, Marie-Joelle Virolle, we aimed to progress in the characterization of this regulation by dealing with three Centre National de la Recherche convergent aspects: the effector enzymes involved, the master regulator CRP, and the Scientifique (CNRS), France dependence on glucose availability. Reviewed by: Abhijit Shukla, Methods: The transcriptional response of E. coli BW25113 was analyzed across 14 Memorial Sloan Kettering Cancer Center, United States relevant scenarios. These conditions arise when the wild type and four isogenic mutants Feng Jiang, (defective in deacetylase CobB, defective in N(ε)-lysine acetyl transferase PatZ, Q- and Chinese Academy of Medical R-type mutants of protein CRP) are studied under three levels of glucose availability Sciences, China (glucose-limited chemostat and glucose-excess or glucose-exhausted in batch culture). *Correspondence: acetylated Néstor V. Torres The Q-type emulates a permanent stage of CRP , whereas the R-type emulates [email protected] a permanent stage of CRPdeacetylated.
    [Show full text]
  • Escherichia Coli
    Escherichia coli “E. coli” redirects here. For the protozoan commensal, 1 Biology and biochemistry see Entamoeba coli. This article is about Escherichia coli as a species. For E. coli in medicine, see Pathogenic Escherichia coli. For E. coli in molecular biology, see Escherichia coli (molecular biology). Escherichia coli (/ˌɛʃᵻˈrɪkiə ˈkoʊlaɪ/;[1] also known as E. coli) is a Gram-negative, facultatively anaerobic, rod- shaped bacterium of the genus Escherichia that is com- monly found in the lower intestine of warm-blooded or- ganisms (endotherms).[2] Most E. coli strains are harm- less, but some serotypes can cause serious food poisoning in their hosts, and are occasionally responsible for product recalls due to food contamination.[3][4] The harmless strains are part of the normal flora of the gut, and can [5] benefit their hosts by producing vitamin K2, and pre- venting colonization of the intestine with pathogenic bac- teria.[6][7] E. coli is expelled into the environment within Model of successive binary fission in E. coli fecal matter. The bacterium grows massively in fresh fe- cal matter under aerobic conditions for 3 days, but its numbers decline slowly afterwards.[8] E. coli and other facultative anaerobes constitute about 0.1% of gut flora,[9] and fecal–oral transmission is the major route through which pathogenic strains of the bac- terium cause disease. Cells are able to survive outside the body for a limited amount of time, which makes them potential indicator organisms to test environmental sam- ples for fecal contamination.[10][11] A growing body of re- search, though, has examined environmentally persistent E.
    [Show full text]
  • Gene Expression Analysis of Escherichia Coli O157:H7 at 10 and 37°C and Under Acidic Conditions Using High Density Oligonucleotide Microarrays
    University of Tennessee, Knoxville TRACE: Tennessee Research and Creative Exchange Doctoral Dissertations Graduate School 12-2007 Gene Expression Analysis of Escherichia coli O157:H7 at 10 and 37°C and Under Acidic Conditions Using High Density Oligonucleotide Microarrays Kristina K. Carter University of Tennessee - Knoxville Follow this and additional works at: https://trace.tennessee.edu/utk_graddiss Part of the Food Science Commons Recommended Citation Carter, Kristina K., "Gene Expression Analysis of Escherichia coli O157:H7 at 10 and 37°C and Under Acidic Conditions Using High Density Oligonucleotide Microarrays. " PhD diss., University of Tennessee, 2007. https://trace.tennessee.edu/utk_graddiss/134 This Dissertation is brought to you for free and open access by the Graduate School at TRACE: Tennessee Research and Creative Exchange. It has been accepted for inclusion in Doctoral Dissertations by an authorized administrator of TRACE: Tennessee Research and Creative Exchange. For more information, please contact [email protected]. To the Graduate Council: I am submitting herewith a dissertation written by Kristina K. Carter entitled "Gene Expression Analysis of Escherichia coli O157:H7 at 10 and 37°C and Under Acidic Conditions Using High Density Oligonucleotide Microarrays." I have examined the final electronic copy of this dissertation for form and content and recommend that it be accepted in partial fulfillment of the requirements for the degree of Doctor of Philosophy, with a major in Food Science and Technology. David A. Golden, Major Professor We have read this dissertation and recommend its acceptance: Michael Davidson, Doris D’Souza, Todd Reynolds, Arnold Saxton Accepted for the Council: Carolyn R. Hodges Vice Provost and Dean of the Graduate School (Original signatures are on file with official studentecor r ds.) To the Graduate Council: I am submitting herewith a dissertation written by Kristina K.
    [Show full text]
  • Glucose in Escherichia Coli Involving Nupc and Nupg Nucleoside Transporters
    SUPPLEMENTARY INFORMATION Title: A cAMP/CRP-controlled mechanism for the incorporation of extracellular ADP- glucose in Escherichia coli involving NupC and NupG nucleoside transporters Authors: Goizeder Almagro1, Alejandro M. Viale2, Manuel Montero1, Francisco José Muñoz1, Edurne Baroja-Fernández1, Hirotada Mori3 and Javier Pozueta-Romero1* Supplementary Table 1: E. coli genes whose deletions caused “glycogen-deficient” or “glycogen-less” phenotypes in E. coli cells cultured in solid KM-ADPG medium. Cellular localization: OM (outer membrane), IM (inner membrane), C (cytoplasm), P (periplasm). The function of each gene for which deletion affects glycogen accumulation was identified by referring to the EchoBASE (http://ecoli-york.org/) and EcoCyc (http://www.ecocyc.org/) databases. aCellular Gene Function Localization Cyclic AMP-activated global transcription factor protein, mediator of crp C catabolite repression cya C Adenylate cyclase, cyclic AMP synthesis glgA C Glycogen synthase glgC C ADPG pyrophosphorylase High-affinity transporter of (deoxy) nucleosides except (deoxy) guanosine, nupC IM + (deoxy) inosine, and xanthosine; Nucleoside:H symporter of the Concentrative Nucleoside Transporter (CNT) family nupG IM High-affinity transporter of all natural purine and pyrimidine (deoxy) nucleosides except xanthosine; Nucleoside:H+ Symporter (NHS) family Lipoamide dehydrogenase, E3 component of pyruvate and 2-oxoglutarate lpd C dehydrogenases complexes. Lpd catalyzes the transfer of electrons to the ultimate acceptor, NAD+ rssA C Predicted phospholipase, patatin-like family. Function unknown ybjL IM Putative AAE family transporter. Function unknown. ycgB C RpoS regulon member. Function unknown yedF C predicted sulfurtransferase (TusA family). Function unknown. yegV C Predicted sugar/nucleoside kinase. yoaE IM Predicted inner membrane protein. Function unknown. Supplementary Table 2: E.
    [Show full text]
  • Platform Develops Antisense Therapies to Counter Multidrug-Resistant Bacteria
    ARTICLE https://doi.org/10.1038/s42003-021-01856-1 OPEN Facile accelerated specific therapeutic (FAST) platform develops antisense therapies to counter multidrug-resistant bacteria Kristen A. Eller1, Thomas R. Aunins1, Colleen M. Courtney1,2, Jocelyn K. Campos1, Peter B. Otoupal1, ✉ Keesha E. Erickson1, Nancy E. Madinger3 & Anushree Chatterjee1,2,4,5 Multidrug-resistant (MDR) bacteria pose a grave concern to global health, which is perpe- tuated by a lack of new treatments and countermeasure platforms to combat outbreaks or antibiotic resistance. To address this, we have developed a Facile Accelerated Specific 1234567890():,; Therapeutic (FAST) platform that can develop effective peptide nucleic acid (PNA) therapies against MDR bacteria within a week. Our FAST platform uses a bioinformatics toolbox to design sequence-specific PNAs targeting non-traditional pathways/genes of bacteria, then performs in-situ synthesis, validation, and efficacy testing of selected PNAs. As a proof of concept, these PNAs were tested against five MDR clinical isolates: carbapenem-resistant Escherichia coli, extended-spectrum beta-lactamase Klebsiella pneumoniae, New Delhi Metallo- beta-lactamase-1 carrying Klebsiella pneumoniae, and MDR Salmonella enterica. PNAs showed significant growth inhibition for 82% of treatments, with nearly 18% of treatments leading to greater than 97% decrease. Further, these PNAs are capable of potentiating antibiotic activity in the clinical isolates despite presence of cognate resistance genes. Finally, the FAST plat- form offers a novel delivery approach to overcome limited transport of PNAs into mammalian cells by repurposing the bacterial Type III secretion system in conjunction with a kill switch that is effective at eliminating 99.6% of an intracellular Salmonella infection in human epithelial cells.
    [Show full text]
  • The Whole Set of the Constitutive Promoters Recognized by Four Minor Sigma Subunits of Escherichia Coli RNA Polymerase
    RESEARCH ARTICLE The whole set of the constitutive promoters recognized by four minor sigma subunits of Escherichia coli RNA polymerase Tomohiro Shimada1,2¤, Kan Tanaka2, Akira Ishihama1* 1 Research Center for Micro-Nano Technology, Hosei University, Koganei, Tokyo, Japan, 2 Laboratory for Chemistry and Life Science, Institute of Innovative Research, Tokyo Institute of Technology, Nagatsuda, Yokohama, Japan a1111111111 ¤ Current address: School of Agriculture, Meiji University, Kawasaki, Kanagawa, Japan a1111111111 * [email protected] a1111111111 a1111111111 a1111111111 Abstract The promoter selectivity of Escherichia coli RNA polymerase (RNAP) is determined by the sigma subunit. The model prokaryote Escherichia coli K-12 contains seven species of the OPEN ACCESS sigma subunit, each recognizing a specific set of promoters. For identification of the ªconsti- Citation: Shimada T, Tanaka K, Ishihama A (2017) tutive promotersº that are recognized by each RNAP holoenzyme alone in the absence of The whole set of the constitutive promoters other supporting factors, we have performed the genomic SELEX screening in vitro for their recognized by four minor sigma subunits of binding sites along the E. coli K-12 W3110 genome using each of the reconstituted RNAP Escherichia coli RNA polymerase. PLoS ONE 12(6): holoenzymes and a collection of genome DNA segments of E. coli K-12. The whole set of e0179181. https://doi.org/10.1371/journal. pone.0179181 constitutive promoters for each RNAP holoenzyme was then estimated based on the loca- tion of RNAP-binding sites. The first successful screening of the constitutive promoters was Editor: Dipankar Chatterji, Indian Institute of 70 Science, INDIA achieved for RpoD (σ ), the principal sigma for transcription of growth-related genes.
    [Show full text]
  • Genome-Wide Analysis of Fis Binding in Escherichia Coli Indicates a Causative Role for A-/AT-Tracts
    Downloaded from genome.cshlp.org on October 1, 2021 - Published by Cold Spring Harbor Laboratory Press Letter Genome-wide analysis of Fis binding in Escherichia coli indicates a causative role for A-/AT-tracts Byung-Kwan Cho,1 Eric M. Knight,1 Christian L. Barrett, and Bernhard Ø. Palsson2 Department of Bioengineering, University of California–San Diego, La Jolla, California 92093-0412, USA We determined the genome-wide distribution of the nucleoid-associated protein Fis in Escherichia coli using chromatin immunoprecipitation coupled with high-resolution whole genome-tiling microarrays. We identified 894 Fis-associated regions across the E. coli genome. A significant number of these binding sites were found within open reading frames (33%) and between divergently transcribed transcripts (5%). Analysis indicates that A-tracts and AT-tracts are an important signal for preferred Fis-binding sites, and that A6-tracts in particular constitute a high-affinity signal that dictates Fis phasing in stretches of DNA containing multiple and variably spaced A-tracts and AT-tracts. Furthermore, we find evidence for an average of two Fis-binding regions per supercoiling domain in the chromosome of exponentially growing cells. Transcriptome analysis shows that ∼21% of genes are affected by the deletion of fis; however, the changes in magnitude are small. To address the differential Fis bindings under growth environment perturbation, ChIP-chip analysis was performed using cells grown under aerobic and anaerobic growth conditions. Interestingly, the Fis-binding regions are almost identical in aerobic and anaerobic growth conditions—indicating that the E. coli genome topology mediated by Fis is superficially identical in the two conditions.
    [Show full text]
  • Gene Regulatory Network
    computational systems biology Computational Systems Biology Regulatory networks and integrated network Hongwu Ma Computational Systems Biology Group 19 February 2010 computational systems biology Outline • The reconstruction • Network structure • Link regulatory network with metabolic network • Network Motif analysis computational systems biology Transcriptional regulation: biological basis • Transcription unit: operon • Sigma factor (rpoD, rpoS, rpoN,) RNA polymerase • Transcription factor (TF) TFs • Promotor Sigma factor • TF binding site Genes Binding site Promotor • More complex in Eukaryotes Transcription unit Interactions between proteins and genes computational systems biology Reconstruction of MN and TRN E. coli A1 B1 + mB 1.2.1.3 TF m A +? A2 B2 P. aeruginosa Are the orthologous genes in different organisms regulated in similar ways? computational systems biology Transcriptional regulatory networks • Can not be reconstructed directly from gene annotation information • data is mainly from database which collect information from literature (for E. coli and B. subtilis) • ChIP on chip technology for high throughput reconstruction (mainly yeast network) • Reverse engineering from Microarray data computational systems biology Available databases • RegulonDB http://regulondb.ccg.unam.mx/ • Ecocyc: ecocyc.org • DBTBS (B. subtilis): dbtbs.hgc.jp • Prodoric Net (Prokaryote): prodoric.tu-bs.de • TRANSFAC (Eukaryotes): www.biobase.de/pages/products/transfac.html Mainly Use E. coli as an example computational systems biology E. coli regulatory
    [Show full text]
  • Rho and Nusg Suppress Pervasive Antisense Transcription in Escherichia Coli
    Downloaded from genesdev.cshlp.org on October 1, 2021 - Published by Cold Spring Harbor Laboratory Press Rho and NusG suppress pervasive antisense transcription in Escherichia coli Jason M. Peters,1,2,5 Rachel A. Mooney,1 Jeffrey A. Grass,1,3 Erik D. Jessen,1,2 Frances Tran,1,3,6 and Robert Landick1,3,4,7 1Department of Biochemistry, 2Department of Genetics, 3Great Lakes Bioenergy Research Center, 4Department of Bacteriology, University of Wisconsin, Madison, Wisconsin 53706, USA Despite the prevalence of antisense transcripts in bacterial transcriptomes, little is known about how their synthesis is controlled. We report that a major function of the Escherichia coli termination factor Rho and its cofactor, NusG, is suppression of ubiquitous antisense transcription genome-wide. Rho binds C-rich unstructured nascent RNA (high C/G ratio) prior to its ATP-dependent dissociation of transcription complexes. NusG is required for efficient termination at minority subsets (~20%) of both antisense and sense Rho-dependent terminators with lower C/G ratio sequences. In contrast, a widely studied nusA deletion proposed to compromise Rho-dependent termination had no effect on antisense or sense Rho-dependent terminators in vivo. Global colocalization of the histone-like nucleoid-structuring protein (H-NS) with Rho-dependent terminators and genetic interactions between hns and rho suggest that H-NS aids Rho in suppression of antisense transcription. The combined actions of Rho, NusG, and H-NS appear to be analogous to the Sen1–Nrd1–Nab3 and nucleosome systems that suppress antisense transcription in eukaryotes. [Keywords: RNA polymerase; Rho-dependent transcription termination; antisense transcription; H-NS; NusG; NusA] Supplemental material is available for this article.
    [Show full text]
  • Escherichia Coli As a Host for Metabolic Engineering
    UCLA UCLA Previously Published Works Title Escherichia coli as a host for metabolic engineering Permalink https://escholarship.org/uc/item/5mz1s1pz Authors Pontrelli, Sammy Chiu, Tsan-Yu Lan, Ethan I. et al. Publication Date 2018-04-01 Peer reviewed eScholarship.org Powered by the California Digital Library University of California Metabolic Engineering xxx (xxxx) xxx–xxx Contents lists available at ScienceDirect Metabolic Engineering journal homepage: www.elsevier.com/locate/meteng Escherichia coli as a host for metabolic engineering Sammy Pontrellia, Tsan-Yu Chiub, Ethan I. Lanc, Frederic Y.-H. Chena,b, Peiching Changd,e, ⁎ James C. Liaob, a Department of Chemical and Biomolecular Engineering, University of California, Los Angeles, USA b Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan c Department of Biological Science and Technology, National Chiao Tung University, Hsinchu, Taiwan d Department of Chemical Engineering, National Tsing Hua University, Hsinchu, Taiwan e Material and Chemical Research Laboratories, Industrial Technology Research Institute, Hsinchu, Taiwan ABSTRACT Over the past century, Escherichia coli has become one of the best studied organisms on earth. Features such as genetic tractability, favorable growth conditions, well characterized biochemistry and physiology, and avail- ability of versatile genetic manipulation tools make E. coli an ideal platform host for development of industrially viable productions. In this review, we discuss the physiological attributes of E. coli that are most relevant for metabolic engineering, as well as emerging techniques that enable efficient phenotype construction. Further, we summarize the large number of native and non-native products that have been synthesized by E. coli, and address some of the future challenges in broadening substrate range and fighting phage infection.
    [Show full text]