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Study of Zymomonas Mobilis Uncoupled Energy Metabolism

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Study of Zymomonas Mobilis Uncoupled Energy Metabolism University of Latvia Faculty of Biology Reinis Rutkis Doctoral Thesis Study of Zymomonas mobilis uncoupled energy metabolism Submitted for the degree of Doctor of Biology Subfield of Microbiology Supervisor: Prof. Uldis Kalnenieks Riga, 2013 ! "! The doctoral thesis was carried out in University of Latvia, Faculty of Biology, and at the Institute of Microbiology and Biotechnology. From 2006 to 2013. This work has been supported by the European Social Fund within the project «Support for Doctoral Studies at University of Latvia». The research was supported by grant 09.1306 of Latvian Council of Science and by Latvian ESF projects European Structural Fund Nr. 2009/0207/1DP/1.1.1.2.0/ 09/APIA/VIAA/128 „Latvian Interdisciplinary Interuniversity Scientific Group of Systems Biology’ and 2009/0138/1DP/1.1.2.1.2/09/IPIA/VIAA/004. The thesis contains the introduction, review of literature, materials and methods, results and discussion chapters, conclusions, theses for defence and reference list. Form of the thesis: Collection of research articles in biology, microbiology subfield Supervisor: Professor Uldis Kalnenieks, Institute of Microbiology and Biotechnology, University of Latvia, Riga, Latvia Reviewers: 1) Professor Indrikis Mui!nieks Faculty of Biology University of Latvia, 2) Professor Raivo Vilu, Department of Chemistry, Tallin University of Technology 3) Professor T"lis Juhna, Institute of Heat, Gas and Water technologies, Riga Technical University The thesis will be defended at the public session of the Doctoral Committee of Biology, University of Latvia, at 13:00, lecture hall nr. 2, on the 8th of April, 2014, at the Faculty of Biology, University of Latvia, Kronvalda Blvd 4, Riga, Latvia. The thesis is available at the Library of the University of Latvia, Kalpaka blvd. 4. This thesis is accepted for the commencement of the degree of Doctor of Biology on 24.10. 2013 by the Doctoral Committee of Biology, University of Latvia. Secretary of the Doctoral Committee _______________/Daina Eze/ Chairman of the Doctoral Committee _______________/Pauls Pump#ns/ © University of Latvia, 2013 © Reinis Rutkis, 2013 ! #! ABSTRACT Facultatively anaerobic, Gram-negative ethanol producing bacterium Z. mobilis is of great interest from a biotechnological perspective. Showing an extremely rapid catabolism, which is quite loosely matched to the needs of cellular biosynthesis, Z. mobilis is considered as a classical example of the ucoupled energy metabolism. Ucoupled energy metabolism or uncoupled growth phenomenon, is largely what makes this bacterium an outstanding ethanol producer. However, the mechanisms of uncoupled growth so far are not fully understood. Although Z. mobilis possess high respiratory capacity, the respiratory system still remains poorly understood and it does not appear to be primarily required for energy conservation. The aim of the dissertation was (I) to examine the structure and function of electron transport chain and its relation to the uncoupled growth, (II) via creating computional kinetic model of the Entner-Doudoroff pathway, to investigate the possible regulatory factors of Z. mobilis rapid catabolism. Results of the research allow to conclude that: (I) the respiratory chain of Z. mobilis contains only one functional NAD(P)H dehydrogenase and at least two electron pathways to oxygen, (II) inhibition of respiration stimulates Z. mobilis aerobic growth due to reduction of toxic acetaldehyde in the media, (III) one of the functions of Z. mobilis respiratory chain might be to prevent endogenous oxidative stress, (IV) The absence of oxidative phosphorylation activity in aerobically growing Z. mobilis primarily results from insufficient degree of energy coupling + between the proton-motive force and the F0F1 type H -dependent ATPase, (V) metabolic control analysis revealed that the majority of flux control resides outside the Entner-Doudoroff pathway, suggesting that glycolytic flux during the uncoupled growth is largely controled by ATP-consuming reactions, (VI) Increase of ATP- consuming reactions within physiological capacity, making growth more uncoupled, might serve as the appropriate strategy to increase the glycolytic flux in Z. mobilis. Work was performed at the Institute of Microbiology and Biotechnology, University of Latvia. ! $! KOPSAVILKUMS Fakultat$vi anaerobajai, Gram-negat$vai, etanolu produc#jo%ai bakt#rijai Z. mobilis ir noz$m$gs biotehnolo&isks potenci"ls. Uzr"dot augstu no biosint#zes neatkar$gu katabolisma "trumu, Z. mobilis bakt#rijas ir uzskat"mas par tipisku atj'gt" ener&#tisk" metabolisma piem#ru. Liel" m#r" tie%i atj'gtais ener&#tiskais metabolisms vai citiem v"rdiem, atj'gt"s aug%anas fenomens padara %o bakt#riju par potenci"li izcilu etanola producentu. Neskatoties uz to, atj'gt"s aug%anas mehanismi l$dz %im ir nepiln$gi izp#t$ti. Turkl"t, lai ar$ Z. mobilis piem$t akt$va elpo%anas (#de, t"s prim"r" loma l$dz galam nav skaidra un visdr$z"k nav saist"ma ar ener&ijas ra!o%anu. )$s disert"cijas m#*(i bija (I) izp#t$t Z. mobilis elpo%anas (#des struktur"l"s un funkcion"l"s $patn$bas un t"s saist$bu ar atj'gto aug%anu, (II) konstru#t Entnera- Dudorova ce+a kin#tisko datormodeli un ar t" pal$dz$bu skaidrot Z. mobilis strauj" katabolisma galvenos regulatoros faktorus. Darb" ieg'tie rezult"ti +auj secin"t, ka: (I) Z. mobilis elpo%anas (#de satur vienu funkcion"li akt$vu NAD(P)H dehidrogen"zi un vismaz divus elektronu transporta atzarus uz sk"bekli, (II) pateicoties zem"kai acetaldeh$da koncentr"cijai vid#, elpo%anas (#des inhib#%ana stimul# Z. mobilis aerobo aug%anu, (III) viena no elpo%anas (#des funkcij"m iesp#jams ir saist"ma ar Z. mobilis aizsardz$bu pret endog#no oksidat$vo stresu, (IV) Oksidat$v"s fosforil#%anas aktivit"tes tr'kums aerobi augo%"s Z. mobilis %'n"s ir skaidrojams ar nepietiekamu + protondzin#jsp#ka un F0F1 tipa H -atkar$g"s ATF"zes ener&#tisku saj'g%anu, (V) Z. mobilis glikol$zes "trums tiek kontrol#ts ne tikai E-D ce+", bet gan galvenok"rt ir atkar$gs no ATF pat#ri,a, kas atj'gt"s aug%anas gad$jum" da+#ji tiek nodro%in"ts ar + F0F1 tipa H -atkar$g"s ATF"zes pal$dz$bu, (VI) ATP pat#r#jo%o reakciju aktivit"tes palielin"%ana Z. mobilis fiziolo&isk"s kapacit"tes robe!"s var kalpot k" efekt$v"k" strat#&ija glikol$zes "tuma palielin"%anai E-D ce+". Darbs ir izstr"d"ts Latvijas Universit"tes Mikrobiolo&ijas un Biotehnlo&ijas instit't". ! %! TABLE OF CONTENTS ABSTRACT . 3 KOPSAVILKUMS . 4 TABLE OF CONTENTS . 5 ABBREVIATIONS . 7 INTRODUCTION . 8 1 LITERATURE REVIEW 1.1 Characteristics of Z. mobilis . 9 1.2 Z. mobilis central metabolism . 9 1.3 Uncoupled metabolism and the Entner-Doudoroff pathway . 12 1.4 Z. mobilis aerobic metabolism and respiratory chain . 14 1.5 Biotechnology and metabolic engineering of Z. mobilis . 15 1.6 Modeling of Z. mobilis central metabolism . 16 2 MATERIALS AND METHODS 2.1 Bacterial strains, media, cultivation and membrane vesicles 2.1.1 Bacterial strains . 19 2.1.2 Media . 19 2.1.3 Cultivation . 19 2.1.4 Preparation of cell-free extracts and membrane vesicles . 20 2.2 Analytical methods 2.2.1 Enzymatic assays . 20 2.2.2 Determination of glucose and ethanol . 21 2.2.3 Monitoring of pO2 and pCO2 . 21 2.2.4 Determination of H2O2 production . 21 2.2.5 Determination of protein concentration . 21 2.2.6 Cell concentration . 22 2.2.7 Cytochrome spectroscopy . 22 2.2.8 FT-IR analysis . 22 2.4 Genetic engineering of mutant strains 2.4.1 DNA extraction and purfication . 23 2.4.2. PCR . 23 2.4.3 qRT- PCR . 24 2.4.4 Cloning techniques . 25 2.5 Metabolic modeling 2.5.1 System characteristics, simplifying assumptions and moieties . 26 ! &! 2.5.2 Enzyme kinetics . 28 2.5.3 Parameter optimisation . 28 2.5.4 Quantifying the flux control . 30 2.5.5 Quantifying ATP homoeostasis . 31 3. RESULTS 3.1 NADH dehydrogenase deficiency results in low respiration rate and improved aerobic growth of Zymomonas mobilis . 33 3.2 Electron transport and oxidative stress in Zymomonas mobilis respiratory mutants . 41 3.3 Application of FT-IR spectroscopy for fingerprinting of Zymomonas mobilis respiratory mutants . 55 3.4 The inefficient aerobic energetics of Zymomonas mobilis: identifying the bottleneck . 63 3.5 Kinetic modeling of Zymomonas mobilis Entner-Doudoroff pathway: insights into control and functionality . 73 4 DISCUSSION 4.1 Structure and function of the respiratory chain . 91 4.2 Aerobic growth of the respiratory mutant strains . 94 4.3 The physiological role of acetaldehyde . 95 4.4 The physiological role of Z. mobilis H+-dependent ATPase . 96 4.5 Kinetic modeling of the E-D pathway . 97 4.6 Do energy-dissipating reactions control glycolytic flux in E-D pathway? 99 4.7 Putative energy dissipating mechanisms . 100 4.8 Approaches to increase of the glycolytic flux in Z. mobilis E-D pathway 101 5 CONCLUSIONS . 103 6 MAIN THESIS FOR DEFENCE . 104 7 LIST OF ORIGINAL PUBLICATIONS . 105 8 APPROBATION OF THE RESEARCH 8.1 International . 106 8.2 National . 107 9 ACKNOWLEDGEMENTS . 108 10 REFERENCES . 109 11 APPENDIX . 121 ! '! ABBREVIATIONS ADH Alcohol dehydrogenase ACET Acetaldehyde AK Adenylate kinase ATPcons ATP consuming reactions A.U. Arbitrary units bPG 1,3-bisphosphoglycerate DCCD Dicyclohexylcarbodiimide E-D pathway Entner-Doudoroff pathway EMP pathway Embden Meyerhof Parnas pathway ENO Enolase ETOHcy Cytoplasmic ethanol ETOHex Extracellular ethanol ETOHexp Ethanol transport FT-IR Fourier transform infrared GAP Glyceraldehyde 3-phosphate GAPD Glyceraldehyde 3-phosphate dehydrogenase GF Glucose facilitator GK Glucokinase GLUCcy Cytoplasmic glucose GLUCex Extracellular glucose
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