Application of metabolic flux and transcriptome analyses to understanding the physiology of engineered Geobacillus thermoglucosidasius Charlotte Emily Ward Imperial College London, Department of Life Sciences 2014 Thesis submitted for the degree of Doctor of Philosophy of Imperial College London The work in this thesis is my own and has not been submitted previously for any award. All other sources of information used in this document have been acknowledged appropriately. 1 The copyright of this thesis rests with the author and is made available under a Creative Commons Attribution Non-Commercial No Derivatives licence. Re- searchers are free to copy, distribute or transmit the thesis on the condition that they attribute it, that they do not use it for commercial purposes and that they do not alter, transform or build upon it. For any reuse or redistribution, researchers must make clear to others the licence terms of this work 2 Abstract Geobacillus thermoglucosidasius has been identified as an organism capable of producing bio- ethanol from lignocellulosic biomass based on its ability to ferment both hexose and pentose sugars. Engineering of the wild-type strain DL33 (wt) has produced a single knock out strain DL44 (∆ldh) and a double knock out strain DL66 (∆ldh∆pfl"pdh), both of which have increased capacity for bioethanol production. The nutritional requirements of the strains under anaerobic conditions are yet to be fully understood. In this study, a systems approach to understanding the metabolism of the wild-type and engi- neered strains has been taken in order to further understand the changes in metabolism resulting from the mutations introduced. For the first time 13C-metabolic flux analysis has been applied to the comparative study of the wild-type and engineered strains using global isotopomer balancing. This has revealed flux through the anaplerotic reactions has reversed from being in the direction of pyruvate / phosphoenolpyruvate in the wild-type, to being in the direction of oxaloacetate / malate in the engineered strains. Alterations in TCA cycle flux between the strains were also seen. Furthermore alanine was found to be produced as a fermentation product in each strain. Analysis of the genome sequence has revealed an unusual oxidative branch of the pentose phosphate pathway, missing 6-phosphogluconolactonase but with genes encoding the rest of the pathway still present, suggesting that flux through this pathway may still proceed, dependent on the themolability of glucono-1,5-lactone-6-phosphate. It has been found that RNA extracted from G. thermoglucosidasius is prone to rapid degrada- tion which may affect the outcome of analysis of the transcriptome by RNA-seq. Nonetheless, it has been possible to apply RNA-seq to the wild-type organism grown aerobically and use this to identify transcripts for the major pathways of central carbon metabolism and the most highly expressed transcripts of the culture. Acknowledgements My initial thanks go to Professor David Leak for providing the opportunity for me to carryout this work and for his continued support and guidance throughout the duration of the project. I would also like to thank Professor Peter Nixon for ac- cepting me as a student in his lab with David Leak’s departure to the University of Bath. My thanks also go to TMO Renewables for their funding, support and assistance during the project, as well as to the BBSRC for funding this project. Thank you to Mr. James Mansfield for all his help with bioreactors, Dr. Jeremy Bartosiak-Jentys for general guidance, Dr. Volker Brehends for help with GC-MS and Matlab and Dr. Shyam Masakapalli for all his help and advice regarding metabolic flux analysis. I would like to thank my colleagues in the Leak and Nixon labs whose support and camaraderie made such a great environment in which to carry out this project. I would especially like to thank Rochelle and Jianfeng who have been great friends and provided invaluable help through scientific discussions. Finally, I would like to thank my Mum, Dad, Jeremy, family and friends who have provided me with so much support and encouragement over the years. 1 Contents List of Figures 10 List of Tables 15 1 Introduction 22 1.1 Systems biology . 22 1.1.1 What is systems biology? . 22 1.1.2 Application of systems biology in metabolic engineering of micro-organisms . 23 1.1.3 Genomic analysis . 27 1.1.4 Transcriptomic analyses . 27 1.1.4.1 Microarray analysis . 28 1.1.4.2 RNA-seq . 29 1.1.4.3 Understanding the prokaryotic transcriptome . 30 1.1.5 Proteomic analyses . 31 1.1.5.1 Mass spectrometry based protein profiling . 31 1.1.6 Metabolic analysis . 32 1.1.6.1 Metabolomics . 32 1.1.7 Fluxomic analyses . 33 1.1.7.1 Flux Balance Analysis . 33 1.1.7.2 Steady state 13C Metabolic flux analysis . 34 2 1.1.7.3 Variants on steady-state metabolic flux analysis . 34 1.2 13C Metabolic flux analysis . 36 1.2.1 Experimental methods in metabolic flux analyis . 38 1.2.1.1 Organism cultivation . 38 1.2.1.2 Isotopomer measurement . 40 1.2.1.3 External metabolite quantification . 40 1.2.1.4 Biomass analysis . 41 1.2.1.5 Genetic analysis . 41 1.2.2 Computational methods in metabolic flux analysis . 41 1.2.2.1 Flux ratio analysis . 42 1.2.2.2 Global isotopomer balancing . 42 1.2.3 Tools for metabolic flux analysis . 45 1.2.4 Creating a model for 13C-MFA global isotopomer balancing 46 1.3 The Genus Geobacillus ....................... 48 1.3.1 Applications of Geobacillus spp. 48 1.3.2 Geobacillus thermoglucosidasius .............. 49 1.3.3 Engineering for bioethanol production . 51 1.4 Aims and objectives . 52 2 Materials and methods 53 2.1 Storage and maintenance of strains . 53 2.1.1 Strains used in this study . 53 2.1.2 Frozen glycerol stocks . 53 2.1.3 Growth of bacterial strains on agar plates . 53 2.1.4 Sterilisation . 54 2.1.5 Centrifugation of bacterial cultures . 54 2.1.6 Optical density analysis . 55 2.2 Media used in this study . 55 3 2.2.1 Tryptone glycerol pyruvate (TGP) medium . 55 2.2.2 Ammonium salts medium (ASM) . 55 2.3 Bacterial cultivation . 56 2.3.1 Cultivation in shake flasks and Hungate tubes . 56 2.3.2 Cultivation in bioreactors . 56 2.3.2.1 1.5 Litre chemostat cultivations . 56 2.3.2.2 Small scale chemostat cultivations . 57 2.4 Molecular biology techniques . 59 2.4.1 Storage of RNA and DNA . 59 2.4.2 Polymerase chain reaction . 59 2.4.3 Primers . 59 2.4.4 Agarose gel electrophoresis . 59 2.4.5 Isolation of genomic DNA . 60 2.4.6 RNA stabilisation . 60 2.4.7 RNA extraction . 61 2.4.7.1 Qiagen RNeasy Bacteria Mini Kit . 61 2.4.7.2 Ambion mirVana miRNA Isolation kit . 61 2.4.7.3 Ambion RiboPure Bacteria kit . 61 2.4.7.4 Extraction in TNS-PAS buffer . 61 2.4.7.5 Hot phenol RNA extraction . 62 2.4.8 DNase treatment of RNA . 63 2.4.9 RNA quality assessment . 63 2.5 Genome sequencing . 63 2.5.1 Genome annotation . 64 2.5.2 Genomic alignment and analysis . 64 2.6 RNA-seq . 64 2.7 Chromatographic techniques . 65 4 2.7.1 High performance liquid chromatography analysis (HPLC) analysis of sugars, organic acids and ethanol . 65 2.7.2 High performance liquid chromatography analysis (HPLC) analysis of amino acids . 66 2.7.3 Gas chromatography mass spectrometry (GC-MS) analy- sis of intracellular proteinogenic amino acids . 66 2.7.3.1 Culture sampling . 66 2.7.3.2 Cell hydrolysis . 66 2.7.3.3 Derivatization of proteinogenic amino acids . 66 2.7.3.4 Gas chromatography mass spectrometry . 67 2.7.4 Gas chromatography mass spectrometry (GC-MS) analy- sis of extracellular compounds . 67 2.7.4.1 Culture sampling . 68 2.7.4.2 Freeze drying . 68 2.7.4.3 Derivatization . 68 2.7.4.4 GC-MS analysis . 68 2.8 Computational methods . 69 2.8.1 Correction for naturally occurring isotopes . 69 2.8.1.1 FiatFlux . 69 2.8.1.2 IsoCor . 69 2.8.1.3 Matlab MS Correction Tool . 70 2.8.2 Model construction . 70 2.8.3 Model simulation . 71 2.8.4 Linear flux fitting procedure . 71 2.8.5 Multiple simulations flux fitting proceedure . 71 2.8.6 Statistical analysis of flux distribution . 72 5 3 Sequencing of the DL33 (wt) genome and defining conditions for 13C metabolic flux analysis 73 3.1 Introduction . 73 3.2 Sequencing of the genome of wild-type DL33 . 74 3.2.1 Isolation of genomic DNA . 74 3.2.2 Genome sequencing . 75 3.2.3 Annotation and curation . 75 3.2.4 Features of the DL33 genome . 76 3.2.5 Contig alignment against an assembled Geobacillus ther- moglucosidasius genome . 80 3.3 Anaerobic growth of G. thermoglucosidasius and nutritional re- quirements . 81 3.4 Defining conditions for aerobic and non aerobic growth in 13C metabolic flux analysis studies . 85 3.5 Assessment of amino acid requirements . 89 3.6 Discussion . 92 4 Isotopic labelling of Geobacillus thermoglucosidasius using [U-13C] glucose 95 4.1 Introduction . 95 4.2 Isotope labelling experiments . 96 4.3 GC-MS analysis of proteinogenic amino acids . 96 4.4 Mass isotopomer fragment validation and extent of labelling analysis 99 4.4.1 Correction for naturally occurring isotopes . 99 4.4.1.1 FiatFlux . 100 4.4.1.2 IsoCor . 101 4.4.1.3 Matlab MS Correction Tool .