PhD Thesis Modelling of Metabolic Pathways of Saccharopolyspora erythraea Using Flux Balance Analysis A Thesis submitted to the University of London for the degree of Doctor of Philosophy By Colin Mark Jaques March 2004 The Advanced Centre for Biochemical Engineering Department of Biochemical Engineering University College London Torrington Place London WC1E 7JE United Kingdom Colin Jaques 1 UMI Number: U602593 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. Dissertation Publishing UMI U602593 Published by ProQuest LLC 2014. Copyright in the Dissertation held by the Author. Microform Edition © ProQuest LLC. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code. ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 PhD Thesis Soli Deo Gloria Colin Jaques 2 PhD Thesis Abstract The objective of this thesis is to use metabolic modelling techniques to investigate primary and secondary metabolism in S. erythraea and from this to identify key factors controlling flux distribution during secondary metabolism. S. erythraea is a member of the actinomycetes a group of bacteria responsible for the production of a number of commercially important small molecules. Actinomycete physiology is considerably more complicated than that seen in "simple" bacteria such as E. coli. The conjecture investigated in this thesis is that metabolic modelling techniques that take into account this extra complexity should be more useful in designing strategies for overproduction of desired metabolites than simpler models. The thesis gives the first detailed description of the dynamic changes in biomass composition seen during the batch cultivation of S. erythraea. It further shows that incorporation of this information into a flux balance model of the organism’s metabolism significantly improves the flux distributions generated especially in the stationary phase. Using this improved technique growth phase and stationary phase metabolism are investigated. Some of the unusual stationary phase behaviour is shown to be the result of glucose uptake being independent of demand. Rigid control of branch points in the metabolic network is not found suggesting that the organism’s metabolism is flexible. A reverse metabolic engineering strategy is applied, two variants of the wild type organism are compared with an industrial strain. The industrial strain is found to have a considerably lower glucose uptake rate than the parental strain. The relationship between TCA cycle flux, oxidative phosphorylation and organic acid secretion is investigated using an uncoupler. This project demonstrates that applied correctly flux balance analysis is a powerful tool for investigating actinomycete physiology. The insights gained are of direct relevance to the commercial production of secondary metabolites in S. erythraea. Colin Jaques 3 PhD Thesis Acknowledgements It is impossible in this day and age to pursue cutting edge science and engineering without a large amount of help. I have received financial, material, intellectual and practical support from many sources and I wish to take this opportunity to thank those who supported me. I am indebted to my supervisors, Yuhong Zhou, Frank Baganz and John Ward for setting up the opportunity for me to study S. erythraea in this way. All their time, thoughts and direction over the course of the project have proved invaluable. I also have much to be thankful for in the officemates I have had in the cloisters office over my time at UCL, with them I have batted back and forth many of the ideas developed in this thesis. Particular thanks go to Misti Ushio for help with modelling and physiology, to Preben Krabben for his willingness to share his huge knowledge of the biochemistry of actinomycetes and to help out with the practical work, to Helen Irvine for help with identifying fumarate and to Jake Hodgson for his general enthusiasm for discussion. Unseen but not unappreciated were James Jameson’s all night sampling efforts when he could have been enjoying his holidays. I am extremely grateful for the financial support I have received from the BBSRC without which the project could not have taken place. Their generous provision has enabled me to live comfortably and has provided me access to first class biochemical engineering research facilities. The quality of these facilities is also in no small part due to the continued determination of the department and the academic staff in pursuing cutting edge research. Thanks are also due to Clive Osborne, Billy Doyle and Ian Buchanan who maintain these facilities. On a more personal note thanks are due to my parents who brought me up to be interested in and to think about the world around me, who taught me to fear the Lord and spoke to me the words of eternal life. More recently they have had to put up with my absent presence as I spent several months with them writing at my desk in the attic. Finally I would like to thank my fiancee Grace who will shortly become my helper, ally, encourager, confidant and companion and who has provided great motivation to finish writing quickly before our wedding. Colin Jaques 4 PhD Thesis Table of Contents 1 INTRODUCTION............................................................................................................................................... 12 1.1 System U nder Investigation ....................................................................................................................13 1.1.1 Introduction............................................................................................................................................. 13 1.1.2 Saccharopolyspora erythraea ............................................................................................................. 14 1.1.3 Polyketides .............................................................................................................................................16 1.2 Synthesis of Erythromycin .................................................................................................................... 18 1.2.1 Macrolactone Biosynthesis ................................................................................................................. 18 1.2.2 Deoxysugar Biosynthesis .....................................................................................................................23 1.2.3 Synthesis of the Red Pigment ...................................... 27 1.3 M etabolic Engineering of P olyketide Biosynthesis .................................................................... 28 1.3.1 Introduction............................................................................................................................................. 28 1.3.2 Manipulating Chain Length................................................................................................................29 1.3.3 Changing the Priming Unit ..................................................................................................................29 1.3.4 Changing the Extender Unit. ............................................................................................................... 29 1.3.5 Changing the Reduction Level............................................................................................................30 1.3.6 Manipulating the Stereochemistry.................................................................................................... 30 1.3.7 Hybrid PKS and Nonribosomal Peptide Systems ...........................................................................31 1.3.8 Post PKS Modifications .......................................................................................................................31 1.3.9 Changing Deoxysugar Groups............................................................................................................31 1.3.10 Combinatorial Biosynthesis ................................................................................................................ 32 1.3.11 Problems with Fusing Proteins .......................................................................................................... 32 1.3.12 Interspecies PKS Transfer................................................................................................................... 33 1.3.13 Conclusions.............................................................................................................................................33 1.4 M etabolic M o d e l l in g ;................................................................................................................... 34 1.4.1 Introduction to Metabolic Modelling ................................................................................................34 1.4.2 Kinetic Models ....................................................................................................................................... 34 1.4.3 Flux balance Analysis ...........................................................................................................................36 1.4.4 Metabolic Network Analysis ................................................................................................................41