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Engineering of the Synthetic Metabolic Pathway for Biodegradation of Environmental Pollutant

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Engineering of the Synthetic Metabolic Pathway for Biodegradation of Environmental Pollutant MASARYK UNIVERSITY FACULTY OF SCIENCE LOSCHMIDT LABORATORIES DEPARTMENT OF EXPERIMENTAL BIOLOGY Engineering of the synthetic metabolic pathway for biodegradation of environmental pollutant Doctoral dissertation Pavel Dvořák Supervisors: Prof. Mgr. Jiří Damborský, Dr. Doc. RNDr. Zbyněk Prokop, Ph.D. BRNO 2014 Poděkování Na tomto místě bych chtěl poděkovat svému školiteli Jiřímu Damborskému za profesionální vedení, schopnost a odhodlání dělat kvalitní, nepodlézavou vědu, ochotu setkávat se, diskutovat a radit, která není ani mezi školiteli postgraduálních studentů zcela automatická, a konečně za jeho víru ve zdárné konce, která mě dovedla až k sepsání této práce. Velmi děkuji i Zbyňku Prokopovi, mému školiteli specialistovi, za jeho optimismus a cenné rady a všem svým současným i minulým kolegům, kteří přispívali a přispívají k tomu, že Loschmidtovy laboratoře jsou nejenom špičkovým vědeckým týmem, ale i příjemným místem pro práci, kvůli kterému se člověk každé pondělní ráno rád přiměje vstát z postele. Největší dík ale patří mé ženě Monice za její lásku, přátelství, toleranci a schopnost vracet mě z badatelských výšin zpátky nohama na zem. Bibliographic entry Author: Mgr. Pavel Dvořák Loschmidt Laboratories Department of Experimental Biology Faculty of Science Masaryk University Title of dissertation: Engineering of the synthetic metabolic pathway for biodegradation of environmental pollutant Study Programme: Biology Field of study: Molecular and Cellular Biology Supervisor: Prof. Mgr. Jiří Damborský, Dr. Supervisor-specialist: doc. RNDr. Zbyněk Prokop, PhD. Year of defence: 2014 Keywords: biocatalysis; biodegradation; kinetic modelling; metabolic engineering; in vitro multi-enzyme reaction; synthetic biology; 1,2,3-trichloropropane Bibliografický záznam Autor: Mgr. Pavel Dvořák Loschmidtovy laboratoře Ústav experimentální biologie Přírodovědecká fakulta Masarykova univerzita Název disertace: Inženýrství syntetické metabolické dráhy pro biodegradaci environmentálního polutantu Studijní program: Biologie Studijní obor: Molekulární a buněčná biologie Školitel: Prof. Mgr. Jiří Damborský, Dr. Školitel specialista: doc. RNDr. Zbyněk Prokop, PhD. Rok obhajoby: 2014 Klíčová slova: biokatalýza; biodegradace; kinetické modelování; metabolické inženýrství; in vitro multi-enzymová reakce; syntetická biologie; 1,2,3-trichlorpropan Scio me nihil scire. (I know that I know nothing) Socrates Success consists of going from failure to failure without loss of enthusiasm. Winston Churchill © Pavel Dvořák, Masaryk University 2014 CONTENT MOTIVATION 1 ABSTRACT 2 ABSTRAKT 4 INTRODUCTION 7 1. Emerging technologies for engineering of metabolic pathways 7 1.1 Metabolic engineering 9 1.1.1 Strategies and tools 10 1.2 Synthetic biology 17 1.2.1 Strategies and tools 18 1.3 Protein engineering 21 1.3.1 Strategies and tools 21 1.4 Perspectives 24 2. Engineering of biodegradation pathways 25 2.1 Strategies and tools 27 2.2 Perspectives 32 3. Engineering of the synthetic metabolic pathway for biodegradation of 1,2,3 trichloropropane 34 3.1 Halogenated hydrocarbons and 1,2,3-trichloropropane 34 3.2 Overview of 1,2,3-trichloropropane pathway engineering 38 CONTRIBUTION TO THE RESULTS 47 CHAPTER 1 In vitro assembly and immobilization of the synthetic pathway for biodegradation of toxic recalcitrant pollutant 1,2,3- trichloropropane 49 SUPPLEMENTARY TABLES AND FIGURES 69 CHAPTER 2 Maximizing the efficiency of in vitro multi-enzyme process by stoichiometry optimization 79 SUPPLEMENTARY TABLES AND FIGURES 93 CHAPTER 3 Computer-assisted engineering of the synthetic pathway for biodegradation of 1,2,3-trichloropropane in heterologous host E. coli 99 SUPPLEMENTARY TABLES AND FIGURES 119 CHAPTER 4 Assembly of the synthetic pathway for biodegradation of 1,2,3-trichloropropane in Pseudomonas putida KT2440 CF1 131 SUPPLEMENTARY TABLES AND FIGURES 145 SUMMARY 147 REFERENCES 148 CURRICULUM VITAE 160 LIST OF PUBLICATIONS 162 LIST OF CONTRIBUTIONS AT CONFERENCES AND SYMPOSIA 163 Motivation MOTIVATION Nature possess a great potential to cope itself with many problems arising from continuously increasing human activity on the Earth. Among others, this potential is hidden in astonishing variability of metabolic pathways of living organisms. For example, it is not completely rare phenomenon that bacterium can adapt its metabolic traits for new substrate and break down a toxic polluting compound. However, in certain cases, the evolution is not fast enough or ends in a deadlock. Such challenges can be possibly solved by state-of-the-art tools of recently established scientific disciplines including metabolic engineering, protein engineering and synthetic biology. Still, the huge complexity of dynamic processes in living cell represents a major bottleneck for any attempts to rationally engineer natural or synthetic metabolic pathways for the purpose of biodegradation of environmental pollutants or biosynthesis of value added chemicals. The complexity can be significantly reduced by reconstructing selected multi-enzyme reactions in vitro or by assembling orthogonal metabolic modules within an organism. Such model studies help us to understand the dynamic behavior of metabolic pathways and the obtained knowledge can be step-by-step utilized for rational engineering of living systems. The objectives of the Ph.D. project and this Thesis: 1. Introduction to the fields of metabolic engineering and synthetic biology with special attention devoted to the knowledge-based engineering of biodegradation pathways; prologue to the selected model system - synthetic metabolic pathway for biodegradation of anthropogenic pollutant 1,2,3-trichloropropane. 2. In vitro reconstruction and immobilization of the model pathway. Development of the biodegradation process based on immobilized enzymes. 3. Detailed kinetic characterization of employed enzymes, development and validation of kinetic model for the pathway in vitro. 4. Optimization of the pathway in vitro by employment of suitable engineered enzymes and balancing of biocatalysts' stoichiometry using kinetic modelling. 5. Application of obtained knowledge and synthetic biology tools for rational engineering of the pathway in heterologous host Escherichia coli and dissection of the pathway bottlenecks in vivo. 6. Selection of suitable microbial host (chassis) for biodegradation of 1,2,3-trichloropropane and further evolution of the synthetic pathway. - 1 - Abstract ABSTRACT This Thesis describes the application of synthetic biology and metabolic engineering approaches for rational redesign of metabolic pathway for biodegradation of important environmental pollutant 1,2,3-trichloropropane (TCP). The emerging technologies for engineering of biosynthetic and biodegradation pathways are described in the Introduction of the Thesis and prologue to the origins of synthetic TCP route is provided. The pathway consisting of three enzymes – haloalkane dehalogenase, haloalcohol dehalogenase and epoxide hydrolase - from two different microorganisms can convert toxic TCP into harmless product glycerol but suffers from several important bottlenecks. The four studies described in the Results section of the Thesis aim at rational dissection of these handicaps, optimization of pathway performance and construction of biocatalyst utilizable for TCP removal from the contaminated sites. Chapter 1 of the Results section describes reconstruction of the model pathway in in vitro conditions and developing of a novel biotechnology for TCP transformation based on immobilized enzymes. The efficiency of the pathway was enhanced by employment of engineered haloalkane dehalogenase with improved activity toward TCP and the route was immobilized in the form of purified enzymes or cell-free extracts. The performance of the three-enzyme system was tested in batch and continuous operations. The study provides the first available report on the use of an immobilized synthetic metabolic pathway employing engineered enzyme for the biotransformation of the toxic industrial waste into desirable commodity chemical. Further improvement of the reaction efficiency can be achieved by tuning enzymes' stoichiometry. Development of the workflow for maximizing the efficiency of in vitro multi-enzyme process by stoichiometry optimization is addressed in Chapter 2. In this study, we employed kinetic modelling to maximize the efficiency of a three-enzyme system based on in vitro assembled TCP pathway. Mathematical modelling and one-pot multi-enzyme laboratory experiments provided detailed insight into pathway dynamics, enabled the selection of suitable engineered enzyme and afforded high yield of the final product glycerol, while minimizing biocatalyst loadings. The study highlights the potential of kinetic modelling for industrial biocatalysis and presents a broadly applicable strategy for optimizing multi-enzyme processes. Chapter 3 describes application of previously developed kinetic model for computer-assisted engineering of TCP pathway in heterologous host Escherichia coli. We assembled TCP route in the laboratory strain E. coli BL21 (DE3), and used it as an orthogonal biological system for thorough investigation of pathway bottlenecks in vivo. Variants of the pathway employing wild-type or engineered haloalkane dehalogenase were designed using modified mathematical model. The E. coli recombinants with optimized and non-optimized stoichiometry of pathway enzymes were constructed and characterized in terms of their viability in presence of TCP and degradation efficiency. The validated model
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