Development of a transcriptional regulator-based bioreporter - towards a generic selection method for novel enzymes Teunke van Rossum Thesis committee Promotor Prof. Dr John van der Oost Personal Chair at the Laboratory of Microbiology Wageningen University & Research Co-promotor Dr Servé W.M. Kengen Assistant Professor at the Laboratory of Microbiology Wageningen University & Research Other members Prof. Dr Willem J.H. van Berkel, Wageningen University & Research Prof. Dr Jan Roelof van der Meer, University of Lausanne, Switzerland Prof. Dr Maarten Merkx, Eindhoven University of Technology Dr Ruud A. Weusthuis, Wageningen University & Research This research was conducted under the auspices of the Graduate School VLAG (Advanced studies in Food Technology, Agrobiotechnology, Nutrition and Health Sciences). Development of a transcriptional regulator-based bioreporter - towards a generic selection method for novel enzymes Teunke van Rossum Thesis submitted in fulfilment of the requirements for the degree of doctor at Wageningen University by the authority of the Rector Magnificus Prof. Dr A.P.J. Mol, in the presence of the Thesis Committee appointed by the Academic Board to be defended in public on Friday 14 September 2018 at 11 a.m. in the Aula. Teunke van Rossum Development of a transcriptional regulator-based bioreporter - towards a generic selection method for novel enzymes, 242 pages. PhD thesis, Wageningen University, Wageningen, the Netherlands (2018) With references, with summary in English ISBN: 978-94-6343-326-6 DOI: https://doi.org/10.18174/456660 Table of contents Chapter 1 General introduction and Thesis outline 7 Chapter 2 Reporter-based screening and selection of enzymes 21 Chapter 3 A growth- and bioluminescence-based bioreporter for 51 the in vivo detection of novel biocatalysts Chapter 4 Modifying the sensor part of a dual bioreporter to broaden 91 its target range Chapter 5 Engineering the ligand specificity of the transcriptional 121 regulator AraC and enrichment of desired variants by combined selection and screening Chapter 6 Inhibitory and stimulatory effects of L-arabinose on growth 163 of Escherichia coli BW25113 Chapter 7 Summary and General discussion 191 Appendices References 218 About the author 232 Publication list 233 Overview of completed training activities 234 Acknowledgements 236 Chapter 1 General introduction and Thesis outline Chapter 1 General introduction A brief history of enzymes Enzymes are active proteins that are required for living organisms to catalyse their biochemical reactions. They speed up a reaction by lowering the activation energy, thereby allowing the equilibrium to be reached more quickly1. More than 8000 years ago, people unknowingly already made use of enzymatic conversions via fermentation by whole-cell microorganisms to make early forms of bread and beer (Fig. 1). The first application of a cell- free enzyme was probably the use of chymosin, a protease that is part of rennet from animal stomachs, for cheese making more than 7000 years ago 2,3 . However, the elucidation of the underlying mechanisms of these processes was comparatively recent. It started in 1830, when Gerardus Johannes Mulder first mentioned the name proteins 4. Only a few years later, two major discoveries were made. Anselme Payen discovered the first enzyme, the starch degrading diastase, and Charles Cagniard de Latour discovered yeast, after being prompted into biology when the French Academy of Science promised a prize of one kilogram of gold for a solution of the mystery of fermentation. The second half of the 19 th century was characterized by the step by step unravelling of what enzymes are and how they function, as well as by the first industrial enzyme production. In 1869, Friedrich Miescher used highly impure pepsin, extracted from pig stomach, in his discovery of DNA 4,5 . Five years later, Christian Hansen marketed the first industrially produced enzymes, crude rennet 6. In 1876, Wilhelm Friedrich Kühne discovered trypsin, a substance in pancreatic juice that degraded other biological substances. One year later, he was the first person to call biological catalysts ‘enzymes’, meaning ‘in yeast’, to distinguish enzymes from the micro-organisms that produce them 1,4 . A crucial step in unravelling the mechanism of enzyme function was made by Emil Fischer in 1894. He postulated the key-lock principle, in which substrate and enzyme fit perfectly to one another, a theory 60 years later extended by the induced fit theory 7. Another major step was made three years later by Eduard Buchner, who demonstrated that fermentation was possible with an extract of yeast in the absence of intact yeast cells. In the 20 th century, enzyme research accelerated and increasingly more enzymes were discovered. In 1909, Wilhelm Johannsen introduced the term ‘gene’ for the carrier of heredity and Sir Archibald Garrod described enzyme deficiencies as cause of certain human diseases 4. In 1913, Leonor Michaelis and Maud Menten showed that the enzyme-catalysed 8 General introduction and Thesis outline <6000 BC Fermented products: beer, bread <5000 BC 1st cell-free enzyme used: rennet (chymosin) in cheese making 1 1830 Proteins 1st mentioned (Gerardus Johannus Mulder) 1833 1st enzyme discovered: diastase (Anselm Payen) 1835 Yeast discovered (Charles Cagniard de Latour ) 1850 1869 Inpure pepsin used for DNA discovery (Friedrich Miescher) 1874 1st industrially produced enzymes: rennet (Christian Hansen) 1877 Biocatalysts called ‘enzymes’ (Wilhelm Friiedrich Kühne) 1894 Key-lock prinicple (Emil Fischer) 1897 Fermentation with yeast extract instead of live yeast (Eduard Buchner) 1900 Term ‘gene’ for hereditary information (Wilhem Johanssen) 1909 + enzyme deficiencies cause human diseases (Sir Archibald Garrod) 1913 Michaelis-Menten equation (Leonor Michaelis+Maud Menten) +1st patent for enzymes in detergents (Otto Röhm) 1926 Urease crystals + enzymes are proteins (James Sumner) <1930 Pectinases used in food industry 1941 ‘One gene codes for one enzyme’ (George Beadle) 1944 DNA is carrier of heredity (Oswald Avery and others) 1950 1953 Structure of DNA (James Watson+Francis Crick) 1955 International Comission of Enzymes for enzyme nomenclature 1959 Protease 1st commercially used in detergents <1960 Lots of enzymes used industrially for starch hydrolysis 1961 Enzyme nomenclature established 1973 Recombinant DNA technology introduced (Stanley Cohen+Herbert Boyen) 1980 1st recombinant DNA company (Genentech) + PCR (Kary Mullis and others) Patent to make bleach-resistant proteases for detergents(Novo Nordisk) 1988 + 1st enzyme from genetically modified source in food industry: chymosin 1990s 1st engineered enzymes applied: subtilisins >1990s Rapid increase in number of available enzymes for industrial applica- Fig 1: Timeline of a summarized history of enzymes reaction rate was proportional to the concentration of the enzyme-substrate complex, leading to the well-known Michealis-Menten equation8. The 20 th century also dates the onset of the industrial application of enzymes with, in 1913, the first patent on pancreatic enzymes in detergents by Otto Röhm 9, and by 1930, the use of pectinases in the food 9 Chapter 1 industry for clarifying fruit juices 2. In the following decades, four important discoveries were made not only for the future of enzyme research, but more importantly for biology in general. James Sumner demonstrated the protein basis of enzymes after he crystallized urease in 1926. In 1941, George Beadle postulated ‘one gene codes for one enzyme’. In 1944, Oswald Avery and co-workers proved that DNA is the carrier of heredity 10 and in 1953, James Watson and Francis Crick unravelled the structure of DNA 11 . These discoveries form the basis of all molecular biology today. Meanwhile, more and more enzymes were discovered, leading to much confusion in enzyme names. As a result, the General Assembly of the International Union of Biochemistry (IUB) decided in 1955 to set up the first International Commission on Enzymes in order to formulate a nomenclature for enzymes. This nomenclature was established in 1961 and since then updated regularly (http://www.sbcs.qmul.ac.uk/iubmb/enzyme/). How is it structured? Enzymes are divided into six main classes based on the reaction type (Table 1) and each class with further subdivisions is indicated with an Enzyme Commission or EC number. The main classes are Oxidoreductases (EC 1), transferases (EC 2), hydrolases (EC 3), lyases (EC 4), isomerases (EC 5) and ligases (EC 6) 12 . With this nomenclature in place, much confusion like equal names for enzymes with different functions was prevented. Table 1: Main enzyme classes 12,13 EC Enzyme name Type of reaction Reaction schema number a EC 1 Oxidoreductases Transfer of electrons (or hydride ions or H Ae- + B Æ A + B e- atoms) EC 2 Transferases Transfer of a functional group A-B + C Æ A + B -C EC 3 Hydrolases Transfer of a functional group to water A-B + H 2O Æ A-OH + B-H EC 4 Lyases Addition of groups to double bonds or A-B-C-D Æ A-D + B=C formation of double bonds by removal of groups EC 5 Isomerases Transfer of groups with molecules, giving A-B Æ B-A isomers EC 6 Ligases Formation of a bond by condensation C + D + ATP Æ C-D + ADP + P i coupled to ATP cleavage aEC number is the Enzyme Commission number. In the second half of the 20 th century, increasingly more enzymes were industrially applied. In 1959, the first commercial protease in detergents was used 4 and by 1960 a lot of enzymes were used in the food industry for starch hydrolysis 2. A very important development for the industrial application of enzymes was the introduction of recombinant 10 General introduction and Thesis outline DNA technology by Stanley Cohen and Herbert Boyer in 1973. This followed the discoveries of DNA modifying enzymes, starting with DNA polymerase I by Arthur Kornberg in 1958. The first recombinant DNA company, Genentech, was founded in 1980. In the same year, Kary 1 Mullis and others developed PCR technology. In 1988, a patent was awarded to Novo Nordisk for a process to make bleach-resistant proteases for the use in detergents; in the same year, the first enzyme from a genetically-modified source, chymosin, was approved for use in the food industry 4.