FACULTY OF SCIENCE UNIVERSITY OF COPENHAGEN PhD thesis Davide Ravasio Flavour compounds in fungi Flavour analysis in ascomycetes and the contribution of the Ehrlich pathway to flavour production in Saccharomyces cerevisiae and Ashbya gossypii Academic advisor: Prof. Steen Holmberg, Department of Biology, University of Copenhagen. Co-supervisor: Prof. Jürgen Wendland, Yeast Genetics Group, Carlsberg Laboratory Submitted: 01/10/14 “There is nothing like looking, if you want to find something. You certainly usually find something, if you look, but it is not always quite the something you were after.” ― J.R.R. Tolkien, The Hobbit Institutnavn: Natur- og Biovidenskabelige Fakultet Name of department: Department of Biology Author: Davide Ravasio Titel: Flavour-forbindelser i svampe. Flavour-analyse i ascomyceter og bidrag fra Ehrlich biosyntesevejen til smagsproduktion i Saccharomyces cerevisiae og Ashbya gossypii Title: Flavour compounds in fungi. Flavour analysis in ascomycetes and the contribution of the Ehrlich pathway to flavour production in Saccharomyces cerevisiae and Ashbya gossypii Academic advisor: Prof. Steen Holmberg, Prof. Jürgen Wendland Submitted: 01/10/14 Table of contents Preface ................................................................................................................................................ 1 List of Papers ..................................................................................................................................... 2 Summary ............................................................................................................................................ 3 Resumé ............................................................................................................................................... 5 Acknowledgments .............................................................................................................................. 7 Abbreviations ................................................................................................................................... 10 1. Introduction .............................................................................................................................. 11 1.1 The Fungi Kingdom ........................................................................................................... 11 1.2 Ascomycota: The Saccharomycotina clade ...................................................................... 12 1.3 Saccharomyces cerevisiae: the model organism ............................................................ 15 1.4 Yeast carbon metabolism ................................................................................................. 18 1.5 The Eremothecium genus: Ashbya gossypii and Eremothecium cymbalariae ............ 20 Ashbya gossypii ....................................................................................................................... 21 Eremothecium cymbalariae .................................................................................................... 23 1.6 Fungal system and their contribution to industrial processes ........................................ 23 1.7 Aroma and flavour definition, chemical type .................................................................. 24 1.8 Flavour additives and natural flavours ............................................................................ 26 1.9 Bioflavour production (Ehrlich pathway, FFAs and lactate) .......................................... 29 Ehrlich pathway ....................................................................................................................... 30 Fatty acids as substrates for flavour formation ....................................................................... 33 Metabolism of lactate and citrate ............................................................................................ 34 1.10 Biological properties of quorum sensing molecules and VOCs as signaling molecules . 35 1.11 Biotechnological application of fungal VOCs .................................................................. 37 1.12 Fungal VOC collection and detection ............................................................................... 38 VOC collection .......................................................................................................................... 38 VOC separation ........................................................................................................................ 39 VOC detection ........................................................................................................................... 39 2. Aim ............................................................................................................................................ 40 3. Objectives and state-of-the-art ................................................................................................ 41 3.1 Functional analysis of the ARO gene family in S.cerevisiae and A. gossypii ................. 41 3.2 Analysis of the different volatile profiles of A. gossypii and E. cymbalariae is correlated to their genetic backgrounds. ...................................................................................................... 43 3.3 Flavour molecules produced in the Saccharomyces clade. ............................................. 44 4. Discussion ................................................................................................................................. 44 4.1 Reporter assay for ARO genes in S. cerevisiae and A. gossypii ...................................... 44 4.2 Use of a lacZ- reporter assay to correlate reporter gene activity with flavour production ........................................................................................................................................... 46 4.3 General flavour differences between A. gossypii and E. cymbalariae ........................... 47 4.4 VOCs produced in the Saccharomyces clade ................................................................... 49 5. Conclusions and Outlook ......................................................................................................... 53 References ........................................................................................................................................ 55 Paper 1 .............................................................................................................................................. 64 Paper 2 ............................................................................................................................................. 65 Paper 3 ............................................................................................................................................. 66 Appendix .......................................................................................................................................... 67 Preface This thesis “Flavour compounds in fungi: Flavour analysis in ascomycetes and the contribution of the Ehrlich pathway to flavour production in Saccharomyces cerevisiae and Ashbya gossypii” represents an overview of my PhD internship carried out at the Carlsberg Laboratory in Denmark. This project was supervised by Prof. Jürgen Wendland, head of the Yeast Genetics Group in the Carlsberg Laboratory and Prof. Steen Holmberg at the Department of Biology, University of Copenhagen. My thesis was funded by the European Union Marie Curie Initial Training Network, Cornucopia. 1 List of Papers I. Davide Ravasio, Andrea Walther, Kajetan Trost, Urska Vrhovsek and Jürgen Wendland (2014). An indirect assay for volatile compound production in yeast strains. Scientific report 4: 3707. II. Davide Ravasio, Jürgen Wendland and Andrea Walther (2014). Major contribution of the Ehrlich pathway for 2-phenylethanol/rose flavour production in Ashbya gossypii. FEMS Yeast Res. doi: 10.1111/1567-1364.12172. III. Davide Ravasio, Silvia Carlin, Teun Boekhout, Urska Vrhovsek, Jürgen Wendland and Andrea Walther (2014). A survey of flavor production among non-conventional yeasts. Manuscript. 2 Summary Fungi produce a variety of volatile organic compounds (VOCs) during their primary or secondary metabolism and with a wide range of functions. The main focus of this research work has been put on flavour molecules that are produced during fermentation processes, mainly esters and alcohols derived from the catabolism of amino acids. These compounds are produced by the Ehrlich pathway. The conversion of amino acids into aroma alcohols is accomplished by three enzymatic steps: i) a transamination, ii) a decarboxylation and iii) a dehydration reaction. The transaminase and decarboxylase enzymes are encoded by the ARO gene family which represents a widely conserved set of genes in the Saccharomyces clade. Comparative genomic analysis revealed conservation of these genes also in the riboflavin over producer Ashbya gossypii, a closely related species belonging to the Eremothecium clade. ARO80 is a transcription factor that represents the key regulator of the ARO gene family. The first part of the thesis will unveil the ARO80-dependent regulation of the Ehrlich pathway in both Saccharomyces cerevisiae and A. gossypii. Promoter analyses of the ARO genes in S. cerevisiae showed that the ScARO9 promoter region is directly regulated by the ScAro80 transcription factor. This interaction has been used to create a lacZ-reporter system to correlate the formation