VTT CREATES BUSINESS FROM TECHNOLOGY Technologyandmarketforesight•Strategicresearch•Productandservicedevelopment•IPRandlicensing Dissertation •Assessments,testing,inspection,certification•Technologyandinnovationmanagement•Technologypartnership VTT PUBLICATIONS 739 • • • VTT PUBLICATIONS 739 IDENTIFICATION Many microorganisms that live on decaying plant material can use D-galacturonate for growth. Eukaryotic catabolic D-galacturonate pathway was not known before. In this thesis work the pathway for D-galacturonate catabolism was identified in the filamentous fungusTrichoderma reesei. The pathway consisted of four enzymes: NADPH- dependent D-galacturonate reductase (GAR1), L-galactonate dehydratase (LGD1), OF L-threo-3-deoxy-hexulosonate aldolase (LGA1) and NADPH-dependent glycer- aldehyde reductase (GLD1). In this pathway D-galacturonate was converted to THE pyruvate and glycerol via L-galactonate, L-threo-3-deoxy-hexulosonate and L- FUNGAL glyceraldehyde. The enzyme activities of GAR1, LGD1 and LGA1 were present in crude mycelial extract only when T. reesei was grown on D-galacturonate. The corresponding genes were identified and cloned. They were functionally expressed inSaccharomyces CATABOLIC cerevisiae, and the enzymes were characterised. GAR1 and LGA1 catalysed reversible reactions, whereas only the forward reactions were observed for LGD1 and GLD1. When gar1, lgd1 or lga1 was deleted in T. reesei the deletion strain was unable to grow with D-galacturonate as the only carbon source, demonstrating that all the D-GALACTURONATE corresponding enzymes were essential for D-galacturonate catabolism and that no alternative D-galacturonate pathway exists in T. reesei. PATHWAY Satu Hilditch Identification of the fungal catabolic D-galacturonate pathway ISBN 978-951-38-7398-1 (soft back ed.) ISBN 978-951-38-7399-8 (URL: http://www.vtt.fi/publications/index.jsp) ISSN 1235-0621 (soft back ed.) ISSN 1455-0849 (URL: http://www.vtt.fi/publications/index.jsp) VTT PUBLICATIONS 739 Identification of the fungal catabolic D-galacturonate pathway Satu Hilditch Faculty of Biosciences Department of Biological and Environmental Sciences Division of Biochemistry University of Helsinki Helsinki, Finland To be presented with the permission of the Faculty of Biosciences of the University of Helsinki, for public criticism in the Auditorium 2041 at the Department of Biosciences, Viikki Biocenter, Viikinkaari 5, Helsinki, on June 11th, 2010 at 12 o’clock noon. ISBN 978-951-38-7398-1 (soft back ed.) ISSN 1235-0621 (soft back ed.) ISBN 978-951-38-7399-8 (URL: http://www.vtt.fi/publications/index.jsp) ISSN 1455-0849 (URL: http://www.vtt.fi/publications/index.jsp) Copyright © VTT 2010 JULKAISIJA – UTGIVARE – PUBLISHER VTT, Vuorimiehentie 5, PL 1000, 02044 VTT puh. vaihde 020 722 111, faksi 020 722 4374 VTT, Bergsmansvägen 5, PB 1000, 02044 VTT tel. växel 020 722 111, fax 020 722 4374 VTT Technical Research Centre of Finland, Vuorimiehentie 5, P.O. Box 1000, FI-02044 VTT, Finland phone internat. +358 20 722 111, fax + 358 20 722 4374 Technical editing Leena Ukskoski Text preparing Raija Sahlstedt Edita Prima Oy, Helsinki 2010 2 Satu Hilditch. Identification of the fungal catabolic D-galacturonate pathway. Espoo 2010. VTT Publications 739. 74 p. + app. 38 p. Keywords Trichoderma reesei, filamentous fungus, D-galacturonate, catabolic pathway, D-galacturonate reductase, L-galactonate dehydratase, L-threo-3-deoxy- hexulosonate aldolase, glyceraldehyde reductase, enzyme activity Abstract Pectin is a natural polymer consisting mainly of D-galacturonic acid monomers. Microorganisms living on decaying plant material can use D-galacturonic acid for growth. Although bacterial pathways for D-galacturonate catabolism had been described previously, no eukaryotic pathway for D-galacturonate catabolism was known at the beginning of this work. The aim of this work was to identify such a pathway. In this thesis the pathway for D-galacturonate catabolism was identified in the filamentous fungus Trichoderma reesei. The pathway consisted of four enzymes: NADPH-dependent D-galacturonate reductase (GAR1), L-galactonate dehydratase (LGD1), L-threo-3-deoxy-hexulosonate aldolase (LGA1) and NADPH-dependent glyceraldehyde reductase (GLD1). In this pathway D- galacturonate was converted to pyruvate and glycerol via L-galactonate, L-threo- 3-deoxy-hexulosonate and L-glyceraldehyde. The enzyme activities of GAR1, LGD1 and LGA1 were present in crude mycelial extract only when T. reesei was grown on D-galacturonate. The activity of GLD1 was equally present on all the tested carbon sources. The corresponding genes were identified either by purifying and sequencing the enzyme or by expressing genes with homology to other similar enzymes in a heterologous host and testing the activities. The new genes that were identified were expressed in Saccharomyces cerevisiae and resulted in active enzymes. The GAR1, LGA1 and GLD1 were also produced in S. cerevisiae as active enzymes with a polyhistidine-tag, and purified and characterised. GAR1 and LGA1 catalysed reversible reactions, whereas only the forward reactions were observed for LGD1 and GLD1. When gar1, lgd1 or lga1 was deleted in T. reesei the deletion strain was unable to grow with D-galacturonate as the only carbon 3 source, demonstrating that all the corresponding enzymes were essential for D- galacturonate catabolism and that no alternative D-galacturonate pathway exists in T. reesei. A challenge for biotechnology is to convert cheap raw materials to useful and more valuable products. Filamentous fungi are especially useful for the conversion of pectin, since they are efficient producers of pectinases. Identification of the fungal D-galacturonate pathway is of fundamental importance for the utilisation of pectin and its conversion to useful products. 4 Preface This study was carried out at VTT Biotechnology in the Metabolic Engineering team. The financial support from Maj and Tor Nessling Foundation, Academy of Finland and the University of Helsinki is greatly appreciated. I am indebted to former Vice President Prof. Juha Ahvenainen, Vice President Prof. Anu Kaukovirta-Norja, Research Professor Hans Söderlund and Technology Manager Dr. Richard Fagerström for providing excellent working facilities. I am grateful to Technology Manager Dr. Tiina Nakari-Setälä, Team Leader Dr. Laura Ruohonen and Research Professor Merja Penttilä for their supportive and encouraging attitude towards this thesis work. My warmest thanks are due to my supervisor Doc. Peter Richard for being the inexhaustible source of ideas and the driving force of this work. I highly admire his enthusiastic and devoted attitude towards science. His constant support and encouragement in all situations have been invaluable over the years. I wish to thank my co-authors Janis Liepins, Suvi Berghäll, Dr. Paula Jouhten, Dos. Peter Richard, Dos. Hannu Maaheimo, Dos. John Londesborough, Prof. Merja Penttilä and Dos. Nisse Kalkkinen for their contributions to the research work and writing of the manuscripts. Additional thanks are addressed to John and Peter for their constructive criticism on the thesis manuscript. Emeritus Prof. Pekka Mäntsälä and Doc. Taina Lundell are warmly thanked for the careful pre-examination of the thesis and for their valuable comments. Michael Bailey is acknowledged for revising the English language. I sincerely thank everyone working in the yeast/mould lab for the friendly and supportive working atmosphere and for all the help they have readily provided on various matters. It has been a privilege to work with such skilled colleagues. Being part of this awesome group of people has meant a lot to me. From the other labs of VTT Biotechnology I especially want to thank Birgit Hillebrandt- Chellaoui for technical assistance with the protein purification and Eila Leino who often helped me with the enzyme activity measurements. I heartily thank 5 Anne, Anu, Eija, Jari, Laura, Mari, Mervi, Mikko, Outi, Ritva and Virve for the companionship and refreshing discussions over the lunch table. I wish to thank all my friends and relatives for encouragement along the way. My special loving thanks are due to my parents, Keijo and Riitta, for their wonderful support which I will cherish always. Above all, I want to thank my dearest ones, Kai and Leo, for their understanding and love and for being the most important source of joy and strength to me. Espoo, May 2010 Satu Hilditch 6 Contents Abstract ................................................................................................................. 3 Preface.................................................................................................................. 5 List of publications................................................................................................. 9 List of symbols .................................................................................................... 10 1. Introduction ................................................................................................... 13 1.1 Pectin and D-galacturonate .............................................................................................. 14 1.2 Pectin degradation and D-galacturonate utilisation .......................................................... 18 1.2.1 Bacterial D-galacturonate pathways................................................................. 19 1.2.2 Metabolism of D-galacturonate in plants and animals ..................................... 23 1.2.3 Earlier observations