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Distaso Shortened Bangor University DOCTOR OF PHILOSOPHY Metagenomics approaches to discover new industrially-relevant enzymes Distaso, Marco Award date: 2019 Awarding institution: Bangor University Link to publication General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. • Users may download and print one copy of any publication from the public portal for the purpose of private study or research. • You may not further distribute the material or use it for any profit-making activity or commercial gain • You may freely distribute the URL identifying the publication in the public portal ? Take down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Download date: 24. Sep. 2021 Bangor University DOCTOR OF PHILOSOPHY Metagenomics approaches to discover new industrially-relevant enzymes Distaso, Marco Award date: 2019 Awarding institution: Bangor University Link to publication General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. • Users may download and print one copy of any publication from the public portal for the purpose of private study or research. • You may not further distribute the material or use it for any profit-making activity or commercial gain • You may freely distribute the URL identifying the publication in the public portal ? Take down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Download date: 17. Apr. 2019 Metagenomics approaches to discover new industrially- relevant enzymes A thesis submitted to Bangor University in candidature for the degree of Philosophiae Doctor by Marco Antonio Distaso B.Sc., M.Sc. School of Natural Sciences Bangor University Bangor, Gwynedd, LL57 2UW United Kingdom 2018 Acknowledgments This thesis is the result of an incredible journey that deeply changed me. I would like to express my gratitude to my supervisor, Peter Golyshin, for the great opportunity he offered me, for the great supervision and the friendly and exciting work environment he created. My thanks to Dr Tran Hai, for all the help, advice and guidance throughout these years. A big hug to Soshila Ramayah, a precious colleague and irreplaceable friend who was always ready to help and encourage me. Thanks for your support, your reassuring words, the spicy food and the moral and spiritual lessons. I would like to thank my fellow colleagues Rafa, Samuel and Cristina for their valuable and fundamental support. Thanks to my family, even from thousands of miles away your encouragement and unconditional love were constantly with me. Finally, I would like to thank all the incredible people I have met during these years. Completely different from each other, from different countries, with very different backgrounds, together we lived many amazing adventures that I will never forget. I’ve learned so much from all of you, and without your support I would not be here now. VI Table of Contents Abstract ................................................................................................................................................. XII List of figures ........................................................................................................................................ XIII List of tables ....................................................................................................................................... XVIII Abbreviations ........................................................................................................................................ XX Chapter I Introduction ................................................................................................................................ 2 1.1 Soil microorganisms and their interactions with plants ............................................................... 2 1.2 Root exudate and root-rhizosphere communication .................................................................. 3 1.3 Allelopathy ................................................................................................................................... 4 1.3.1 Allelopathy for weed management ....................................................................................... 6 1.4 Sorgoleone discovery and herbicidal activity ............................................................................... 8 1.4.1 Activity of sorgoleone .......................................................................................................... 10 1.4.2 Release of sorgoleone into the rhizosphere ........................................................................ 12 1.4.3 Mineralization of sorgoleone and interaction with soil microbes ....................................... 14 1.4.4 Biosynthesis of sorgoleone .................................................................................................. 14 1.5 Genetic pathway of sorgoleone synthesis .................................................................................. 15 1.5.1 Fatty acid desaturase from sorghum ................................................................................... 18 1.5.2 Alkylresorcinol synthase from sorghum .............................................................................. 18 1.5.3 O-methyltransferase from sorghum .................................................................................... 20 1.5.4 Cytochrome P450 monooxygenases from sorghum ............................................................ 21 1.6 Chemical synthetic route for sorgoleone production ................................................................. 22 1.7 Novel enzymatic route for sorgoleone production ..................................................................... 23 1.8 Cashew Nut Shell Liquid as a potential feedstock for sorgoleone production ........................... 24 1.9 Enzyme discovery from metagenomes ....................................................................................... 27 1.10 Aim and objectives of the project ............................................................................................. 27 1.11 References................................................................................................................................. 29 Chapter II Metagenomic mining of enzyme diversity ................................................................................... 40 2.1 Introduction ................................................................................................................................ 40 2.2 An industrial perspective ............................................................................................................ 42 2.2.1 Mining biocatalysis from extreme environments ................................................................ 44 VII 2.3 Metagenomics and its approaches ............................................................................................. 46 2.3.1 Screening methods .............................................................................................................. 47 2.3.1.1 Sequence-based enzyme discovery ............................................................................. 47 2.3.1.2 Function-based enzyme discovery ............................................................................... 49 2.3.1.3 Cloning vectors/vehicles and library construction ...................................................... 49 2.4 Limitation in the metagenomic enzyme discovery process ........................................................ 51 2.4.1 Coverage, representation and need for enrichment ........................................................... 51 2.4.2 Need for multiple hosts ....................................................................................................... 54 2.5 High-throughput and single cell genomics.................................................................................. 54 2.5.1 Single-cell genomics ............................................................................................................. 55 2.5.2 SIGEX .................................................................................................................................... 56 2.5.3 Microarrays .......................................................................................................................... 57 2.5.2 The way forward – from enzyme discovery to the preparation of ready-to-use biocatalysts ................................................................................................................................... 57 2.6 Conclusions and research needs ................................................................................................. 59 2.7 References................................................................................................................................... 60 Chapter III Taxonomic and functional annotation of CNSL-enriched microbial
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