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Engineering Yeast Metabolism for Production of Sesquiterpenes

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Engineering Yeast Metabolism for Production of Sesquiterpenes THESIS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY Engineering Yeast Metabolism for Production of Sesquiterpenes STEFAN TIPPMANN Department of Biology and Biological Engineering CHALMERS UNIVERSITY OF TECHNOLOGY Gothenburg, Sweden 2016 Engineering Yeast Metabolism for Production of Sesquiterpenes Stefan Tippmann ISBN 978-91-7597-459-0 © STEFAN TIPPMANN, 2016. Doktorsavhandlingarnar vid Chalmers Tekniska Högskola Ny serie nr 4140 ISSN 0346-718X Department of Biology and Biological Engineering Chalmers University of Technology SE-412 96 Gothenburg, Sweden Telephone +46 (0)31 772 1000 The cover illustrates production of sesquiterpenes by fermentation of the yeast Saccharomyces cerevisiae. Printed by Chalmers Reproservice Gothenburg, Sweden 2016 Engineering Yeast Metabolism for Production of Sesquiterpenes Stefan Tippmann Department of Biology and Biological Engineering Chalmers University of Technology Abstract Sesquiterpenes belong to the large and diverse group of isoprenoids, which are ubiq- uitous in the plant kingdom and have various applications in the chemical indus- try as fragrances, pharmaceuticals and as substitutes for petroleum-based gasoline, diesel and jet fuels. In this project, production of sesquiterpenes was studied in Sac- charomyces cerevisiae using farnesene as an example with the objective to gain new insights into the synthesis of these compounds and to evaluate different metabolic engineering strategies. Farnesyl diphosphate (FPP) represents the universal precursor for all sesquiter- penes and different strategies were addressed to increase production of this interme- diate and to allow for its efficient conversion to farnesene. As FPP is provided by the mevalonate pathway, we aimed at increasing the flux through the pathway by incor- poration of malonyl-CoA using a recently identified acetoacetyl-CoA synthase from Streptomyces sp. strain CL190. While the enzyme had detrimental effects on growth as well as on product formation, it was able to compensate for the loss of the essen- tial, endogenous acetoacetyl-CoA thiolase. Additionally, a homologous enzyme with superior efficiency could be identified. Secondly, as FPP is required as substrate for different pathways and represents a metabolic branch point, a novel tool for enzyme co-localization was developed to divert more flux towards farnesene. The system uti- lizes scaffolds based on affibodies and could improve product yields by more than twofold. Furthermore, the role of terpene synthases on the production of farnesene was elucidated by comparing farnesene synthases with different plant origins, i.e. Malus domestica, Citrus junos and Artemisia annua. While the selected candidates produced similar amounts of farnesene (up to 170 mg/L), these enzymes appeared to be superior in comparison to other sesquiterpene synthases, i.e. santalene synthase. Lastly, the response to increased product formation was investigated using transcrip- tome and metabolome analysis, which highlighted changes across various metabolic pathways and identified pantothenic acid as a potential target for future engineering strategies. In conclusion, the study evaluates different metabolic engineering strategies for production of sesquiterpenes in S. cerevisiae and provides new insights into the cellu- lar response during their production. Additionally, utilization of affibodies as a novel tool in metabolic engineering to increase chemical production in S. cerevisiae was highlighted. Keywords: Saccharomyces cerevisiae, metabolic engineering, sesquiterpenes, isoprenoids, affibodies, metabolomics, transcriptomics iii Acknowledgements I would like to acknowledge all the people who contributed directly or indirectly to the work presented in this thesis. First and foremost, I would like to thank my supervisors Jens Nielsen and Verena Siewers for all their contributions, support and patience as well as for always taking time to help and discuss. It was a great experience to work with you. Besides, I am particularly grateful to Sakda Khoomrung, with whom I have been working on several different projects and who has contributed to this work in many different ways. I also gratefully acknowledge Yun Chen and Raphael Ferreira, who have been working with on the acetoacetyl-CoA synthase expression in yeast as well as Intawat Nookaew and Partho Sarathi Sen for analyzing transcriptome and metabolome data with me. In this respect, I would also like to thank Francesco Gatto for his support and for helping me to understand the “basic bioinformatics”, as he would phrase it. Moreover, I gratefully acknowledge Josefine Anfelt and Paul Hudson from KTH in Stockholm, with whom I have been working on affibody scaffolds for several years. Thank you for all the efforts to finish the project successfully! Likewise, I thank Florian David and Jacqueline Rand for their help and valuable discussions of the project. Furthermore, I would like to thank António Roldão for the perpetual troubleshooting and assistance during numerous yeast fermentations. The Systems & Synthetic Biology Division has been a great work environment and I am very grateful to all the people in the group for the daily discussions, advice and support. In addition, I wish to thank Rahul Kumar for his encouragements and very valuable advice regarding the directions of my project as well as Andreas Hellström for all the good discussions and help in the lab. Lastly, I wish to thank my family for their continuous support. Thanks to you I was able to take on this challenge in the first place. Throughout the last years you kept telling me, you don’t really understand what all of this is about and I hope this thesis will provide some answers. At the end, a very special thanks to my greatest supporter, “min sambo”, Melanie. The past years have been very adventurous: by the time I started, Paul was born, Emil was only 4 years old and we had just moved to Sweden. Organization became a crucial skill to live up to the standards for PhD students in the group and to keep up the family life simultaneously. It often felt impossible to manage both and I know I missed out on some end. Thank you for all your support! v List of Publications The thesis is based on the following publications and manuscripts: I Tippmann S, Chen Y, Siewers V and Nielsen J (2013) From fragrances and phar- maceuticals to advanced biofuels: Production of isoprenoids in Saccharomyces cerevisiae. Biotechnology Journal 8(12):1435-1444 II Tippmann S, Scalcinati G, Siewers V and Nielsen J (2016) Production of far- nesene and santalene by Saccharomyces cerevisiae using fed-batch cultivations with RQ-controlled feed. Biotechnology and Bioengineering 113(1):72-81 III Tippmann S, Khoomrung S, Sarathi Sen P, Nookaew I and Nielsen J Metabolic and transcriptomic response to production of farnesene in Saccharomyces cere- visiae. Manuscript in preparation. IV Tippmann S*, Anfelt J*, David F, Rand JM, Siewers V, Uhlén M, Nielsen J and Hudson EP Affibody scaffolds improve production of sesquiterpenes in Saccha- romyces cerevisiae. Accepted for Publication in ACS Synthetic Biology doi:10.1021/acssynbio.6b00109. *equal contribution V Tippmann S, Ferreira R, Siewers V, Nielsen J and Chen Y Effects of acetoacetyl- CoA synthase expression on production of sesquiterpenes in Saccharomyces cere- visiae. Under review. VI Tippmann S, Nielsen J and Khoomrung S (2016) Improved quantification of farnesene during microbial production from Saccharomyces cerevisiae in two- liquid-phase fermentations. Talanta 146:100-106 Additional publications not included in the thesis: VII Khoomrung S, Martinez JL, Tippmann S, Jansa-Ard S, Buffing MF, Nicastro R and Nielsen J (2015) Expanded metabolite coverage of Saccharomyces cere- visiae extract through improved chloroform/methanol extraction and tert-butyl- dimethylsilyl derivatization. Analytical Chemistry Research 6:9-16 VIII Khoomrung S, Tippmann S, Martinez JL, Nookaew I, Moritz T, and Nielsen J Comprehensive analysis of industrial yeast metabolome by high-resolution mass spectrometry. Manuscript in Preparation. vii Contribution Summary I Performed the literature study and wrote the manuscript. II Designed the study, performed all experiments on farnesene production and wrote the manuscript. III Designed the study, performed the experiments, processed and analyzed the metabolome data, analyzed the transcriptome data and wrote the manuscript. IV Assisted in designing the study, performed all experiments in Saccharomyces cerevisiae and wrote the manuscript. V Designed the study, performed all experiments and wrote the manuscript. VI Designed the study, performed all experiments and wrote the manuscript. Preface This dissertation is submitted for the partial fulfillment of the degree of doctor of philosophy. It is based on work carried out between December 2011 and June 2016 in the Division for Systems and Synthetic Biology at the Department of Biology and Biological Engineering, Chalmers University of Technology under the supervision of Professor Jens Nielsen. The research was funded by the Knut and Alice Wallenberg Foundation, FORMAS and the Chalmers Foundation. Stefan Tippmann October 2016 ix Contents Abstract .......................................... iii Acknowledgements ................................... v List of Publications ................................... vii 1 Introduction ..................................... 1 1.1 Chemistry, Origin and Industrial Applications . 1 1.2 Biosynthesis of Sesquiterpenes . 4 1.3 Metabolic Engineering for Sesquiterpene Production . 8 1.4 Outline and Objectives
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