This document is downloaded from DR‑NTU (https://dr.ntu.edu.sg) Nanyang Technological University, Singapore. Metabolic engineering yeast cells for medium‑chained biofuel synthesis Li, Xiang 2015 Li, X. (2015). Metabolic engineering yeast cells for medium‑chained biofuel synthesis. Doctoral thesis, Nanyang Technological University, Singapore. https://hdl.handle.net/10356/62904 https://doi.org/10.32657/10356/62904 Downloaded on 02 Oct 2021 17:55:26 SGT METABOLIC ENGINEERING YEAST CELLS FOR MEDIUM METABOLIC ENGINEERING YEAST CELLS FOR MEDIUM-CHAINED BIOFUEL SYNTHESIS - CHAINED BIOFUEL SYNTHESIS LI XIANGLI 201 LI XIANG 5 SCHOOL OF CHEMICAL AND BIOMEDICAL ENGINEERING 2015 METABOLIC ENGINEERING YEAST CELLS FOR MEDIUM-CHAINED BIOFUEL SYNTHESIS LI XIANG LI XIANGLI School of Chemical and Biomedical Engineering A thesis submitted to the Nanyang Technological University in partial fulfillment of the requirement for the degree of Doctor of Philosophy 2015 Acknowledgements ACKNOWLEDGEMENTS Four years at Nanyang Technological University were unforgettable. NTU provided me a platform to pursue my degree and opportunity to get to know the most frontier areas and so many outstanding researchers who generously offered me sincere help. Herein, I would like to extend my deep appreciations to them. First and foremost, I would like to thank my supervisor, Prof. Chen Wei Ning William. He kindly provided me the opportunity to pursue my PhD, which has been a great honor. He has always been so kind and supportive and the family-like atmosphere in our research group really has made the four years enjoyable. His professional guidance, enlightening instructions and patient supervisions supported me in every stage in the process of generating this essay. I shall express my appreciations to my colleagues: Dr. Feng Huixing, Dr. Zhang Jianhua, Dr. Wang Mingxuan, Dr. Bai Jing, Dr. Zhou Yusi, Dr. Laleh Sadrolodabaee, Dr. Tan Yi Lin Jane, Mr. Tan Kee Yang, Ms. Tang Xiaoling, Ms. Shi Jiahua, Ms. Chen Liwei, Ms. Zhao Guili, Ms. Lee Jie Lin Jaslyn and Ms. Toh Shi Hui, for their constructive suggestions and kind encouragement, for their support and friendship. Then I shall express my highly grateful to the professional officer Dr Fang Ning, Dr Ong Teng Teng, Dr. Wang Xiujuan, Ms. Tan Lay Yian and Ms. Gan Chew Mei Jessica, for their professional technician support. Acknowledgements Last but not least, I would like to express my love to my parents and my fiancée, to whom this dissertation is dedicated to. Their unconditional love and trust have supported me and would always be my greatest wealth. Finally, I appreciate the financial support from Competitive Research Program that funded the research in this dissertation. Contents CONTENTS LIST OF FIGURES ............................................................................................................. i LIST OF TABLES ............................................................................................................. ix ABBREVIATIONS ........................................................................................................... xi ABSTRACT ...................................................................................................................... xv CHAPTER 1 ....................................................................................................................... 1 INTRODUCTION .............................................................................................................. 1 1.1 Background ........................................................................................................... 1 1.1.1 Development of biofuel ............................................................................. 1 1.1.2 Renewable jet fuel ...................................................................................... 2 1.2 Biofuel production by metabolic engineering ....................................................... 4 1.3 Fatty acids metabolism in S. cerevisiae ................................................................ 5 1.4 Proteomics ............................................................................................................. 7 1.4.1 Introductions on proteomics ...................................................................... 7 1.4.2 Basic introduction of LC-MS ..................................................................... 8 1.4.3 Proteomics basing on LC-MS .................................................................... 9 1.4.4 Stable-isotope technique .......................................................................... 15 1.5 Basic introduction of GC .................................................................................... 20 CHAPTER 2 ..................................................................................................................... 23 OBJECTIVES ................................................................................................................... 23 CHAPTER 3 ..................................................................................................................... 25 RESEARCH DESIGN AND METHODS ........................................................................ 25 3.1 Strains and media ................................................................................................ 25 3.2 Double deletion strain construction .................................................................... 25 I Contents 3.3 Cloning target genes ........................................................................................... 32 3.3.1 LOX and HPL cloning ............................................................................. 32 3.3.2 ADC cloning ............................................................................................ 34 3.4 Construction of functional S. cerevisiae strains .................................................. 35 3.5 Growth curve test ................................................................................................ 37 3.6 Protein extraction and Western-blot analysis ..................................................... 37 3.6.1 Gel electrophoresis ................................................................................... 38 3.6.2 Membrane transfer ................................................................................... 41 3.6.3 Blocking ................................................................................................... 42 3.6.4 Detection .................................................................................................. 42 3.7 Biotransformation and identification .................................................................. 43 3.7.1 GC-FID .................................................................................................... 43 3.7.2 GC-MS ..................................................................................................... 44 3.8 Proteomics ........................................................................................................... 44 3.9 Protein identification and data analysis .............................................................. 46 3.10 Protein quantification and data analysis ........................................................... 48 CHAPTER 4 ..................................................................................................................... 51 METABOLICALLY ENGINEERED YEAST CELLS AND MEDIUM- CHAINED HYDROCARBON BIOFUEL PRECURSORS SYNTHESIS ...................... 51 4.1 Introduction ......................................................................................................... 51 4.1.1 The lipoxygenase pathway ....................................................................... 51 4.1.2 Lipoxygenase ........................................................................................... 54 4.1.3 Hydroperoxide lyase ................................................................................ 55 4.2 Experiment procedure ......................................................................................... 56 4.3 Results ................................................................................................................. 56 II Contents 4.3.1 Construction of recombinant plasmid 9LHP ........................................... 56 4.3.2 Construction of double deletion strain ..................................................... 57 4.3.3 Construction of functional strains ............................................................ 60 4.3.4 Growth curve test ..................................................................................... 62 4.3.5 Western blot ............................................................................................. 63 4.3.6 Proteomics ................................................................................................ 64 4.3.7 GC-FID detection ..................................................................................... 78 4.4 Conclusions ......................................................................................................... 82 4.5 Future directions ................................................................................................. 84 CHPATER 5 ..................................................................................................................... 87 METABOLICALLY ENGINEERED YEAST CELLS AND MEDIUM- CHAINED HYDROCARBON BIOFUEL SYNTHESIS (PRELIMINARY) ................. 87 5.1 Introduction 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