ENGINEERING OF THE AROMATIC AMINO ACID BIOSYNTHETIC PATHWAYS IN ESCHERICHIA COLI FOR THE PRODUCTION OF VALUE-ADDED CHEMICALS by YUHENG LIN (Under the Direction of YAJUN YAN) ABSTRACT Aromatic amino acids (AAAs) L-phenylalanine, L-tyrosine and L-tryptophan serve not only as the building blocks for protein synthesis, but also as the precursors of secondary metabolites in organisms. A number of AAA-derived metabolites have shown pharmaceutical or industrial importance. However, these molecules usually occur in nature at low abundance, which greatly limits their efficient isolation, production and broad applications. In past decades, the development of metabolic engineering and synthetic biology facilitated the construction of ‘microbial chemical factories’ that convert renewable carbon sources to various valuable molecules. This dissertation focuses on the engineering of the E. coli AAA biosynthetic pathways to achieve the heterologous production of pharmaceutically and industrially important chemicals derived from AAAs or its biosynthetic intermediates. After careful evaluation, we select several important target compounds, including caffeic acid, simple coumarins, salicylic acid, muconic acid and 5-hydroxytryptophan and explore their production in E. coli. For this purpose, we designed, reconstituted and optimized their respective artificial biosynthetic pathways, and also conducted the modification of the E. coli native shikimate pathway via combinatorial metabolic engineering approaches. This research established powerful microbial platforms for the production of AAA related value-added chemicals, exhibiting great potential for commercialized production. INDEX WORDS: Metabolic Engineering; Aromatic Amino Acids Biosynthesis; Microbial Production; Biocatalysts; Value-added Chemicals ENGINEERING OF THE AROMATIC AMINO ACID BIOSYNTHETIC PATHWAYS IN ESCHERICHIA COLI FOR THE PRODUCTION OF VALUE-ADDED CHEMICALS by YUHENG LIN BE, China Agricultural University, China, 2007 MS, Graduate University Chinese Academy of Sciences, China, 2010 A Dissertation Submitted to the Graduate Faculty of The University of Georgia in Partial Fulfillment of the Requirements for the Degree DOCTOR OF PHILOSOPHY ATHENS, GEORGIA 2014 © 2014 Yuheng Lin All Rights Reserved ENGINEERING OF THE AROMATIC AMINO ACID BIOSYNTHETIC PATHWAYS IN ESCHERICHIA COLI FOR THE PRODUCTION OF VALUE-ADDED CHEMICALS by YUHENG LIN Major Professor: Yajun Yan Committee: Michael W. W. Adams Jim Kastner Ramaraja Ramasamy Electronic Version Approved: Julie Coffield Interim Dean of the Graduate School The University of Georgia December 2014 DEDICATION I would like to dedicate this dissertation to my beloved grandfather Mr. Wenqing Lin. 谨以此博士论文献给我最亲爱的祖父林文清先生。 iv ACKNOWLEDGEMENTS I would like to express the deepest appreciation to my major professor Dr. Yajun Yan, who continually and convincingly conveyed a spirit of ambition, perseverance and open-mindedness in regard to research and life. Without his guidance and persistent help this dissertation would not have been possible. I would also like to thank my committee members, Dr. Michael W. W. Adams, Dr. Jim Kastner and Dr. Ramaraja Ramasamy, who helped me improve my research work and dissertation. Finally, I would like to thank my family for their long-lasting and selfless support. v TABLE OF CONTENTS Page ACKNOWLEDGEMENTS .......................................................................................................................... v LIST OF TABLES ..................................................................................................................................... viii LIST OF FIGURES ..................................................................................................................................... ix CHAPTER 1 INTRODUCTION .................................................................................................................... 1 2 BIOSYNTHESIS OF CAFFEIC ACIS BY METABOLICALLY ENGINEERED ESCHERICHIA COLI ............................................................................................................... 4 2.1 Abstract ........................................................................................................................ 5 2.2 Background .................................................................................................................. 6 2.3 Materials and Methods ................................................................................................. 8 2.4 Results and Discussion .............................................................................................. 14 2.5 Summary .................................................................................................................... 22 3 COMBINATORIAL BIOSYNTHESIS OF PLANT-SPECIFIC SIMPLE COUMARINS IN ESCHERICHIA COLI ............................................................................................................ 23 3.1 Abstract ....................................................................................................................... 24 3.2 Background ................................................................................................................. 24 3.3 Materials and Methods ................................................................................................ 30 3.4 Results ......................................................................................................................... 36 3.5 Discussion ................................................................................................................... 45 4 MICROBIAL SYNTHESIS OF THE ANTICOAGULANT DRUG PRECURSOR 4- HYDROXYCOUMARIN ...................................................................................................... 48 4.1 Abstract ...................................................................................................................... 49 vi 4.2 Background ................................................................................................................ 49 4.3 Results ........................................................................................................................ 53 4.4 Discussion .................................................................................................................. 61 4.5 Materials and Methods ............................................................................................... 64 4.6 Supporting Information .............................................................................................. 70 5 MICROBIAL PRODUCTION OF SALICYLIC ACID AND MUCONIC ACID THROUGH METABOLIC ENGINEERING APPROACHES .................................................................. 87 5.1 Abstract ....................................................................................................................... 88 5.2 Background ................................................................................................................ 88 5.3 Materials and Methods ................................................................................................ 91 5.4 Results ......................................................................................................................... 96 5.5 Discussion ................................................................................................................. 105 6 EFFICIENT PRODUCTION OF 5-HYDROXYTRYPTOPHAN THROUGH COMBINATORIAL METABOLIC AND PROTEIN ENGINEERING .............................. 109 6.1 Abstract ..................................................................................................................... 110 6.2 Background .............................................................................................................. 110 6.3 Materials and Methods .............................................................................................. 112 6.4 Results ....................................................................................................................... 117 6.5 Discussion ................................................................................................................. 129 7. CONCLUSION ....................................................................................................................... 132 REFERENCES ........................................................................................................................................ 135 vii LIST OF TABLES Page Table 2.1: Strains and plasmids used in Chapter 2 .................................................................................... 10 Table 2.2: Primers used in Chapter 2 .......................................................................................................... 11 Table 2.3: In vitro activity of 4HPA3H complex ....................................................................................... 15 Table 2.4: Comparison of in vitro activity of RcTAL and RsTAL ............................................................. 18 Table 2.5: Production of caffeic acid and tyrosine by engineered E. coli strains ....................................... 20 Table 3.1: Strains and plasmids used in Chapter 3 ..................................................................................... 30 Table 3.2: Primers used in Chapter 3 .......................................................................................................... 32 Table 3.3: Substrate Specificities of 4-coumarate:CoA ligases (4CLs) .....................................................