Engineering of the Plant Abscisic Acid Biosynthetic Pathway in Escherichia coli by Dominic Ludovice A thesis submitted in conformity with the requirements for the degree of Master of Science Cell and Systems Biology University of Toronto © Copyright by Dominic Ludovice 2016 Engineering of the Plant Abscisic Acid Biosynthetic Pathway in Escherichia coli Dominic Ludovice Master of Science Cell and Systems Biology University of Toronto 2016 Abstract Abscisic acid (ABA) is a phytohormone involved in many aspects of plant growth and development, including the regulation of seed development, dormancy, germination, and plant responses to environmental stress. Escherichia coli is an ideal system in which to engineer the ABA biosynthetic pathway since the early steps have been successfully reproduced in E. coli by Misawa and colleagues (1990) using the carotenoid biosynthesis gene cassette from Pantoea ananatis. In this study, Arabidopsis thaliana genes were used to build the pathway starting from the precursor zeaxanthin. The next biosynthetic enzyme, zeaxanthin epoxidase (ZEP), was successfully expressed in E. coli, leading to the effective production of violaxanthin. However, the enzyme responsible for catalyzing the subsequent step has yet to be identified. This system is now being used to identify uncharacterized genes of the pathway. ii Acknowledgments First and foremost, I would like to thank my supervisor, Professor Eiji Nambara, without whom this project would not have been possible. I am grateful for the support and help he has given me throughout this project. I would also like to thank the current and former members of the Nambara Lab, who treated me like family. Our Post Doctoral Fellow, Dr. Dawei Yan, for helping me in almost all of my lab experiments. Lisza Duermeyer, one of our PhD. students, for keeping the lab in order. Catalina Leoveanu, the other Msc. student, for keeping the office lively. Dr. Masato Otani, our previous Post Doctoral Fellow, for helping me with the HPLC. Ayako Nambara, our lab technician, for keeping the lab from running out of supplies. Ehsan Khodapanahi, one of our PhD. students, for helping fix the automatic pipettor. Yumi Tanaka, our previous lab technician, for the Gibson Assembly mix recipe. I would also like to extend my thanks to the members of the McCourt Lab, Dr. Shigeo Toh, Dr. Shelly Lumba, Duncan Holbrook-Smith, and Eric Nam for their input during lab meeting, which helped shape this project. I would like to extend my thanks my committee members, Professor Daphne Goring and Professor Peter McCourt for helping to guide and direct this project. Finally yet importantly, I would like to thank my friends and family who have kept me sane throughout the two years of this project. I would like to thank my mom, dad and sister for their unconditional love and support throughout this arduous endeavor. iii Table of Contents Acknowledgments .......................................................................................................................... iii Table of Contents ........................................................................................................................... iv List of Tables ................................................................................................................................. vi List of Figures ............................................................................................................................... vii Abbreviations ............................................................................................................................... viii 1 Introduction ................................................................................................................................ 1 1.1 ABA biosynthesis ............................................................................................................... 3 1.2 Engineering the ABA biosynthetic pathway in to Escherichia coli ................................... 4 1.3 A. thaliana genes will supplant the missing steps of the ABA biosynthetic pathway in engineered E. coli. ............................................................................................................... 5 1.4 The short-term goal is to identify uncharacterized genes of the ABA biosynthetic pathway. .............................................................................................................................. 6 1.5 Xanthoxin and beyond ........................................................................................................ 9 2 Materials and Methods ............................................................................................................... 9 2.1 Growing engineered E. coli producing carotenoids for HPLC analysis ............................. 9 2.2 Harvesting and extraction of carotenoids ......................................................................... 10 2.3 HPLC condition ................................................................................................................ 11 2.4 Violaxanthin standard ....................................................................................................... 11 2.5 Cloning .............................................................................................................................. 11 2.5.1 AtZEP classical cloning ........................................................................................ 12 2.5.2 Cloning of genes of interest .................................................................................. 12 2.5.3 AtNXD1 cloning .................................................................................................... 14 2.5.4 AtNCED3 Cloning ................................................................................................ 14 2.5.5 Gibson Assembly of pACHP-Zea(Spec) .............................................................. 14 2.5.6 AtZEP cloning by Gibson Assembly .................................................................... 15 iv 2.6 Strains of E. coli ................................................................................................................ 15 2.7 Plasmids ............................................................................................................................ 15 2.7.1 Duet Plasmid System ............................................................................................ 15 2.7.2 Plasmid list ............................................................................................................ 17 2.8 Western Blot ..................................................................................................................... 18 2.8.1 Growth of E. coli harboring pET-AtZEP for protein extraction ........................... 18 2.8.2 Growth of E. coli harboring pCOLA-AtNXD1 for protein extraction .................. 18 2.8.3 Gel electrophoresis and Western blotting ............................................................. 18 3 Results ...................................................................................................................................... 18 3.1 Construction and expression of pACHP-Zea(Spec) ......................................................... 18 3.2 AtZEP expression produces all-trans-violaxanthin .......................................................... 20 3.3 Expression of carotenoid enzyme-related genes in E. coli harboring pACHP(Spec)- AtZEP ................................................................................................................................ 24 3.4 AtNXD1 expression produces a novel peak ..................................................................... 28 3.5 Expression of AtNCED3 is unable to produce xanthoxin in violaxanthin-producing E. coli ..................................................................................................................................... 31 4 Discussion ................................................................................................................................ 33 4.1 Violaxanthin production in E. coli .................................................................................... 33 4.2 Search for the enzyme that catalyzes violaxanthin isomerization to 9-cis-violaxanthin .. 34 4.3 AtNXD1 could be the missing neoxanthin synthase ........................................................ 36 4.4 Xanthoxin could not be detected when AtNCED3 was expressed in all-trans- violaxanthin-producing E. coli .......................................................................................... 37 4.5 Colorimetric assay for screening for the violaxanthin isomerase ..................................... 38 References ..................................................................................................................................... 39 Appendix ....................................................................................................................................... 45 v List of Tables Table 1. HPLC conditions for carotenoid detection. .................................................................... 11 Table 2. Genes inserted in pCOLADuet-1 plasmid. ..................................................................... 13 Table 3. Novagen Duet plasmids. ................................................................................................. 15 Table 4. Plasmids and their descriptions used for this project. ..................................................... 17