In planta expression of exocellulase enzymes for Bio-ethanol production A thesis submitted in fulfilment of the requirements for the degree of Doctor of Philosophy Parsu Ram Budathoki Degree of Bachelor of Science, University of North Bengal, India Master of Science in Biotechnology, Bangalore University, India School of Science College of Science, Engineering and Health RMIT University March 2018 Declaration I certify that except where due acknowledgement has been made, the work is that of the author alone; the work has not been submitted previously, in whole or in part, to qualify for any other academic award; the content of the thesis is the result of work which has been carried out since the official commencement date of the approved research program; any editorial work, paid or unpaid, carried out by a third party is acknowledged; and, ethics procedures and guidelines have been followed. Parsu Ram Budathoki Date: 13/03/2018 i ACKNOWLEDGEMENTS I wish to thank my senior supervisor, Professor Trevor Stevenson for his help and advice during the period of research. I also thank my secondary supervisor Dr Gregory Nugent for his guidance and assistance. I thank Professor David Stalker for many useful discussions. I am indebted to Mrs. Kim Stevenson for her assistance and guidance in my laboratory work. I also thank Dr. Chung Hong Chen (former CSIRO Scientist) for providing me with plasmids and helping me in the cloning vectors. I acknowledge Dr. Chaitali Dekiwadia for assistance in sample preparation and imaging of TEM work at RMIT Microscopy and Microanalysis Facility. The research described in this thesis was carried on while I held a research scholarship from the ARC Linkage grant in partnership with Biomass Conversion Technologies, was funded from Chevron Technology Ventures in USA is gratefully acknowledged. I sincerely appreciate the help and support from front desk staffs; Dr. Lisa Dias, Leeanne Bickford, Claire Bayly, David Heathcote, Shannon Fernandes and other teaching and non teaching staffs. I would also like to thank my collegues; Jonathan O'brien, Khaled Saleh A Allemailem, Mohamed Said, Layla Alhasan, Mohamed Taha and other fellow students from RMIT for their support and cooperation. This PhD thesis would not been possible without the help of Professor Manfred Ringhofer from Osaka Sangyo University Japan who was my mentor, sponsor and guardian starting from year 12, three years of Bachelor Degree and two years Masters Degree from India. Professor Manfred had visited my house twice during my PhD and updated about the progress of my writing and provided feedback. ii I owe my sincere thanks and gratitude to my past and present teachers, experts and well wishers who contributed for the successful completion of my endeavour. I thank Mr. Bhanu Kumar Baral, former Principle of Kumudini Homes Kalimpong School for his guidance and support. My great respect and love to former biology teacher Madam Kasang Bhutia. I thank Fr. Kinley Tshering, St. Joseph’s College Darjeeling for the financial support during my studies in Darjeeling. I remain in debt with late principle C. B Rai from St. Joseph’s College Darjeeling for the college scholarship for Bachelor degree of Science. I thank Dr. Karim Karipodi from Garden City College Bangalore India, for providing Scholarship for two years of Master of Science course. I thank all my school friends for their support. I am always greatful to my school mates (Nir Maya Rai and Abir Rai) currently resettled in Sacramento, California, USA for their love, compassion and support. I also thank my friend Dr. Jitendra Mehta from Rajestan India for this valuable discussion, support and encouragement. I thank Mr. Khem Khanal from Melton for his moral support. I thank my sisters (Sabitra Thapa and Rupa Rai) and brother (Yadu Prasad Budathoki) and sister-in-laws (Meg Thapa and Mon Bahadur Rai) for their support and encouragement. Special thanks to my nephews (Purna Thapa, Indra Thapa, Raj Thapa and Sayal Rai) and nieces (Rosey Rai and Ashika Thapa, Bedha, Gita and Bandana) for supporting my work. Baby Arron (my brother’s son) was born while I was writing my thesis. I love you baby Arron and you had brought happiness to our family. This thesis is dedicated to my mother (Kamal Maya Budathoki), Father (Bal Bahadur Budathoki), my wife Leela Maya Thapa, my beloved son Amir Budathoki and gorgeous daughter Amira Budathoki for always believing in me and encouraging me to follow my dreams. For Amir and Amira, I love you. Thank you so much for trusting me, understanding me and allowing me to complete my stuides. Above all I am proud to be your dad. I thank almighty for his blessings in my work. iii Table of Contents Chapter 1: Project background ................................................ 1 1.1 History of cellulosic ethanol production .................................................. 1 1.2 Challenges of cellulosic ethanol production .......................................... 2 1.3 Types of Biofuels ...................................................................................... 3 1.3.1 First generation biofuels ........................................................................................... 3 1.3.2 Second generation biofuels: Cellulosic ethanol .................................................... 4 1.3.3 Biomass recalcitrance: Principal challenge of second generation biofuel production ............................................................................................................................. 5 1.4.Cellulose .................................................................................................... 7 1.4.1 Hemicellulose ............................................................................................................. 8 1.4.2 Pectin ........................................................................................................................... 9 1.4.3 Lignin ........................................................................................................................... 9 1.5 Overview of cellulosic ethanol production ............................................ 10 1.5.1 Biomass pretreatment technology ........................................................................ 11 1.5.2 Economic analysis of the cellulosic ethanol production utilising the cellulases produced via microbial fermentation .............................................................................. 12 1.6 Cellulase producing organisms ............................................................. 13 1.6.1 Cellulases system .................................................................................................... 15 1.6.1.1 Free enzymes …..……………….……………………………………………..15 1.6.2 Industrially important cellulolytic fungi .......................................................... 17 1.6.2.2 Termite gut protozoan cellulases can digest wood………………………..20 1.7 Cellulase structure and function ............................................................ 21 1.7.1 Catalytic module ...................................................................................................... 22 1.7.2 Carbohydrate-binding module (CBM) .................................................................. 23 1.8. Enzymatic hydrolysis of cellulose ........................................................ 24 1.8.1 Binding mechanism of cellulases to cellulose ..................................................... 25 1.9 Heterologous expression of cellulases................................................. 25 1.9.1 Cellulase expression in bacterial systems ........................................................... 26 1.9.2 Yeast expression of cellulases .............................................................................. 28 1.10 Heterologous expression of termite cellulases ................................. 30 iv 1.11 In planta expression of cellulases ....................................................... 30 1.11.1 Host plants for heterologous expression of cellulases .................................... 34 1.11.2 Expressing cellulases in plants through nuclear transformation.................... 34 1.11. 3 Expressing cellulases through plastid transformation .................................... 35 1.11.4 Challenges of cellulase gene expression in plants .......................................... 37 1.12 Project overview ................................................................................... 37 1.13 Research questions ............................................................................. 38 1.14 Project Aim ............................................................................................ 39 Chapter 2: Materials and methods ........................................ 40 2.1 Growth curve for E. coli strains containing cbhI in plastid expression vectors ..................................................... 40 2.2 Plant materials for stable transformation ............................................. 40 2.3 Plant material for transient assays ....................................................... 41 2.4 Agrobacterium tumefaciens culture preparation ................................. 41 2.5 Agroinfiltration ......................................................................................... 41 2.6 Bacterial strain used as a host for plastid transformation vector ...... 42 2.7 Generation of the GFP expression vector ........................................... 42 2.8 Design of cbhI expression cassettes for CBHI. ..................................