ABSTRACT OZDEMIR, INCI. Role of S-Layer Homology Domain
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ABSTRACT OZDEMIR, INCI. Role of S-layer Homology Domain Proteins in Plant Biomass Conversion to Biofuels by Extremely Thermophilic Caldicellulosiruptor species. (Under the direction of Robert M. Kelly.) Second-generation biofuels require the conversion of lignocellulose from agricultural waste, non-edible parts of crops, and grasses into fermentable sugars. Lignocellulose is composed of pectin, hemicellulose, cellulose and lignin, all of which form a matrix that must be penetrated and deconstructed by physical, chemical and biological methods to facilitate conversion to biofuels. Certain microorganisms have the capacity to extract the carbohydrate content of lignocellulose through the coordinated action of glycoside hydrolases and other metabolic processes, which together can contribute to biofuel production. Of interest here are extremely thermophilic bacteria from the genus Caldicellulosiruptor. One of the intriguing features of these bacteria is that they do not produce cellulosomes but rather produce multi-domain (hemi)cellulolytic enzymes. Some of these biocatalysts contain S-layer homology (SLH) domains, anchoring the enzymes to the cell envelope, and also have carbohydrate-binding domains (CBM), which bind the enzyme to the substrate. This can be a very efficient way of degrading plant biomass since enzymes are localized in close proximity of the cell where control its local microenvironment is possible. Six different groups of SLH-domain proteins with GH domains could be identified in the genome sequences of Caldicellulosiruptor species. Representatives from each group were produced recombinantly in E. coli and examined with respect to biochemical and biophysical properties. Csac_0678 (bifunctional cellulase/xylanase from C. saccharolyticus with homologs in all eight genome- sequenced Caldicellulosiruptor species), Calkr_2245 (xylanase from C. kristjanssonii), Calhy_0060 ( -1,3-1,4-glucanase from C. hydrothermalis), Calkro_0111 (bifunctional -1,3 glucanase/ -1,3-1,4-glucanase from C. kronotskyensis), Calhy_1629 ( -xylosidase from C. hydrothermalis), and Calhy_2383 ( -1,3 glucanase from C. hydrothermalis) were identified in the sequenced genomes of Caldicellulosiruptor species as the multi-domain SLH- domain containing enzymes with different conserved domain organizations. These enzymes were characterized biochemically (except Calhy_2383 since no plant biomass-based substrate could be identified) and all exhibited hemicellulolytic activity; Csac_0678 had both hemicellulolytic and cellulolytic activity. The optimum temperature and pH of these enzymes were found to be between 68-75 C and 5-7, respectively. Csac_2722, encoded in a 2593 amino acid open reading frame (ORF) in C. saccharolyticus, did not contain a GH domain but had several sugar binding domains and SLH domains, which were also characterized. Csac_2722 and Csac_0678 bound to complex carbohydrates, such as crystalline cellulose and xylan. A truncation mutant of Csac_0678, lacking its CBM28 binding domain, did not bind to the carbohydrates, confirming the necessisity of this motif in anchoring to plant biomass substrates. Polyclonal antibodies raised against Csac_0678 (without the SLH domains) enabled confocal microscopy and fluorescence imaging analysis that confirmed that Csac_0678 was localized on the cell surface of C. saccharolyticus. The cellular localization and functional biochemical properties indicate roles for Csac_0678 and Csac_2722 in recruitment and hydrolysis of complex polysaccharides and the deconstruction of lignocellulosic biomass. The results from this study indicate that SLH-domain proteins with GHs and CBM motifs are important for Caldicellulosiruptor species in deconstructing lignocellulose. From the biotechnological perspective, these SLH-domain containing glycoside hydrolases are potentially important in reducing the recalcitrance of crystalline cellulose and facilitating its conversion to fermentable sugars. Furthermore, this work revealed that Caldicellulosiruptor species utilize cell envelope-associated enzymes and proteins, in addition to GHs present in the cytoplasm and secretome, to process complex polysaccharides. © Copyright 2012 by Inci Ozdemir All Rights Reserved Role of S-layer Homology Domain Proteins in Plant Biomass Conversion to Biofuels by Extremely Thermophilic Caldicellulosiruptor species by Inci Ozdemir A dissertation submitted to the Graduate Faculty of North Carolina State University in partial fulfillment of the requirements for the Degree of Doctor of Philosophy Chemical Engineering Raleigh, North Carolina 2012 APPROVED BY: ______________________________ ______________________________ Robert M. Kelly David F. Ollis Chair of Advisory Committee _______________________________ ________________________________ Amy M. Grunden Balaji M. Rao ii DEDICATION To my parents, my aunt and my uncle iii BIOGRAPHY Inci Ozdemir was born and raised in Izmir, Turkey. She received a Bachelor of Science degree in Chemical Engineering with a concentration in Biology in 2006 at the Middle East Technical University (Ankara, Turkey). After finishing her undergraduate education with high honors she moved to the USA to pursue a Doctorate Degree in the Department of Chemical Engineering at the North Carolina State University with a minor in Biology. After completing her PhD studies in Robert M. Kelly’s group, Inci will be joining Pfizer, Inc. as a Senior Scientist to utilize her expertise in Culture Process Development group in June, 2012. iv ACKNOWLEDGMENTS First of all I would like to thank Dr. Robert M. Kelly for giving me the opportunity to work in his lab and for his guidance and support throughout the years. I also would like to thank him for sending me to various international and national scientific conferences to present my work and learn from other scientists. I will never forget the Thermophiles conference and the Yellowstone National Park trip I took with all the Kelly group members. I also would like to thank him for his help and support while I was job hunting; it was very much appreciated. I truly enjoyed working in the Kelly lab with its recent members: Jaspreet Notey, Andrew Frock, Arpan Mukherjee, Hong Lian, Aaron Hawkins, Andrew Loder, Garrett Wheaton, Jeff Zurawski, and Yejun Han. Special thanks go to Sara Blumer- Schuette - she was a great mentor, teacher, friend, and a great problem solver over the past four years. I also would like to thank the past members of the Kelly lab: Morgan Harris for his friendship and his help in my first years in the lab, Amy VanFossen and Jason Nichols for their mentoring. Thanks to my international friends: Nimish Gera, Mahmud Hussain, Haiyan Liu, Prasenjit Sarkar, Jaspreet Notey (Thank you again!), and Arpan Mukherjee (Thank you again!) for understanding me, for listening to me, and for the parties we organized where we danced and ate wonderful homemade international food. Grad school would not be v the same without you all. I also would like to thank Dr. Saad Khan and Sandra Bailey for their help and support. Last but not least, I would like to thank my parents for their unfailing love and support. Thanks to my Turkish friends: Hande Ertorer, Funda Gunes, and Nurcan Tezcan Ergin for their endless support and special thanks to Carlos Reyes for his love, support, and encouragement. vi TABLE OF CONTENTS LIST OF TABLES ...................................................................................................... x LIST OF FIGURES ....................................................................................................xi CHAPTER 1. EXTREMOPHILES .............................................................................. 1 ABSTRACT ............................................................................................................ 2 1. Extremophilic Microorganisms and Enzymes ..................................................... 3 2. Structure, Function, and Stability of Extremophilic Enzymes ............................. 4 2.1. Enzymes from Extreme Thermophiles ............................................................ 4 2.1.1. Extreme thermophiles ............................................................................... 4 2.1.2. Adaptations to extreme temperatures ....................................................... 5 2.1.3. Industrial applications of extremely thermophilic enzymes ....................... 7 2.2 Enzymes from Psychrophiles ......................................................................... 16 2.2.1. Psychrophiles ......................................................................................... 16 2.2.2. Adaptations to cold temperatures ........................................................... 16 2.2.3. Industrial applications of psychrophilic enzymes .................................... 18 2.3. Enzymes from Alkaliphiles ............................................................................. 19 2.3.1. Alkaliphiles .............................................................................................. 19 2.3.2. Adaptations to high pH............................................................................ 20 2.3.3. Biotechnological applications of alkaliphilic enzymes ............................. 21 2.4. Enzymes from Halophiles .............................................................................. 23 2.4.1. Halophiles ............................................................................................... 23 2.4.2. Adaptations to high salt concentration ...................................................