Isolation of fungal cellulase gene transcript from Penicillium spinulosum A Master’s Thesis Presented to the faculty of The College of Science and Mathematics Colorado State University – Pueblo In Partial Fulfillment of the requirements for the degree of Master of Science in Biochemistry By Srivatsan Parthasarathy Colorado State University – Pueblo May, 2018 ACKNOWLEDGEMENTS I would like to thank my research mentor Dr. Sandra Bonetti for guiding me through my research thesis and helping me in difficult times during my Master’s degree. I would like to thank Dr. Dan Caprioglio for helping me plan my experiments and providing the lab space and equipment. I would like to thank the department of Biology and Chemistry for supporting me through assistantships and scholarships. I would like to thank my wife Vaishnavi Nagarajan for the emotional support that helped me complete my degree at Colorado State University – Pueblo. III TABLE OF CONTENTS 1) ACKNOWLEDGEMENTS …………………………………………………….III 2) TABLE OF CONTENTS …………………………………………………….....IV 3) ABSTRACT……………………………………………………………………..V 4) LIST OF FIGURES……………………………………………………………..VI 5) LIST OF TABLES………………………………………………………………VII 6) INTRODUCTION………………………………………………………………1 7) MATERIALS AND METHODS………………………………………………..24 8) RESULTS………………………………………………………………………..50 9) DISCUSSION…………………………………………………………………….77 10) REFERENCES…………………………………………………………………...99 11) THESIS PRESENTATION SLIDES……………………………………………...113 IV ABSTRACT Cellulose and cellulosic materials constitute over 85% of polysaccharides in landfills. Cellulose is also the most abundant organic polymer on earth. Cellulose digestion yields simple sugars that can be used to produce biofuels. Cellulose breaks down to form compounds like hemicelluloses and lignins that are useful in energy production. Industrial cellulolysis is a process that involves multiple acidic and thermal treatments that are harsh and intensive. Enzymatic breakdown of cellulose is regarded as the most productive method for cellulolysis as it can occur under environmentally benign conditions. Cellulolytic enzymes, cellulases, are produced naturally by several organisms and synthesized industrially for commercial applications. Fungi are major producers of cellulases because of their requirement to contact and utilize plant products through cellulolysis. Our lab identified a cellulase synthesized by Penicillium spinulosum that has a higher activity as compared to other conventional fungal sources of cellulase. The study described here focuses on isolating the gene that codes for this fungal cellulase using a genetic approach. Cellulase gene targets were identified using degenerate primer PCRs and sequences having similarity to cellulases were selected. These PCR primers were then used to identify regions in fungal RNA that encoded cellulase. These regions were used to identify a possible coding sequence that can be constructed using cDNA libraries to yield a gene that codes for a functional cellulolytic protein. This study improves the understanding of the genetics of a cellulase gene in P. spinulosum and yields products that can be used to produce the high-activity cellulase in vitro. V LIST OF FIGURES Figure No. Figure Name Page Number 1. Penicillium spinulosum on cellulose 4 2. Electroin micrograph of Penicillium mycelia 5 3. Penicillin structure 6 4. Cellulose monomer structure 9 5. Mechanism of action of cellulose oxidases 11 6. Mechanism of action of cellulose phosphorylase 12 7. Cellulolysis reaction 12 8. Fungal cellulase crystal structure 14 9. 5’ 3’ RACE reaction overview 18 10. Biofuel production pipeline 22 11. a Genomic DNA agarose gel electrophoresis 55 11. b DNA quantification 55 12. a Annealing temperature optimization 56 12. b Annealing temperature for all primer sets 56 12. c Expanded view of gradient PCR 57 13. a RNA agarose gel electrophoresis 61 13. b Analysis of two different RNA isolations 61 13. c pH optimization for RNA isolation 62 13. d Optimal RNA bands 62 14. a cDNA in agarose gel electrophoresis 63 14. b RT-gradient PCR in agarose gel electrophoresis 65 14. c RT-PCR of all primer sets 65 15 E. coli colonies after transformation 67 16. a Screening of transformants using primer A 68 16. b Screening of transformants using primer B 68 16. c Screening of transformants using primer C 68 16. d Screening of transformants using primer D 68 17. a RACE reaction in primer G 71 17. b RACE reaction repeated using same G primers 71 18 PCR using mixed batch of primers 72 19. a Genomic DNA contamination in RNA isolation 74 19. b Effect of pH of phenol in RNA isolation 74 19. c Effect of incubation times in RNA isolation 75 19. d Effect of number of ethanol washes in isolation 75 19. e Optimal RNA prep. in gel electrophoresis 76 20. Comparison of genomic DNA isolation 83 21. Overview of TA cloning 91 22. TA cloning strategy 92 23. RACE reaction overview 94 VI LIST OF TABLES Table No. Table Name Page Number 1. Standard Growth media composition 25 2. Probe sequences of cellulase DNA 29 3. PCR reaction mixture 31 4. RT master mix composition 37 5. Cloning reaction components 38 6. RACE buffer mix 42 7. 5’ RACE cDNA reaction 42 8. 3’ RACE cDNA reaction 43 9. RACE master mix 43 10. RACE product PCR 44 11. Nested Mixed batch PCR mix 45 12. Optimal annealing temperatures 58 13. BLAST analysis of amplified sequence 69 14. Optimization of RNA isolation protocol 90 Scheme 1. P. spinulosum gene isolation strategy 79 VII INTRODUCTION 1 Cellulose is the most abundant carbohydrate polymer on earth and comprises a major portion of the biomass in landfills. It has been found that plants annually generate 180 billion tons of cellulose worldwide (Sticklen 2008). According to the Environmental Protection Agency (EPA), up to 1,600 landfill sites have been created in the USA which accommodate up to 250 million tons of cellulosic waste in 2010. The use of cellulose and cellulosic byproducts in modern times is extensive and therefore, the modification of cellulose and its byproducts and the disposal of these materials in a cost- and energy-effective manner are a major topic in environmental and biomass conversion literature. Cellulose is notably resistant to depolymerization by chemical means. Much of the recent literature deals with methods that treat cellulose from different sources with a combination of chemical and enzymatic treatments to reduce its crystallinity and size (Cheng, Varanasi et al. 2011). Most of these applications warrant the breakdown of cellulose, a polymer of glucose subunits linked through β-1,4-linkages. One of the most efficient ways to breakdown cellulose is by using cellulases, which are enzymes found in bacteria isolated from ruminant stomachs and fungi. Use of these enzymes not only increase the yield of the reaction but also helps in minimizing the hazardous effluents that are obtained as a result of other harsher processes like using high temperature and pressures (Deguchi 2006) or hydrolysis using very low pH acids (Fan 1987). Previous research in Dr. Bonetti’s lab detailed the isolation of a fungal cellulase protein from Penicillium spinulosum. A P. spinulosum protein band (ca. 105 kDa) containing β-glucosidase activity, obtained from size exclusion chromatography, was characterized by HPLC/MS/MS. This band showed homology to two P. chrysogenum peptides (Wheeler et al. 2011). This led to the hypothesis that isolation of the transcribing portions of a P. spinulosum cellulase gene may help reconstruct the gene sequence and improve the bioavailability of the enzyme using molecular cloning strategies. This knowledge can then be used in several applications that have immense 2 potential and benefit in environmental and industrial applications. Examples of these cellulase applications include digestion of landfill wastes and reduction of landfill size, conversion of cellulosic wastes to biofuel feedstock, and modification of carbohydrate-rich food, paper or wood products. FUNGI Fungi are a group of organisms that belong to a kingdom of eukaryotic organisms. Although the classification of fungi as a separate kingdom started in the early 18th century, the importance of mycology grew only during the late 19th century (Bruns 2006). Fungi can range from being microscopic to macroscopic. The most important characteristic that demarcates fungi from other kingdoms like plants, animals, protists and bacteria is the presence of a cell wall made of chitin. There are other characteristics that make it a monophyletic group, which is a group with a common ancestor (Hibbett, Binder et al. 2007). In the field of mycology, all fungal organisms come under a group of “eumycetes” that differ from myxomycetes (slime molds) and oomycetes (water molds) (McGinnis and Tyring 1996). Previous classification of organisms into different kingdoms was based on certain morphological and observable features but with the advances in molecular techniques, classification started to be based on the ribosomal genes. Recent classifications of fungi and other organisms are also based on DNA sequences (Hibbett, Binder et al. 2007). Most fungi grow as hyphae which are cylindrical structures that contain nuclei and are the basic unit of every fungus. These hyphae form interconnected networks through apical budding and branching that lead to the formation of mycelia (Harris 2008). Even though hyphae and mycelia are microscopic, the network becomes extensive which makes the mycelia visible or macroscopic. These mycelia usually bear a spore-bearing fruiting body called sporangia at their tips. The fungi 3 are