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INVESTIGATOR: O`Malley, M. A.

PERFORMING INSTITUTION:
Massachusetts Institute of Technology
Cambridge, MASSACHUSETTS 02139

GENETIC IDENTIFICATION AND CHARACTERIZATION OF CELLULASES AND CELLULOLYTIC COMPLEXES FROM FUNGI

NON-TECHNICAL SUMMARY: The energy contained within plant biomass is the most promising renewable resource available for biofuel development, and is an attractive alternative to petroleum-based fuels. Most of the energy in plant material is contained within cellulose, a complex polymer of sugars in plant cell walls, which can be broken down into simple sugars for fermentation into energy-rich organic fuels (e.g. ethanol). However, the complicated structure of cellulose renders this approach quite challenging due to difficulties associated with enzymatic conversion of cellulose to simple sugars. Fortunately, nature has evolved several enzymes over millions of years that work together to break down plant material in this fashion. These enzymes can be found within bacteria and fungi that thrive in cellulose-rich environments (e.g., the digestive tract of grazing animals, compost piles, and soil). In order to accelerate the development of plant-based biofuels, this project aims to discover new cellulolytic enzymes from fungi that reside within the digestive tract of large herbivores, and to exploit them for bioprocessing. Novel enzymes will be annotated based on analyzing real-time protein production within the fungi when supported on plant material in isolated culture. Attractive cellulolytic enzymes will be produced in Saccharomyces cerevisiae (baker's yeast) to screen their reactivity and optimize their performance against plant biomass. This project will serve to significantly expand the number and diversity of known enzymes to facilitate industrial-scale cellulose breakdown, and will improve the economic feasibility of plant-based biofuels.

OBJECTIVES: This project aims to accelerate the development of cellulosic biofuels by applying powerful genomic and biochemical tools towards the discovery of novel cellulose-degrading enzymes that originate from fibrolytic anaerobic fungi. These fungi reside within the rumen and hindgut of large herbivorous animals that naturally thrive on a lignocellulose-rich diet, yet remarkably few enzymes have been identified and characterized from these microbes. Specific objectives for this project are to: (1) develop a new molecular genetic platform to identify novel fungal cellulases by illuminating differences in relative transcription levels when grown on varied carbon sources; (2) screen and evaluate activity of anaerobic fungal cellulases identified via catabolic regulation studies through heterologous expression in the yeast S. cerevisiae; and (3) optimize catalytic performance of fungal cellulases expressed in yeast to achieve maximal lignocellulase breakdown for fermentation into ethanol. Expected experimental outcomes from this project include the creation of a novel bioinformatics-based platform for cellulase discovery and the isolation of full DNA coding sequences for non-catalytic adapter complexes and cellulolytic enzymes from anaerobic fungi. Using this information, combinatorial yeast strains harboring recombinant fungal cellulases for synergistic action against lignocellulose will also be constructed and evaluated to facilitate Consolidated BioProcessing (CBP).

APPROACH: The methods and approaches employed in this project will involve the use of cutting-edge genomic and biochemical tools geared toward novel protein discovery and analysis. In order to identify novel cellulolytic enzymes from anaerobic fungi, a new bioinformatics platform will be developed based on catabolic regulation. This platform will be able to decipher intracellular transcriptional regulation patterns within fungi in order to identify proteins that are upregulated when the organism is grown on cellulose, and repressed when grown on a simpler carbon source (e.g. glucose, fructose). To establish this methodology, catabolic regulation experiments will first be performed on anaerobic fungi to determine the time frame and extent of catabolic regulation by following the transcription and translation of known cellulases from these organisms. Once such parameters are known, novel enzymes will be revealed following mRNA purification and computational transcriptome assembly, where genes that exhibit a pattern of regulation similar to known cellulases are likely involved in cellulose hydrolysis. As a complementary approach to bioinformatic discovery, biochemical chromatography methods will also be employed to isolate components of fungal cellulosomes (large, multi-protein cellulolytic complexes) based on sequence knowledge of cellulosome components. Following the identification of new candidate cellulolytic genes from fungi, these genes will be codon-optimized and ligated into yeast-specific vectors for heterologous expression and evaluation in the yeast Saccharomyces cerevisaie. This host system will serve as an ideal platform to examine enzyme production, maturation, and trafficking through the yeast secretory pathway, and is amenable to large-scale bioprocessing applications. In order to improve synergistic action of recombinant fungal cellulases expressed in yeast, mutagenesis and protein engineering approaches will be used to improve the activity of individual enzymes and cellulosome complexes. With knowledge of cellulase expression behavior in a recombinant yeast system, combinatorial yeast strains will be developed and optimized to hydrolyze model cellulosic substrates using the new enzymes, and eventually more complex lignocellulosic biomass.

PROGRESS: 2011/09 TO 2013/08
OUTPUTS: During the course of this project, several methods were developed to aid in the extraction, classification, and culture of anaerobic gut fungi to contribute to lignocellulose depolymerization and renewable biofuel production. A novel RNA extraction technique was developed to facilitate the collection of regulated mRNA for use in downstream deep sequencing efforts for fungal transcriptome assembly and determination, and a novel gut fungal isolate was extracted from the digestive tract of a horse. Relevant genetic information obtained during the project will be deposited to GenBank as the final publication from this work is submitted. This project formed the basis for an international collaboration with researchers in the UK, and the PD of this project spent approximately one month learning fundamental methods for gut fungal isolation and characterization in the laboratory of Michael K. Theodorou. Throughout this project, research findings were disseminated to the wider community through scientific publication and presentation at research conferences. Specifically, the PD presented the findings of this project at the American Institute of Chemical Engineers annual meeting in 2011 and 2012, and in public seminars in the Department of Chemical Engineering at several institutions throughout 2011 (UC-Santa Barbara, Case Western Reserve, Notre Dame, University of Maryland, UC-Riverside, and the University of Texas). PARTICIPANTS: The PD for this project was a postdoctoral fellow in the Department of Biology at MIT, and resources/instrumentation to facilitate this project were provided by MIT and the Broad Institute of MIT and Harvard. The PD traveled to the lab of Michael K. Theodorou (University of Durham/Centre for Process Innovation) to learn techniques relevant to anaerobic gut fungal culture, which were critical to completion of this project. While conducting this research, the PD also received training in course development and implementation, as she designed and taught the undergraduate elective course "Fueling Sustainability: Engineering Microbial Systems for Biofuel Production" in 2011. TARGET AUDIENCES: In order to augment the fellow's research training as it relates to cellulosic biofuels, she developed and instructed a new course at MIT entitled "Fueling Sustainability: Engineering Microbial Systems for Biofuel Production" (MIT Course 7.347). The goal of this course was to equip advanced undergraduate students at MIT with the necessary skills to read and evaluate the primary research literature as it pertains to the bioenergy field. Material discussed in this course examined technological advances in microbiology, biochemical engineering, and industrial processing that have contributed to the current state of cellulosic biofuels in the United States, while approaching the subject from a multi-disciplinary standpoint. Since enrollment was open to all undergraduate students at MIT, the class consisted of a diverse mixture of scientists, engineers, and humanities majors, which further enhanced classroom discussions while inspiring the exchange of ideas between students of different disciplines. Instruction of this course was also of utmost training value to the fellow, as she plans to continue teaching this course in her independent faculty position following completion of the fellowship. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

IMPACT: 2011/09 TO 2013/08
This project led to the isolation of a novel anaerobic gut fungus from the digestive tract of a horse, from which approximately 27,000 novel transcripts were identified. These transcripts have revealed hundreds of novel cellulase, xylanase, and other lignocellulose-active gene products, which can be directly utilized in bioprocessing efforts for renewable biofuel production. Building from this finding, it was determined that the novel fungal isolate exhibits substrate-specific catabolic regulation of its lignocellulolytic enzymes. Anaerobic gut fungal enzymes were produced in the yeast Saccharomyces cerevisiae in order to screen their reactivity and determine their enzymatic efficiency against traditional cellulosic substrates. Through these efforts, it was determined that the production of functional fungal cellulases in yeast can be achieved, though reactivity of these enzymes is not predictable. Future efforts will focus on determining the bottlenecks associated with recombinant production of functional fungal cellulases in yeast to aid in consolidated bio-processing of lignocellulose.

PUBLICATIONS (not previously reported): 2011/09 TO 2013/08
M. A. OMalley, M. K. Theodorou, C. A. Kaiser, Evaluating expression and catalytic activity of anaerobic fungal fibrolytic enzymes native to Piromyces sp E2 in Saccharomyces cerevisiae, Environmental Progress and Sustainable Energy, 31(1): 37-46 (2012).