DOI
https://doi.org/10.25772/33TF-YZ40
Defense Date
2011
Document Type
Dissertation
Degree Name
Doctor of Philosophy
Department
Engineering
First Advisor
Stephen Fong
Abstract
Currently, one of the hurdles hindering efficient production of cellulosic biofuel is the recalcitrant nature of cellulose to hydrolysis. A wide variety of cellulase enzymes are found natively in microorganisms that can potentially be used to effectively hydrolyze cellulose to fermentable sugars. Thermobifida fusca is a high G-C content, thermophilic, gram-positive soil actinobacterium with high cellulolytic activity. The phenomenological and mechanistic parameters affecting cellulase activity were studied in T. fusca and two mechanisms have been found: 1) transcriptions of cellulase-related genes were not closely associated with measured differences in cellulase activity and 2) cellular energetics (intracellular ATP) correlated more closely to changes in specific cellulase activity. In T. fusca, CelR is thought to act as the primary regulator of cellulase gene expression by binding to a 14-bp inverted repeat: 5’-(T)GGGAGCGCTCCC(A) that is upstream of many known cellulase genes. An efficient procedure for creating precise chromosomal gene replacements has been developed and this procedure was demonstrated by generating a celR deletion strain. Measurements of mRNA transcript levels in both the celR deletion strain and the wild-type strain indicated that the CelR potentially acts as a repressor for some cellulase genes and as an activator for other cellulase genes. Based on the protocol of disrupting celR gene, the direct conversion of untreated cellulosic biomass to 1-propanol in aerobic growth conditions using an engineered strain of T. fusca was demonstrated. Based upon computational predictions, a bifunctional butyraldehyde/alcohol dehydrogenase (encoded by adhE2) was added to T. fusca leading to production of 1-propanol during growth on glucose, cellobiose, cellulose (Avicel), switchgrass, and corn stover. The highest 1-propanol titer (0.48 g/L) was achieved for growth on switchgrass. The adaptive evolution of T. fusca was conducted to find a high cellulase-yield strain. The evolved strains of T. fusca were generated for two different scenarios: continuous exposure to cellobiose (strain muC with specialist phenotype) or alternating exposure to cellobiose and glucose (strain muS with generalist phenotype). Characterization of cellular phenotypes and whole genome re-sequencing were conducted for both the muC and muS strains and 18 and 14 point mutations in the muC and muS strains, respectively were verified. Among these mutations, the site mutation of Tfu_1867 was found to contribute the specialist phenotype and the site mutation of Tfu_0423 was found to contribute the generalist phenotype. The experiment results were used to test genome-scale metabolic model of T. fusca built in this study.
Rights
© The Author
Is Part Of
VCU University Archives
Is Part Of
VCU Theses and Dissertations
Date of Submission
May 2011