Bioprospecting for thermostable glycoside hydrolases : a metatranscriptomic approach

M.S. University of Hawaii at Manoa 2012.Includes bibliographical references.The conversion of cellulosic material from linear form to monomer is the rate-limiting step in current biofuels production technologies from lignocellulosic material. The renewed focus on clean, sustainable, carbon-neutral fuels has resulted in increased interest in novel cellulolytic enzymes and microbial strains, with the aim to increase the efficiency of the above conversion. However, the availability of suitable cellulolytic enzymes has been restricted by the limited number of cellulolytic microbial strains in which these enzymes can be procured. One approach to increase efficiency has been to bioprospect novel thermophilic microbial strains, the logic being that an increase in production temperature will result in increased rates of lignocellulosic hydrolysis. Unfortunately, microbiologists have had limited success in cultivating cellulolytic thermophiles with only a small number of strains isolated. The goal of this project was to circumvent the need to cultivate cellulolytic thermophiles by applying a metatranscriptomic approach to discover new thermostable cellulases commonly referred to as glycoside hydrolases (GH-enzymes that hydrolyze the glycosidic linkages between sugar molecules). This was achieved by employing a novel in situ enrichment technique to enrich for thermostable GHs directly from a geothermal environment. These GHs were being actively expressed by the resident microbial population to hydrolyse lignocellulose. This approach not only eliminated the need for cultivation, but also selected for the actively expressed GHs unregulated in response to the lignocellulosic material feedstock. This strategy removed the emphasis on identifying potentially relevant and substrate-active GHs from genomic data via genomic analysis of a cultivated microorganism or from environmental metagenomic surveys