Engineering E. coli toward consolidated bioprocessing of cellulose
Rutter, Charles David
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Cellulosic biomass is an incredibly abundant resource and a capable feedstock for production of energy, biofuels, and commodity chemicals. Current technologies for bioprocessing of cellulose utilize a three-step process in which enzymes capable of cellulose hydrolysis are expressed and purified, cellulose is hydrolyzed, and then product is formed in separate processes. This multi-step processing increase costs. As such, one approach to lowering these costs it to develop on consolidated system in which all three of these processes occur in a single step. Toward this aim, the three main goals of this dissertation are (1) characterization of a new hydrolytic enzyme and its application to fermentation of relevant sugars, (2) selection of proteins capable of intracellular cellobiose transport, and (3) development of a minimal set of cellulases capable of extensive hydrolysis under physiological conditions. A mixture of cellodextrins is produced by enzymatic hydrolysis of cellulose and Ced3A, a cellodextrinase, was shown to hydrolyze all of these completely to glucose and confer the ability to metabolize these sugars to E. coli when expressed. Activity on cellobiose, however, was lower than on other species. Co-expression of Cep94A, a cellobiose phosphorylase, and Ced3A was shown to improve the cellobiose metabolism of E. coli. In order to facilitate conversion of cellobiose to glucose by Cep94A, cellobiose must be transported into the cytoplasm. Three cellobiose permease enzymes, LacY, CP1, and CP2, were expressed in E. coli. It was shown that each protein has affinity for cellobiose transport and expression of each 126 allowed fermentation of cellobiose by E. coli strains expressing a cytoplasmic cellobiase. All three proteins are likely suitable for cellobiose transport during a consolidated bioprocess. Finally, a system of three cellulase enzymes Cel5H, Cel9R, and Cel48S were evaluated at E. coli physiological conditions and it was shown that extensive hydrolysis occurred at over half of the compositions tested. Additionally, when strains expressing cellulases were grown in binary culture with strains previously engineered for cellodextrin metabolism substantial product formation was observed, representing suitable performance of a consolidated cellulose bioprocess. This dissertation presents successful performance of all three components necessary for consolidated bioprocessing both individually and when working in tandem. Furthermore, the technologies developed in this dissertation demonstrate the capacity for consolidated bioprocessing of cellulose.