Genomics of cellulolytic clostridia and development of rational metabolic engineering strategies
Carere, Robert Carlo
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Consolidated bioprocessing, a process in which cellulase production, substrate hydrolysis, and fermentation occur simultaneously, offers the potential for lower biofuel production costs than traditional approaches and is an economically attractive near-term goal for fermentative production of ethanol and/or hydrogen (H2) as biofuels. Current yields fall short of theoretical maxima, vary considerably between species, and are influenced by the highly branched metabolic pathways utilized by fermentative organisms. For fermentative ethanol/ H2 production to become practical, yields must be increased either through intelligent species selection, a manipulation of culture conditions, or via the implementation of rational metabolic engineering strategies. A comparative genomics approach amoungst select members of the Firmicutes, Euryarchaeota, and Thermotogae was used to identify genes relevent to ethanol and H2 production. Growth, end-product synthesis, enzyme activities and the associated transcription of select genes were studied in the cellulolytic anaerobe, Clostridium thermocellum ATCC 27405, during batch fermentation of cellobiose to determine the effect of elevated N2 and H2 sparging on end-product distribution. The absence of genes encoding acetaldehyde dehydrogenase and bifunctional acetaldehyde/alcohol dehydrogenase (AdhE) correlates with elevated H2 yields and low ethanol production. The type(s) of encoded hydrogenases appear to have minimal impact on H2 production in organisms that do not encode ethanologenic pathways, however, they do influence reduced end-product yields in those that do. We also find that while gas sparging can be used to effectively shift carbon and electron flow, the observed shifts at the pyruvate branch-point are likely principally influenced by the availability of reduced electron carriers (NAD, NADP, ferredoxin) and thermodynamic considerations. Finally, both electrotransformation and conjugative plasmid protocols were developed and evaluated for thermophilic species C. thermocellum and Thermoanaerobacter pseudethanolicus 39E, and the mesophilic bacterium, Clostridium termitidis CT1112. The efficiency of transformation for C. thermocellum strain ATCC 27405 is consistently low whereas transformation frequencies were ~100-fold higher in C. termitidis. Observed frequencies of plasmid transfer, via conjugation, were similar in both C. thermocellum and C. termitidis suggesting the transfer of single stranded DNA may circumvent aggressive restriction methylation systems.