(567br) A Combinatorial Approach to Genetic Library Enrichment of C. Cellulolyticum On Lignocellulose

Clostridium cellulolyticum (ATCC 35319) contains a fully functional cellulosome enabling the degradation of cellulose and hemicellulose for fermentation. Ultimately, this organism is of consideration for biofuels production in a consolidated bioprocess. However, substantial improvements in cell growth rate are still needed before the organism can prove useful for an industrial fermentation. Creating a C. cellulolyticum genomic DNA (gDNA) library and subjecting enriched cells to a growth competition assay provides a means to rapidly screen for genes, regulatory sequences, or DNA fragments that result in increased cell growth. C. cellulolyticum gDNA spanning the entire genome was fractured into 700-3000 bp fragments and cloned into the clostridial expression vectors pIMP1 (no promoter) and pSOS95 (strong promoter). The new plasmids were transfected into wild-type C. cellulolyticum cultures. Library enrichment was performed on cellulose and hemicellulose substrates, self-selecting for the inserts most beneficial to growth rate. A single round of library enrichment led to a strain of C. cellulolyticum which overcomes the metabolic bottlenecks present in the parent strain, leading to more rapid lignocellulose uptake, faster cellular growth rate, and increased tolerance to alcohol fermentation products. Here, we also report the findings of transgenic library enrichment. A genomic library was also prepared from the industrially-viable C. acetobutylicum (ATCC 824) gDNA and enriched in C. cellulolyticum cultures. We have used the results from dual library enrichments to further identify metabolic bottlenecks of C. cellulolyticum and identify strategies to engineer higher growth rates in this organism. These represent significant steps in developing an economically viable cellulolytic organism for the production of biofuels in a consolidated bioprocess.