Improving the Growth Rate of Cellulolytic Clostridia Through Genomic Library Enrichment

Clostridium cellulolyticum (ATCC 35319) contains a fully functional cellulosome enabling the degradation of cellulose and hemicellulose for fermentation. We ultimately plan to use this organism 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 enriching collections of mutants through a growth competition assay allows for rapid self-selection of genes, regulatory sequences, or DNA fragments that yield increased cell growth. Here, we also report the findings of transgenic library enrichment. A genomic library was 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 and natural selection through continuous culture bioreactor allowed us to identify a similar genomic fragment within both organisms which, when overexpressed in C. cellulolyticum, gives a significant growth advantage over organisms containing control plasmids.  These represent significant steps in developing an economically viable cellulolytic organism for the production of biofuels in a consolidated bioprocess.  Here, we provide significant details of the enrichment results and assess the capabilities of C. cellulolyticum in a consolidated bioprocess.