Engineering the Metabolic Profile of Clostridium cellulolyticum with Genomic DNA Libraries

Abstract

Clostridium cellulolyticum H10 (ATCC 35319) has the ability to ferment cellulosic substrates into ethanol and weak acids. The growth and alcohol production rates of the wild-type organism are low and, therefore, targets of metabolic engineering. A genomic DNA expression library was produced by a novel application of degenerate oligonucleotide primed PCR (DOP-PCR) and was serially enriched in C. cellulolyticum grown on cellobiose in effort to produce fast-growing and productive strains. The DNA library produced from DOP-PCR contained gene-sized DNA fragments from the C. cellulolyticum genome and from the metagenome of a stream bank soil sample. The resulting enrichment yielded a conserved phage structural protein fragment (part of Ccel_2823) from the C. cellulolyticum genome that, when overexpressed alone, enabled the organism to increase the ethanol yield by 250% compared to the plasmid control strain. The engineered strain showed a reduced production of lactate and a 250% increased yield of secreted pyruvate. Significant changes in growth rate were not seen in this engineered strain, and it is possible that the enriched protein fragment may be combined with the existing rational metabolic engineering strategies to yield further high-performing cellulolytic strains.