1-30: Advances in the development of a plasmid transfer system suitable for cellulolytic Clostridial species

Enhancing biofuel (H₂, ethanol) yields during the fermentation of lignocellulosic substrates by anaerobes such as Clostridium thermocellum is of paramount importance if production costs are to become economically competitive.  Currently, a lack of robust transformation strategies has limited the metabolic engineering of cellulolytic anaerobes to only a handful of studies. The following outlines our progress in a developing a suitable transformation system for Clostridial species.  Our findings show that the efficiency of transformation for C. thermocellum strain ATCC 27405 is consistently low with values of approximately 4.3 x 10² cfu/ug DNA.  Consequently, screening for mutants, obtained via single-crossover homologous recombination, has proven to be a laborious process. While transformants containing the replicative plasmid pNW33mob may be subcultured for multiple passages, plasmid loss remains a concern that may be related to aggressive restriction modification systems.  Bioinformatic analysis of C. thermocellum strains ATCC 27405, DSM 4150, DSM 2360 and DSM 1313 as well as mesophilic species Clostridium cellulolyticum H10 and Clostridium termitidis for the presence of restriction modification systems reveals that C. thermocellum strain 27405 possesses numerous endogenous nucleases compared to these other strains and species.  Given the difficulties associated with isolating recombinant mutants using suicide vectors in Clostridial species, an alternative strategy (ClosTron) that uses replicative plasmids and elements derived from the broad host range Ll.LtrB intron of Lactococcus lactis is being investigated for use in C. termitidis.  Insights gained from these studies will be vital to future efforts aimed at the alteration of existing metabolic pathways within cellulolytic species.