Markerless deletion of the putative type I and III restriction-modification systems in the cellulolytic bacterium Clostridium cellulovorans using a codBA-based counterselection technique.

Cellulose from lignocellulosic biomass (LB) is of increasing interest for the production of commodity chemicals. However, its use as substrate for fermentations is a challenge due to its structural complexity. In this context, the highly cellulolytic Clostridium cellulovorans has been considered an interesting microorganism for the breakdown of LB. C. cellulovorans does not naturally produce solvents in useful concentrations, but this could be achieved by metabolic engineering. Unfortunately, this is hampered by the lack of tools for genetic engineering. We describe a genetic system that allows strain engineering by the allelic-coupled exchange method. First, the Gram-positive origin of pUB110 was identified as a suitable clostridial 'pseudo-suicide' origin of replication for the construction of deletion vectors. Second, an efficient counterselection strategy based on a codBA cassette and the use of 5-fluorocytosine as the counterselective compound was employed. Third, since the prevention of DNA transfer by host restriction-modification (RM) systems is a critical barrier to genome engineering, deletion plasmids containing flanking regions for the putative type I (Clocel_1114) and III (Clocel_2651) RM systems were constructed and transferred into C. cellulovorans. The restriction-less strains C. cellulovorans ΔClocel_1114 and C. cellulovorans ΔClocel_2651 exhibit high conjugation efficiency and can be easily used for further metabolic engineering.