Development of a markerless gene knock-out system forMannheimia succiniciproducensusing a temperature-sensitive plasmid

A temperature-sensitive derivative of the Mannheimia varigena plasmid pMVSCS1 was constructed by hydroxylamine treatment for use in the development of a markerless gene knock-out system for Mannheimia succiniciproducens. The temperature-sensitive plasmid pMVSCS1-ts was stably maintained at 30 degrees C, but failed to replicate at 42 degrees C. DNA sequencing of the replication origin revealed a single base substitution as being responsible for its temperature sensitivity. The region of replication origin was amplified by PCR to construct an Escherichia coli-M. succiniciproducens shuttle vector pME19-ts to further examine the thermosensitivity. To make markerless mutants of M. succiniciproducens, the Cre-lox system with the variant lox66 and lox71 sites was used to prevent the instability caused by multiple loxP sites in the genome. The transient cre expression was carried out using the temperature-sensitive plasmid pCRX5, which was consequently cured after the verification of the markerless mutant by growing cells at 42 degrees C. For the demonstration of the markerless deletion of multiple genes using this method, the ldhA gene and the oadGAB operon of M. succiniciproducens encoding lactate dehydrogenase and oxaloacetate decarboxylase, respectively, were successfully deleted sequentially. This markerless deletion method should be useful for further metabolic engineering of M. succiniciproducens, which is a promising industrial bacterium for succinic acid production from renewable resources.