Metabolic Engineering of Cofactor F420 Production in Mycobacterium smegmatis

Cofactor F-420 is a unique electron carrier in a number of microorganisms including Archaea and Mycobacteria. It has been shown that F-420 has a direct and important role in archaeal energy metabolism whereas the role of F-420 in mycobacterial metabolism has only begun to be uncovered in the last few years. It has been suggested that cofactor F-420 has a role in the pathogenesis of M. tuberculosis, the causative agent of tuberculosis. In the absence of a commercial source for F-420, M. smegmatis has previously been used to provide this cofactor for studies of the F-420-dependent proteins from mycobacterial species. Three proteins have been shown to be involved in the F420 biosynthesis in Mycobacteria and three other proteins have been demonstrated to be involved in F-420 metabolism. Here we report the over-expression of all of these proteins in M. smegmatis and testing of their importance for F-420 production. The results indicate that co-expression of the F-420 biosynthetic proteins can give rise to a much higher F-420 production level. This was achieved by designing and preparing a new T7 promoter-based co-expression shuttle vector. A combination of co-expression of the F-420 biosynthetic proteins and fine-tuning of the culture media has enabled us to achieve F-420 production levels of up to 10 times higher compared with the wild type M. smegmatis strain. The high levels of the F-420 produced in this study provide a suitable source of this cofactor for studies of F-420-dependent proteins from other microorganisms and for possible biotechnological applications.