title: Metabolic engineering of cofactor F420 production in Mycobacterium smegmatis.
abstract: 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 F(420) 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.
authors.affiliation: Structural Biology Laboratory, School of Biological Sciences, The University of Auckland, Auckland, New Zealand. g.bashiri@auckland.ac.nz
authors.forename: Ghader
authors.initials: G
authors.lastname: Bashiri
authors.forename: Aisyah M
authors.initials: AM
authors.lastname: Rehan
authors.forename: David R
authors.initials: DR
authors.lastname: Greenwood
authors.forename: James M J
authors.initials: JM
authors.lastname: Dickson
authors.forename: Edward N
authors.initials: EN
authors.lastname: Baker
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry; D017123:Bacteriophage T7 / Q000378:metabolism; D001483:Base Sequence; D018360:Crystallography, X-Ray / Q000379:methods; D004583:Electrons; D005821:Genetic Techniques; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D020102:Mycobacterium smegmatis / Q000378:metabolism*; D009169:Mycobacterium tuberculosis / Q000378:metabolism*; D016366:Open Reading Frames; D010957:Plasmids / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D012256:Riboflavin / Q000031:analogs & derivatives* / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; C015084:coenzyme F420; D012256:Riboflavin