Metabolic Engineering of Cofactor F420 Production in Mycobacterium smegmatis

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

Determination of environmental impacts of antimicrobial usage for US Northern Great Plains swine-production facilities: a life-cycle assessment approach

Purpose This study used life-cycle assessment (LCA) methodology to examine the environmental effects associated with sub-therapeutic tylosin and chlortetracycline (CTC) antimicrobial use within US Northern Great Plains (NGP) swine-production facilities. Antimicrobial feed-additive use is widespread within this industry and is expected to play an integral role within future carbon-management strategies due to its ability to increase feed efficiency and control disease. Materials and methods The LCA model system boundaries for this study were: (1) antimicrobial manufacturing; (2) feed manufacturing; (3) transport of antimicrobials to the feed-mill and completed feed to the swine-production facility; (4) electricity and propane use associated with swine-production operations; and (5) swine enteric and manure-storage and handling emissions. The functional unit is the growth life cycle of one head of swine from starter (7 kg) to finisher (111 kg market weight; “wean-to-finish”) production stages. Environmental impacts considered include global warming, acidification and eutrophication, ecotoxicity, and fossil-fuel use following EcoIndicator 99 assessment methodology. Results and discussion High-estimated energy requirements associated with CTC and tylosin manufacturing, coupled with the large transportation distances to the feed manufacturing and swine-production facilities increased climate change and ecotoxicity impacts compared with a no antimicrobial-use scenario. However, feeding CTC resulted in several local positive changes including increased feed utilization, lower producer costs due to shortened production times, and reduced manure greenhouse gas emissions. These positive changes in the local environment however did not offset negative global impacts associated with material manufacturing and transport for the specific scenarios analyzed. Increased use of renewable-energy sources for both swine and antimicrobial production resulted in net environmental enhancement. Conclusions This study demonstrates both the beneficial and negative environmental aspects associated with sub-therapeutic antimicrobial within the swine-production industry, and provides swine producers and environmental practitioners with tangible alternatives for meeting both livestock-health management and future carbon-management constraints within a reduced-carbon-emission consumer and regulatory marketplace