Identification of CODH Transcription Regulator in Eubacterium limosum KIST612 using GUS Reporter System

Abstract

In prokaryotes, most metabolic systems regulate the transcription and expression of genes according to a specific substrate. In general, a factor called a transcription regulator affects the promoter region of a specific gene according to the environment or substrate to regulate transcription. In the case of strains such as E. coli, almost all regulate mechanisms have been identified, but there are many unidentified strains such as acetogen. Acetogen are anaerobic microorganisms, which can conserve ATP and metabolite through Wood-Ljungdahl Pathway. E. limosum KIST612 is an acetogen, which can conserve ATP through either autotrophic Wood-Ljungdahl Pathway or heterotrophic conditions, and it produces acetate as a prime metabolite and butyrate as well. CODH is a key-enzyme in carbon monoxide consumption at the Wood-Ljungdahl Pathway. It seems that the transcription of CODH is regulated by the presence of carbon monoxide. In acetogen, CODH transcription regulators have not been identified. The GUS reporter system is an expression system capable of identifying the presence and level of expression of a specific promoter. In order to see how transcription regulation work out in metabolic system of E. limosum KIST612, the GUS reporter systems; pECPH2::uidA (ß-glucuronidase) were constructed in E. coli and E. limosum KIST612 strains. transcription regulator identification system for identifying a regulatory system is constructed by inserting a gene's promoter that is expected to be regulated and a transcription regulator into a GUS reporter system. With the transcription regulator identification system, it would be possible to confirm the presence of transcription regulation and its level. Also, the presence of regulation of the native promoter and expression level of the strain used can be identified. Through this, it is possible to secure a strain that is not affected by the substrate and has a minimal effect on the flux in the cell.