Genetic engineering system for syngas-utilizing acetogen, Eubacterium limosum KIST612

Acetogens are capable of fixing inorganic gases to organic acids with/without bio-alcohols via the Wood-Ljungdahl pathway. Because of their potential to produce value-added metabolites, acetogens have received attention in the field of biorefinery. Eubacterium limosum KIST612 is one of the promising acetogens, which has been well studied for its syngas utilizing ability, and its genomic data are available. A strain-specific genetic engineering system for maximal benefits has not yet been studied in the strain, though. In the present study, we first developed a foreign gene expression system using a native constitutive promoter and the CRISPR/Cas9-based gene editing system for KIST612. Transcriptomics-based candidate selection of native promoter and GUS reporter gene assay enabled the identification of promoter of rubredoxin oxidoreductase (P_rbo) as a strong constitutive promoter in the strain. Application of Cas9-mediated genome editing was able to effectively delete the pyrF gene in the chromosome of KIST612.