Development of highly characterized genetic bioparts for efficient gene expression in CO₂-fixing Eubacterium limosum

Acetogenic bacteria demonstrate industrial potential for utilizing carbon dioxide (CO₂) for biochemical production using the Wood-Ljungdahl pathway. However, the metabolic engineering of acetogenic bacteria has been hampered by the limited number of available genetic bioparts for gene expression. Here, we integrated RNA sequencing, ribosome profiling, differential RNA sequencing, and RNA 3'-end sequencing results of Eubacterium limosum to establish genetic bioparts, such as promoters, 5' untranslated regions, and transcript terminators, to regulate transcriptional and translational expression of genes composing of biosynthetic pathways. In addition, a transformation method for the strain was developed to efficiently deliver the obtained genetic bioparts into cells, resulting in a transformation efficiency of 2.5 × 10⁵ CFU/μg DNA. Using this method, the genetic bioparts were efficiently introduced, and their strengths were measured, which were then applied to optimize the heterologous expression of acetolactate synthase and acetolactate decarboxylase for non-native biochemical acetoin production. The strategy developed in this study is the first report on integrating multi-omics data for biopart development of CO₂ or syngas utilizing acetogenic bacteria, which lays a foundation for the efficient production of biochemicals from CO₂ or syngas as a carbon feedstock under autotrophic growth conditions.