A Comprehensive Study of Eubacterium callanderi KIST612: From Reclassification to Tunable Expression, Butyrate and CODH/ACS Metabolism

Abstract

The increasing demand for sustainable energy solutions has driven research into microbial bioconversion processes that utilizing acetogens. Acetogens are anaerobic bacteria capable of converting C1 gases, such as carbon monoxide (CO), carbon dioxide (CO2), and hydrogen (H2), into valuable biofuels and chemicals via the reductive acetyl-CoA pathway. Among these, Eubacterium callanderi KIST612 has emerged as a promising candidate due to its high CO tolerance (up to 200 kPa) and its metabolic versatility. Before this strain could be utilized for industrial applications, its characteristics were analyzed. Comparative genomic analyses, including 16S rRNA, housekeeping gene, and core-proteome comparisons, confirmed that KIST612 shares 100% sequence identity for rpoB and gyrA with E. callanderi DSM 3662, but only 96.0% and 95.8% identity with E. limosum ATCC 8486, respectively. Average nucleotide identity (ANI) analysis further supported its reclassification, with KIST612 exhibiting an ANI value of 98.7% compared to 100% for E. callanderi DSM 3662. Metabolic profiling revealed that KIST612 produces butyrate under H2/CO2 conditions, distinguishing it from E. limosum ATCC 8486. Following this, the study aimed to enhance the industrial applicability of E. callanderi KIST612 through a three-step approach: (1) the development of a genetic toolkit, (2) an understanding of metabolic pathways, and (3) the identification of transcriptional regulation mechanisms. First, a tunable gene expression system was developed for E. callanderi KIST612 using a synthetic promoter library and CRISPR interference (CRISPRi). A total of 325 synthetic promoters with varying strengths were constructed, and their activities were quantified using GFP assays, demonstrating an 85.4-fold range in promoter strength. CRISPRi-mediated gene knockdown, utilizing the strongest synthetic promoter, achieved nearly complete repression (hyper-knockdown) of target gene expression, demonstrating a strong correlation between promoter strength and knockdown efficiency. Next, to investigate alternative butyrate biosynthesis pathways, acetate kinase promiscuity was examined. The specific activity of butyryl-phosphate to butyrate conversion by acetate kinase was confirmed to be 154.0 U mg-1. Based on these results, the enzyme promiscuity of acetate kinase was revealed. Comparative sequence analysis identified conserved motifs among butyrate-producing acetogens, suggesting an evolutionary basis for this promiscuity. Finally, the transcriptional regulation of the Carbon Monoxide Dehydrogen¬ase/Acetyl-CoA Synthetase (CODH/ACS) was studied, identifying Rex as a key transcription regulator. Electrophoretic mobility shift assays (EMSA) confirmed that Rex binds to the promoter regions of the CODH/ACS cluster, demonstrating its role in redox-dependent (NAD+/NADH) transcriptional regulation. By integrating strain reclassification, genetic engineering, metabolic analysis, and regulatory optimization, this research provides a comprehensive framework for improving acetogen-based bioprocesses. The findings contribute to the advancement of sustainable C1 gas bioconversion, thereby facilitating the industrial-scale production of biofuels and biochemical.