Transcriptomic analysis reveals adhE2-dominant expression in early-butanol-producing Clostridium acetobutylicum ATCC 824 (pCD07239)

Abstract

Clostridium acetobutylicum ATCC 824 (pCD07239), designated CD07239 in this study, was previously constructed by introducing the Clostridioides difficile CD630_0723–CD630_0729 gene cluster into the parental ATCC 824 strain. The CD07239 strain initiates butanol production during the early growth phase. To investigate the transcriptional basis of this phenotype, we performed RNA-seq-based transcriptome analysis using phenotype-defined RNA samples collected during batch fermentation. At 3 h, CD07239 had already produced approximately 0.1 g/L butanol, whereas butanol was not detected in ATCC 824. At 9 h, both strains produced butanol, but CD07239 accumulated a substantially higher level than ATCC 824. Principal component analysis revealed that the transcriptomes were separated according to fermentation stage and strain background. In ATCC 824, major solventogenic genes, including adhE1, ctfA, ctfB, and adc, were strongly induced at 9 h compared with 3 h. In contrast, early butanol production in CD07239 at 3 h was not accompanied by premature induction of these genes. Instead, adhE2 showed markedly high expression in CD07239, with log₂ fold changes of 6.4 at 3 h and 6.6 at 9 h compared with ATCC 824. Additional adhE2 expression in ATCC 824 did not reproduce early butanol production, suggesting that adhE2 alone is insufficient to induce this phenotype. These results indicate that early butanol production in CD07239 is associated with an adhE2-dominant transcriptional state distinct from the conventional solventogenic transition of ATCC 824.

Keywords

adhE2 / Butanol / Early butanol production / Clostridium acetobutylicum / Transcriptome

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Funding

National Research Foundation of Korea(2022R1A2C1006214 and RS-2024-00439872)

RIGHTS & PERMISSIONS

Jiangnan University