Efforts to install a heterologous Wood-Ljungdahl pathway in Clostridium acetobutylicum enable the identification of the native tetrahydrofolate (THF) cycle and result in early induction of solvents

Here, we report the construction of a Clostridium acetobutylicum strain ATCC 824 (pCD07239) by heterologous expression of carbonyl branch genes (CD630_0723∼CD630_0729) from Clostridium difficile, aimed at installing a heterologous Wood-Ljungdahl pathway (WLP). As part of this effort, in order to val...

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Bibliographic Details
Published in:Metabolic engineering 2023-05, Vol.77, p.188-198
Main Authors: Jang, Yu-Sin, Kim, Won Jun, Im, Jung Ae, Palaniswamy, Sampathkumar, Yao, Zhuang, Lee, Haeng Lim, Yoon, Ye Rin, Seong, Hyeon Jeong, Papoutsakis, Eleftherios T., Lee, Sang Yup
Format: Article
Language:English
Subjects:
Acidogenic phase
Butanol
Butanols - metabolism
Clostridium acetobutylicum
Clostridium acetobutylicum - genetics
Clostridium acetobutylicum - metabolism
Fermentation
Solvents
Tetrahydrofolate
Wood
Wood-Ljungdahl pathway
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Summary:Here, we report the construction of a Clostridium acetobutylicum strain ATCC 824 (pCD07239) by heterologous expression of carbonyl branch genes (CD630_0723∼CD630_0729) from Clostridium difficile, aimed at installing a heterologous Wood-Ljungdahl pathway (WLP). As part of this effort, in order to validate the methyl branch of the WLP in the C. acetobutylicum, we performed 13C-tracing analysis on knockdown mutants of four genes responsible for the formation of 5-methyl-tetrahydrofolate (5-methyl-THF) from formate: CA_C3201, CA_C2310, CA_C2083, and CA_C0291. While C. acetobutylicum 824 (pCD07239) could not grow autotrophically, in heterotrophic fermentation, it began producing butanol at the early growth phase (OD600 of 0.80; 0.162 g/L butanol). In contrast, solvent production in the parent strain did not begin until the early stationary phase (OD600 of 7.40). This study offers valuable insights for future research on biobutanol production during the early growth phase. •THF cycle to yield CH3-THF was evaluated in C. acetobutylicum.•Methyl branch of Wood-Ljungdahl pathway was validated by 13C-tracing.•Heterologous Wood-Ljungdahl pathway was installed in C. acetobutylicum.•Butanol production at early growth phase was achieved in engineered strain.
ISSN:1096-7176
1096-7184
DOI:10.1016/j.ymben.2023.04.005