A characterized promoter suite enables metabolic rewiring of the acetogen Acetobacterium woodii toward ethanol production.

Abstract

Acetogenic bacteria are promising platforms for converting gaseous and liquid one-carbon (C1) substrates such as carbon monoxide, carbon dioxide (CO2), formate and methanol into fuels and chemicals, but their application is limited by sparse genetic tools and poor control over metabolic fluxes. Here, we developed a modular plasmid assembly platform and a suite of characterized native, heterologous and methanol-inducible promoters for the model acetogen Acetobacterium woodii, enabling rapid and tunable control of gene expression together with CRISPR-based genome editing. Using an anaerobe-compatible fluorescent reporter (pFAST), promoter strength was quantified, from medium to high expression levels. Subsequently, a minimal promoter replacement at the native adhE (bifunctional aldehyde-alcohol dehydrogenase) locus along with deletion of the neighboring LysR-type regulator was done employing the newly characterized promoters, demonstrating that a single promoter replacement in the genome was sufficient to shift A. woodii from a primarily acetogenic toward a partly ethanologenic phenotype. The engineered strain was able to produce ethanol not only from fructose but also from the C1 substrates methanol + CO2 and formate + syngas. Together with the promoter replacement strategy, these findings underline that ethanol formation in A. woodii is constrained both by energy conservation and by endogenous transcriptional control. Relieving energetic bottlenecks through the choice of energetically favorable C1 substrates and decoupling adhE expression from its native promoter suffices to enable ethanol production from methanol + CO2 and formate + syngas, directly linking the Wood-Ljungdahl pathway to ethanol formation. These results illustrate how small, targeted genome edits can significantly reshape carbon flux in acetogens and positions A. woodii as a viable chassis for C1-based bioproduction.