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Background  Lignocellulosic biomass is a promising resource of renewable biochemicals and biofuels. However, the presence of inhibitors existing in lignocellulosic hydrolysates (LCH) is a great challenge to acetone-butanol-ethanol (ABE) fermentation by  Clostridium acetobutylicum . In particular, phenolic compounds (PCs) from LCH severely block ABE production even at low concentrations. Thus, it is urgent to gain insight into the intracellular metabolic disturbances caused by phenolic inhibitors and elucidate the underlying mechanisms to identify key industrial bottlenecks that undermine efficient ABE production.    Results  In this study, a time-course of ABE fermentation by  C. acetobutylicum  in the presence of four typical PCs (syringaldehyde, vanillin, ferulic acid, and  p -coumaric acid) was characterized, respectively. Addition of PCs caused different irreversible effects on ABE production. Specifically, syringaldehyde showed the greatest inhibition to butanol production, followed by vanillin, ferulic acid, and  p -coumaric acid. Subsequently, a weighted gene co-expression network analysis (WGCNA) based on RNA-sequencing data was applied to identify metabolic perturbations caused by four LCH-derived PCs, and extract the gene modules associated with extracellular fermentation traits. The hub genes in each module were subjected to protein–protein interaction analysis and enrichment analysis. The results showed that functional modules were PC-dependent and shared some unique features. Specifically,  p -coumaric acid caused the most extensive transcriptomic disturbances, particularly affecting the gene expressions of ribosome proteins and the assembly of flagella, DNA replication, repair, and recombination; the addition of syringaldehyde caused significant metabolic disturbances on the gene expressions of ribosome proteins, starch and sucrose metabolism; vanillin mainly disturbed purine metabolism, sporulation and signal transduction; and ferulic acid caused a metabolic disturbance on glycosyl transferase-related gene expressions.    Conclusion  This study uncovers novel insights into the inhibitory mechanisms of PCs for the first time and provides guidance for future metabolic engineering efforts, which establishes a powerful foundation for the development of phenol-tolerant strains of  C. acetobutylicum  for economically sustainable ABE production with high productivity from lignocellulosic biomass.

biotechnology for biofuels

Gene coexpression network analysis reveals a novel metabolic mechanism of Clostridium acetobutylicum responding to phenolic inhibitors from lignocellulosic hydrolysates