Abstract
Clostridium thermocellum is a premier thermophilic bacterium utilized in consolidated bioprocessing (CBP) for the direct conversion of lignocellulosic biomass into bioethanol. However, wild-type strains suffer from low ethanol tolerance and suboptimal yields due to competing metabolic pathways. In this study, we conducted a comparative genomic analysis of two highly efficient mutant strains, CTE-M1 and CTE-M2, obtained through adaptive laboratory evolution under high ethanol stress and cellulosic substrates. Whole-genome resequencing revealed key single nucleotide variants (SNVs) and insertions/deletions (indels) that distinguish these mutants from the wild-type DSM 1313 strain. Notably, both mutants harbored a crucial non-synonymous mutation in the bifunctional acetaldehyde/alcohol dehydrogenase (adhE) gene, which altered the catalytic site to favor ethanol synthesis over acetate. Additionally, CTE-M2 exhibited a promoter deletion in the hydrogenase maturation helper gene (hydG), correlating with a significant reduction in hydrogen production and a redirection of reducing equivalents toward ethanol. Fermentation profiling confirmed that CTE-M1 and CTE-M2 achieved ethanol yields of 0.38 g/g and 0.42 g/g glucan, respectively, representing a substantial increase over the wild-type. These genomic insights provide a clear genetic blueprint for rational metabolic engineering of thermophilic biocatalysts to advance second-generation biofuel production.
