Bioinformatic Characterization of Mutations Associated with Enhanced Acetate Metabolism In Esherichia coli

Abstract

The conversion of non-food lignocellulosic biomass, such as from trees or grasses, into sugars that can be converted into ethanol by fermentation is key to the future U.S. energy infrastructure. The accumulation of acetate during lignocellulosic biomass pretreatment inhibits microbial fermentation and limits bioethanol yields. This study aimed to characterize Escherichia coli strains engineered to efficiently utilize acetate as a sole carbon source to support acetate detoxification prior to yeast fermentation. Eight mutant strains were generated using either spontaneous or ethyl methanesulfonate (EMS) mutagenesis, then characterized using whole-genome sequencing and comparative bioinformatic analysis. Variant detection was performed using two pipelines, Bowtie2 + bcftools and Bowtie2 + DeepVariant, to evaluate differences in sensitivity and precision. The mutants exhibited enhanced growth on acetate despite lacking mutations in acetate metabolism genes (acs, pta, ackA). Instead, changes occurred in genes associated with replication and potentially global regulation of gene expression (dnaA), acid resistance (gadX), glycogen metabolism (glgX), membrane stability (ytcA), and proton export (ECOLC_RS20895). These mutations collectively improved stress tolerance, pH homeostasis, and metabolic efficiency. The results suggest that global physiological remodeling, rather than direct enzymatic modification, underlies enhanced acetate assimilation in E. coli, providing a genetic foundation for designing robust microbial strains for bioethanol production.