LAUSR

Production of 1-butanol from microorganisms has garnered significant interest due to its prospect as a drop-in biofuel and precursor for a variety of commercially relevant chemicals. Previously, high 1-butanol titer has been reported in Escherichia coli strain JCL166, which contains a modified clostridial 1-butanol pathway. Although conventional and metabolomics-based strain improvement strategies of E. coli strain JCL166 have been successful in improving production in rich medium, 1-butanol titer was severely limited in minimal medium. To further improve growth and consequently 1-butanol production in minimal medium, adaptive laboratory evolution (ALE) using mutD5 mutator plasmid was done on JCL166. Comparative metabolomics analysis of JCL166 and BP1 revealed global perturbations in the evolved strain BP1 compared to JCL166 (44 out of 64 metabolites), encompassing major metabolic pathways such as glycolysis, nucleotide biosynthesis, and CoA-related processes. Collectively, these metabolic changes in BP1 result in improved growth and, consequently, 1-butanol production in minimal medium. Furthermore, we found that the mutation in ihfB caused by ALE had a significant effect on the metabolome profile of the evolved strain. This study demonstrates how metabolomics was utilized for characterization of ALE-developed strains to understand the overall effect of mutations acquired through evolution.