Enhancing the heat tolerance of homolactic Escherichia coli through reverse metabolic engineering.

Improving Escherichia coli's thermotolerance through rational engineering is hindered by limited knowledge of the molecular mechanisms involved in supraoptimal thermal adaptation. To address this issue, we applied a reverse metabolic engineering strategy to develop a D-homolactic E. coli strain under non-aerated conditions. We first characterized the thermal reaction norms of the kinetic and stoichiometric parameters in strains evolved through continuous adaptive laboratory evolution, identifying those that maintained parental volumetric productivity and the lactate/glucose yield at high temperatures. Then, the genomic analysis of two thermally adapted strains was performed to determine the mutations acquired during thermal adaptation. Thermally adapted strains revealed convergent mutations in regulatory genes, notably metJ, lrp, and components of the RNA polymerase complex. Introducing these point mutations into the parental strain demonstrated that individual mutations in rpoB and metJ significantly improved growth at high temperatures, despite the presence of complex epistatic interactions in combinatorial analyses. KEY POINTS: • ALE significantly enhanced the thermotolerance of homolactic Escherichia coli • Mutations in rpoB and metJ notably enhance growth at high temperatures • Lactate productivity and yield stayed similar to the parental strain.