Abstract
Isobutanol, in spite of its significant superiority over ethanol as a biofuel, remains commercially non-viable due to the non-availability of a suitable chassis which can handle the solvent toxicity associated with its production. To meet this challenge, we chose Lactococcus lactis which is known for its ability to handle environmental stress and carried out Adaptive laboratory evolution (ALE) in a continuous stirred tank reactor (CSTR) to evolve an isobutanol tolerant strain. The strain was grown for more than 60 days (> 250 generations) while gradually increasing the selection pressure, i.e. isobutanol concentration, in the feed. This led to the evolution of a strain that had an exceptionally high tolerance of up to 40 g/l of isobutanol even though a scanning electron microscope (SEM) study as well as analysis of membrane potential revealed only minor changes in cellular morphology. Whole genome sequencing which was done to confirm the strain integrity also showed comparatively few mutations in the evolved strain. However, the criticality of these mutations was reflected in major changes that occurred in the transcriptome, where gene expression levels from a wide range of categories that involved membrane transport, amino acid metabolism, sugar uptake and cell wall synthesis were significantly altered. Analysing the synergistic effect of these changes that lead to the complex phenotype of isobutanol tolerance can help in the construction of better host platforms for isobutanol production.