An adaptive evolution approach utilizing reversible mutS inactivation for development of Lactobacillus casei strains with enhanced lactic acid tolerance at low pH

Adaptive evolution has been applied to microorganisms to increase industrially desirable phenotypes. In this study, we explored reversible inactivation of the gene encoding the DNA mismatch repair enzyme MutS as a tool for adaptive evolution of Lactobacillus casei strains with enhanced lactic acid tolerance at low pH. ΔmutS derivatives of L. casei 12A and ATCC 334 were constructed by two-step gene inactivation. The wild-type cells and ΔmutS derivatives were subjected to serial passage in MRS broth adjusted with lactate to successively lower pH values (pH 5.5 to 4.0) over a 100-day period. At the end of the adaptive evolution treatment, the ΔmutS lesion in the 12A and ATCC 334 derivatives was repaired by gene replacement in one representative isolate. Growth studies at pH 4.0 confirmed all four adapted cultures grew more rapidly and to higher cell densities than untreated wild-type cells. Moreover, the adapted ΔmutS derivatives grew significantly more rapidly and to higher cell densities than the respective adapted wild-type cells. Genome sequence analysis of the ΔmutS derivatives revealed L. casei ATCC 334 had suffered lesions in additional genes associated with replication fidelity, while 12A had not.  This finding indicated that restoration of the mutS gene in 12A would yield a stable platform, and illustrates possible risks of using MutS as a target for inducing hypermutation. Work is underway to analyze the rate of lactic acid production at pH 3.8 by the restored 12A derivative, and to characterize the mutations responsible for its highly acid-tolerant phenotype.