Adaptive Laboratory Evolution as a Means To Generate Lactococcus lactis Strains with Improved Thermotolerance and Ability To Autolyze

Lactococcus lactis subsp. (referred to here as L. lactis) is a model lactic acid bacterium and one of the main constituents of the mesophilic cheese starter used for producing soft or semihard cheeses. Most dairy L. lactis strains grow optimally at around 30°C and are not particularly well adapted to the elevated temperatures (37 to 39°C) to which they are often exposed during cheese production. To overcome this challenge, we used adaptive laboratory evolution (ALE) in milk, using a setup where the temperature was gradually increased over time, and isolated two evolved strains (RD01 and RD07) better able to tolerate high growth temperatures. One of these, strain RD07, was isolated after 1.5 years of evolution (400 generations) and efficiently acidified milk at 41°C, which has not been reported for industrial L. lactis strains until now. Moreover, RD07 appeared to autolyze 2 to 3 times faster than its parent strain, which is another highly desired property of dairy lactococci and rarely observed in the L. lactis subspecies used in this study. Model cheese trials indicated that RD07 could potentially accelerate cheese ripening. Transcriptomics analysis revealed the potential underlying causes responsible for the enhanced growth at high temperatures for the mutants. These included downregulation of the pleiotropic transcription factor CodY and overexpression of genes, which most likely lowered the guanidine nucleotide pool. Cheese trials at ARLA Foods using RD01 blended with the commercial Flora Danica starter culture, including a 39.5°C cooking step, revealed better acidification and flavor formation than the pure starter culture. In commercial mesophilic starter cultures, L. lactis is generally more thermotolerant than Lactococcus cremoris, whereas is more prone to autolysis, which is the key to flavor and aroma formation. In this study, we found that adaptation to higher thermotolerance can improve autolysis. Using whole-genome sequencing and RNA sequencing, we attempt to determine the underlying reason for the observed behavior. In terms of dairy applications, there are obvious advantages associated with using L. lactis strains with high thermotolerance, as these are less affected by curd cooking, which generally hampers the performance of the mesophilic starter. Cheese ripening, the costliest part of cheese manufacturing, can be reduced using autolytic strains. Thus, the solution presented here could simplify starter cultures, make the cheese manufacturi