Characterization of Lactococcus lactis mutants with improved performance at high temperatures and potential dairy applications

Lactococcus lactis (L. lactis) is a Gram-positive mesophile, which has considerable importance in the dairy industry for production of cheese and butter milk, and which carries the “GRAS” (generally recognized as safe) designation.
Temperature has a great impact on dairy fermentation processes through its effect on the starter culture. The optimum and maximum temperature for most L. lactis strains are approximately 30ºC and 38ºC, respectively. Increasing the fermentation temperature could have several beneficial effects, e.g. reduce bacteriophage attacks and increase acidification rate, the latter because the increased energy consumption at high temperatures potentially could stimulate glycolysis. However in many cases the fitness is affected and mostly negative effects on productivity are observed.
In this study, the non-GMO approach, experimental adaptation, was employed for isolating thermo-tolerant L. lactis. The adaptation was carried out using a serial-transfer regime at steadily increasing temperatures, and the strain used was L. lactis subsp. cremoris MG1363, which is a well-characterized dairy isolate. After exposure to increasing temperatures over 900 generations, one mutant (TM29) capable of growing at 40ºC was successfully isolated. By determining the temperature dependent growth rate profile, a shift of the optimum temperature from 30ºC to 36ºC was observed for TM29. Meanwhile, metabolic flux analysis revealed that TM29 was able to hold higher glucose consumption and lactate production rates when compared to MG1363 at high temperatures.
Whole genome re-sequencing identified 13 SNPs, one DIP and one large deletion in TM29, and additional sequencing of the isolated intermediates indicated dynamic accumulation of mutations with rising fitness in a temporal order. DNA microarray analysis revealed apparent differences in the transcriptional response to heat between the mutant and parent. It was found that SNPs preceding gene groESL and ribU resulted in over-expression of chaperone proteins GroES-GroEL, and membrane associated riboflavin transporter protein RibU in TM29, respectively. Moreover, a large deletion in TM29 caused the inactivation of 10 genes (llmg_1349-llmg_1358).
Through allelic replacement and gene knockout followed by fitness assessment, four main positive mutations were eventually discovered. The SNP preceding groESL and deletion of llmg_1349-llmg_1358 contributed to 30% and 10% increase in the growth rate of MG1363 at 38ºC, respectively. The over-expression that was caused by the SNP preceding ribU relived FAD starvation, which results in insufficient pyruvate dehydrogenase and NADH oxidase at high temperatures. Through replacing the mutated rpoC allele, that encodes the β’ subunit of the RNA polymerase, MG1363 exhibited extended maximum growth temperature with concomitant phenotypic changes, e.g. with respect to morphology and cellular fatty acid composition.
At last, acidification capability in milk was compared at different temperatures, for the endpoint mutant TM29 and the wild-type MG1363. It was found that TM29 was able acidify milk at 40ºC, whereas MG1363 could not. Tradeoffs, as a consequence of using a synthetic medium for the evolution, were also observed. TM29 acidified milk slower than MG1363 at 36ºC, which was not in accordance with growth in synthetic medium.