Propionibacterium freudenreichii (P. freudenreichii) is a Gram-positive bacterium that plays a major role in Swiss-type cheese ripening. The main metabolic products of this species, namely propionic acid, acetic acid and CO2, along with aromatic compounds released from the transamination of amino acids and lipolysis, contribute to the development of the characteristic sweet and nutty flavor of this cheese variety, as well as to eye formation. In the initial phases of cheesemaking, the lactic acid starters Streptococcus thermophilus and Lactobacillus delbrueckii subsp. lactis degrade the lactose present in the curd, releasing L-lactate and D lactate, respectively. The key factor ensuring the growth of P. freudenreichii in the cheese matrix is its ability to utilize DL-lactate as a carbon source. The first step in DL-lactate catabolism is its oxidation to pyruvate, which is commonly described as facilitated by DL lactate dehydrogenases. Subsequently, pyruvate is either reduced to propionate via the Wood Werkman cycle or oxidized to acetate and CO2. In recent years, it has been observed that the presence of a D-lactate dehydrogenase in the genome is not required in order for P. freudenreichii to catabolize D-lactate under anoxic conditions. Moreover, the role of lactate dehydrogenases in this species has been proposed to be that of producing lactate, rather than oxidizing it. Considering these premises, it was aim of this thesis to shed light on the genetic background of DL-lactate utilization in P. freudenreichii, focusing on the first oxidation step of this catabolic pathway. First, two P. freudenreichii strains unable to degrade L-lactate were sourced from the Agroscope strain collection and phenotypically characterized. After an incubation time of 72 h in YEL broth, no D lactate was detectable in the medium, while L-lactate was only minimally depleted. The genetic background of this peculiar phenotype was investigated through whole genome sequencing and variant calling, using FAM-14222, a genotypically closely related strain, as a reference. An SNV in the lutB gene, causing an amino acid substitution from arginine to cysteine, was identified. This gene is part of the lutABC operon, which has been previously linked to lactate utilization in other bacterial species. Subsequently, the strains’ ability to utilize L-lactate was successfully restored by following an adaptive laboratory evolution approach, which involved repeated subculturing in a medium containing L-lactate as the main carbon source. Sequencing of the lutB gene confirmed that isolates with a restored ability to utilize L-lactate had also reverted the mutation back to wild-type, supporting the involvement of the lutABC operon in L-lactate catabolism in P. freudenreichii. In order to further characterize the role of the lutB gene in L-lactate utilization, a single crossover gene knockout strategy was implemented, though unsuccessfully. Instability issues of the plasmid insertion could not be overcome, and the development of a more stable, double-crossover-based strategy was unsuccessful, due to difficulties in the counter-selection of double-crossover mutants. As a strategy to gain further insights in both L-lactate and D-lactate utilization, RNA sequencing was performed on P. freudenreichii DSM 20271T. The transcriptomic profile of this strain during L-lactate utilization was compared to that of D-lactate utilization, including a control without lactate and three sampling time points. Differential gene expression analysis between different media highlighted the upregulation of the lutABC operon in L-lactate, which further supports our previous findings. The two L-lactate dehydrogenases present in the genome were not differentially expressed, which is in accordance with the hypothesis that they may be involved in L-lactate production, rather than consumption. As of D-lactate, no homologue of the D lactate dehydrogenase annotated in strain CIRM-BIA1 was found in the genome of strain DSM 20271T; nevertheless, DSM 20271T is able to degrade D-lactate, showing that D lactate dehydrogenases are not involved in this process. Surprisingly, a single gene was upregulated in D-lactate compared to the L-lactate condition, namely PdxT, which encodes the pyridoxal 5'-phosphate (PLP) synthase subunit PdxT. No oxidoreductases, racemases, isomerases, or genes with a lactate-related annotation were found to be differentially expressed. Although the general involvement of the lutABC operon in L-lactate utilization is clear and plausible, some questions remain open, including the enantiospecificity of these proteins, and the roles of the single genes composing the operon. To answer these questions, further efforts for obtaining stable knockouts of the lutABC genes will be beneficial. This may be achieved with the implementation of a protocol for double-crossover gene knockouts, or with the use of the CRISPR-Cas9 system.