Identification of the major ldh gene related to D-lactic acid synthesis in Fructobacillus tropaeoli CRL 2034

J. BLECKWEDEL; L.G. RUIZ RODRÍGUEZ; F. MOZZI; R. R. RAYA

Simposio; 12th International Symposium on Lactic Acid Bacteria; 2017

Fructobacillus tropaeoli CRL 2034 is a lactic acid bacterium isolated from fig that converts pyruvate mainly into D-lactic acid by using the enzyme D(−)-lactate dehydrogenase (LDH). The aim of this study was to identify the gene(s) responsible for lactic acid formation in this organism. A genomic analysis of F. tropaeoli CRL 2034 revealed that 3 genes encode lactate-related dehydrogenases: two d-ldh genes (named ldh1 and ldh2) and one l-ldh gene (named ldh3). The two d-ldh genes, which are homologous (79% identity) and organized in a bicistronic operon (ldh1ldh2), encode two D-LDH proteins of 331 aa each (identities 74%; positives 87%); while the ldh3 gene is monocistronic and encodes a protein of 308 aa. ldh mutants were constructed by insertion of the non-replicative plasmid pRV300 carrying an internal fragment of each gene. To study the effect of the different mutations on cell growth, all strains were grown in MRS broth containing glucose (2 %, w/v) and fructose (2 %, w/v). While growth of mutants ldh2 and ldh3 was comparable to control cells, the doubling time of ldh1 mutant was markedly reduced, possibly due to the inability of these cells to efficiently re-oxidate NAD(P)H through reduction of pyruvate to lactate. These results suggest that LDH-1 is the main enzyme responsible for the conversion of pyruvate into lactic acid.