Metabolic engineering of Lactobacillus fermentum for production of mannitol and pure L-lactic acid or pyruvate

Abstract

For production of mannitol in combination with pure L-lactic acid or pyruvate, the D- and L-lactate dehydrogenase genes (ldhD and ldhL) of a mannitol-producing Lactobacillus fermentum strain were cloned and stepwise inactivated. For inactivation of both ldh genes by a gene replacement technique, deletion constructs removing a 0.4-kb fragment from the promoter and the 5' end region of the ldh genes were used. The first inactivation mutant, designated L. fermentum GRL1030, carried the deletion in ldhD (DeltaldhD). A double mutant, DeltaldhD-DeltaldhL, was constructed by the inactivation of the ldhL gene of strain GRL1030, resulting in strain L. fermentum GRL1032. The correctness of the both mutants was confirmed at the DNA level by polymerase chain reaction, as shown by the absence of ldh transcripts by northern blotting and as a lack of the corresponding enzyme activity. In bioreactor cultivations, the single mutant GRL1030 produced mannitol and L-lactic acid as expected. Mannitol and lactic acid yields and productivities were practically unaffected by deletion of the ldhD gene. The double mutant GRL1032 produced mannitol and pyruvate as expected. However, although the yield of mannitol from fructose remained high, its volumetric productivity was reduced. The double mutation negatively affected the glucose consumption rate, resulting in reduced cellular growth. In addition to pyruvate, the double mutant produced 2,3-butanediol. More surprisingly, some lactic acid was still produced.