Identification of human D lactate dehydrogenase deficiency.

Phenotypic and biochemical categorization of humans with detrimental variants can provide valuable information on gene function. We illustrate this with the identification of two different homozygous variants resulting in enzymatic loss-of-function in LDHD, encoding lactate dehydrogenase D, in two unrelated patients with elevated D-lactate urinary excretion and plasma concentrations. We establish the role of LDHD by demonstrating that LDHD loss-of-function in zebrafish results in increased concentrations of D-lactate. D-lactate levels are rescued by wildtype LDHD but not by patients' variant LDHD, confirming these variants' loss-of-function effect. This work provides the first in vivo evidence that LDHD is responsible for human D-lactate metabolism. This broadens the differential diagnosis of D-lactic acidosis, an increasingly recognized complication of short bowel syndrome with unpredictable onset and severity. With the expanding incidence of intestinal resection for disease or obesity, the elucidation of this metabolic pathway may have relevance for those patients with D-lactic acidosis.

Elimination of D-lactate synthesis increases poly(3-hydroxybutyrate) and ethanol synthesis from glycerol and affects cofactor distribution in recombinant Escherichia coli.

The effect of eliminating D-lactate synthesis in poly(3-hydroxybutyrate) (PHB)-accumulating recombinant Escherichia coli (K24K) was analyzed using glycerol as a substrate. K24KL, an ldhA derivative, produced more biomass and had altered carbon partitioning among the metabolic products, probably due to the increased availability of carbon precursors and reducing power. This resulted in a significant increase of PHB and ethanol synthesis and a decrease in acetate production. Cofactor measurements revealed that cultures of K24K and K24KL had a high intracellular NADPH content and that the NADPH/NADP(+) ratio was higher than the NADH/NAD(+) ratio. The ldhA mutation affected cofactor distribution, resulting in a more reduced intracellular state, mainly due to a further increase in NADPH/NADP(+). In 60-h fed-batch cultures, K24KL reached 41.9 g·liter⁻¹ biomass and accumulated PHB up to 63% ± 1% (wt/wt), with a PHB yield on glycerol of 0.41 ± 0.03 g·g⁻¹, the highest reported using this substrate.

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

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.