NAD-Independent L-Lactate Dehydrogenase Is Required for L-Lactate Utilization in Pseudomonas stutzeri SDM

Abstract

BackgroundVarious Pseudomonas strains can use l-lactate as their sole carbon source for growth. However, the l-lactate-utilizing enzymes in Pseudomonas have never been identified and further studied.Methodology/Principal FindingsAn NAD-independent l-lactate dehydrogenase (l-iLDH) was purified from the membrane fraction of Pseudomonas stutzeri SDM. The enzyme catalyzes the oxidation of l-lactate to pyruvate by using FMN as cofactor. After cloning its encoding gene (lldD), l-iLDH was successfully expressed, purified from a recombinant Escherichia coli strain, and characterized. An lldD mutant of P. stutzeri SDM was constructed by gene knockout technology. This mutant was unable to grow on l-lactate, but retained the ability to grow on pyruvate.Conclusions/SignificanceIt is proposed that l-iLDH plays an indispensable function in Pseudomonas l-lactate utilization by catalyzing the conversion of l-lactate into pyruvate.

NAD-Independent l-Lactate Dehydrogenase Required for l-Lactate Utilization in Pseudomonas stutzeri A1501

“…The PCR product was cloned into EcoRI-and HindIII-digested pK18mobsacB, a mobilizable plasmid that does not replicate in Pseudomonas, to form pK18mobsacB-⌬lldA. To transfer the plasmid pK18mobsacB-⌬lldA into P. stutzeri A1501, a triparental filter mating was performed as previously described using E. coli DH10B (pK18mobsacB-⌬lldA) as the donor strain, E. coli HB101(pRK2013) as the helper strain, and P. stutzeri A1501 as the recipient strain (29). Integration of the plasmid pK18mobsacB-⌬lldA into the chromosome of P. stutzeri A1501 by the first crossover was selected on an LB plate supplemented with 50 g ml Ϫ1 kanamycin.…”

“…The result PCR products were cloned into HindIII/ PstI-cut pBBRMSC-5, a broad-host-range plasmid. The resulting plasmids were, respectively, conjugated into P. stutzeri A1501 via a triparental mating method as previously described (29).…”

“…Utilization of lactate has also been studied in different Pseudomonas strains (29,30). The genes similar to lldD of E. coli exist in the genomes of most Pseudomonas species ( Fig.…”

Genome Sequence of the Lactate-Utilizing Pseudomonas aeruginosa Strain XMG

“…ertain Pseudomonas species, such as Pseudomonas aeruginosa, P. putida, and P. stutzeri, can use lactate as the sole carbon and energy source for growth (6,7,11,15). NAD-independent L-lactate dehydrogenase (L-iLDH) and NAD-independent D-lactate dehydrogenase (D-iLDH) catalyze the oxidation of L-lactate and D-lactate into pyruvate, respectively.…”

“…NAD-independent L-lactate dehydrogenase (L-iLDH) and NAD-independent D-lactate dehydrogenase (D-iLDH) catalyze the oxidation of L-lactate and D-lactate into pyruvate, respectively. They play essential roles in the utilization of lactate in these species (6,7,14,15). Some potential applications of L-iLDH and D-iLDH, such as 2-oxobutyrate production (4), pyruvate production (2,5,10,19), and kinetic resolution of 2-hydroxy acids racemic mixtures (3,8), have been also studied in recent years.…”