Pyruvate Formate-Lyase Is Essential for Fumarate-Independent Anaerobic Glycerol Utilization in the Enterococcus faecalis Strain W11

Abstract

Although anaerobic glycerol metabolism in Enterococcus faecalis requires exogenous fumarate for NADH oxidation, E. faecalis strain W11 can metabolize glycerol in the absence of oxygen without exogenous fumarate. In this study, metabolic end product analyses and reporter assays probing the expression of enzymes involved in pyruvate metabolism were performed to investigate this fumarate-independent anaerobic metabolism of glycerol in W11. Under aerobic conditions, the metabolic end products of W11 cultured with glycerol were similar to those of W11 cultured with glucose. However, when W11 was cultured anaerobically, most of the glucose was converted to L-lactate, but glycerol was converted to ethanol and formate. During anaerobic culture with glycerol, the expression of the L-lactate dehydrogenase and pyruvate dehydrogenase E1␣␤ genes in W11 was downregulated, whereas the expression of the pyruvate formate-lyase (Pfl) and aldehyde/alcohol dehydrogenase genes was upregulated. These changes in the expression levels caused the change in the composition of end products. A pflB gene disruptant (⌬pfl mutant) of W11 could barely utilize glycerol under anaerobic conditions, but the growth of the ⌬pfl mutant cultured with either glucose or dihydroxyacetone (DHA) under anaerobic conditions was the same as that of W11. Glucose metabolism and DHA generates one NADH molecule per pyruvate molecule, whereas glycerol metabolism in the dehydrogenation pathway generates two NADH molecules per pyruvate molecule. These findings demonstrate that NADH generated from anaerobic glycerol metabolism in the absence of fumarate is oxidized through the Pfl-ethanol fermentation pathway. Thus, Pfl is essential to avoid the accumulation of excess NADH during fumarate-independent anaerobic glycerol metabolism. E nterococci are the most common lactic acid bacteria in the human intestinal tract, and some strains are well-known opportunistic pathogens (1, 2). Physiological studies have shown that Enterococcus faecalis can use glycerol as a carbon source (3). Microorganisms utilize glycerol through a dehydrogenation pathway, a phosphorylation pathway, or both. The dehydrogenation pathway begins with the dehydrogenation of glycerol to produce dihydroxyacetone (DHA), which is catalyzed by glycerol dehydrogenase (GldA). The DHA produced is then phosphorylated by DHA kinase (4). In the phosphorylation pathway, glycerol is first phosphorylated to glycerol-3-phosphate (glycerol-3P) by a glycerol kinase and then oxidized to dihydroxyacetone phosphate by a glycerol-3P oxidase or a glycerol-3P dehydrogenase (4). In either pathway, the resulting dihydroxyacetone phosphate enters glycolysis. Lactic acid bacteria, such as several Lactobacillus species and Pediococcus pentosaceus, generally utilize glycerol through the phosphorylation pathway, rather than the dehydrogenation pathway (5, 6), although E. faecalis strains can utilize glycerol through either the dehydrogenation pathway or the phosphorylation pathway (7).In some E. faecalis strains, glycerol metabolis...

l -Lactate Production from Biodiesel-Derived Crude Glycerol by Metabolically Engineered Enterococcus faecalis: Cytotoxic Evaluation of Biodiesel Waste and Development of a Glycerol-Inducible Gene Expression System

“…The E. faecalis strains were cultured in 40 ml of culture medium in 200-ml Erlenmeyer flasks, and the cell extract was prepared as described previously (14). The assay conditions for lactate dehydrogenase (Ldh) activity were a modification of the published method (17).…”

“…Construction of a glycerol-inducible ldhL1 gene expression plasmid for E. faecalis. The pflB-pflA gene promoter region of W11 (P pflB ) amplified by PCR was cloned into XbaI-SalI-digested pAM401 to generate pAM-P pflB (14). The Lactobacillus pentosus NBRC 106467 T L-lactate dehydrogenase gene (ldhL1 LP ) lacking a translation start codon was amplified by PCR using KOD FX (Toyobo Co. Ltd., Osaka, Japan), the primers listed in Table 2, and total DNA as a template; digested with XhoI; and cloned into SalI-NruI-digested pAM-P pflB to generate pPFL-ldhL1 LP .…”

“…The Lactobacillus pentosus NBRC 106467 T L-lactate dehydrogenase gene (ldhL1 LP ) lacking a translation start codon was amplified by PCR using KOD FX (Toyobo Co. Ltd., Osaka, Japan), the primers listed in Table 2, and total DNA as a template; digested with XhoI; and cloned into SalI-NruI-digested pAM-P pflB to generate pPFL-ldhL1 LP . The plasmid was inserted into E. faecalis strains using the electroporation method, as described previously (14).…”

“…Alternatively, glycerol is converted to DHAP via glycerol-3-phosphate (glycerol-3P) by glycerol kinase and glycerol-3P oxidase/dehydrogenase in the phosphorylation pathway. Enterococcus faecalis is a lactic acid bacterium that can use glycerol as a carbon source through one or both of the pathways described above (12,13) and produces acetate, acetoin, ethanol, and lactate as end products (14). This indicates that E. faecalis is one of the bacteria that can convert crude glycerol to other valuable materials, such as ethanol and D/L-lactate.…”

“…Our previous study showed that the E. faecalis strain W11 consumes 100 mM glycerol within 16 h with or without oxygen (14), which is a higher rate than those of other E. faecalis strains (13). In addition, although the transformation efficiency of enterococci is strongly dependent on species and strains (15,16), introduction of plasmid DNA into W11 by electroporation allowed W11 to express other genes and enabled the creation of a gene disruptant designated ⌬pfl (14). Thus, these backgrounds suggest the possibility that a bioconversion process using metabolically engineered W11 can convert glycerol to other materials at a high conversion efficiency.…”