The las Enzymes Control Pyruvate Metabolism in Lactococcus lactis during Growth on Maltose

Abstract

The fermentation pattern of Lactococcus lactis with altered activities of the las enzymes was examined on maltose. The wild type converted 65% of the maltose to mixed acids. An increase in phosphofructokinase or lactate dehydrogenase expression shifted the fermentation towards homolactic fermentation, and with a high level of expression of the las operon the fermentation was homolactic.In Lactococcus lactis sugars can be metabolized either by homolactic fermentation, producing lactate, or by mixed acid fermentation, producing formate, acetate, and ethanol. The former mode of fermentation is favored during growth on readily metabolizable sugars, such as glucose and lactose, whereas growth on slowly fermentable sugars, such as maltose (11) and galactose (17), usually results in mixed acid fermentation.The mechanisms regulating this shift in metabolism are still unclear. It has been observed that under sugar starvation conditions the level of fructose-1,6-bisphosphate (FBP) is lowered (16), and since FBP is an allosteric activator of lactate dehydrogenase (LDH) (19), this could explain a shift in fermentation pattern (4, 16) (Fig. 1). The level of the triose phosphates dihydroxyacetone phosphate and glyceraldehyde-3-phosphate in L. lactis is also lowered during growth on slowly fermentable sugars (17). Since the triose phosphates are allosteric inhibitors of pyruvate formate lyase (PFL) (14), this may also be part of the explanation of the shift in fermentation mode (Fig. 1). Other investigators have suggested that the NADH/NAD ϩ ratio regulates the shift in metabolism. A correlation was found between this ratio and the glycolytic flux; a high ratio gave a high flux and vice versa (5). These investigators also determined that glyceraldehyde-3-phosphate dehydrogenase was activated and LDH was deactivated at low ratios, whereas the opposite was the case at high ratios. This type of regulation could, in combination with the above-mentioned effect of the triose phosphates, explain the shift in pyruvate metabolism. A more recent study indicated that both the level of FBP and the NADH/NAD ϩ ratio may be important, but with large variations between strains (18).In recent years the glucose metabolism in L. lactis has been studied extensively with respect to the glycolytic rate and product formation. One study focusing on the las operon, coding for phosphofructokinase (PFK), pyruvate kinase (PYK), and LDH (9), revealed that LDH has a strong negative effect on the mixed acid flux; the flux control coefficient was less than Ϫ1, meaning that a 10% reduction in LDH leads to a Ͼ10% increase in the mixed acid flux (1). Another study revealed that PYK exhibits strong positive control of formate and acetate production (flux control coefficient for PYK on the formate and acetate flux were both close to 1) (7). The positive control by PYK and the negative control by LDH almost cancelled out, resulting in very little effect of modulation of the entire operon on the fermentation pattern. In this paper we describe a control analysis of ...

Characterization of Three Lactic Acid Bacteria and Their Isogenic ldh Deletion Mutants Shows Optimization for Y ATP (Cell Mass Produced per Mole of ATP) at Their Physiological pHs

“…E. faecalis showed mixed acid fermentation under almost all conditions but showed clearly more homolactic acid fermentation at low pH. Both S. pyogenes and L. lactis mainly showed homolactic acid fermentation, as was shown previously (24), under all conditions and showed no pH dependency with respect to mixed acid versus homolactic acid fermentation. This is in contrast to previous observations, caused by the strong differences in the amount of arginine in the medium (28) (data not shown).…”

“…While the complex rich media M17 and THY contain a variety of different potential carbon sources, in CDM-LAB medium, glucose is the only sugar component. It has been shown previously that utilization of sugars like maltose or galactose as the carbon source by LAB led to changes in product formation compared to that from growth on glucose (24,25,29). By screening a large variety of carbon sources, we could show that the ldh deletion mutants are not able to utilize all carbon sources as efficiently as their cognate wild types.…”

“…L. lactis NZ9000 and the lactate dehydrogenase (LDH)-deficient strain NZ9010 (11,17), E. faecalis V583 and V583 ⌬ldh-1 (12), and S. pyogenes M49 591 and M49 591 ⌬ldh were grown in batch cultures at 37°C in 96-well plates in either Todd-Hewitt broth supplemented with 0.5% (wt/vol) yeast extract (Oxoid) (THY medium) or a chemically defined medium (CDM) specifically designed to support the growth of all three LAB (pH 7.4). The CDM-LAB medium (12) contained the following per liter: 1 g K 2 HPO 4 , 5 g KH 2 PO 4 , 0.6 g ammonium citrate, 1 g acetate, 0.25 g tyrosine, 0.24 g alanine, 0.125 g arginine, 0.42 g aspartic acid, 0.13 g cysteine, 0.5 g glutamic acid, 0.15 g histidine, 0.21 g isoleucine, 0.475 g leucine, 0.44 g lysine, 0.275 phenylalanine, 0.675 g proline, 0.34 g serine, 0.225 g threonine, 0.05 g tryptophan, 0.325 g valine, 0.175 g glycine, 0.125 g methionine, 0.1 g asparagine, 0.2 g glutamine, 10 g glucose, 0.5 g L-ascorbic acid, 35 mg adenine sulfate, 27 mg guanine, 22 mg uracil, 50 mg cystine, 50 mg xanthine, 2.5 mg D-biotin, 1 mg vitamin B 12 , 1 mg riboflavin, 5 mg pyridoxamine-HCl, 10 g p-aminobenzoic acid, 1 mg pantothenate, 5 mg inosine, 1 mg nicotinic acid, 5 mg orotic acid, 2 mg pyridoxine, 1 mg thiamine, 2.5 mg lipoic acid, 5 24 . Both media were buffered with either 100 mM morpholineethanesulfonic acid buffer or 100 mM morpholinepropanesulfonic acid buffer for growth at pH 6.5 and 7.5, respectively.…”

“…lactis is by far the best-studied lactic acid bacterium (3,13,14,23,24,30), and a kinetic model for its complete glycolysis, including some branching pathways, has been developed (2, 10). For the other two lactic acid bacteria, less information is readily available.…”

Effects of Glucose and Starch on Lactate Production by Newly Isolated Streptococcus bovis S1 from Saanen Goats

“…Observations of cell morphology showed an aspheric shape with a diameter of approximately 1 m and aggregations similar to strings of beads, consistent with the original definition of S. bovis by Orla-Jensen in 1919 (29). The 16S rRNA gene (1,461 bp) sequence of the isolate shared high homology with the rRNA gene of S. bovis ATCC 27960 (GenBank accession number AB002481.1) isolated from swine and S. bovis JB1 (AF104109.1) isolated from the rumen of cows (30,31). While the taxonomic status of S. bovis and close relatives such as S. equinus has been the subject of much discussion over many years (32), the S1 isolate in our study also shared a high affinity with the S. equinus-like strain HC5 (KF573427.1) and strain NRIC1535 (AB362710.1).…”