Control of the shift from homolactic acid to mixed-acid fermentation in Lactococcus lactis: predominant role of the NADH/NAD+ ratio.

During batch growth of Lactococcus lactis subsp. lactis NCDO 2118 on various sugars, the shift from homolactic to mixed-acid metabolism was directly dependent on the sugar consumption rate. This orientation of pyruvate metabolism was related to the flux-controlling activity of glyceraldehyde-3-phosphate dehydrogenase under conditions of high glycolytic flux on glucose due to the NADH/NAD+ ratio. The flux limitation at the level of glyceraldehyde-3-phosphate dehydrogenase led to an increase in the pool concentrations of both glyceraldehyde-3-phosphate and dihydroxyacetone-phosphate and inhibition of pyruvate formate lyase activity. Under such conditions, metabolism was homolactic. Lactose and to a lesser extent galactose supported less rapid growth, with a diminished flux through glycolysis, and a lower NADH/NAD+ ratio. Under such conditions, the major pathway bottleneck was most probably at the level of sugar transport rather than glyceraldehyde-3-phosphate dehydrogenase. Consequently, the pool concentrations of phosphorylated glycolytic intermediates upstream of glyceraldehyde-3-phosphate dehydrogenase decreased. However, the intracellular concentration of fructose-1,6-bisphosphate remained sufficiently high to ensure full activation of lactate dehydrogenase and had no in vivo role in controlling pyruvate metabolism, contrary to the generally accepted opinion. Regulation of pyruvate formate lyase activity by triose phosphates was relaxed, and mixed-acid fermentation occurred (no significant production of lactate on lactose) due mostly to the strong inhibition of lactate dehydrogenase by the in vivo NADH/NAD+ ratio.     

Conversion of Lactococcus lactis from homolactic to homoalanine fermentation through metabolic engineering.

We report the engineering of Lactococcus lactis to produce the amino acid L-alanine. The primary end product of sugar metabolism in wild-type L. lactis is lactate (homolactic fermentation). The terminal enzymatic reaction (pyruvate + NADH-->L-lactate + NAD+) is performed by L-lactate dehydrogenase (L-LDH). We rerouted the carbon flux toward alanine by expressing the Bacillus sphaericus alanine dehydrogenase (L-AlaDH; pyruvate + NADH + NH4+ -->L-alanine + NAD+ + H2O). Expression of L-AlaDH in an L-LDH-deficient strain permitted production of alanine as the sole end product (homoalanine fermentation). Finally, stereospecific production (>99%) of L-alanine was achieved by disrupting the gene encoding alanine racemase, opening the door to the industrial production of this stereoisomer in food products or bioreactors.     

The pool of ADP and ATP regulates anaerobic product formation in resting cells of Lactococcus lactis

Lactococcus lactis grows homofermentatively on glucose, while its growth on maltose under anaerobic conditions results in mixed acid product formation in which formate, acetate, and ethanol are formed in addition to lactate. Maltose was used as a carbon source to study mixed acid product formation as a function of the growth rate. In batch and nitrogen-limited chemostat cultures mixed acid product formation was shown to be linked to the growth rate, and homolactic fermentation occurred only in resting cells. Two of the four lactococcal strains investigated with maltose, L. lactis 65.1 and MG1363, showed more pronounced mixed acid product formation during growth than L. lactis ATCC 19435 or IL-1403. In resting cell experiments all four strains exhibited homolactic fermentation. In resting cells the intracellular concentrations of ADP, ATP, and fructose 1,6-bisphosphate were increased and the concentration of P(i) was decreased compared with the concentrations in growing cells. Addition of an ionophore (monensin or valinomycin) to resting cultures of L. lactis 65.1 induced mixed acid product formation concomitant with decreases in the ADP, ATP, and fructose 1,6-bisphosphate concentrations. ADP and ATP were shown to inhibit glyceraldehyde-3-phosphate dehydrogenase, lactate dehydrogenase, and alcohol dehydrogenase in vitro. Alcohol dehydrogenase was the most sensitive enzyme and was totally inhibited at an adenine nucleotide concentration of 16 mM, which is close to the sum of the intracellular concentrations of ADP and ATP of resting cells. This inhibition of alcohol dehydrogenase might be partially responsible for the homolactic behavior of resting cells. A hypothesis regarding the level of the ATP-ADP pool as a regulating mechanism for the glycolytic flux and product formation in L. lactis is discussed.     

Effects on product formation in Lactococcus lactis 65.1 in continuous culture with complete cell recycling

Lactococcus lactis 65.1 was cultivated in a batch culture, which underwent starvation for 3 days, continuous culture and continuous culture with complete cell recycling. The objective was to study the product formation and intracellular protein pattern. Changes from homofermentative to heterofermentative metabolism were observed in continuous culture at the lower dilution rates as well as continuous cultures with complete cell recycling at a fixed dilution rate (D=0.4 h^–1). Changes in intracellular protein pattern were observed when starving the cells in a batch culture and also when recycling the cells in a continuous culture. Some changes were the same in these two cases. The data collected from these experiments show how the fermentation technique can affect the products of the microorganism being cultured and gives some interesting information on the complete cell recycling technique, which is of great interest in fermentation processes.     

Effect of temperature and pH on growth and product formation of Lactococcus lactis ssp. lactis ATCC 19435 growing on maltose

Lactococcus lactis ssp. lactis ATCC 19435 is known to produce mixed acids when grown on maltose. A change in fermentation conditions only, elevated temperatures (up to 37 °C) and reduced pH values (down to 5.0) resulted in a shift towards homolactic product formation. This was accompanied by decreased growth rate and cell yield. The results are discussed in terms of redox balance and maintenance, and the regulation of lactate dehydrogenase and pyruvate formate-lyase. Received: 14 December 1998 / Received revision: 12 January 1999 / Accepted: 22 January 1999     

Control analysis as a tool to understand the formation of the las operon in Lactococcus lactis

In Lactococcus lactis the enzymes phosphofructokinase (PFK), pyruvate kinase (PK) and lactate dehydrogenase (LDH) are uniquely encoded in the las operon. We used metabolic control analysis to study the role of this organization. Earlier studies have shown that, at wild-type levels, LDH has no control over glycolysis and growth rate, but high negative control over formate production (C(Jformate)LDH=-1.3). We found that PFK and PK exert no control over glycolysis and growth rate at wild-type enzyme levels but both enzymes exert strong positive control on the glycolytic flux at reduced activities. PK exerts high positive control over formate (C(Jformate)PK=0.9-1.1) and acetate production (C(Jacetate)PK=0.8-1.0), whereas PFK exerts no control over these fluxes at increased expression. Decreased expression of the entire las operon resulted in a strong decrease in the growth rate and glycolytic flux; at 53% expression of the las operon glycolytic flux was reduced to 44% and the flux control coefficient increased towards 3. Increased las expression resulted in a slight decrease in the glycolytic flux. At wild-type levels, control was close to zero on both glycolysis and the pyruvate branches. The sum of control coefficients for the three enzymes individually was comparable with the control coefficient found for the entire operon; the strong positive control exerted by PK almost cancels out the negative control exerted by LDH on formate production. Our analysis suggests that coregulation of PFK and PK provides a very efficient way to regulate glycolysis, and coregulating PK and LDH allows cells to maintain homolactic fermentation during glycolysis regulation.     

Cooperation between Lactococcus lactis and nonstarter lactobacilli in the formation of cheese aroma from amino acids

In Gouda and Cheddar type cheeses the amino acid conversion to aroma compounds, which is a major process for aroma formation, is essentially due to lactic acid bacteria (LAB). In order to evaluate the respective role of starter and nonstarter LAB and their interactions in cheese flavor formation, we compared the catabolism of phenylalanine, leucine, and methionine by single strains and strain mixtures of Lactococcus lactis subsp. cremoris NCDO763 and three mesophilic lactobacilli. Amino acid catabolism was studied in vitro at pH 5.5, by using radiolabeled amino acids as tracers. In the presence of alpha-ketoglutarate, which is essential for amino acid transamination, the lactobacillus strains degraded less amino acids than L. lactis subsp. cremoris NCDO763, and produced mainly nonaromatic metabolites. L. lactis subsp. cremoris NCDO763 produced mainly the carboxylic acids, which are important compounds for cheese aroma. However, in the reaction mixture containing glutamate, only two lactobacillus strains degraded amino acids significantly. This was due to their glutamate dehydrogenase (GDH) activity, which produced alpha-ketoglutarate from glutamate. The combination of each of the GDH-positive lactobacilli with L. lactis subsp. cremoris NCDO763 had a beneficial effect on the aroma formation. Lactobacilli initiated the conversion of amino acids by transforming them mainly to keto and hydroxy acids, which subsequently were converted to carboxylic acids by the Lactococcus strain. Therefore, we think that such cooperation between starter L. lactis and GDH-positive lactobacilli can stimulate flavor development in cheese.     

Properties of 2,3-Butanediol Dehydrogenases from Lactococcus lactis subsp. lactis in Relation to Citrate Fermentation

Two 2,3-butanediol dehydrogenases (enzymes 1 and 2; molecular weight of each, 170,000) have been partially purified from Lactococcus lactis subsp. lactis (Streptococcus diacetylactis) D10 and shown to have reductase activity with either diacetyl or acetoin as the substrate. However, the reductase activity with 10 mM diacetyl was far greater for both enzymes (7.0- and 4.7-fold for enzymes 1 and 2, respectively) than with 10 mM acetoin as the substrate. In contrast, when acetoin and diacetyl were present together, acetoin was the preferred substrate for both enzymes, with enzyme 1 showing the more marked preference for acetoin. meso-2,3-Butanediol was the only isomeric product, with enzyme 1 independent of the substrate combinations. For enzyme 2, both the meso and optical isomers of 2,3-butanediol were formed with acetoin as the substrate, but only the optical isomers were produced with diacetyl as the substrate. With batch cultures of strain D10 at or near the point of citrate exhaustion, the main isomers of 2,3-butanediol present were the optical forms. If the pH was sufficiently high (>pH 5), acetoin reduction occurred over time and was followed by diacetyl reduction, and meso-2,3-butanediol became the predominant isomer. Interconversion of the optical isomers into the meso isomer did occur. The properties of 2,3-butanediol dehydrogenases are consistent with diacetyl and acetoin removal and the appearance of the isomers of 2,3-butanediol.     

Proteome analysis of the purine stimulon from Lactococcus lactis

A comparative expression proteome analysis was carried out by analyzing differential expression patterns of pulse-labelled proteins on two-dimensional gels under standard conditions and during purine nucleotide starvation, followed by mass spectrometric identification of regulated proteins. Based upon the expression patterns, three stimulons could be identified in Lactococcus lactis subsp. cremoris. The Psu proteins (purine starvation up-regulated) had increased synthesis during purine depletion in a purine auxotroph. Among these proteins were enzymes of the purine biosynthesis pathways (PurE, PurS, PurM, PurL), and enzymes involved in the generation of C1 units (GlyA, Fhs). C1 units are primarily required for purine biosynthesis. Upon analysis of the nucleotide sequence preceding the structural genes for these proteins in the L. lactis IL1403 genome sequence showed that all contained PurBox-Pribnov box structures resembling the PurR activated promoters for the purDEK and purCSQLF operons. Most, and possibly all members of the Psu stimulon are thus members of the PurR regulon. Five Psu proteins could not be identified. The second stimulon, the Psd stimulon (purine starvation decreased), whose members are down-regulated during purine depletion, contained proteins related to protein synthesis (PpsB, EF-TS, trigger factor), or to GTPases (FtsZ, EF-TS); or are involved in energy metabolism (GapB, CcpA). No common regulatory elements could be found for members of this stimulon. Two Psd proteins escaped identification. The last, Dcu (decoynine up-regulated), stimulon contained proteins whose synthesis escaped the severe general depression during inhibition of the GMP synthetase by decoynine. This regulon was comprised of mostly glycolytic enzymes (fructose bisphosphate aldolase, enolase, pyruvate kinase) and translation elongation factors (GTPases: EF-TU, EF-G). Two Dcu proteins could not be identified. Out of 28 proteins subjected to mass spectrometry, 19 could be readily identified despite the fact that only the genome sequence of a strain of L. lactis subsp. lactis was available. The two subspecies share about 85% sequence identity, comparable to the genetic distance between Escherichia coli and Salmonella typhimurium. A success rate of 68% indicates that it may be feasible to perform proteomics based upon genomic sequences of relatives outside the genus.     

Plasmid transfer via transduction from Streptococcus thermophilus to Lactococcus lactis

Using Streptococcus thermophilus phages, plasmid transduction in Lactococcus lactis was demonstrated. The transduction frequencies were 4 orders of magnitude lower in L. lactis than in S. thermophilus. These results are the first evidence that there is phage-mediated direct transfer of DNA from S. thermophilus to L. lactis. The implications of these results for phage evolution are discussed.     

Comparison of cell wall proteinases from Lactococcus lactis subsp. cremoris AC1 and Lactococcus lactis subsp. lactis NCDO 763

Summary The cell wall proteinases of Lactococcus lactis subsp. lactis NCDO 763 and L. lactis subsp. cremoris AC1 hydrolyse -casein with a similar specificity even though some quantitative differences can be observed for a few degradation products analysed by reverse phase HPLC and sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The main peptides soluble in 1.1% trifluoroacetic acid and liberated by the two proteinases were identified and have been found to be the same for the two enzymes. They are located in two areas of the -casein sequence (53–93 and the C-terminal part: 129–209) and they include bitter tasting or physiologically active fragments. No narrow specificity was observed for these proteinases. However, glutamine and serine residues are more frequently encountered in position P1 and P1 of the sensitive peptide bond and the close environment (position P2 to P4 and P2 to P4) of the cleaved bond is mainly hydrophobic.     

Purification and characterization of pullulanase from Lactococcus lactis

This paper describes a simple and efficient method of isolation of a plullulanase type I from amylolytic lactic acid bacteria (ALAB). Extracellular pullulanase type I was purified from a cell-free culture supernatant of Lactococcus lactis IBB 500 by using ammonium sulfate fractionation and dialysis (instead of ultrafiltration), and ion-exchange chromatography with CM Sepharose FF followed by gel filtration chromatography with Sephadex G-150 as the final step. A final purification factor of 14.36 was achieved. The molecular mass of the enzyme was estimated as 73.9 kD. The optimum temperature for the enzyme activity was 45°C and the optimum pH was 4.5. Pullulanase activity was increased by addition Co(2+) and completely inhibited by Hg(2+). The enzyme activity was specifically directed toward α-1,6 glycosidic linkages of pullulan giving maltotriose units. Enzymatic hydrolysis of starch and amylose produced a mixture of maltose and maltotriose.     

Plasmid involvement in the formation of a spontaneous bacteriophage insensitive mutant of Lactococcus lactis

Lactococcus lactis subsp. lactis biovar. diacetylactis DPC721 is a spontaneous bacteriophage insensitive mutant of strain DPC220, isolated after challenge with an industrial bacteriophage, phi D1. Plasmid analysis demonstrated that the bacteriophage insensitivity was associated with the absence of two native DPC220 plasmids (pAH82 and pAH33), and the presence of a novel plasmid (pAH90) in DPC721. The plasmids were transferred by conjugative mobilization to a plasmid free background where it was confirmed by restriction mapping that pAH90 is a co-integrate formed by the precise recombination of pAH82 and pAH33. The resistance phenotype encoded by pAH90 was also active against two bacteriophage homologous for the plasmid-free strain. Plasmid pAH90 was shown to encode at least two independent resistance mechanisms, including an adsorption-inhibition mechanism and a restriction and modification system. The adsorption-inhibition mechanism encoded by the co-integrate plasmid was specific for one of the phage used in this study.     

The Lactic Acid Stress Response of Lactococcus lactis subsp. lactis

The lactic acid tolerance response (LATR) of the lactic acid bacterium Lactococcus lactis subsp. lactis has been studied. A dramatic increase in survival to a severe acid stress (pH 3.9) was obtained by preexposing the cells for 30 min to a mildly acid shock at pH 5.5. Whole-cell protein extract analysis revealed that during the acid tolerance response 33 polypeptides are induced over the level of naive cells. Among these are the major heat shock proteins DnaK and GroEL. In conjunction with a previous report (Hartke et al. 1994), the results establish that L. lactis can adapt to lactic acid exposure in two different ways: a logarithmic phase LATR, which may be activated by protons, and a stationary-phase LATR, which needs no activation by protons. Both systems are independent of de novo protein synthesis. Received: 8 February 1996 / Accepted: 11 March 1996     

Overproduction and purification of the CcpA protein from Lactococcus lactis

In this work we present cloning and overexpression of lactococcal CcpA protein in Escherichia coli Xl1blue strain as a fusion with 6 x His tag. A high yield of the CcpA protein was obtained when the cells were cultured in liquid medium LB with 100 microg/ml ampicillin at 37 degrees C and subsequently for 4 h after induction by IPTG. The procedure let us obtain 5 mg of homogenous CcpA protein. Glutaraldehyde crosslinking analysis indicated the formation of dimer or tetramer forms of the CcpA protein.