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PubMed1997

Development and Use of a Screening Procedure for Production of (alpha)-Acetolactate by Lactococcus lactis subsp. lactis biovar diacetylactis Strains.

A method was developed to screen and isolate mutagenized Lactococcus lactis subsp. lactis biovar diacetylactis strains accumulating (alpha)-acetolactate. This compound is accumulated by (alpha)-acetolactate decarboxylase-deficient strains and undergoes spontaneous degradation into diacetyl on agar plates. The diacetyl produced is detected by a colorimetric reaction yielding a red halo around the colonies.

Journal Article↗
PubMed1997

Metabolism and Energetics of Lactococcus lactis during Growth in Complex or Synthetic Media.

When Lactococcus lactis was grown in various complex or synthetic media, the fermentation of glucose remained homolactic whatever the medium used, with a global carbon balance of about 87%. Moreover, the nitrogen balance was not equilibrated, indicating that some amino acids led to the production of unknown nitrogen-containing carbon compounds while part of the glucose might contribute to anabolic pathways. In minimal medium containing six amino acids, a high concentration of serine was deaminated to pyruvate. This did not occur in more complete media, suggesting the presence of a regulation of this phenomenon by an amino acid. Ammonia produced during serine consumption was partly reconsumed after serine exhaustion. The values for biomass yield and biomass yield relative to ATP (Y(infATP)), the maximal growth rate, the specific rate of glucose consumption, and the corresponding rate of ATP synthesis all increased with the complexity of the medium, amino acid composition having the most pronounced effect. The Y(infATP) values were shown to range from 6.6 to 17.6 g of biomass(middot)mol of ATP(sup-1) on minimal and complex media.

Journal Article↗
PubMed1998

Analysis of the bacteriolytic enzymes of the autolytic lactococcus lactis subsp. cremoris strain AM2 by renaturing polyacrylamide gel electrophoresis: identification of a prophage-encoded enzyme

Lactococcus lactis subsp. cremoris AM2 was previously shown to lyse early and extensively during cheese ripening (M.-P. Chapot-Chartier, C. Deniel, M. Rousseau, L. Vassal, and J.-C. Gripon, Int. Dairy J. 4:251-269, 1994). We analyzed the bacteriolytic activities of autolytic strain AM2 by using renaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis performed with two different substrates in the gel, Micrococcus lysodeikticus and L. lactis autoclaved cells. Several lytic activities were detected in L. lactis AM2; a major lytic activity, designated A2 (46 kDa), was found only with the L. lactis cell substrate. This activity appears to be different from major peptidoglycan hydrolase AcmA characterized previously (G. Buist, J. Kok, K. J. Leenhouts, M. Dabrowska, G. Venema, and A. J. Haandrickman, J. Bacteriol. 177:1554-1563, 1995), which has a similar molecular mass. The two enzymes differ in substrate specificity as well as in sensitivity to pH and different chemical compounds. L. lactis AM2 is lysogenic and mitomycin C inducible. Enzyme A2 was shown to be inducible by mitomycin C and to be prophage encoded. It was identified as an enzyme similar to the lysin encoded by lactococcal small isometric temperate bacteriophages. A prophage-cured derivative of L. lactis AM2 was obtained, and this isolate exhibited different autolytic properties than AM2. After prolonged incubation in the stationary phase after growth on M17 medium, the extent of lysis of an AM2 culture was 60%, whereas over the same period there was almost no lysis in a prophage-cured derivative strain culture. These results suggest that the prophage lytic system is involved in the strain AM2 lysis observed in liquid medium and that it could also be involved in the lysis observed during cheese ripening.

Journal Article↗
PubMed1998

Use of a genetically enhanced, pediocin-producing starter culture, lactococcus lactis subsp. lactis MM217, To control listeria monocytogenes in cheddar cheese

Cheddar cheese was prepared with Lactococcus lactis subsp. lactis MM217, a starter culture which contains pMC117 coding for pediocin PA-1. About 75 liters of pasteurized milk (containing ca. 3.6% fat) was inoculated with strain MM217 (ca. 10(6) CFU per ml) and a mixture of three Listeria monocytogenes strains (ca. 10(3) CFU per ml). The viability of the pathogen and the activity of pediocin in the cheese were monitored at appropriate intervals throughout the manufacturing process and during ripening at 8 degreesC for 6 months. In control cheese made with the isogenic, non-pediocin-producing starter culture L. lactis subsp. lactis MM210, the counts of the pathogen increased to about 10(7) CFU per g after 2 weeks of ripening and then gradually decreased to about 10(3) CFU per g after 6 months. In the experimental cheese made with strain MM217, the counts of L. monocytogenes decreased to 10(2) CFU per g within 1 week of ripening and then decreased to about 10 CFU per g within 3 months. The average titer of pediocin in the experimental cheese decreased from approximately 64,000 arbitrary units (AU) per g after 1 day to 2,000 AU per g after 6 months. No pediocin activity (<200 AU per g) was detected in the control cheese. Also, the presence of pMC117 in strain MM217 did not alter the cheese-making quality of the starter culture, as the rates of acid production, the pH values, and the levels of moisture, NaCl, and fat of the control cheese and the experimental cheese were similar. Our data revealed that pediocin-producing starter cultures have significant potential for protecting natural cheese against L. monocytogenes.

Journal Article↗
PubMed1998

Influence of reduced water activity on lactose metabolism by lactococcus lactis subsp. cremoris At different pH values

The influence of reduced water activity (aw) on lactose metabolism by Lactococcus lactis subsp. cremoris 2254 and 2272 was studied at different pH values. In control incubations (aw, 0.99) with nongrowing cells in pH-controlled phosphate buffer, the levels of carbon recovered as L-(+)-lactate were 92% at pH 6.1 and 5.3 and 78% at pH 4.5. However, the levels of recovery decreased to approximately 50% at all pH values tested when the aw was 0.88 (with glycerol as the humectant). When growing cells in broth controlled at pH 6.3 were used, a reduction in the aw from 0.99 to 0.96 resulted in a decrease in the level of lactose carbon recovered as L-(+)-lactate from 100 to 71%. Low levels of L-(+)-lactate carbon recovery (<50%) were also observed with cells resuspended in pH-uncontrolled reconstituted skim milk at aw values of 0.99 and 0. 87 and in young cheese curds. The missing lactose carbon could not be accounted for by acetate, ethanol, formate, acetaldehyde, or pyruvate. Attempts were made to determine where the missing lactose carbon was diverted to under the stress conditions used. Some of the missing lactose carbon was recovered as galactose (0.1 to 2.5 mM) in culture supernatants. Decreasing either the aw or the pH resulted in increased galactose accumulation by nongrowing cells; adjusting both environmental factors together potentiated the effect. The sensitivities of the two lactococcal strains tested were different; strain 2272 was more prone to accumulate galactose under stress conditions. A methyl pentose(s) and additional galactose were found in acid-hydrolyzed supernatants from cultures containing both growing and nongrowing cells, indicating that a saccharide(s) rich in these components was formed by lactococci under low-aw and low-pH stress conditions.

Journal Article↗
PubMed1998

Glutamate Biosynthesis in Lactococcus lactis subsp. lactis NCDO 2118

Unlike other lactic acid bacteria, Lactococcus lactis subsp. lactis NCDO 2118 was able to grow in a medium lacking glutamate and the amino acids of the glutamate family. Growth in such a medium proceeded after a lag phase of about 2 days and with a reduced growth rate (0.11 h-1) compared to that in the reference medium containing glutamate (0.16 h-1). The enzymatic studies showed that a phosphoenolpyruvate carboxylase activity was present, while the malic enzyme and the enzymes of the glyoxylic shunt were not detected. As in most anaerobic bacteria, no alpha-ketoglutarate dehydrogenase activity could be detected, and the citric acid cycle was restricted to a reductive pathway leading to succinate formation and an oxidative branch enabling the synthesis of alpha-ketoglutarate. The metabolic bottleneck responsible for the limited growth rate was located in this latter pathway. As regards the synthesis of glutamate from alpha-ketoglutarate, no glutamate dehydrogenase was detected. While the glutamate synthase-glutamine synthetase system was detected at a low level, high transaminase activity was measured. The conversion of alpha-ketoglutarate to glutamate by the transaminase, the reverse of the normal physiological direction, operated with different amino acids as nitrogen donor. All of the enzymes assayed were shown to be constitutive.

Journal Article↗
PubMed1999

Influence of carbohydrate starvation and arginine on culturability and amino acid utilization of lactococcus lactis subsp. lactis.

Two strains of Lactococcus lactis subsp. lactis were used to determine the influence of lactose and arginine on viability and amino acid use during carbohydrate starvation. Lactose provided energy for logarithmic-phase growth, and amino acids such as arginine provided energy after carbohydrate exhaustion. Survival time, cell numbers, and ATP concentrations increased with the addition of arginine to the basal medium. By the onset of lactose exhaustion, the concentrations of glycine-valine and glutamate had decreased by as much as 67% in L. lactis ML3, whereas the serine concentration increased by 97% during the same period. When no lactose was added, the concentrations of these amino acids remained constant. Similar trends were observed for L. lactis 11454. Without lactose or arginine, L. lactis ML3 was nonculturable on agar but was viable after 2 days, as measured by fluorescent viability stains and intracellular ATP levels. However, L. lactis 11454 without lactose or arginine remained culturable for at least 14 days. These data suggest that lactococci become viable but nonculturable in response to carbohydrate depletion. Additionally, these data indicate that amino acids other than arginine facilitate the survival of L. lactis during carbohydrate starvation.

Journal Article↗
PubMed1999

Specificity of lactococcus lactis subsp. cremoris SK11 proteinase, lactocepin III, in low-water-activity, high-salt-concentration humectant systems and its stability compared with that of lactocepin I

Marked changes in the specificity of hydrolysis of alphas1-, beta-, and kappa-caseins by lactocepin III from Lactococcus lactis subsp. cremoris SK11 were found in humectant systems giving the equivalent water activity (aw) and salt concentration of cheddar cheese. Correlations were noted between certain peptides produced by the activity of lactocepin III in the humectant systems and peptides found in cheddar cheese. The stability of lactocepin III was compared with that of lactocepin I from L. lactis subsp. cremoris HP in the humectant systems at different pHs. Significant differences between the stability of each of the lactocepin types were evident. The relationship between stability and humectant type, aw, pH, and NaCl concentration was complex. Nevertheless, in those systems where aw, pH, and NaCl concentration were equivalent to those in cheddar cheese, lactocepin I was generally more stable than lactocepin III. It was concluded that differences in the specificity and/or stability of various lactocepin types are likely to persist in cheese itself and therefore potentially contribute to differences in the peptide composition of ripened cheese.

Journal Article↗
PubMed2000

Cloning, characterization, controlled overexpression, and inactivation of the major tributyrin esterase gene of Lactococcus lactis.

The gene encoding the major intracellular tributyrin esterase of Lactococcus lactis was cloned using degenerate DNA probes based on 19 known N-terminal amino acid residues of the purified enzyme. The gene, named estA, was sequenced and found to encode a protein of 258 amino acid residues. The transcription start site was mapped 233 nucleotides upstream of the start codon, and a canonical promoter sequence was identified. The deduced amino acid sequence of the estA product contained the typical GXSXG motif found in most lipases and esterases. The protein was overproduced up to 170-fold in L. lactis by use of the nisin-controlled expression system recently developed for lactic acid bacteria. The estA gene was inactivated by chromosomal integration of a temperature-sensitive integration vector. This resulted in the complete loss of esterase activity, which could then be recovered after complementation of the constructed esterase-deficient strain with the wild-type estA gene. This confirms that EstA is the main enzyme responsible for esterase activity in L. lactis. Purified recombinant enzyme showed a preference for short-chain acyl esters, surprisingly also including phospholipids. Medium- and long-acyl-chain lipids were also hydrolyzed, albeit less efficiently. Intermediate characteristics between esterases and lipases make intracellular lactococcal EstA difficult to classify in either of these two groups of esterolytic enzymes. We suggest that, in vivo, EstA could be involved in (phospho)lipid metabolism or cellular detoxification or both, as its sequence showed significant similarity to S-formylglutathione hydrolase (FGH) of Paracoccus denitrificans and human EstD (or FGH), which are part of a universal formaldehyde detoxification pathway.

Amino Acid Sequence↗
PubMed2002

Mutations in the Lactococcus lactis Ll.LtrB group II intron that retain mobility in vivo.

BACKGROUND: Group II introns are mobile genetic elements that form conserved secondary and tertiary structures. In order to determine which of the conserved structural elements are required for mobility, a series of domain and sub-domain deletions were made in the Lactococcus lactis group II intron (Ll.LtrB) and tested for mobility in a genetic assay. Point mutations in domains V and VI were also tested. RESULTS: The largest deletion that could be made without severely compromising mobility was 158 nucleotides in DIVb(1-2). This mutant had a mobility frequency comparable to the wild-type Ll.LtrB intron (DeltaORF construct). Hence, all subsequent mutations were done in this mutant background. Deletion of DIIb reduced mobility to approximately 18% of wild-type, while another deletion in domain II (nts 404-459) was mobile to a minor extent. Only two deletions in DI and none in DIII were tolerated. Some mobility was also observed for a DIVa deletion mutant. Of the three point mutants at position G3 in DV, only G3A retained mobility. In DVI, deletion of the branch-point nucleotide abolished mobility, but the presence of any nucleotide at the branch-point position restored mobility to some extent. CONCLUSIONS: The smallest intron capable of efficient retrohoming was 725 nucleotides, comprising the DIVb(1-2) and DII(ii)a,b deletions. The tertiary elements found to be nonessential for mobility were alpha, kappa and eta. In DV, only the G3A mutant was mobile. A branch-point residue is required for intron mobility.

Journal Article↗
PubMed2005

Industrial-scale production and purification of a heterologous protein in Lactococcus lactis using the nisin-controlled gene expression system NICE: the case of lysostaphin.

BACKGROUND: The NIsin-Controlled gene Expression system NICE of Lactococcus lactis is one of the most widespread used expression systems of Gram-positive bacteria. It is used in more than 100 laboratories for laboratory-scale gene expression experiments. However, L. lactis is also a micro-organism with a large biotechnological potential. Therefore, the aim of this study was to test whether protein production in L. lactis using the NICE system can also effectively be performed at the industrial-scale of fermentation. RESULTS: Lysostaphin, an antibacterial protein (mainly against Staphylococcus aureus) from S. simulans biovar. Staphylolyticus, was used as a model system. Food-grade lysostaphin expression constructs in L. lactis were grown at 1L-, 300-L and 3000-L scale and induced with nisin for lysostaphin production. The induction process was equally effective at all scales and yields of about 100 mg/L were obtained. Up-scaling was easy and required no specific effort. Furthermore, we describe a simple and effective way of downstream processing to obtain a highly purified lysostaphin, which has been used for clinical phase I trials. CONCLUSION: This is the first example that shows that nisin-regulated gene expression in L. lactis can be used at industrial scale to produce large amounts of a target protein, such as lysostaphin. Downstream processing was simple and in a few steps produced a highly purified and active enzyme.

Journal Article↗
PubMed2005

Optimization of the Lactococcus lactis nisin-controlled gene expression system NICE for industrial applications.

BACKGROUND: The nisin-controlled gene expression system NICE of Lactococcus lactis is one of the most widely used expression systems in Gram-positive bacteria. Despite its widespread use, no optimization of the culture conditions and nisin induction has been carried out to obtain maximum yields. As a model system induced production of lysostaphin, an antibacterial protein (mainly against Staphylococcus aureus) produced by S. simulans biovar. Staphylolyticus, was used. Three main areas need optimization for maximum yields: cell density, nisin-controlled induction and protein production, and parameters specific for the target-protein. RESULTS: In a series of pH-controlled fermentations the following parameters were optimized: pH of the culture, use of NaOH or NH4OH as neutralizing agent, the addition of zinc and phosphate, the fermentation temperature, the time point of induction (cell density of the culture), the amount of nisin added for induction and the amount of three basic medium components, i.e. yeast extract, peptone and lactose. For each culture growth and lysostaphin production was followed. Lysostaphin production yields depended on all parameters that were varied. In the course of the optimization a three-fold increase in lysostaphin yield was achieved from 100 mg/l to 300 mg/l. CONCLUSION: Protein production with the NICE gene expression system in L. lactis strongly depends on the medium composition, the fermentation parameters and the amount of nisin added for induction. Careful optimization of key parameters lead to a significant increase in the yield of the target protein.

Journal Article↗
PubMed2005

Protein secretion in Lactococcus lactis : an efficient way to increase the overall heterologous protein production.

Lactococcus lactis, the model lactic acid bacterium (LAB), is a food grade and well-characterized Gram positive bacterium. It is a good candidate for heterologous protein delivery in foodstuff or in the digestive tract. L. lactis can also be used as a protein producer in fermentor. Many heterologous proteins have already been produced in L. lactis but only few reports allow comparing production yields for a given protein either produced intracellularly or secreted in the medium. Here, we review several works evaluating the influence of the localization on the production yields of several heterologous proteins produced in L. lactis. The questions of size limits, conformation, and proteolysis are addressed and discussed with regard to protein yields. These data show that i) secretion is preferable to cytoplasmic production; ii) secretion enhancement (by signal peptide and propeptide optimization) results in increased production yield; iii) protein conformation rather than protein size can impair secretion and thus alter production yields; and iv) fusion of a stable protein can stabilize labile proteins. The role of intracellular proteolysis on heterologous cytoplasmic proteins and precursors is discussed. The new challenges now are the development of food grade systems and the identification and optimization of host factors affecting heterologous protein production not only in L. lactis, but also in other LAB species.

Journal Article↗
PubMed2006

Heterologous expression of Brucella abortus GroEL heat-shock protein in Lactococcus lactis.

BACKGROUND: Brucella abortus is a facultative intracellular pathogen that mainly infects cattle and humans. Current vaccines rely on live attenuated strains of B. abortus, which can revert to their pathogenic status and thus are not totally safe for use in humans. Therefore, the development of mucosal live vaccines using the food-grade lactic acid bacterium, Lactococcus lactis, as an antigen delivery vector, is an attractive alternative and a safer vaccination strategy against B. abortus. Here, we report the construction of L. lactis strains genetically modified to produce B. abortus GroEL heat-shock protein, a candidate antigen, in two cellular locations, intracellular or secreted. RESULTS: Only the secreted form of GroEL was stably produced in L. lactis, suggesting a detrimental effect of GroEL protein when intracellularly produced in this bacterium. Only trace amounts of mature GroEL were detected in the supernatant fraction of induced lactococcal cultures, and the GroEL precursor remained stacked in the cell fraction. Attempts to raise the secretion yields were made, but even when GroEL was fused to a synthetic propeptide, secretion of this antigen was not improved. CONCLUSION: We found that L. lactis is able to produce, and to secrete, a stable form of GroEL into the extracellular medium. Despite the low secretion efficiency of GroEL, which suggest that this antigen interacts with the cell envelope of L. lactis, secretion seems to be the best way to achieve both production and protein yields, regardless of cellular location. The L. lactis strain secreting GroEL has potential for in vivo immunization.

Journal Article↗
PubMed2001

4-Hydroxy-2,5-dimethyl-3(2H)-furanone (HDMF) production in simple media by lactic acid bacterium, Lactococcus lactis subsp. cremoris IFO 3427.

4-Hydroxy-2,5-dimethyl-3(2H)-furanone (HDMF) was detected in simple media prepared with heated sugar and amino acid solution as a result of the Maillard reaction. However, an increase in the amount of HDMF was observed in the media fermented by Lactococcus lactis subsp. cremoris IFO 3427. 4-Hydroxy-2(or 5)-ethyl-5(or 2)-methyl-3(2H)-furanone (HEMF) was not detected in all the simple media examined in this study.

Journal Article↗
PubMed2002

Synchronized fresh cell bioreactor system for continuous L-(+)-lactic acid production using Lactococcus lactis IO-1 in hydrolysed sago starch.

An efficient bioreactor, termed a 'synchronized fresh cell bioreactor', was developed and consisted of a pH-dependent substrate feed system coupled with cross flow filtration and turbidity control. The effect of high dilution rate and high cell density coupled with high cell viability on the production of l-lactic acid in continuous culture by Lactococcus lactis IO-1 in enzyme-hydrolysed sago starch medium was investigated. For all changes in dilution rate, cells responded in a synchronized way to the addition of glucose by increasing the rate of biomass formation. Consequently, a glucose-free feed solution was required to maintain the cell concentration at a particular pre-set value. This set-up facilitated the maintenance of the cells in a permanent log phase. At a cell concentration of 15 gl(-1) and a feed glucose concentration of 53 gl(-1), volumetric LA productivities of 8.2, 19.3 and 33.1 gl(-1)h(-1) were obtained at dilution rates of 0.21, 0.50 and 1.1 h(-1), respectively. The respective residual glucose concentrations in the spent medium were 1.90, 0.24 and 3.80 gl(-1). By increasing the cell density, the volumetric productivity increased proportionally. At high cell density, higher dilution rates resulted in lower lactate concentrations in the culture medium resulting in higher productivity. This reactor facilitated efficient operation with high cell viability by maintaining the cells in continuous growth phase for long-term fermentation. Therefore, the growth rate (mu) was calculated according to the Monod equation. Using this system, high specific productivities can be obtained which guarantees high commercial productivity at economical cost with only a small investment for setting up the sago industry.

Journal Article↗
PubMed1997

Cloning of a gene encoding nisin resistance from Lactococcus lactis subsp. lactis M189 which is transcribed from an extended 210 promoter.

A 56-kb plasmid was identified in Lactococcus lactis subsp. lactis (L. lactis) M189 which encodes resistance to nisin (Nis(R)) following mobilization of the plasmid into L. lactis LM0230. The Nis(R) determinant was localized on a 1.6-kb HindIII fragment by DNA restriction fragment deletion and subcloning. An open reading frame (ORF) of 957 bases was identified by sequence analysis and its transcription start site was mapped by primer extension. The ORF is flanked by two regions which exhibit complete homology to parts of the inverted repeat sequences of IS981 and ISS1T. The promoter for transcription was found to consist of an extended -10 site (TgTGtTATAAT) that lacks a -35 site. Function of the extended -10 promoter was demonstrated by its ability to express the promoterless cat gene from Staphylococcus aureus. Base substitution analysis revealed that the TgTGt extension is essential for promoter efficiency in L. lactis.

Journal Article↗