Host specificity of nisin production by Lactococcus lactis

Kinetics of nisin production have been investigated in terms of endogenous features of the producer organism, Lactococcus lactis. Nisin-producing transposons (Tn Nip) were transferred to different hosts by conjugation. Constructs were cultivated in batch cultures and nisin produced was measured. The proteinase function of C2Prt (Tn Nip)-1 was eliminated by plasmid curing, resulting in the construct C2Prt - (Tn Nip)-1. C2Prt - (Tn Nip)-1 produced nisin to a higher concentration compared to C2Prt (Tn Nip)-1 and was able to maintain the maximum concentration till the end of cultivation. The final concentration of nisin produced was host-specific, because when different constructs carrying the same Tn Nip were cultivated they produced nisin to different concentrations. However, when the same host carried Tn Nip transposons derived from different donors the concentration of nisin produced was similar, suggesting that the two Tn Nip transposons may be similar.     

High nisin Z production by Lactococcus lactis UL719 in whey permeate with aeration

The influence of controlled pH (5.0–6.5) and initial dissolved oxygen level (0–90% air saturation) on nisin Z production in a yeast extract/Tween 80-supplemented whey permeate (SWP) was examined during batch fermentations with citrate positive Lactococcus lactis subsp. lactis UL719. The total activity corresponding to the sum of soluble and cell-bound activities, as measured by a critical dilution method, was more than 50% lower at pH 5.0 than in the range 5.5–6.5, although the specific production decreased as pH increased. A maximum nisin Z activity of 8200 AU/ml (4100IU/ml) was observed in the supernatant after 8h of culture for pH ranging from 5.5 to 6.5. Prolonging the culture beyond 12h decreased this activity at pH 6.0 and 6.5 but not at pH 5.5 or 5.0. A corresponding increase in cell-bound activity was probably due to adsorption of soluble bacteriocin to the cell wall. Aeration increased cell-bound and total activity to maximum values of 32800 and 41000 AU/ml (16400 and 20500IU/ml), respectively, with an initial level of 60% air saturation after 24h of incubation at pH 6.0. The specific production at 60% or 90% initial air saturation was eight-fold higher than at 0%.     

Modelling the production of nisin by Lactococcus lactis in fed-batch culture

Nisin production in batch culture and fed-batch cultures (sucrose feeding rates were 6, 7, 8, and 10 g l^–1 h^–1, respectively) by Lactococcus lactis subsp. lactis ATCC 11454 was investigated. Nisin production showed primary metabolite kinetics, and could be improved apparently by altering the feeding strategy. The nisin titer reached its maximum, 4,185 IU ml^–1, by constant addition of sucrose at a feeding rate of 7 g l^–1 h^–1; an increase in 58% over that of the batch culture (2,658 IU ml^–1). Nisin biosynthesis was affected strongly by the residual sucrose concentration during the feeding. Finally, a mathematical model was developed to simulate the cell growth, sucrose consumption, lactic acid production and nisin production. The model was able to describe the fermentation process in all cases.     

Genes involved in immunity to the lantibiotic nisin produced by Lactococcus lactis 6F3.

The lantibiotic nisin is produced by several strains of Lactococcus lactis. The complete gene cluster for nisin biosynthesis in L. lactis 6F3 comprises 15 kb of DNA. As described previously, the structural gene nisA is followed by the genes nisB, nisT, nisC, nisI, nisP, nisR, and nisK. Further analysis revealed three additional open reading frames, nisF, nisE, and nisG, adjacent to nisK. Approximately 1 kb downstream of the nisG gene, three open reading frames in the opposite orientation have been identified. One of the reading frames, sacR, belongs to the sucrose operon, indicating that all genes belonging to the nisin gene cluster of L. lactis 6F3 have now been identified. Proteins NisF and NisE show strong homology to members of the family of ATP-binding cassette (ABC) transporters, and nisG encodes a hydrophobic protein which might act similarly to the immunity proteins described for several colicins. Gene disruption mutants carrying mutations in the genes nisF, nisE, and nisG were still able to produce nisin. However, in comparison with the wild-type strain, these mutants were more sensitive to nisin. This indicates that besides nisI the newly identified genes are also involved in immunity to nisin. The NisF-NisE ABC transporter is homologous to an ABC transporter of Bacillus subtilis and the MbcF-MbcE transporter of Escherichia coli, which are involved in immunity to subtilin and microcin B17, respectively.     

Heterologous expression and purification of NisA, the precursor peptide of lantibiotic nisin from Lactococcus lactis

The lantibiotic nisin is a ribosomally synthesised and post-translationally modified antimicrobial peptide produced by strains of Lactococcus lactis, and used as safe and natural preservative in food industry. The nisA structural gene encodes ribosomally synthesised and biologically inactive a 57 amino acid precursor peptide (NisA) which undergoes several post-translational modifications. In this study, we report the expression of precursor nisin as a His6-tagged peptide in Escherichia coli and its purification using a nickel affinity column. The technique of spliced-overlap extension PCR was used to amplify the nisA gene and the T7 promoter region of pET-15b vector. This approach was used to introduce six histidine residues at the C-terminus of prenisin. The identity of the expressed peptide was confirmed by N-terminal sequencing. The expressed His-tagged prenisin was purified under denaturing conditions, and named as prenisin-His6. The purified prenisin-His6 was analyzed by SDS-PAGE, Western blotting and mass spectroscopy. These results showed that the nisin precursor peptide can be successfully produced using an E. coli expression system.     

The kinetics of end-product inhibition of l-lactate production from xylose and glucose by Lactococcus lactis IO-1

Summary The relative contributions of lactate inhibition and the generation of sterile (undividing) cells to the low xylose utilisation rate of Lactococcus lactis IO-1 was investigated. The lactate inhibition constant of xylose grown cells was shown to be 9.3 times more than that of glucose grown cells. However, the sterile cell production rate and LDH inactivation rate of the xylose cultures were at least 10 times less than the glucose cultures. Thus, it is suggested that the slower substrate consumption rate in xylose medium is caused mainly by the large inhibition constant for the end product.     

Improving nisin production by increasing nisin immunity/resistance genes in the producer organism Lactococcus lactis

The effect on nisin production of increasing nisin immunity/resistance genes in Lactococcus lactis subsp. lactis MG1363 was investigated. The 60-kb nisin immunity/resistance plasmid pND300, which was isolated from a non-nisin-producing strain, encodes five genes involved in nisin immunity/resistance, which are very similar to those of the immunity/resistance system encoded by the nisin-production transposon. The introduction of pND300 into MG1363(TnNip) resulted in the construct being able to produce significantly more nisin than the parent MG1363(TnNip). The introduction of pND314, which contains the nisin immunity/resistance genes subcloned into pSA3, into MG1363(TnNip) allowed the strain to grow more rapidly than the parent MG1363(TnNip) with a concomitant increase in the rate of nisin production. This work illustrates that introduction of pND300 and a derivative containing the nisin immunity/resistance system of pND300 into MG1363 (TnNip) can result in significant alterations to the kinetics of nisin production. These observations indicate approaches that may be used successfully to improve the economics of nisin production. Received: 11 February 1998 / Received revision: 25 June 1998 / Accepted: 27 June 1998     

Enhanced production of pediocin PA-1 and coproduction of nisin and pediocin PA-1 by Lactococcus lactis.

The production and secretion of class II bacteriocins share a number of features that allow the interchange of genetic determinants between certain members of this group of antimicrobial peptides. Lactococcus lactis IL1403 encodes translocatory functions able to recognize and mediate secretion of lactococcin A. The ability of this strain to also produce the pediococcal bacteriocin pediocin PA-1, has been demonstrated previously by the introduction of a chimeric gene, composed of sequences encoding the leader of lactococcin A and the mature part of pediocin PA-1 (N. Horn, M. I. Martínez, J. M. Martínez, P. E. Hernández, M. J. Gasson, J. M. Rodríguez, and H. M. Dodd, Appl. Environ. Microbiol. 64:818-823, 1998). This heterologous expression system has been developed further with the introduction of the lactococcin A-dedicated translocatory function genes, lcnC and lcnD, and their effect on bacteriocin yields in various lactococcal hosts was assessed. The copy number of lcnC and lcnD influenced production levels, as did the particular strain employed as host. Highest yields were achieved with L. lactis IL1403, which generated pediocin PA-1 at a level similar to that for the parental strain, Pediococcus acidilactici 347, representing a significant improvement over previous systems. The genetic determinants required for production of pediocin PA-1 were introduced into the nisin-producing strain L. lactis FI5876, where both pediocin PA-1 and nisin A were simultaneously produced. The implications of coproduction of these two industrially relevant antimicrobial agents by a food-grade organism are discussed.     

Identification and characterization of the lantibiotic nisin Z, a natural nisin variant

Lactococcus lactis strain NIZO 22186 produces an extracellular, lanthionine-containing 3.5-kDa polypeptide with antimicrobial activity. Its retention time on reversed-phase (RP) HPLC and its amino acid composition showed high similarities but no complete identity to nisin. The gene for this lantibiotic, designated nisZ, has been cloned and its nucleotide sequence was found to be identical to that of the precursor nisin gene apart from a single mutation resulting in the substitution His27Asn in the mature polypeptide. NMR studies of the natural nisin variant, which has been designated nisin Z, confirmed the His27Asn substitution and indicated that it has a similar structure to nisin.     

Note : Genetic and biochemical characterization of nisin Z produced by Lactococcus lactis ssp. lactis biovar. diacetylactis UL 719

The bacteriocin produced by Lactococcus lactis ssp. lactis biovar. diacetylactis UL 719 was purified and characterized. Two peaks exhibiting antimicrobial activity were obtained after purification. Primary structure of the peptide of major peak 2 was identical to that of nisin Z when determined by Edman degradation and confirmed by DNA sequence analysis. The molecular mass as determined by mass spectrometry was 3346·39 ± 0·40 Da for peak 1 and 3330·39 ± 0·27 Da for peak 2, which suggests that peak 1 may correspond to an oxidized form of nisin Z. The two purified peaks exhibiting xrantimicrobial activity appear to correspond with the oxidized and native forms of nisin Z.     

Identification of the lantibiotic nisin Q,a new natural nisin variant produced by Lactococcus lactis 61-14 isolated from a river in Japan

Lactococcus lactis 61-14 isolated from river water produced a bacteriocin active against a wide range of Gram-positive bacteria. N-terminal amino acid sequencing, mass spectral analysis of the purified bacteriocin, and genetic analysis using nisin-specific primers showed that the bacteriocin was a new natural nisin variant, termed nisin Q. Nisin Q and nisin A differ in four amino acids in the mature peptide and two in the leader sequence.     

Potential application of the nisin Z preparation of Lactococcus lactis W8 in preservation of milk

Aims: The aim of the study is to evaluate the effectiveness of the preparation of nisin Z from Lactococcus lactis W8‐fermented milk in controlling the growth of spoilage bacteria in pasteurized milk. Methods and Results: Spoilage bacteria isolated from pasteurized milk at 8 and 15°C were identified as Enterococcus italicus, Enterococcus mundtii, Enterococcus faecalis, Bacillus thuringiensis, Bacillus cereus, Lactobacillus paracasei, Acinetobacter sp., Pseudomonas fluorescens and Enterobacter aerogenes. These bacteria were found to have the ability to survive pasteurization temperature. Except Enterobacter aerogenes, the spoilage bacteria were sensitive to the nisin Z preparation of the L. lactis W8. Addition of the nisin Z preparation to either the skim milk or fat milk inoculated with each of the spoilage bacteria reduced the initial counts (about 5 log CFU ml^?1) to an undetectable level within 8–20 h. The nisin Z preparation extended the shelf life of milk to 2 months under refrigeration. Conclusions: The nisin Z preparation from L. lactis W8‐fermented milk was found to be effective as a backup preservative to counteract postpasteurization contamination in milk. Significance and Impact of the Study: A rapid inhibition of spoilage bacteria in pasteurized skim and fat milk with the nisin Z preparation of L. lactis W8 is more significant in comparison with the commercially available nisin (nisin A). The nisin Z preparation can be used instead of commercial nisin, which is not effective in fat milk.     

Influence of the carbon source on nisin production in Lactococcus lactis subsp. lactis batch fermentations.

Nisin production by Lactococcus lactis subsp. lactis NIZO 22186 was studied in batch fermentation using a complex medium. Nisin production showed primary metabolite kinetics: nisin biosynthesis took place during the active growth phase and completely stopped when cells entered the stationary phase. A stringent correlation could be observed between the expression of the prenisin gene (nisA) and the synthesis of the post-translationally enzymically modified and processed mature nisin peptide. Moreover, it seemed likely that nisin had a growth control function. A physiological link is proposed between sucrose fermentation capacity and nisin production ability. Carbon source regulation appears to be a major control mechanism for nisin production.     

Properties of nisin Z and distribution of its gene, nisZ, in Lactococcus lactis.

Two natural variants of the lantibiotic nisin that are produced by Lactococcus lactis are known. They have a similar structure but differ in a single amino acid residue at position 27; histidine in nisin A and asparagine in nisin Z (J.W.M. Mulders, I.J. Boerrigter, H.S. Rollema, R.J. Siezen, and W.M. de Vos, Eur. J. Biochem, 201:581-584, 1991). The nisin variants were purified to apparent homogeneity, and their biological activities were compared. Identical MICs of nisin A and nisin Z were found with all tested indicator strains of six different species of gram-positive bacteria. However, at concentrations above the MICs, with nisin Z the inhibition zones obtained in agar diffusion assays were invariably larger than those obtained with nisin A. This was observed with all tested indicator strains. These results suggest that nisin Z has better diffusion properties than nisin A in agar. The distribution of the nisin variants in various lactococcal strains was determined by amplification of the nisin structural gene by polymerase chain reaction followed by direct sequencing of the amplification product. In this way, it was established that the nisZ gene for nisin Z production is widely distributed, having been found in 14 of the 26 L. lactis strains analyzed.     

Modeling of growth and lactate fermentation by Lactococcus lactis subsp. lactis biovar. diacetylactis in batch culture

The kinetic behaviour of Lactococcus lactis subsp. lactis biovar. diacetylactis was studied in batch culture under non-limiting conditions that allow high growth and product formation. A model based on laboratory results is proposed for growth and l-lactate fermentation. It shows the necessity for differentiating biomass into three physiological states, two active, X_g (growth + acidification) and X_ng (acidification), and one inactive, X_i. The kinetic theory of the model demonstrates the non-competitive nature of fermentation end-product inhibition on growth and acidification, and describes the passage from one physiological state to another. Satisfying simulations were obtained for batch fermentations, and the use of this type of model for determining and optimizing fermentation parameters is discussed.Correspondence to: C. Diviès     

Fine tuning of the lactate and diacetyl production through promoter engineering in Lactococcus lactis

Lactococcus lactis is a well-studied bacterium widely used in dairy fermentation and capable of producing metabolites with organoleptic and nutritional characteristics. For fine tuning of the distribution of glycolytic flux at the pyruvate branch from lactate to diacetyl and balancing the production of the two metabolites under aerobic conditions, a constitutive promoter library was constructed by randomizing the promoter sequence of the H(2)O-forming NADH oxidase gene in L. lactis. The library consisted of 30 promoters covering a wide range of activities from 7,000 to 380,000 relative fluorescence units using a green fluorescent protein as reporter. Eleven typical promoters of the library were selected for the constitutive expression of the H(2)O-forming NADH oxidase gene in L. lactis, and the NADH oxidase activity increased from 9.43 to 58.17-fold of the wild-type strain in small steps of activity change under aerobic conditions. Meanwhile, the lactate yield decreased from 21.15 ± 0.08 mM to 9.94 ± 0.07 mM, and the corresponding diacetyl production increased from 1.07 ± 0.03 mM to 4.16 ± 0.06 mM with the intracellular NADH/NAD(+) ratios varying from 0.711 ± 0.005 to 0.383 ± 0.003. The results indicated that the reduced pyruvate to lactate flux was rerouted to the diacetyl with an almost linear flux variation via altered NADH/NAD(+) ratios. Therefore, we provided a novel strategy to precisely control the pyruvate distribution for fine tuning of the lactate and diacetyl production through promoter engineering in L. lactis. Interestingly, the increased H(2)O-forming NADH oxidase activity led to 76.95% lower H(2)O(2) concentration in the recombinant strain than that of the wild-type strain after 24 h of aerated cultivation. The viable cells were significantly elevated by four orders of magnitude within 28 days of storage at 4°C, suggesting that the increased enzyme activity could eliminate H(2)O(2) accumulation and prolong cell survival.