Nisins

This invention relates to the production of protein-engineered nisins.
Reference to FIG. 2 herein, refers to FIGS. 2A and 2B collectively. Reference to FIG. 6 herein, refers to FIGS. 6A and 6B collectively. Reference to FIG. 11 herein, refers to FIGS. 11A and 11B collectively.
Nisin is a highly modified peptide antibiotic produced, for example, by certain strains of Lactococcus lactis. It is of great interest to the food industry because of its efficient antimicrobial activity against a wide range of gram-positive organisms including many spoilage bacteria and food pathogens, for example, Listeria, Clostridia and Bacillus species (12,25).
The chemical structure of nisin is well established (6,17,34,45, FIG. 9) (SEQ ID NO:19). It is a member of the family of antibiotics termed lantibiotics. These unusual polycyclic peptides share the structural features of dehydro-residues and intrachain sulphide bridges forming lanthionine and .beta.-methyllanthionine rings. The atypical residues are introduced by post-translational modification of amino acids serine, threonine and cysteine in the primary sequence of a precursor peptide (lantibiotics are the subject of a recent extensive review, 26). Biosynthesis of nisin thus involves genes for both the inactive precursor of nisin, known as prenisin, (nisA) and also the modifying enzymes responsible for nisin maturation. The mature nisin molecule is based on a sequence of 34 amino acids (SEQ ID NO:19). The protein encoded by nisA includes a 23 amino acid N terminal signal sequence which is cleaved off during secretion of nisin. The conversion of prenisin, encoded by nisA, into mature nisin involves cleavage of the leader and the modification of individual amino acids. The nisA gene has been cloned and characterised (1,27,11) and shown to have a chromosomal location (11,42). A number of additional, as yet uncharacterised, genes involved in the enzymatic modification of prenisin, translocation and immunity are encoded by nisin producing strains (42). These determinants, along with nisA, are thought to be clustered together as has been described recently for the lantibiotics subtilin (28) and epidermin (41). It has been known for some time that nisin determinants can be transferred by conjugation (14) and it has now been established that this ability is due to their carriage on a large conjugative transposon, Tn5301 (22,38).
Established protein engineering techniques can be used to introduce changes to the amino acid sequence of nisin. This involves modifying the coding region of the nisin structural gene, nisA, for example by site-directed or random mutagenesis. Expression of these changes is complicated by the fact that nisin is post-translationally modified.
Variant nisins may be constructed by the expression of variant nisA genes in a host strain which encodes the necessary maturation machinery, and thus can process the modified precursor peptide. The simplest approach is to transform a nisin producing strain with a recombinant plasmid encoding a variant nisA gene. In this background the host's maturation enzymes are available to process both the resident prenisin and its plasmid-encoded variant. A strategy of this type has been reported for a strain that carries the wild-type nisin transposon (29). However, the disadvantage of this system is that both the host's nisin and the engineered variant are synthesised together, making complex chemical separation procedures necessary prior to analysis of the properties of the novel peptide. Such a procedure would be particularly undesirable for industrial scale production of a variant nisin.
According to a first aspect of the present invention there is provided an organism which does not secrete its natural nisin, but is capable of expressing genes for nisin modification, immunity and translocation out of the cell.
By "an organism that does not secrete its natural nisin" we include an organism which does not naturally encode a nisin. Examples of organisms that do not naturally encode a nisin are Bacillus subtilis and Escherichia coli.
Preferably the organism is a lactococcal strain, most preferably Lactococcus lactis.
A second aspect of the present invention provides the organism described above transformed with a coding sequence for a variant prenisin and, if necessary, appropriate regulatory sequences for expression thereof, such that the organism is capable of secreting the corresponding variant nisin.
A third aspect of the present invention provides a process for producing a nisin comprising fermenting this organism and obtaining the nisin produced thereby.
By "nisin" it is meant a peptide antibiotic produced by some naturally occurring nisin producing strains of bacteria. The mature molecule is based on a sequence of amino acids encoded by a gene nisA.
By "variant nisin" it is meant a protein engineered variant of a natural nisin in which changes to the amino acid sequence of the nisin have been introduced as a result of site-directed or random mutagenesis of the nisA gene.
Site-directed mutations of the nisA gene may be made, for example, by the oligonucleotide-directed mutagenesis technique of Zoller & Smith (1983) Meth. Enzymol. 100, 468-500 and Zoller & Smith (1984) DNA 3, 479-480 which uses mismatched oligonucleotide primers to introduce the mutation.
It is convenient to use a method for improving the yield of mutants, for example, the dut-ung method described by Kunkel (1985) Proc. Natl. Acad. Sci. USA 82, 488-492. Alternatively, the polymerase chain reaction (PCR) may be used to generate mutants using mismatched oligonucleotides (Saiki et al (1988) Science 239, 487-491).
Random mutants of the nisA gene can be made chemically using, for example, sodium bisulphite or hydroxylamine as the mutagen. Alternatively, random mutations can be introduced into the nisA gene using enzymatic misincorporation using a DNA polymerase with relatively low fidelity, for example AMV reverse transcriptase or Taq DNA polymerase or by using mixtures of oligonucleotides, spiked during synthesis, to incorporate a small amount of each different bases at each position. These methods are well known in the art.
The derivation of an organism that expresses the maturation genes for nisin biosynthesis, but is deficient in its natural nisA gene product, thereby provides an effective means to produce variant nisins. The transformation of such a strain with a plasmid vector carrying an individual nisA gene containing a site-directed or random mutation generates a strain that produces exclusively a variant nisin.
As the gene for nisA is only one of a coordinately expressed group of genes that also provide the nisin modification, immunity and translocation functions, simple inactivation of the nisA gene does not yield a strain that is able to convert the product of a plasmid-encoded nisA gene into mature nisin.
A fourth aspect of the present invention provides a method of constructing an organism that does not secrete its natural nisin but is capable of expressing the other nisin genes. The method comprises selecting a nisin producing organism and selectively deleting the coding sequence for its natural nisA gene product or otherwise preventing the secretion of the nisin polypeptide, for example, by modifying an amino acid in the prenisin leader sequence, preferably amino acid 4.
A preferred method comprises insertionally inactivating the nisA gene and restoring the activity of the genes for nisin modification, immunity and translocation out of the cell. The restoration of activity may be achieved by selection in media containing nisin at inhibitory levels.
In another method the nisA gene is deleted or a mutation is introduced which prevents its translation and the expression of the associated nisin genes is maintained.
It will be appreciated by a person skilled in the art that an organism which does not naturally secrete nisin, and that does not naturally contain genes for nisin modification, immunity and translocation out of the cell may be converted into an organism useful in the practice of the present invention by transferring into it the said genes for nisin modification, immunity and translocation out of the cell. Thus, one embodiment of the invention provides a means of converting an organism which is not useful in the practice of the invention to one that is.
For example, it is possible to transfer the nisin modification, immunity and translocation genes from the Lactococcus lactis strain FI 7332 (as disclosed in the Example) to an organism that does not contain these genes, nor contains a nisA gene, for example E. coli or B. subtilis or a suitable lactococcal strain, by conjugation or transduction or by gene cloning technology. It is preferred if the organism is B. subtilis or a suitable lactococcal strain.
Furthermore, it is possible to transfer the nisin modification, immunity and translocation genes from a strain not able to produce a nisin (for example Lactococcus lactis strains FI 7300 and FI 7304 as disclosed in the Example) to an organism that does not contain these genes, introduce a nisA gene and then select for nisin production (that is, using similar methods to those disclosed in the Examples for the production of Lactococcus lactis FI 7300). Such a transfer of genes may also be by conjugation or transduction or gene cloning technology (including transformation in the case of the nisA gene). It is preferred if the organism is B. subtilis or a suitable lactococcal strain.
According to a fifth aspect of the present invention, the above-outlined method further comprises transforming the organism with a coding sequence for a variant prenisin and, if necessary, appropriate regulatory sequences for expression thereof. The organism is capable of post-translational modification of said prenisin and secretion of the corresponding variant nisin.
A sixth aspect of the present invention provides a nisin having a residue other than dehydroalanine corresponding to the residue in the first ring of natural nisin derived from the serine residue of unmodified prenisin. Said first ring of mature nisin is shown as ring a in FIG. 9 (SEQ ID NO:15).
A seventh aspect provides a nisin which does not have a dehydroalanine residue in the first ring. This variant nisin is preferably produced using the expression system of the present invention. A study of nisin breakdown during storage has revealed that activity is lost when the first ring of mature nisin becomes opened at amino acid 5 (5,34). This residue is dehydroalanine in mature nisin and it is derived by dehydration of a serine residue in prenisin. It is believed that the opening of the ring is due to the fact that the dehydroalanine is labile. A site-directed mutant of the nisA gene may be constructed in which the codon for serine at residue 5of the natural prenisin is changed to one that encodes an amino acid other than serine. For example, codon for serine 5 may be changed to encode alanine, valine, threonine, leucine, isoleucine, glycine, histidine, arginine, lysine, aspartate, glutamate, asparagine, glutamine, proline, methionine, cysteine, phenylalanine, tyrosine or tryptophan. Alanine is preferred. The expression of the engineered nisA gene in a nisin producing organism in accordance with the present invention leads to the production of a variant mature nisin. In the case of the preferred mutation to an alanine codon, the variant mature nisin is named nisinA S5A. Investigations indicate that this variant nisin has greater stability than natural nisin A and is therefore an example of the exclusive production of an improved nisin.
The variant nisin not having dehydroalanine at position 5 may have other variations as well, although it is preferable to retain the dehydroalanine residue at position 33.
The amino acids replacing serine at position 5 may be modified post-translationally to form an amino acid not coded for directly, for example as disclosed herein for threonine residues.
Evidently, it is also possible to produce this variant nisin by expressing the engineered variant nisA gene on a plasmid vector that is transformed into a normal nisin producing organism. Mature variant nisin is produced as well as normal nisin.
It may also be possible to chemically synthesise the variant nisin peptides wherein Serine 5 is replaced by another amino acid residue.
Peptides, such as the variant nisins, may be synthesised by the Fmoc-polyamide mode of solid-phase peptide synthesis as disclosed by Lu et al (1981) J. Org. Chem. 46, 3433 and references therein. Temporary N-amino group protection is afforded by the 9-fluorenylmethyloxycarbonyl (Fmoc) group. Repetitive cleavage of this highly base-labile protecting group is effected using 20% piperidine in N,N-dimethylformamide. Side-chain functionalities may be protected as their butyl ethers (in the case of serine threonine and tyrosine), butyl esters (in the case of glutamic acid and aspartic acid), butyloxycarbonyl derivative (in the case of lysine and histidine), trityl derivative (in the case of cysteine) and 4-methoxy-2,3,6-trimethylbenzenesulphonyl derivative (in the case of arginine). Where glutamine or asparagine are C-terminal residues, use is made of the 4,4'-dimethoxybenzhydryl group for protection of the side chain amido functionalities. The solid-phase support is based on a polydimethylacrylamide polymer constituted from the three monomers dimethylacrylamide (backbone-monomer), bisacryloylethylene diamine (cross linker) and acryloylsarcosine methyl ester (functionalising agent). The peptide-to-resin cleavable linked agent used is the acid-labile 4-hydroxymethyl-phenoxyacetic acid derivative. All amino acid derivatives are added as their preformed symmetrical anhydride derivatives with the exception of asparagine and glutamine, which are added using a reversed N,N-dicyclohexyl-carbodiimide/1-hydroxybenzotriazole mediated coupling procedure. All coupling and deprotection reactions are monitored using ninhydrin, trinitrobenzene sulphonic acid or isotin test procedures. Upon completion of synthesis, peptides are cleaved from the resin support with concomitant removal of side-chain protecting groups by treatment with 95% trifluoroacetic acid containing a 50% scavenger mix. Scavengers commonly used are ethanedithiol, phenol, anisole and water, the exact choice depending on the constituent amino acids of the peptide being synthesised. Trifluoroacetic acid is removed by evaporation in vacuo, with subsequent trituration with diethyl ether affording the crude peptide. Any scavengers present are removed by a simple extraction procedure which on lyophilisation of the aqueous phase affords the crude peptide free of scavengers. Reagents for peptide synthesis are generally available from Calbiochem-Novabiochem (UK) Ltd, Nottingham NG7 2QJ, UK. Using this chemical synthetic method it is possible to introduce non-natural amino acids into the synthetic nisin variant. Purification may be effected by any one, or a combination of, techniques such as size exclusion chromatography, ion-exchange chromatography and (principally) reverse-phase high performance liquid chromatography. Analysis of peptides may be carried out using thin layer chromatography, reverse-phase high performance liquid chromatography, amino-acid analysis after acid hydrolysis and by fast atom bombardment (FAB) mass spectrometric analysis.
Alternatively, nisin variants, including those with an "unnatural" amino acid replacing serine at position 5, may be produced by in vitro cell-free translation.
Other variant nisins may also be exclusively produced using the expression system of the present invention. Retaining a dehydroalanine residue at positions 5 or 33 is preferred.
An active natural variant of nisin, known as nisin Z, has asparagine instead of histidine at amino acid 27 (15,37). A variant of nisin Z, for example, in which the amino acid corresponding to asparagine is changed to glutamine may be produced using a site-directed mutant of the nisA gene in the expression system. This variant nisin, named nisinA H27Q, has full biological activity.
Further aspects of the present invention provide the variant nisins produced using the expression system of the present invention, and the use of such nisins as antimicrobial agents.
The variant nisins of the present invention are particularly useful as antimicrobial agents in those conditions where the natural nisin is ring-opened and loses its antimicrobial properties. Such conditions include those of high pH present in many food situations and where the activity of normal nisin is limited. Preferred organisms to be killed include Listeria monocytogenes and Gram negative bacteria.

So that the invention may be more readily understood, preferred aspects will now be illustrated by way of example, and with reference to the accompanying drawings in which:
FIG. 1 illustrates the strategy for nisA gene replacement.
It shows a) a map of the gene replacement vector pFI283 and maps showing equivalent regions of chromosome encoding nisA and flanking sequences in strains b) FI5876, c) FI7181 and d) FI7300. The thin line represents plasmid DNA and the thick line represents lactococcal chromosomal DNA. The nisA and nisB genes are indicated by black boxed regions and DNA sequences containing the erythromycin resistance (Em.sup.r) determinant are shown as shaded boxes. The direction of transcription of the genes is indicated by arrows above the maps. The small numbered arrows below the maps represent primers used in PCR analysis. A single recombination event between lactococcal sequences to the left of the Em.sup.r determinant on pFI283 and homologous sequences on FI5876 (X) results in Campbell integration of the plasmid with the organisation of sequences as shown in FI7181. Recombination between pFI283 sequences on both sides of the Em.sup.r determinant and homologous FI5876 sequences (X and Y) leads to gene replacement, as found with FI7300.
FIG. 2 shows the double stranded nucleotide sequence of the nisA gene and flanking regions (SEQ ID NO:1)
Coding regions preceded by ribosome binding sites (RBS) are indicated and the primary translation products (SEQ ID NO:2) are given below the sequence. The arrow between the arginine (R) at codon 154-156 and isoleucine (I) at codon 157-159 shows the point of cleavage of the N-terminal leader sequence of prenisin. Shaded regions represent the nucleotide sequences of synthetic oligomers employed as primers for PCR-mediated site-directed mutagenesis. The 3' end of the primers and the direction in which PCR proceeds is indicated by numbered arrows. The numbers refer to those primers as listed in methods. Specific mismatches included in the primers are represented by shaded nucleotides above or below the sequence. Primers 7 and 8 (SEQ ID NOS 14and 15, respectively) are designed to substitute a glutamine (Q) codon for a histidine (H) codon (coordinates 235-237) Primer 11 (SEQ ID NO:18) contains an alanine (A) codon in place of a serine (S) codon (coordinates 169-171). The primers containing restriction sites at their 5' ends define the termini of either PCR generated SacI/EcoRI restriction fragments (primers 5 and 6) (SEQ ID NOS 12 and 13, respectively) or BamHI/SacI restriction fragments (primers 9 and 10 or 9 and 11) (SEQ ID NOS 16 and 17, or SEQ ID NOS 16 and 18, respectively). The base changes mentioned above are incorporated in these fragments, which are then cloned into the expression vectors (see FIGS. 3 and 4) to reconstitute nisA genes containing the specific mutation.
FIG. 3 shows the construction of nisA expression vectors which use the natural nisin gene promoter.
The linear plasmid maps are of a) pFI172, b) pFI354, c) pFI378 and e) pFI411. Cloned Lactococcal DNA is represented by a thick line and vector sequences are indicated by a thin line. Genes within the cloned sequences are boxed and relevant restriction sites are given above the maps. d) illustrates the strategy employed for site-directed mutagenesis of the nisA gene to introduce the amino acid substitution His.sup.27. Double stranded DNA sequence between the SacI and EcoRI sites shown in pFI378 (c) is represented by a double line. The sites at which primers anneal are indicated by arrows above and below the lines and the fragments which are amplified by PCR using combinations of these primers are represented by single thin lines. The mismatches incorporated in primers 7 and 8 (SEQ ID NOS 14 and 15, respectively) (see FIG. 2), and the mutation they generate in pFI411 (e), are indicated with an asterisk.
FIG. 4 shows the construction of nisA expression vectors for site-directed mutagenesis throughout the nisA gene using the lac promoter.
A PCR generated BamHI fragment, encoding the lacr gene and the divergently transcribed lacR and lacA promoters (18, ref), was digested with XmnI. The 0.45 kb restriction fragment (see FIG. 5) was subcloned into pTG262 to generate pFI451(a). Fragments N and C containing the N- and C-terminal regions of the nisA gene respectively, were constructed by PCR and cloned into pTG262 to generate pFI446(b). The nisA cassette contains the uninterrupted nisA gene preceded by 60 bp, including the RBS, and followed by an intergenic gap and the start of the nisB gene (SEQ ID NO:1as shown in FIG. 2). Vector pFI449 (c) contains the nisA cassette cloned into pFI451 as a BamHI/EcoRI restriction fragment. Vector pFI480 (d) contains the N-fragment of the nisA cassette cloned into pFI451 as a BamHI/SacI fragment. Vector pFI487 (e) contains the C-fragment of the nisA cassette cloned into pFI451 as a SacI/EcoRI fragment.
FIG. 5 shows the nucleotide sequence of the lactose operon promoter (SEQ ID NO:33)
The XmnI/BamHI fragment contains the lacA promoter and start of the nisA gene in the nisA cassette.
FIG. 6 shows plate diffusion bioassays.
Agar is seeded with the indicator strain Lactobacillus helveticus CH-1 and the wells contain samples of supernatants from various Lactococcus lactis subsp. lactis strains. Plate 1 shows that nisin A is produced by the FI7332 expression system as a result of complementation of the hosts nisA deficiency by plasmid encoded nisA genes. Furthermore, biological activity of the variant nisin A/H27Q is demonstrated. Wells contain supernatants from FI7330 (FI7332/pFI172 plasmid encoded nisA); FI5876 (nisin producing parent strain); MG1614 (non-nisin producing strain); FI7332/pFI411 (plasmid encoded nisA/H27Q); FI7332/pFI378 (plasmid encoded nisA); FI7332 plasmid free; and FI7332/pFI354 (plasmid vector for construction of variant nisA genes). Plate 2 demonstrates nisin activity by strains encoding nisA and the variant nisA/S5A gene under the control of the lacA promoter. Wells contain supernatants from FI5876 (nisin producing parent strain); FI7332 (plasmid free); FI7332/pFI378 (plasmid encoded nisA, natural promoter); FI7332/pFI449 (plasmid encoded nisA, lacA promoter); and FI7332/pFI493 (plasmid encoded nisA/S5A, lacA promoter). 100 .mu.l of the following nisin standards (dissolved in 0.02M HCL) were loaded into some wells: 500 U/ml; 300 U/ml; 200 U/ml; 100 U/ml; and 0 U/ml.
FIG. 7 shows the construction of a host strain for the exclusive expression of mutant nisA genes.
Maps are of equivalent chromosomal regions of a) the nisin producing strain FI5876 and derivatives b) FI7300, c) FI7304 and d) FI7332. The nisA gene and the start of nisB gene are indicated by black boxes. The insertions in nisA are signified by a shaded box (Em.sup.r, erythromycin resistance) and an open box (IS905). Primers employed for analysis of nisA insertional inactivation are shown as numbered arrows below the maps. Lines connecting primers represent the amplified fragment with sizes given in kilobases.
FIG. 8 shows PCR analysis of the constructed host strains.
PCR fragments generated with primers 1 and 2 (SEQ ID NOS 8 and 9, respectively) are separated by agarose gel electrophoresis. Template DNA from the non-nisin producing strain MG1614 is not amplified (track 2) whereas a band of 0.9 kb is generated from the nisin producing parent strain FI5876 (track 3). This band is also present when DNA from FI7181 is employed as template. However, Campbell integration of the gene replacement vector pFI283 in this strain (see FIG. 1c) results in the generation of a second band of 1.9 kb (track 4). In FI7330 (track 5) the nisA gene has been insertionally inactivated as a result of gene replacement (see FIG. 1d) and the 0.9 kb band is replaced with the 1.9 kb band containing the integrated Em.sup.r marker. This band is increased in size by a further 1.3 kb to 3.2 kb in FI7304 (track 6) as a result of insertion of IS905 in the Em.sup.r gene (see FIG. 7c). Strain FI7332 has undergone a 200 bp deletion at one end of IS905 (FIG. 7d) and a corresponding decrease in the equivalent PCR generated fragment results in a 3.0 kb band (track 7). Tracks 1 shows .lambda.DNA digested with BglI included as a size standard. The 1.2% agarose gel was run for 2.5 hours at 100 volts.
FIG. 9 shows the structure of nisin A (SEQ ID NO:19).
The modified residues are dehydroalanine (Dha), dehydrobutyrine (Dhb), aminobutyrate (Abu), lanthionine (Ala-S-Ala) and .beta.-methyllanthionine (Abu-S-Ala). The predicted molecular alterations in Nisin A/H27Q and Nisin A/S5A are shown as amino-acid substitutions in ring e and ring a respectively.
FIG. 10 illustrates the presence of multiple copies of DNA in the lactococcal genome.
Southern transfer hybridizations of restriction enzyme digested chromosomal DNA from MG1614 (tracks 1 & 3) and FI5876 (tracks 2 & 4). Restriction endonucleases HincII (tracks 1 & 2) and PvuII (tracks 3 & 4) were used. The filter was probed with a .sup.32 P-labelled gel-purified PCR fragment generated with primers 3 and 4 (SEQ ID NOS 10 and 11, respectively) using FI7304 DNA as template (see FIG. 7c). Multiple bands indicate that there are several regions that display homology to the probe which is characteristic of multicopy IS elements in the chromosome of these Lactococcal strains.
FIG. 11 shows the single stranded nucleotide sequence of insertion sequence IS905 (SEQ ID NO: 4) and its encoded polypeptide sequence (SEQ ID NO: 5).
The inverted repeats which define the termini of the element are underlined.
FIG. 12 shows the potential promoter active sequences at junctions generated by IS905 insertion in strains a) FI 7304 (SEQ ID NOS:6 and 20, respectively) and b) FI 7332 (SEQ ID NOS 7 and 21, respectively)

EXAMPLE
Microbiological techniques and strains used: Most Lactococcus lactis subsp. lactis strains, generated in the course of this work, were derived from the nisin producing strain L. lactis FI5876 (11,22). The construction of the derivative strains and their relevant properties are described in the results and in Table 1. The plasmid-free, non-nisin producing strain MG1614 (13) was included as a control. L. lactis strains were routinely grown at 30.degree. C. in M17 media (43) supplemented with 0.5% (w/v) glucose (GM17). Selection for antibiotic resistance markers was as follows:- chloramphenicol resistance (Cm.sup.r), 5 .mu.g/ml; erythromycin resistance (Em.sup.r) was induced at the subinhibitory level of 50 ng/ml followed by selection at 5 .mu.g/ml.
TABLE 1______________________________________Characterisation of nisin producing strain FI5876 and derivatives. Nisin Nisin Immunity.sup.bStrain Em.sup.r Production.sup.a (IU/ml)______________________________________MG1614 - - <10FI5876 - + 1-3 .times. 10.sup.3FI7300 + - 2-5 .times. 10.sup.2FI7304 - - 1-3 .times. 10.sup.3FI7332 - - 1-3 .times. 10.sup.3______________________________________ .sup.a nisin production was determined by the plate diffusion assay. .sup.b levels at which strains were immune to nisin are given between two values. At the lower level growth was unaffected but inhibition of growth was evident at the upper level.
The Escherichia coli strain MC1022 (3) was the host strain used for construction of recombinant plasmids. Cultures were propagated at 37.degree. C. in L broth (30). Selection for ampicillin resistance (Ap.sup.r) was at 100 .mu.g/ml and Cm.sup.r was at 15 .mu./ml.
Determination of nisin production by L. lactis strains was based on the plate diffusion assay of Tramer and Fowler (44). Lactobacillus helveticus CH-1(Chr. Hansens Labs, Danmark A/S) was used as the nisin sensitive indicator strain. 0.5 ml of an overnight culture, grown in MRS media (9), was used to seed 50 ml MRS agar (pH 6.0) containing 1 ml Tween-20/Ringers solution (50:50). Wells were loaded with 100 .mu.l of test sample and the plates incubated at 4.degree. C. for a minimum of 3 hours (to allow diffusion) prior to overnight incubation at 42.degree. C.
Nisin immunity was determined by streaking a loopful of stationary-phase cells on plates containing varying amounts of nisin. Nisin (Koch-Light), dissolved in 0.2 M HCl, was added to GM17 agar up to a maximum concentration of 5.times.10.sup.3 U/ml at which growth of all strains was inhibited. L. lactis strains were considered to be immune to the highest level of nisin at which growth was evident throughout the streak.
Plasmid curing was achieved by growth in the presence of acriflavine (36).
Transformation: Recombinant plasmids were recovered by transformation of E. coli by the method of Cohen et al, 1972 (8) with the modification of Humphreys et al, 1979 (24). L. lactis strains were transformed by electroporation as described by Holo and Nes, 1989 (19) with the following modifications. Cells were grown in GM17 broth supplemented with 2% glycine and selection was made on GM17 plates containing antibiotic. Sucrose was omitted from the initial growth media and the selection plates. Electroporation was performed using the Gene Pulser apparatus (Bio-Rad).
Molecular techniques: Total genomic DNA from L. lactis strains was prepared according to the method of Lewington et al, 1987 (33). Plasmid DNA was isolated by the SDS alkaline lysis method. Purification of CCC DNA was by CsCl/EtBr gradient centrifugation (35). Restriction enzymes and other DNA modifying enzymes from various sources were used according to the suppliers recommendations. Conditions employed for PCR analysis were as described previously (22). The following primers were used in this study:
______________________________________1. 5'-AAGAATCTCTCATGAGT; (SEQ ID NO:8)2. 5'-CCATGTCTGAACTAACA; (SEQ ID NO:9)3. 5'-GTGGAATACGGGTTTG; (SEQ ID NO:10)4. 5'-TAAATAATTTATAGCTATTG; (SEQ ID NO:11)5. 5'-CAGAGCTCTGATGGGTTG (SacI site underlined); (SEQ ID NO:12)6. 5'-GTAGAATTCCGTTTATCGTTTGGAG (EcoRI site underlined); (SEQ ID NO:13)7. 5'-GCAACTTGTCAGTGTAGTATTCAC; (SEQ ID NO:14)8. 5'-GTGAATACTACACTGACAAGTTGC; (SEQ ID NO:15)9. 5'-AACGGATCCGATTAAATTCTGAAGTTTG (BamHI site underlined); (SEQ ID NO:16)10. 5'-TCAGAGCTCCTGTTTTACAA (SacI site underlined); (SEQ ID NO:17)11. 5'-TCAGAGCTCCTGTTTTACAACCGGGTGTACATAGTGCAAT (SacI site underlined); (SEQ ID NO:18)______________________________________
All PCR amplified fragments generated were initially cloned into pUC18 (47) and the nucleotide sequence confirmed prior to vector construction. Nucleotide sequence determination of plasmid DNA was performed by the dideoxy chain termination method (40). Sequenase Version 2.0 was used according to the suppliers recommendations (United States Biochemical Corp.).
Site-directed mutagenesis was carried out using either PCR-mediated overlap extension or by incorporation of a specific mismatch into the particular primer used in the nisA fragment amplification.
Southern blot hybridisation and labelling of probes was performed according to published techniques (22).
Results
Insertional inactivation of the chromosomally located nisA gene: The gene replacement vector pFI283 was constructed to insertionally inactivate the chromosomally encoded nisA gene. It carries a cloned nisA gene which is disrupted by the insertion of an erythromycin resistance gene (FIG. 1a). Plasmid construction involved the following steps:
The nisA gene of FI5876 was cloned into the shuttle vector pTG262 to generate pFI172 which has been described previously (11,22). A 2 kb AccI/SalI fragment from this construct, containing nisA and the start of nisB, was subcloned into the pBR322 based vector, pMTL23P (4,46). A 1 kb fragment encoding the erythromycin resistance gene of the staphylococcal plasmid pE194 (20) was inserted into the unique SacI site within the cloned nisA gene. This insertion resulted in disruption of the nisA gene. The erythromycin resistance gene in this construct was transcribed in the same direction as the nisA gene and was flanked on either side by approximately 1 kb of lactococcal DNA sequences. A unique EcoRV site in the adjacent polylinker of the vector sequences was used to insert a 2 kb fragment carrying the chloramphenicol resistance gene originating from the staphylococcal plasmid pC194 (21). A map of the resulting recombinant plasmid, pFI283, is shown in FIG. 1a.
The nisin producing strain L. lactis FI5876 was transformed with pFI283 and erythromycin resistant transformants were obtained. The plasmid does not encode a replication origin functional in Lactococcus and hence the recovery of erythromycin resistance in these transformants required integration of this marker into the recipient chromosome.
Reciprocal recombination between homologous sequences on pFI283 (FIG. 1a) and the chromosome of FI5876 (FIG. 1b) could generate two types of transformants. A single cross-over event would result in the entire plasmid integrating in the chromosome (Campbell integration). A double cross-over event, one on either side of the erythromycin resistance gene in pFI283, would exchange the wild-type chromosomal nisA gene for the insertionally inactivated copy, with subsequent loss of chloramphenicol resistance encoded by the non-replicating plasmid (gene replacement). Both recombination mechanisms have been shown to operate in Lactococcus lactis (7,31,32). The two alternative types of recombination could be distinguished phenotypically in the transformants obtained by screening for chloramphenicol resistance. Transformants with only erythromycin resistance were recovered in which gene replacement had taken place (FIG. 1d). Strain FI7300 is typical of this construct.
The chromosomal rearrangement was confirmed by PCR analysis using primers 1 and 2 (SEQ ID NOS 8 and 9, respectively) which specifically amplify a 0.9 kb fragment from FI5876 chromosomal sequences containing the nisA gene and flanking regions (FIG. 1b; FIG. 8, track 3). When DNA from FI7300, was used as template a 1.9 kb fragment was amplified by primers 1 and 2 (SEQ ID NOS 8 and 9, respectively) (FIG. 8, track 5). A 1 kb increase in size of this fragment would be expected if the erythromycin resistance gene was integrated in this part of the chromosome (FIG. 1d) and is consistent with the proposal that gene replacement had substituted the parental wild-type nisA gene with the insertionally inactivated copy.
Strain FI7300 having lost the parental nisA gene as a consequence of gene replacement no longer produced nisin. Furthermore the insertion in the nisA gene in this strain affected the nisin immunity level which was reduced to below 500 U/ml (Table 1).
In an attempt to recover nisin production in the nisA deficient host FI7300 the strain was transformed with pFI172, which encodes nisA, and six transformants were tested for nisin production. The bioassays gave negative results indicating that the chromosomal mutation in this host could not be complemented by provision of the nisA gene product in trans (Table 2).
Activation of genes for nisin immunity and modification: Reduction in nisin immunity due to the insertional inactivation of nisA in FI7300 may be caused by a polar effect on downstream genes. This may also result in reduced expression of genes required for modification thus preventing complementation of the nisA mutation in this host. In order to select derivatives of FI7300 in which the immunity and maturation genes were fully active mutation to wild-type levels of nisin immunity was selected by growth in media containing nisin at inhibitory levels. Colonies which grew on agar plates containing 10.sup.3 .mu.g/ml nisin were picked and the cells grown on in media containing the same level of nisin. One such mutant, designated FI7304, expressed wild-type levels of nisin immunity, did not produce nisin and furthermore was no longer resistant to erythromycin (Table 1).
PCR analysis of FI7304 DNA, using primers 1 and 2 (SEQ ID NOS 8 and 9, respectively) resulted in amplification of a 3.2 kb fragment (FIG. 8, track 6), 1.3 kb larger than the equivalent FI7300 fragment generated by the same primers (FIG. 8, track 5). This suggested that loss of erythromycin resistance was not caused by a deletion in this region of the genome. The retention of the erythromycin resistance gene sequences was confirmed by PCR using primers 3 and 4(SEQ ID NOS 10 and 11, respectively), which are specific for a region at the 3' end of that gene and amplify a 0.4 kb fragment (FIG. 7b). As these primers generated an FI7304 fragment 1.7 kb in size it was concluded that an additional 1.3 kb of DNA was inserted in this region of the erythromycin resistance gene (FIG. 7c). This insert results in loss of erythromycin resistance with concurrent recovery of nisin immunity (see below). A comparison of fragments from FI7300 and FI7304 generated by amplification between primers 3 and 2 (SEQ ID NOS 10 and 9, respectively) and primers 3 and 4 (SEQ ID NOS 10 and 11, respectively) was consistent with this interpretation (FIG. 7b and c).
The extra DNA sequences gained by FI7304 were amplified with primers 3 and 4 (SEQ ID NOS 10 and 11, respectively) (FIG. 7c) and this PCR fragment was used to probe a southern blot of restriction enzyme digested genomic DNA from the parent strain, FI5876. A number of fragments hybridised to the probe (FIG. 10) indicating that the additional DNA in FI7304 is present in multiple copies in the genome of this strain. Further investigation has revealed that these repeated sequences represent a new lactococcal insertion sequence designated IS905, the sequence of which is presented in FIG. 11. As has been demonstrated for other IS elements (2,39,48), transcriptional read-through from a potential promoter within IS905 may lead to turn-on of downstream genes. The IS element IS905 was found to have significant homology with IS256, which is known to express an adjacent antibiotic resistance gene from an internal promoter (2,39). Such promoter activity could account for the observed increase in nisin immunity exhibited by FI7304 which is equivalent to that of the parent strain, FI5876 (Table 1) and it may also have restored the expression of genes required for processing of prenisin to a level sufficient to facilitate nisA complementation. The DNA sequences of the junction of IS905 and the Em gene in FI 7304 and of IS905 and the nisA gene in FI 7332 are shown in FIG. 12.
Expression and maturation of plasmid encoded nisA: FI7304 was transformed with the nisA encoding plasmid, pFI172. Transformants were obtained at low frequency and the majority did not produce nisin in bioassays. One transformant, designated FI7330, was found to yield nisin at a level approximately 50% that of the parent strain FI5876 (FIG. 6). It was presumed that FI7330 had undergone a spontaneous mutation, either within the plasmid or the chromosome sequences, which resulted in nisin production. Isolation of plasmid DNA from FI7330 yielded a molecule indistinguishable from pFI172 on the basis of restriction enzyme analysis. Curing FI7330 of plasmid DNA, to generate the plasmid free strain FI7332, resulted in loss of nisin production (FIG. 6). However, when plasmid pFI172 was introduced back into the latter strain high transformation frequencies were obtained (FIG. 12) and all transformants produced nisin in bioassays.
When DNA from the plasmid free strain, FI7332, was analysed by PCR using primers 1 and 2 (SEQ ID NOS 8 and 9 respectively) a small size reduction (200 bp) was observed in the amplified fragment. The deletion was in the vicinity of the IS905 insertion in FI7304 (FIG. 8, cf tracks 5 and 6). Primers 3 and 4 (SEQ ID NOS 10 and 11 respectively) (derived from the erythromycin resistance gene sequences, (FIG. 7b) did not generate a fragment with this template (FIG. 7d) indicating that the deleted sequences in FI7332 included a region at the 3' end of the erythromycin resistance gene into which IS905 had inserted. The deletion does not extend beyond the nisA gene as primer 2 (SEQ ID NO:9) (specific for sequences at the end of nisA), together with either primers 1 or 3 (SEQ ID NOS 8 and 10 respectively), resulted in fragment amplification (FIG. 7d). The small chromosomal rearrangement in FI7332 has not affected nisin immunity which is conferred at a level indistinguishable from that of the parent strain (Table 1).
Expression of variant nisA genes: To produce engineered nisin molecules PCR-mediated site-directed mutagenesis was carried out on the nucleotide sequence of the nisA gene. The expression vectors pFI354 (FIG. 3b) and pFI451 (FIG. 4a) were designed for the expression of site-directed mutants of the nisA gene.
Plasmid pFI354 was constructed by deletion of a 1.25 kb SacI fragment from pFI172. It thus encodes only the N-terminal part of nisA (FIG. 3b). Using PCR the C-terminal part of the nisA gene was amplified as a 254 bp SacI/EcoRI fragment. By cloning this fragment into pFI354 an intact nisA gene could be reconstituted as in pFI378 (FIG. 3c). Adaptation of this procedure enabled predetermined substitutions to be introduced into the C-terminal region of the nisA gene (using site-directed mutagenesis, see below). These pFI354 derivatives utilize the natural promoter of the nisin operon which lies between the SalI site defining one end of the cloned chromosomal fragment and the start of the nisA gene (FIG. 3). In this system the incorporation of site-directed mutations is limited to sequences in the nisA coding region downstream of the SacI site (FIG. 2) (SEQ ID NOS 1 and 2, respectively).
Initially the nisA codon selected for alteration was Histidine.sup.27 which lies within ring 5 of the mature nisin A molecule (FIG. 9) (SEQ ID NO:19). A naturally occurring variant of nisin A, termed nisin Z, has been identified which contains an Asparagine residue in place of Histidine.sup.27 (15,23,37). The engineered incorporation of a similarly charged Glutamine in place of Asparagine at residue 27 would thus represent a conservative substitution in the nisin Z amino acid sequence. The mutation involved a single base pair change in the PCR generated SacI/EcoRI fragment carrying the C-terminal end of nisA (FIG. 2) (SEQ ID NOS 1 and 2, respectively). The reconstituted gene, containing a Glutamine codon (CAG) in place of a Histidine codon (CAT. FIG. 2) (SEQ ID NOS 1 and 2, respectively) was designated nisA/H27Q in accordance with agreed nomenclature (10). Site-directed mutagenesis was carried out using PCR-mediated overlap extension (FIG. 3d) as follows:
Terminal primers 5 and 6 (SEQ ID NOS 12 and 13, respectively) contain a SacI site and an EcoRI site respectively and define the ends of a 254bp fragment encoding the C-terminal 20 amino acids of prenisin (FIG. 2) (SEQ ID NOS 1 and 2, respectively). A pair of overlapping complementary primers (7 and 8) (SEQ ID NOS 14 and 15, respectively) were designed from sequences within the C-terminal region of nisA which included a single base change from the original nisA sequence (FIG. 2) (SEQ ID NOS 1 and 2, respectively). These were used in PCR amplifications in conjunction with one of the terminal primers (FIG. 3d) to create two partially complementary fragments with the specific mutation located in the overlapping region. The fragments were annealed to provide a template for subsequent PCR involving the same terminal primers that determined the two ends of the SacI/EcoRI fragment (primers 5 and 6, (SEQ ID NOS 12 and 13, respectively) FIG. 3d). The final PCR generated fragment containing the specific mutation was purified by isolation from an agarose gel using DEAE-NA 45 membrane (Schleicher and Schuell). Modification of ragged ends was carried out using T4 DNA polymerase and polynucleotide kinase. The blunt ended fragment was cloned into the SmaI site of pUC18 and the nucleotide sequence of the manipulated region determined to confirm that the selected mutation was present. A SacI/EcoRI fragment from the pUC18 derivatives was then subcloned into pFI354 to recover an uninterrupted nisA reading frame containing the predetermined mutation. This plasmid was designated pFI411 (FIG. 3e).
Vectors pFI480 and pFI487 were constructed in which expression of the nisA gene was under the control of the lacA promoter (FIG. 4). In order to introduce changes throughout the entire nisin molecule a nisA gene cassette was constructed. Using PCR with primers 9 and 10 (SEQ ID NOS 16 and 17, respectively) (FIG. 2) the N-terminal part of the nisA gene was amplified as a 200 bp BamHI/SacI fragment. This fragment was cloned into plasmid pFI354 upstream of the 254bp SacI/EcoRI fragment encoding the C-terminal part of nisA. The latter was generated by PCR using primers 5 and 6 (SEQ ID NOS 12 and 13, respectively) as described above. In this way an intact coding region for the whole nisA gene, lying within a 448 bp BamHI/EcoRI fragment, was reconstituted from two PCR generated fragments which encoded the N and C parts of the nisin molecule. The resultant plasmid was designated pFI446 (FIG. 4). The nisA cassette includes the upstream nisA ribosome binding site and the start of the nisB gene (FIG. 2) (SEQ ID NO:1).
As the natural promoter of nisA is not part of this cassette, the inducible lacA promoter of the lactococcal lactose operon (18, FIG. 5) (SEQ ID NO:3) was employed for expression of nisA and subsequent variant nisA genes. Vector construction involved cloning a XmnI/BamHI fragment containing the lacA promoter, but excluding the lacA ribosome binding site, into pTG262 to generate pFI451 (FIG. 4). Into the adjacent polylinker was cloned the entire nisA cassette on a BamHI/EcoRI fragment to generate pFI449. The individual components of the nisA gene described above ie the 200 bp BamHI/SacI N-fragment and the 254 bp SacI/EcoRI C-fragment, were also separately cloned into pFI451 to generate pFI480 and pFI487, respectively (FIG. 4). To recover an intact nisA gene in these plasmids, insertion of the missing N or C fragment is required. These fragments can be generated by PCR and using appropriate primers, site-specific substitutions can be incorporated into the reconstituted gene (see below).
Using this expression system the amino acid initially selected for alteration was Serine.sup.5. In mature nisin A, Serine.sup.5 is modified to a dehydroalanine residue in ring A (FIG. 9) (SEQ ID NO:19). The aim was to replace this with an Alanine residue by incorporating the required mutation in the N-terminal fragment of the nisA cassette using PCR methodology. Primer 11 (SEQ ID NO:18), which contains an alanine codon (TGC) in place of the Serine.sup.5 codon (CGA), was used together with primer 9 (SEQ ID NO:16) (FIG. 2) to generate an amino-terminal fragment carrying this specific mutation. Primer 11 (SEQ ID NO:18) comprises primer 10 (SEQ ID NO:17) sequences with an additional 20 nucleotides including an alanine codon in place of the wild type serine codon (FIG. 2). Thus, PCR amplification of nisA using primers 9 and 11 (SEQ ID NOS 16 and 18, respectively) generated an N-terminal fragment carrying the specific substitution and subsequent insertion of this BamHI/SacI fragment into pFI487 resulted in a variant nisA gene containing the desired site-specific mutation. The plasmid encoding this variant nisA gene (nisA/S5A) was designated pFI493.
The Nis.sup.- strain FI7332 was transformed with plasmids which encode the nisA gene under the control of either its own promoter (pFI378, FIG. 3c) or the lacA promoter (pFI449, FIG. 4c). When transformants were tested in plate diffusion assays biological activity was detected at a level approximately 50% that of the wild-type parent strain FI5876 (FIG. 6., Table 2). The difference in the expression vector promoters did not appear to significantly affect the level of biological activity (FIG. 6, Table 2).
Transformants of FI7332 containing derivatives of the expression vectors encoding variant nisA genes were also tested for biological activity. Strains encoding nisA/H27Q and nisA/S5A exhibited nisin activity at levels lower than that of FI5876, but comparable to those levels achieved in the expression systems involving nisA complementation (ie FI7332/pFI378 and FI7332/pFI451; Table 2, FIG. 6).
TABLE 2______________________________________Expression of nisA and variant nisA genes in different host strains. NisinStrain Plasmid Nisin gene activity.sup.a______________________________________FI5876 -- nisA ++MG1614 -- -- -" pFI172 nisA -FI7300 -- -- -" pFI172 nisA -FI7304 -- -- -FI7330.sup.b pFI172 nisA +FI7332 -- -- -" pFI172 nisA +" pFI354 -- -" pFI378 nisA +" pFI411 nisA/H27G +" pFI499.sup.c nisA +" pFI493.sup.c nisA/S5A +______________________________________ .sup.a nisin activity was measured by the plate diffusion assay. Zones of inhibition of growth were: ++, 24 mm and +, 18-21 mm (including the 8 mm bore of the well). .sup.b FI7330 was generated by transforming FI7304 with pFI172 followed b a chromosomal deletion (see text). As the host strain is no longer FI7304 a new strain number was allocated. .sup.c plasmids are derivatives of pFI451 (FIG. 4a) in which the nisA cassette is preceded by the lacA promoter. All other plasmids utilise the natural nisA promoter.
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__________________________________________________________________________# SEQUENCE LISTING- (1) GENERAL INFORMATION:- (iii) NUMBER OF SEQUENCES: 21- (2) INFORMATION FOR SEQ ID NO:1:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 448 base (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: cDNA- (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: join(88..258 - #, 369..446)- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:- AACGGNTCNG ATTAAATTCT GAAGTTTGTT AGATACAATG ATTTCGTTCG AA - #GGAACTAC 60#TTT AAC TTG 111 ACTCAAA ATG AGT ACA AAA GAT# Met Ser Thr Ly - #s Asp Phe Asn Leu# 5 1- GAT TTG GTA TCT GTT TCG AAG AAA GAT TCA GG - #T GCA TCA CCA CGC ATT 159Asp Leu Val Ser Val Ser Lys Lys Asp Ser Gl - #y Ala Ser Pro Arg Ile# 20- ACA AGT ATT NNN CTA TGT ACA CCC GGT TGT AA - #A ACA GGA GCT CTG ATG 207Thr Ser Ile Xaa Leu Cys Thr Pro Gly Cys Ly - #s Thr Gly Ala Leu Met# 40- GGT TGT AAC ATG AAA ACA GCA ACT TGT CAN TG - #T AGT ATT CAC GTA AGC 255Gly Cys Asn Met Lys Thr Ala Thr Cys Xaa Cy - #s Ser Ile His Val Ser# 55- AAA TAACCAAATC AAAGGATAGT ATTTTGTTAG TTCAGACATG GATACTATC - #C 308Lys- TATTTTTATA AGTTATTTAG GGTTGCTAAA TAGCTTATAA AAATAAAGAG AG - #GAAAAAAC 368- ATG ATA AAA AGT TCA TTT AAA GCT CAA CCG TT - #T TTA GTA AGA AAT ACA 416Met Ile Lys Ser Ser Phe Lys Ala Gln Pro Ph - #e Leu Val Arg Asn Thr# 70# 448 CCA AAC GAT AAA CGG ANT TNT AC - #Ile Leu Ser Pro Asn Asp Lys Arg Xaa Xaa# 80- (2) INFORMATION FOR SEQ ID NO:2:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 83 amino (B) TYPE: amino acid (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:- Met Ser Thr Lys Asp Phe Asn Leu Asp Leu Va - #l Ser Val Ser Lys Lys# 15- Asp Ser Gly Ala Ser Pro Arg Ile Thr Ser Il - #e Xaa Leu Cys Thr Pro# 30- Gly Cys Lys Thr Gly Ala Leu Met Gly Cys As - #n Met Lys Thr Ala Thr# 45- Cys Xaa Cys Ser Ile His Val Ser Lys Met Il - #e Lys Ser Ser Phe Lys# 60- Ala Gln Pro Phe Leu Val Arg Asn Thr Ile Le - #u Ser Pro Asn Asp Lys# 80- Arg Xaa Xaa- (2) INFORMATION FOR SEQ ID NO:3:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 546 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: DNA (genomic)- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:- GAAGACTTTC TTTCATAAAG TAATTTTTTT CCAAAGATAA TTCTCTTTTA AT - #TGTATCAT 60- AAAAGATAAT ATTTTCAAGG TAAAACAAAC AATTTCAAAC AAAAACAAAC GT - #TAGATGAT 120- GAAATAAGAA CAGAGGATTG ACGTATATTA GCTTAGGTCA GATTTTGTAT AA - #GACGAAAA 180- TAAAGTAGGA CCTCTTAATC AGTAAGTTAT AGAAAGTAAA AGACTTTTGT AA - #TACCTGAA 240- TAGATATTTC ACGTCCATTT TGTGATGGAT TAAATGAACA AAAATGAACA AT - #AATTTAAC 300- GGTGTTATCT ATTTTTTAAA AAAACAAATA AAAAAAAACA AAAAATTAAC AA - #AAATAGTT 360- GCGTTTTGTT TGAATGTTTG ATATCATATA AACAAAGAAA TGATGAAAAC GT - #TATCTTGA 420- ACATTTTGCA AAATATTTTC TACTTCTACG TAGCATTTCG GATCCGATTA AA - #TTCTGAAG 480- TTTGTTAGAT ACAATGATTT CGTTCGAAGG AACTACAAAA TAAATTATAA GG - #AGGCACTC 540# 546- (2) INFORMATION FOR SEQ ID NO:4:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 1313 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: cDNA- (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 90..1262- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:- GGTAGTGTAA AATAAGTTGT GTAAACACAA AAAGGAATAA ATCCGTTATA GT - #AGAGTTGC 60- GAAACATTAC TAGAAAGAGA TTTATTCCT ATG ACT CAG TTT ACC - # ACA GAA CTA 113# Met Thr G - #ln Phe Thr Thr Glu Leu# 5 1- CTT AAC TTC CTA GCC CAA AAG CAA GAT ATT GA - #T GAA TTT TTC CGT ACT 161Leu Asn Phe Leu Ala Gln Lys Gln Asp Ile As - #p Glu Phe Phe Arg Thr# 20- TCT CTT GAA ACT GCT ATG AAT GAT CTG CTT CA - #A GCA GAG TTA TCA GCC 209Ser Leu Glu Thr Ala Met Asn Asp Leu Leu Gl - #n Ala Glu Leu Ser Ala# 40- TTT TTA GGG TAT GAA CCT TAC GAT AAA GTA GG - #C TAT AAT TCT GGG AAT 257Phe Leu Gly Tyr Glu Pro Tyr Asp Lys Val Gl - #y Tyr Asn Ser Gly Asn# 55- AGT CGT AAC GGA AGC TAT TCA CGG CAA TTT GA - #A ACC AAA TAT GGG ACT 305Ser Arg Asn Gly Ser Tyr Ser Arg Gln Phe Gl - #u Thr Lys Tyr Gly Thr# 70- GTT CAG TTG AGC ATT CCT AGA GAT CGT AAT GG - #G AAC TTT AGT CCA GCT 353Val Gln Leu Ser Ile Pro Arg Asp Arg Asn Gl - #y Asn Phe Ser Pro Ala# 85- TTG CTT CCC GCT TAT GGA CGT CGA GAT GAC CA - #C TTG GAA GAG ATG GTT 401Leu Leu Pro Ala Tyr Gly Arg Arg Asp Asp Hi - #s Leu Glu Glu Met Val# 100- ATC AAA CTC TAT CAA ACC GGT GTA ACG ACT CG - #A GAA ATT AGT GAT ATC 449Ile Lys Leu Tyr Gln Thr Gly Val Thr Thr Ar - #g Glu Ile Ser Asp Ile105 1 - #10 1 - #15 1 -#20- ATC GAG CGA ATG TAT GGT CAT CAC TAT AGT CC - #T GCC ACA ATT TCT AAT 497Ile Glu Arg Met Tyr Gly His His Tyr Ser Pr - #o Ala Thr Ile Ser Asn# 135- ATC TCA AAA GCA ACT CAG GAG AAT GTC GCT AC - #T TTT CAT GAG CGA AGC 545Ile Ser Lys Ala Thr Gln Glu Asn Val Ala Th - #r Phe His Glu Arg Ser# 150- TTA GAA GCC AAT TAC TCT GTT TTA TTT CTT GA - #C GGA ACC TAT CTT CCA 593Leu Glu Ala Asn Tyr Ser Val Leu Phe Leu As - #p Gly Thr Tyr Leu Pro# 165- TTA AGA CGT GGA ACC GTT AGT AAA GAA TGT AT - #T CAT ATC GCA CTT GGC 641Leu Arg Arg Gly Thr Val Ser Lys Glu Cys Il - #e His Ile Ala Leu Gly# 180- ATT ACA CCA GAA GGA CAG AAG GCT GTT CTT GG - #A TAT GAA ATC GCC CCA 689Ile Thr Pro Glu Gly Gln Lys Ala Val Leu Gl - #y Tyr Glu Ile Ala Pro185 1 - #90 1 - #95 2 -#00- AAT CAA AAT AAT GCT TCT TGG TCC ACC CTG TT - #A GAC AAG CTT CAA AAC 737Asn Gln Asn Asn Ala Ser Trp Ser Thr Leu Le - #u Asp Lys Leu Gln Asn# 215- CAA GGA ATC CAA CAG GTT TCT CTT GTA GTG AC - #C GAT GGC TTC AAG GGG 785Gln Gly Ile Gln Gln Val Ser Leu Val Val Th - #r Asp Gly Phe Lys Gly# 230- CTT GAA CAG ATT ATC AGT CAG GCT TAC CCA TT - #A GCT AAA CAA CAA CGT 833Leu Glu Gln Ile Ile Ser Gln Ala Tyr Pro Le - #u Ala Lys Gln Gln Arg# 245- TGC TTA ATT CAT ATT AGT CGA AAT CTA GCT AG - #T AAA GTG AAA CGA GCA 881Cys Leu Ile His Ile Ser Arg Asn Leu Ala Se - #r Lys Val Lys Arg Ala# 260- GAT AGA GCG GTT ATT CTG GAG CAA TTT AAA AC - #G ATT TAT CGT GCT GAA 929Asp Arg Ala Val Ile Leu Glu Gln Phe Lys Th - #r Ile Tyr Arg Ala Glu265 2 - #70 2 - #75 2 -#80- AAT TTA GAA ATG GCA GTG CAA GCT TTA GAG AA - #C TTT ATC GCC GAA TGG 977Asn Leu Glu Met Ala Val Gln Ala Leu Glu As - #n Phe Ile Ala Glu Trp# 295- AAA CCA AAG TAT AGG AAA GTC ATG GAA AGT CT - #G GAG AAT ACG GAT AAT1025Lys Pro Lys Tyr Arg Lys Val Met Glu Ser Le - #u Glu Asn Thr Asp Asn# 310- CTT TTA ACT TTT TAT CAG TTT CCC TAC CAG AT - #T TGG CAC AGC ATT TAT1073Leu Leu Thr Phe Tyr Gln Phe Pro Tyr Gln Il - #e Trp His Ser Ile Tyr# 325- TCG ACA AAC CTC ATT GAG TCT CTT AAC AAA GA - #A ATC AAA CGT CAA ACG1121Ser Thr Asn Leu Ile Glu Ser Leu Asn Lys Gl - #u Ile Lys Arg Gln Thr# 340- AAA AAG AAG GTT CTT TTT CCT AAC GAG GAG GC - #T CTG GAA CGT TAC TTA1169Lys Lys Lys Val Leu Phe Pro Asn Glu Glu Al - #a Leu Glu Arg Tyr Leu345 3 - #50 3 - #55 3 -#60- GTT ACT TTG TTT GAA GAT TAT AAT TTC AAG CA - #A AGT CAA CGC ATC CAT1217Val Thr Leu Phe Glu Asp Tyr Asn Phe Lys Gl - #n Ser Gln Arg Ile His# 375- AAA GGG TTT GGC CAA TGT GCT GAC ACA CTT GA - #A AGC TTA TTT GAT1262Lys Gly Phe Gly Gln Cys Ala Asp Thr Leu Gl - #u Ser Leu Phe Asp# 390# 1313CTCTACT TGAGTGTTTA CACATAATTA TTGACAGTAT C- (2) INFORMATION FOR SEQ ID NO:5:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 391 amino (B) TYPE: amino acid (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:- Met Thr Gln Phe Thr Thr Glu Leu Leu Asn Ph - #e Leu Ala Gln Lys Gln# 15- Asp Ile Asp Glu Phe Phe Arg Thr Ser Leu Gl - #u Thr Ala Met Asn Asp# 30- Leu Leu Gln Ala Glu Leu Ser Ala Phe Leu Gl - #y Tyr Glu Pro Tyr Asp# 45- Lys Val Gly Tyr Asn Ser Gly Asn Ser Arg As - #n Gly Ser Tyr Ser Arg# 60- Gln Phe Glu Thr Lys Tyr Gly Thr Val Gln Le - #u Ser Ile Pro Arg Asp# 80- Arg Asn Gly Asn Phe Ser Pro Ala Leu Leu Pr - #o Ala Tyr Gly Arg Arg# 95- Asp Asp His Leu Glu Glu Met Val Ile Lys Le - #u Tyr Gln Thr Gly Val# 110- Thr Thr Arg Glu Ile Ser Asp Ile Ile Glu Ar - #g Met Tyr Gly His His# 125- Tyr Ser Pro Ala Thr Ile Ser Asn Ile Ser Ly - #s Ala Thr Gln Glu Asn# 140- Val Ala Thr Phe His Glu Arg Ser Leu Glu Al - #a Asn Tyr Ser Val Leu145 1 - #50 1 - #55 1 -#60- Phe Leu Asp Gly Thr Tyr Leu Pro Leu Arg Ar - #g Gly Thr Val Ser Lys# 175- Glu Cys Ile His Ile Ala Leu Gly Ile Thr Pr - #o Glu Gly Gln Lys Ala# 190- Val Leu Gly Tyr Glu Ile Ala Pro Asn Gln As - #n Asn Ala Ser Trp Ser# 205- Thr Leu Leu Asp Lys Leu Gln Asn Gln Gly Il - #e Gln Gln Val Ser Leu# 220- Val Val Thr Asp Gly Phe Lys Gly Leu Glu Gl - #n Ile Ile Ser Gln Ala225 2 - #30 2 - #35 2 -#40- Tyr Pro Leu Ala Lys Gln Gln Arg Cys Leu Il - #e His Ile Ser Arg Asn# 255- Leu Ala Ser Lys Val Lys Arg Ala Asp Arg Al - #a Val Ile Leu Glu Gln# 270- Phe Lys Thr Ile Tyr Arg Ala Glu Asn Leu Gl - #u Met Ala Val Gln Ala# 285- Leu Glu Asn Phe Ile Ala Glu Trp Lys Pro Ly - #s Tyr Arg Lys Val Met# 300- Glu Ser Leu Glu Asn Thr Asp Asn Leu Leu Th - #r Phe Tyr Gln Phe Pro305 3 - #10 3 - #15 3 -#20- Tyr Gln Ile Trp His Ser Ile Tyr Ser Thr As - #n Leu Ile Glu Ser Leu# 335- Asn Lys Glu Ile Lys Arg Gln Thr Lys Lys Ly - #s Val Leu Phe Pro Asn# 350- Glu Glu Ala Leu Glu Arg Tyr Leu Val Thr Le - #u Phe Glu Asp Tyr Asn# 365- Phe Lys Gln Ser Gln Arg Ile His Lys Gly Ph - #e Gly Gln Cys Ala Asp# 380- Thr Leu Glu Ser Leu Phe Asp385 3 - #90- (2) INFORMATION FOR SEQ ID NO:6:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 100 base (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: other nucleic acid#= "promoter" DESCRIPTION: /desc- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:- ACGTGCTATC ACTTGAAGCT TATTGATTAA TATTCTTCAA CTCTACTTGA GT - #GTTTACAC 60# 100 TCCA ATTCTTATCT CTTTTCAATA- (2) INFORMATION FOR SEQ ID NO:7:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 100 base (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: other nucleic acid#= "promoter" DESCRIPTION: /desc- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:- ACGTGCTATC ACTTGAAGCT TATTGATTAA TATTCTTCAA CTCTACTGCA AC - #TTGTCATT 60# 100 CAAA TAACCAAATC AAAGGATAGT- (2) INFORMATION FOR SEQ ID NO:8:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 17 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: other nucleic acid#= "primer"A) DESCRIPTION: /desc- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:# 17 T- (2) INFORMATION FOR SEQ ID NO:9:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 17 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: other nucleic acid#= "primer"A) DESCRIPTION: /desc- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:# 17 A- (2) INFORMATION FOR SEQ ID NO:10:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 16 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: other nucleic acid#= "primer"A) DESCRIPTION: /desc- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:# 16- (2) INFORMATION FOR SEQ ID NO:11:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 20 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: other nucleic acid#= "primer"A) DESCRIPTION: /desc- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:# 20 ATTG- (2) INFORMATION FOR SEQ ID NO:12:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 18 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: other nucleic acid#= "primer"A) DESCRIPTION: /desc- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:# 18 TG- (2) INFORMATION FOR SEQ ID NO:13:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 25 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: other nucleic acid#= "primer"A) DESCRIPTION: /desc- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:# 25 CGTT TGGAG- (2) INFORMATION FOR SEQ ID NO:14:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 24 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: other nucleic acid#= "primer"A) DESCRIPTION: /desc- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:# 24GTAT TCAC- (2) INFORMATION FOR SEQ ID NO:15:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 24 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: other nucleic acid#= "primer"A) DESCRIPTION: /desc- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:# 24CAAG TTGC- (2) INFORMATION FOR SEQ ID NO:16:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 28 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: other nucleic acid#= "primer"A) DESCRIPTION: /desc- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:# 28 TTCT GAAGTTTG- (2) INFORMATION FOR SEQ ID NO:17:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 20 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: other nucleic acid#= "primer"A) DESCRIPTION: /desc- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:# 20 ACAA- (2) INFORMATION FOR SEQ ID NO:18:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 40 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: other nucleic acid#= "primer"A) DESCRIPTION: /desc- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:# 40 ACAA CCGGGTGTAC ATAGTGCAAT- (2) INFORMATION FOR SEQ ID NO:19:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 34 amino (B) TYPE: amino acid (C) STRANDEDNESS: (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: peptide- (ix) FEATURE: (A) NAME/KEY: Modified-sit - #e (B) LOCATION: 2#/product= "Dhb"HER INFORMATION:- (ix) FEATURE: (A) NAME/KEY: Modified-sit - #e (B) LOCATION: 5#/product= "Dha or Ala"ORMATION:- (ix) FEATURE: (A) NAME/KEY: Modified-sit - #e (B) LOCATION: 8#/product= "Abu"HER INFORMATION:- (ix) FEATURE: (A) NAME/KEY: Modified-sit - #e (B) LOCATION: 13#/product= "Abu"HER INFORMATION:- (ix) FEATURE: (A) NAME/KEY: Modified-sit - #e (B) LOCATION: 23#/product= "Abu"HER INFORMATION:- (ix) FEATURE: (A) NAME/KEY: Modified-sit - #e (B) LOCATION: 25#/product= "Abu"HER INFORMATION:- (ix) FEATURE: (A) NAME/KEY: Modified-sit - #e (B) LOCATION: 27#/product= "His or Gln"ORMATION:- (ix) FEATURE: (A) NAME/KEY: Modified-sit - #e (B) LOCATION: 33#/product= "Dha"HER INFORMATION:- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:- Ile Xaa Ala Ile Xaa Leu Ala Xaa Pro Gly Al - #a Lys Xaa Gly Ala Leu# 15- Met Gly Ala Asn Met Lys Xaa Ala Xaa Ala Xa - #a Ala Ser Ile His Val# 30- Xaa Lys- (2) INFORMATION FOR SEQ ID NO:20:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 100 base (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: other nucleic acid#= "promoter" DESCRIPTION: /desc- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:- TATTGAAAAG AGATAAGAAT TGGATACTGT CAATAATTAT GTGTAAACAC TC - #AAGTAGAG 60# 100 AATA AGCTTCAAGT GATAGCACGT- (2) INFORMATION FOR SEQ ID NO:21:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 100 base (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: other nucleic acid#= "promoter" DESCRIPTION: /desc- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:- ACTATCCTTT GATTTGGTTA TTTCGTTACG TGAATGCTAC AATGACAAGT TG - #CAGTAGAG 60# 100 AATA AGCTTCAAGT GATAGCACGT__________________________________________________________________________

Number	Name	Date	Kind
4990452	Bryan et al.	Feb 1991
5218101	Hansen et al.	Jun 1993

Number	Date	Country
0 137 869 B1	Apr 1985	EPX
WO 9218633	Oct 1992	EPX

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Non-Patent Literature Citations (8)

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Entry
Kuipers et al. "Expression of wild type and mutant nisin genes in Lactococcus lactis", pp. 250-259 in Jung and Sahl (ed.), Nisin and Novel Lantbiotics. ESCOM, Leiden, The Netherlands, 1991.
Chopin et al., "Insertion and amplification of foreign genes in the Lactococcus lactis subsp. lactis chromosome" Appl. Environ. Microbiol. 55: 1769-1774, Jul. 1989.
Lian et al., "Solution structures of nisin A and its two majordegradation products determined by n.m.r." Biochem J. 283: 413-420, 1992.
Gutterson et al., "Replacement and amplification of bacterial genes with sequences altered in vitro", Proc. Natl. Acad. Sci. USA 80: 4894-4898, Aug. 1983.
Kozak et al., "The effect of proflavin, ethidium bromide and an elevated temperature on the appearance of nisin-negative clones in nisin-producing strains of Streptococcus lactis", J. Gen. Microbiol. 83: 295-302, 1974.
Agriculture & Food Council et al., International Search Report, Int'l Appln. No. PCT/GB93/00676, Int'l Filing Date Apr. 1, 1993.
The Journal of Biological Chemistry, "Structure, Expression, and Evolution of a Gene Encoding the Precursor of Nisin, a Small Protein Antibiotic," George W. Buchman, Sharmila Banerjee and J. Norman Hansen (Nov. 5, 1988).
Applied and Environmental Microbiology, "A Lactococcal Expression System for Engineered Nisins," Helen M. Dodd, Nikki Horn, Zhang Hae and Michael J. Gasson (Nov. 1992).