Nisins

Details Nisins Nisins

1996-12-24

2000-08-08

Priebe, Scott D.

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Recombinant dna technique included in method of making a...

4353201, 435471, 4352523, C12N 121, C12N 1531, C12N 1574

Patent

active

061000566

DESCRIPTION:

BRIEF SUMMARY
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

REFERENCES:
patent: 4990452 (1991-02-01), Bryan et al.
patent: 5218101 (1993-06-01), Hansen et al.
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).

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