RU2441915C9 - Genes and proteins for biosynthesis of lantibiotic 107891 - Google Patents

Abstract

FIELD: biosynthesis.

SUBSTANCE: invention refers to isolation of nucleic acid molecules that mediate enzymes required for biosynthesis route of lantibiotic 107891 and its homologs. NC contains nucleotide sequence (NS) selected out of a) mlb gene cluster with a certain sequence or b) sequence that corresponds to the sequence a) due to genetic code degeneracy and mediates the same polypeptides, or c) any of ORF NS that mediate polypeptides with the determined amino acid sequence or their combinations. Recombinant vector for transformation of a host cell capable of producing lantibiotic 107891, its homologs and precursors were discovered in the invention. Lantibiotic 107891 or its derivatives are received by culture of host cell transformed by the vector in conditions suitable for cell growth, gene expression and production of the lantibiotic ot its derivative. The isolated polypeptide involved in biosynthesis of lantibiotic 107891 and its homologs is described. Lantibiotic 107891 is beneficial as food supplement and for antibacterial agent preparation.

EFFECT: antimicrobial activity, particularly, against gram-positive bacteria, including methicillin- and vancomycin-resistant strains.

34 cl, 4 dwg, 1 tbl, 6 ex

Description

Summary of the invention

The present invention relates to the field of lantibiotics and, more specifically, to the isolation of nucleic acid molecules that encode the enzymes necessary for the biosynthesis of lantibiotic 107891 and its homologs. The functions of the gene products involved in the production of 107891 are disclosed. The present invention provides new biosynthetic genes necessary for the production of 107891, encoded polypeptides, recombinant vectors containing nucleic acid sequences that encode these polypeptides, host cells transformed with these vectors, and production methods lantibiotics using these transformed host cells, including methods for producing 107891, its predecessor, its derivative th or modified lantibiotic, different from 107891 or its predecessor.

State of the art

The constant increase in pathogenic bacteria resistant to existing antibiotics is a global health hazard, and thus there is an urgent need to detect and develop a new compound that is active against resistant bacteria. The latter leads to a renewed interest in natural products, which in the past have been a rich source of antibiotics such as penicillins, macrolides and glycopeptides. Antimicrobial peptides are also attractive candidates, among them peptides designated as lantibiotics, i.e. lanthionine-containing antibiotics. Lantibiotics form a special group among antimicrobial peptides and differ in several features, such as the characteristics of the primary and spatial structure, unique pathways of biosynthesis and peptide modification reactions, and strong antibacterial activity. They are a group of peptide antimicrobial compounds secreted by gram-positive bacteria, and primarily act on gram-positive bacteria. Lantibiotics are synthesized on ribosomes in the form of prepropeptides, which are post-translationally modified to their biologically active forms. The prepeptide consists of an N-terminal leader sequence that does not undergo any post-translational modification and is cleaved during or after secretion from the cell, and a C-terminal region (propeptide) that is modified post-translationally. Lantibiotics are produced by various bacteria: a common property of these compounds is the presence of one or more than one lanthionine residue, which consists of two alanine residues covalently crosslinked by a thioether bond. A thioether bridge is formed when the cysteine residue reacts with a dehydroalanine or dehydrobutyrin group to form a lanthionine or methylanthionine residue, respectively. Residues of dehydroamino acids, in turn, are formed by dehydration of serine and threonine, respectively. Based on the structural and functional properties, lantibiotics are usually divided into two groups, type A and type B. Lantibiotics type A are elongated cationic peptides with a length varying from 20 to 34 amino acid residues: nisin, subtilin, epidermine and Pep5 are members of this group. Type B is globular peptides with a common negative charge: examples of this group of lantibiotics are mersacidin, cinnamycin, lacticin 481 and actagardine. These structural differences are reflected in the mechanism of action. Type A compounds carry out their antimicrobial activity by blocking the biosynthesis of the cell wall and the formation of pores in cell membranes through a mechanism that may or may not be promoted by prior fixation on target cell lipid II. Type B lantibiotics also show their antimicrobial activity by inhibiting the biosynthesis of peptidoglycans, but these compounds do not form pores after binding to lipid II.

Lantibiotics have been shown to be effective and useful as dietary supplements and antibacterial agents. Nisin, the most studied lantibiotic, is produced by Lactococcus lactis and is active at low concentrations (low nanomolar MICs (minimum inhibitory concentrations)) against many gram-positive bacteria, including drug-resistant strains and food pathogens Clostridium botulinum and Listeria monocytogenes. It is widely used as a food preservative without significant development of bacterial resistance. Other lantibiotics exhibit interesting biological activities: for example, epidermine is highly effective against Propionibacterium acnes; cinnamycin and duramycin inhibit phospholipase A ₂ and angiotensin-converting enzyme, providing potential applications as anti-inflammatory agents and for regulating blood pressure, respectively; and mersacidin inhibits many gram-positive bacteria, including methicillin-resistant Staphylococcus aureus (MRSA).

The genes responsible for the biosynthesis of lantibiotics are organized into clusters denoted by the lan locus symbol, with a more specific genotypic designation for each lantibiotic (for example, nis for nisin, gdm for gallidermin, cin for cinnamycin). Many lan genes were sequenced, demonstrating a high level of similarity in gene organization. Each cluster includes: a lanA structural gene encoding a prepeptide; and the gene (s) necessary (s) for the dehydration of Ser and Thr residues in the LanA propeptide region and for the formation of a thioether. For type A lantibiotics, LanB carries out dehydration reactions, and LanC is involved in the formation of a thioether bridge, whereas in type B lantibiotics, the only LanM enzyme catalyzes both reactions. Additional genes are usually present in lantibiotic clusters: lanT encodes an ABC transporter for secretion of the lantibiotic, often in combination with a second transport system encoded by lanEFG genes; lanP encodes a protease for processing, but some clusters lack such a gene that can be a part of lanT, or its function can be provided by a cellular protease; and lanI encodes a protein involved in self-defense; and lanKR is responsible for regulating lan gene expression.

There is potential and benefit for obtaining improved lantibiotics by manipulating naturally occurring compounds. However, lantibiotics are structurally complex peptides, and their availability for chemistry is limited by several positions in the molecule. One of the main limitations for chemistry is to change the type or order of amino acids present in the peptide backbone. In light of the above, it may be desirable to have genes and enzymes useful for redirecting these steps to the formation of a lantibiotic in order to obtain derivatives that are difficult or impossible to obtain by chemical means. Thus, general methods for creating new derivatives of lantibiotics directly using fermentation processes using precisely designed strains would be highly desirable.

In fact, only unusual amino acids in lantibiotics contribute to their biological activity, as well as enhance their structural stability. Enzymes involved in the biosynthesis of lantibiotics present a high potential for constructing peptides by introducing unusual amino acids into the desired peptides.

Lantibiotic 107891 shows antibacterial activity against gram-positive bacteria, including methicillin and vancomycin-resistant strains, but shows limited activity against gram-negative bacteria (for example, some Moraxella, Neisseria and Haemophilus spp.). 107891 were isolated after fermentation of Microbispora sp. PTA-5024 (WO 2005/04628 A1). It consists of a complex of closely related factors A1 and A2, the structure of which can be converted into a 24 amino acid peptide skeleton containing lanthionine and methylanthionine as components. In addition to this, a chlorine atom and one or two -OH residues are present in the molecule. The structure of the components of complex 107891 is represented by formula 1 in FIG. 3, where R is [OH] with factor A ₁ (R = OH), factor A ₂ (R = - (OH) ₂ ). 107891, apparently, combines the elements of type A and B lantibiotics: rings A and B in 107891 are closely related to equivalent rings in compounds of type A; however, as in type B lantibiotics, 107891 is rather globular, it does not contain flexible C-terminal sequences and is devoid of charged amino acid residues. Therefore, it cannot be predicted whether the lan cluster involved in 107891 formation encodes a single LanM enzyme or individual LanB and LanC proteins. In addition, there is no prerequisite for chlorine-containing lantibiotics, so the genes responsible for this post-translational modification cannot be predicted from the available data.

The scheme of industrial methods for the production of antibiotics was relatively successful, leading to large-scale fermentations with antibiotic titers reaching levels of several grams per liter. This is achieved mainly through the following empirical, experimental, and erroneous approaches and is devoid of a rational basis. Thus, developing new methods and improving existing technology is time consuming and can result in bacterial cultures that are unstable, incompatible and accumulate undesirable by-products. In recent years, rational methods have been successfully applied to increase the level of antibiotic produced by Streptomyces spp., Which is often involved in the manipulation of key regulatory elements present in the gene cluster of interest, or overexpression of speed-limiting stages in this way. Thus, genes encoding such cluster-associated regulators or limiting steps in the synthesis can be effective means for improving yield. However, the cluster-associated regulators identified to date in actinomycetes belong to several different families of proteins. Even within the same family, there is a significant variation in sequence identity. Thus, the existence, nature, quantity and sequence of cluster-associated regulators cannot be predicted by comparison with another cluster, even a cluster that determines a related antibiotic. As an example, the tylosin gene cluster encodes four different regulators, while none of the regulators are found in the cluster defining the related macrolide antibiotic erythromycin. Similarly, the nature and prerequisite for a speed-limiting stage in the path of biosynthesis cannot be established a priori.

Thus, means to increase the yield of 107891 may be highly desirable. However, there are no examples of clusters from other members of the Microbispora genus. Thus, mechanism (s) cannot be predicted (s) by which the producer strain protects itself against the action of 107891, controls the expression of other lan genes, or coordinates the expression of lan genes with other cellular processes. Information about them will be very useful for optimizing the production method.

Description of the invention

The present invention provides a set of isolated polynucleotide molecules necessary for the biosynthesis of lantibiotic 107891 in microorganisms.

Thus, in accordance with one of its aspects, the present invention relates to polynucleotide molecules that are selected from a continuous DNA sequence (SEQ ID NO: 1), which is an mlb gene cluster isolated from Microbispora sp. PTA-5024, and consists of 17 ORFs (open reading frame) encoding the polypeptides necessary for the formation of 107891.

The amino acid sequences of the polypeptide encoded by these 17 ORFs are shown in SEQ ID NO: 2-18.

The present invention also provides an isolated nucleic acid comprising a nucleotide sequence selected from the group consisting of:

a) the mlb gene cluster encoding the polypeptides necessary for the synthesis of 107891 and its homologs (SEQ ID NO: 1);

b) the nucleotide sequence encoding the same polypeptides encoded by the mlb gene cluster (SEQ ID NO. 1), but different from the nucleotide sequence of the mlb gene cluster itself;

C) any of the nucleotide sequences of ORF 1-17 mlb encoding the polypeptides encoded by SEQ ID NO: 2-18;

g) the nucleotide sequence encoding the same polypeptide encoded by any of the ORF 1-17 mlb (SEQ ID NO: 2-18), but different from the nucleotide sequence of the specified ORF.

Another objective of the invention is to provide an isolated nucleic acid containing a nucleotide sequence selected from the group consisting of:

e) a nucleotide sequence encoding a polypeptide that is at least 65%, preferably 86%, more preferably 90%, most preferably 95% or more identical in amino acid sequence to the polypeptide encoded by any of the ORF 1- 17 mlb (SEQ ID NO: 2-18).

In one embodiment, the isolated nucleic acids of the invention comprise an ORF selected from ORF 1-17 (SEQ ID NO: 2-18) that encodes a polypeptide necessary for the synthesis of prepropeptide 107891.

In another embodiment, the nucleic acid comprises an ORF selected from ORF 1-17 (SEQ ID NO: 2-18), which encodes a polypeptide necessary for the synthesis of the dehydratase enzyme 107891. In yet another embodiment, the nucleic acid comprises an ORF selected from ORF 1-17 (SEQ ID NO: 2-18), which encodes the polypeptide necessary for the synthesis of lanthionine and methylanthionine residues 107891.

According to yet another embodiment, a nucleic acid of the invention provides an ORF selected from ORF 1-17 (SEQ ID NO: 2-18) that encodes a polypeptide necessary for the chlorination of the tryptophan residue of amino acid 4 in 107891.

In yet another embodiment, a nucleic acid comprising an ORF selected from ORF 1-17 (SEQ ID NO: 2-18) that encodes a polypeptide necessary for the hydroxylation of a proline residue of amino acid 14 in 107891 is provided.

In yet another embodiment, a nucleic acid is provided comprising an ORF selected from ORF 1-17 (SEQ ID NO: 2-18) that encodes a flavoprotein necessary for oxidative decarboxylation, which results in S - [(Z) -2-aminovinyl ] - (3S) -3-methyl-D-cysteine (AviMeCys) residues present at positions 21 and 24 in 107891.

According to yet another embodiment, a nucleic acid of the invention provides an ORF selected from ORF 1-17 (SEQ ID NO: 2-18) that encodes a polypeptide necessary for the recovery of flavoprotein.

In accordance with yet another embodiment, nucleic acids are provided that comprise combinations of ORFs selected from ORFs 1-17 (SEQ ID NO: 2-18) encoding the polypeptides necessary for export of 107891 and resistance to 107891.

In yet another embodiment, nucleic acids are provided that comprise combinations of ORFs selected from ORF 1-17 (SEQ ID NO: 2-18) encoding the polypeptides necessary to regulate expression of the mlb gene cluster.

Those skilled in the art will recognize that the present invention, which provides nucleotide sequences encoding polypeptides from the biosynthesis pathway 107891, also provides nucleotides encoding fragments derived from such polypeptides. According to the invention, one skilled in the art will understand that since the genetic code is degenerate, the same polypeptides encoded by SEQ ID NO: 2-18 can be encoded by natural or artificial variants of ORF 1-17, i.e. nucleotide sequences that differ from the genomic nucleotide sequences defined by ORF 1-17, but which encode the same polypeptides.

In addition, it is also clear that naturally occurring or artificially obtained variants may consist of polypeptides encoded by SEQ ID NO: 2-18, where these variants have the same function (s) as the above-mentioned starting polypeptides, but contain the addition, deletion or replacement of an amino acid that is not essential for folding or catalytic function, or a conservative replacement of essential amino acids.

Those skilled in the art will also understand that while providing the nucleotide sequence of the entire cluster necessary for the biosynthesis of 107891, the present invention also provides nucleotide sequences necessary for the expression of the genes represented in said cluster. Such regulatory sequences include, but are not limited to, promoter and enhancer sequences, antisense sequences, transcriptional terminator and antiterminator sequences. These sequences are useful for regulating the expression of genes present in the mlb gene cluster. Cells carrying said nucleotide sequences, single or fused to other nucleotide sequences, are also within the scope of the present invention.

In one aspect of the present invention, there are provided isolated nucleic acids comprising nucleotide sequences encoding an ORF6 polypeptide (SEQ ID NO: 7), or naturally occurring variants or derivatives of said polypeptide encoding prepropeptide 107891.

In another aspect of the present invention, there are provided nucleic acids comprising nucleotide sequences encoding an ORF7 polypeptide (SEQ ID NO: 8), or naturally occurring variants or derivatives of said polypeptide useful for the dehydration of serine and threonine residues of the lantibiotic precursor.

In yet another aspect of the present invention, there is provided a nucleic acid comprising nucleotide sequences encoding an ORF8 polypeptide (SEQ ID NO: 9), or naturally occurring variants or derivatives of said polypeptide useful for forming lanthionine and methylanthionine in a lantibiotic precursor.

In another aspect of the present invention, there are provided nucleic acids comprising nucleotide sequences encoding an ORF9 polypeptide (SEQ ID NO: 10), or naturally occurring variants or derivatives of said polypeptide useful for forming AviMeCys in an antibiotic precursor. In another aspect of the present invention, there are provided nucleic acids comprising nucleotide sequences encoding an ORF15 polypeptide (SEQ ID NO: 16), or naturally occurring variants or derivatives of said polypeptide useful for chlorinating a tryptophan residue in a lantibiotic precursor.

In yet another aspect, the present invention provides nucleic acids comprising nucleotide sequences encoding an ORF2 polypeptide (SEQ ID NO: 3), or naturally occurring variants or derivatives of said polypeptide useful for hydroxylating a proline residue in a lantibiotic precursor.

In another aspect of the present invention, there are provided nucleic acids comprising nucleotide sequences encoding polypeptides defined by ORF 1, 10-11, 13-14 and 17 (SEQ ID NO: 2, 11-12, 14-15 and 18), or occurring in nature or artificially obtained variants or derivatives of these polypeptides useful for export from 107891 cells or 107891 precursor.

In another aspect of the present invention, there are provided nucleic acids comprising nucleotide sequences encoding ORFs of 3 and 5 polypeptides (SEQ ID NOs: 4 and 6), or naturally occurring or artificially derived variants or derivatives of these polypeptides useful for regulating lantibiotic production.

In one embodiment of the present invention, there is provided a lantibiotic-producing strain bearing extracopies of nucleotide sequences defining at least one ORF selected from any of ORF 1-17 (SEQ ID NO: 2-18).

In one preferred embodiment, such a lantibiotic-producing strain is any strain of the Actinomycetales order.

In another preferred embodiment, such a lantibiotic-producing strain is a member of the Microbispora genus.

In one preferred embodiment of the present invention, there is provided a Microbispora strain comprising one or more variants of the nucleotide sequence defined in SEQ ID NO: 1, which variant results in increased or decreased expression of one or more ORF 1-17 (SEQ ID NO: 2-18).

In one preferred embodiment of the present invention, there are provided nucleic acids containing the nucleotide sequence defined in SEQ ID NO: 1, or a portion thereof located on one or more vectors, useful for producing 107891, one or more of its precursors, or a derivative thereof by another cell.

In one preferred embodiment, said nucleotide sequence or portion thereof is in the same vector. A suitable vector is any cosmid, phosmid, BAC, PAC, ESAC vector capable of carrying a complete mbl cluster as defined in this application. Suitable vectors are well known to those skilled in the art and are described in the literature (Kieser et al., 2000, and the references therein).

In one aspect of the present invention, a method for increasing the production of 107891, comprising the following stages:

- transformation with a recombinant DNA vector of a microorganism that produces 107891 or its homologs, or precursors of 107891 or its homologues in a biosynthetic manner, where the specified vector contains a DNA sequence selected from any of ORF 1-17 (SEQ ID NO: 2-18), which encodes activity that is speed-limiting in the specified path;

- culturing said microorganism transformed with said vector under conditions suitable for cell growth, expression of said gene and production of said antibiotic or antibiotic precursor.

A suitable host cell is Actinomycetales, belongs to the families Streptosporangiaceae, Micromonosporaceae, Pseudonocardiaceae and Streptomycetaceae, to the genus Microbispora, Actinoplanes, Planomonospora, Streptomyces and the like.

In another aspect of the present invention, a method for producing derivatives of 107891 and its homologues, comprising the following stages:

- cloning in a suitable vector a segment selected from the nucleotide sequence defined in SEQ ID NO: 1, where the specified segment contains at least a portion of one of ORF 1-17 (SEQ ID NO: 2-18), where the specified ORF encodes a polypeptide, which catalyzes the stage of biosynthesis, which it is desirable to bypass;

- inactivation of the specified ORF by removing or replacing one or more codons that are specific for amino acids that are indispensable for the activity of the specified polypeptide;

- transformation of the indicated recombinant DNA vector of the microorganism that produces 107891 or its homologs, or the precursor 107891 biosynthetically;

- screening of the obtained transformants in relation to those in which the indicated DNA sequence is replaced by a mutant copy;

- cultivation of mutant cells under conditions suitable for cell growth, expression of the indicated pathway and the production of an analogue of the specified pathway.

In another aspect of the present invention, a method for producing new lantibiotics, comprising the following stages:

- transformation with a recombinant vector of DNA of a microorganism that produces a lantibiotic or its homologues or precursor in a biosynthetic manner, where the specified vector contains one or more ORF selected from ORF 1-17 (SEQ ID NO: 2-18), which encodes the enzyme (s ) capable of (e) modifying said lantibiotic or lantibiotic precursor;

- culturing the specified microorganism, transformed by the specified vector under conditions suitable for cell growth, expression of the specified gene and the production of the specified lantibiotic, or its homologues, or the predecessor of the lantibiotic.

In another aspect of the present invention, a method for producing new lantibiotics, comprising the following stages:

- transformation of a microorganism DNA with a recombinant vector, wherein said vector contains one or more ORFs selected from ORF 1-17 (SEQ ID NO: 2-18), which encodes an enzyme (s) capable of modifying the lantibiotic or lantibiotic precursor ;

- culturing the specified microorganism, transformed with the specified vector, under conditions suitable for cell growth, expression of the specified gene, in the presence of the specified lantibiotic or precursor lantibiotic.

In another aspect of the present invention, a method for producing new lantibiotics, comprising the following stages:

- transformation of a microorganism DNA with a recombinant vector, wherein said vector contains one or more than one ORF selected from ORF 1-17 (SEQ ID NO: 2-18), which encodes one or more polypeptides that modify a lantibiotic or a lantibiotic precursor;

- obtaining a cell extract or cell fraction of the specified microorganism under conditions suitable for the presence of the active (s) polypeptide (s), where the specified cell extract or cell fraction contains at least the specified (s) polypeptide (s);

- adding a lantibiotic or lantibiotic precursor to said cell extract or cell fraction and incubating said mixture under conditions in which said polypeptide (s) can (ut) modify said lantibiotic or lantibiotic precursor.

Another aspect of the invention includes an isolated polypeptide involved in the biosynthesis pathway 107891 selected from:

- the ORF polypeptide encoded by any of the ORF 1-17 mlb (SEQ ID NO: 2-18) and

a polypeptide that is at least 65%, preferably 95% or more identical in amino acid sequence to the polypeptide encoded by any of ORF 1-17 mlb (SEQ ID NO: 2-18), preferably any of ORF 1 -3, 5-11, 13-17 mlb (SEQ ID NO: 2-4, 6-12, 14-18).

A preferred group of polypeptides comprises any ORF polypeptide encoded by any of ORF 1-3, 5-11, 13-17 mlb (SEQ ID NO: 2-4, 6-12, 14-18), or any polypeptide that is at least over its entire length, 65%, preferably 86%, more preferably 90%, most preferably 95% or more, is identical in amino acid sequence to the polypeptide encoded by any of these ORF mlb.

DEFINITIONS

The term "isolated nucleic acid" in this application refers to a DNA molecule, genomic DNA or complementary DNA (cDNA), which may be single or double stranded, having a natural or synthetic origin. This term also refers to an RNA molecule of natural or synthetic origin.

The term "nucleotide sequence" in this application refers to the full-size or partial sequences of ORF and intergenic regions disclosed in this application.

The term "nucleotide sequence" in this application also refers to and / or contains any of the nucleotide sequences of the invention, presented in the list of sequences. Any of the nucleotide sequences from the sequence listing is:

A) coding sequence

B) an RNA molecule resulting from transcription (A),

B) a coding sequence in which the degeneracy of the genetic code is used to encode an identical polypeptide,

D) intergenic region containing promoters, enhancers, terminator and antiterminator sequences.

The terms "gene cluster", "cluster" and "biosynthetic cluster" in this application designate a continuous segment of the genome of a microorganism containing all the genes necessary for the synthesis of a secondary metabolite.

The term "mlb" in this application refers to the genetic element responsible for the biosynthesis of 107891 in Microbispora sp. PTA-5024. The term is an acronym for Lantibiotic Microbispora.

The term "ORF" in this application refers to a genomic nucleotide sequence that encodes a single polypeptide. In the context of the present invention, the term ORF is synonymous with "gene".

The term "ORF polypeptide" in this application refers to a polypeptide encoded by ORF.

The term "ORF mlb" in this application refers to ORF containing the mlb gene cluster.

The term "secondary metabolite" in this application refers to a biologically active substance produced by a microorganism by expression of a set of genes defined by a gene cluster.

The term "vector", as defined in this application, includes, among other things, any plasmid, cosmid, phage that can transform a prokaryotic host by integration into the cellular genome or exist outside the chromosome (for example, an autonomously replicating plasmid with the origin of religion).

The term "host producer", "host cell" in this application is a microorganism in which the formation of a secondary metabolite is controlled by a gene cluster originating from a donor organism.

The term “homologue” as used herein refers to a polypeptide encoded by a biosynthetic gene cluster that has at least 65% sequence identity with any of the polypeptides encoded by the mlb cluster over its entire length.

BRIEF DESCRIPTION OF GRAPHIC MATERIALS

Figure 1 shows the selected DNA segments derived from the chromosome Microbispora sp. PTA-5024. The thick line denotes the segment described in SEQ ID NO: 1. Cosmids carrying said isolated DNA segments are designated 1G6 and 6H6.

Figure 2 shows the genetic organization of the mlb cluster. Each ORF is represented by an arrow and numbered as in Table 1 (Figure 4). The orientation is the same as in FIG. 1. The numbers on the scales indicate the coordinates of the sequence (in kb).

Figure 3 shows the structure of the components of the complex 107891.

Figure 4 shows the main characteristics of ORF.

A. mlb genes Isolated from Microbispora

107891 is a complex of closely related peptide antibiotics produced by Microbispora sp. PTA-5024. The present invention provides nucleic acid sequences and characteristics of the mlb gene cluster for biosynthesis 107891. The physical organization of the mlb gene cluster along with flanking DNA sequences is shown in FIG. 1, which illustrates the physical map of the 20 kb genomic segment. from the genome of Microbispora sp. PTA-5024 along with two cosmids defining such a segment. The genetic organization of the DNA segment that controls the biosynthesis of 107891 is shown in FIG. 2, and its nucleotide sequence is shown as SEQ ID NO: 1.

The exact boundary of the cluster can be determined based on the functions of its gene products. Thus, at the left end (FIG. 1), the mlb cluster is bounded by ORF1 mlb encoding the ABC transporter (SEQ ID NO: 2) involved in export 107891. At the right end, the mlb cluster is bounded by ORF17 mlb, a membrane ionic antiporter (SEQ ID NO : eighteen). The mlb cluster spans approximately 20,000 base pairs and contains 17 ORFs, designated ORF1 mlb - ORF17 mlb. The continuous nucleotide sequence of SEQ ID NO: 1 (20,000 base pairs) encodes 17 proteins listed in SEQ ID NO: 2-18.

ORF1 (SEQ ID NO: 2) represents 300 amino acids established by translation of SEQ ID NO: 1 from nucleotide 67 to 969.

ORF2 (SEQ ID NO: 3) represents 414 amino acids identified by translation of SEQ ID NO: 1 from nucleotide 966 to 2210.

ORF3 (SEQ ID NO: 4) represents 260 amino acids established by translation of SEQ ID NO: 1 from nucleotide 2941 to 3723.

ORF4 (SEQ ID NO: 5) represents 221 amino acids established by translation of SEQ ID NO: 1 from nucleotide 3948 to 4614.

ORF5 (SEQ ID NO: 6) represents 220 amino acids established by translation of SEQ ID NO: 1 from nucleotide 5283 to 4621 on a complementary chain.

ORF6 (SEQ ID NO: 7) represents 57 amino acids identified by translation of SEQ ID NO: 1 from nucleotide 5414 to 5587.

ORF7 (SEQ ID NO: 8) represents 1115 amino acids identified by translation of SEQ ID NO: 1 from nucleotide 5706 to 9053.

ORF8 (SEQ ID NO: 9) represents 475 amino acids established by translation of SEQ ID NO: 1 from nucleotide 9080 to 10507.

ORF9 (SEQ ID NO: 10) represents 215 amino acids established by translation of SEQ ID NO: 1 from nucleotide 10537 to 11184.

ORF10 (SEQ ID NO: 11) represents 316 amino acids established by translation of SEQ ID NO: 1 from nucleotide 11181 to 12131.

ORF11 (SEQ ID NO: 12) represents 242 amino acids identified by translation of SEQ ID NO: 1 from nucleotide 12253 to 12981.

ORF12 (SEQ ID NO: 13) represents 211 amino acids identified by translation of SEQ ID NO: 1 from nucleotide 13357 to 13992.

ORF13 (SEQ ID NO: 14) represents 249 amino acids identified by translation of SEQ ID NO: 1 from nucleotides 14795 to 15544.

ORF14 (SEQ ID NO: 15) represents 236 amino acids established by translation of SEQ ID NO: 1 from nucleotide 15546 to 16256.

ORF15 (SEQ ID NO: 16) represents 541 amino acids identified by translation of SEQ ID NO: 1 from nucleotide 16370 to 17995.

ORF16 (SEQ ID NO: 17) represents 178 amino acids identified by translation of SEQ ID NO: 1 from nucleotide 17992 to 18528.

ORF17 (SEQ ID NO: 18) represents 430 amino acids identified by translation of SEQ ID NO: 1 from nucleotide 18525 to 19817.

Genomic organization and primary sequence of the mlb 107891 cluster in type A lantibiotics

A comparison of mlb and other clusters of lantibiotic genes revealed important differences. In fact, the mlb cluster is characterized by the presence of several ORFs that do not show homologs in other clusters of lantibiotics. These include ORF 1-6, 12, 15-18 mlb (SEQ ID NO: 2-7, 13, 16-19). In conclusion, the organization of the mlb cluster described in this application is essentially different from the organization of other clusters involved in the synthesis of another lantibiotic. Thus, it represents the first example of a cluster having such a genomic organization.

B. ROLE OF GENES mlb

The present invention has disclosed the DNA sequence responsible for the synthesis of the prepropeptide precursor 107891. The prepropeptide 107891 consists of a leader peptide of 33 amino acids in length and a propeptide of 24 amino acids in length. The nucleic acid sequences referred to in this application are sequences encoding prepropeptide 107891 or fragments thereof. Prepropeptide 107891 with a length of 57 amino acids is a new element that shows along its entire length only 41% identity with the access number in UniProt P21838, which is the lantibiotic prepropeptide gallidermin from Staphylococcus gallinarum.

Other genes present in the mlb cluster are new genetic elements useful for increasing the production of 107891 or for the synthesis of new metabolites. Among them, ORF 7-8 mlb (SEQ ID NO: 8-9) encode proteins involved in the post-translational modification of the ORF6 mlb translation product to introduce lanthionine residues into mature 107891. In particular, the ORF7 mlb polypeptide is responsible for the dehydration of Ser and Thr residues in prepropeptide plot 107891 with the formation of dehydroalanine and degibutirin residues, respectively. The ORF8 mlb polypeptide catalyzes the nucleophilic attack of cysteine residues in the prepropeptide on dehydroamino acid residues. These genes can be cloned and expressed in a heterologous host to form active enzymes capable of introducing the lanthionine residue into other prepropeptides.

Other preferred nucleic acid molecules of the present invention include ORF9 mlb (SEQ ID NO: 10), which encodes a protein involved in oxidative decarboxylation to produce S - [(Z) -2-aminovinyl] - (3S) -3-methyl-D β-cysteine residue (Formula I).

Other preferred nucleic acid molecules of the present invention include ORF16 mlb (SEQ ID NO: 17), which encodes a tryptophanalogenase responsible for adding a chlorine atom to amino acid 4 in 107891. ORF16 mlb is a new and unique genetic element not previously reported in other clusters of lantibiotics. Indeed, chlorination is rather a unique property of 107891 among known lantibiotics. This gene can be cloned and expressed in a heterologous host to produce an active enzyme capable of chlorinating tryptophan residues of lantibiotic molecules. Alternatively, mlb ORF16 may be inactivated in the producer strain, resulting in the formation of 107891 derivatives lacking chlorine attached to amino acid 4.

Other preferred nucleic acid molecules of the present invention include ORF2 mlb (SEQ ID NO: 3, which encodes cytochrome P450 hydroxylase, responsible for post-translational modification of prepropeptide 107891 by adding one or two oxygen atoms to the proline residue at position 14. ORF2 mlb is a new and a unique genetic element that has never been reported in other clusters of lantibiotics Indeed, the proline hydroxylation profile of 107891 is rather unique among lantibiotics. It can be cloned and expressed in a heterologous host to produce an active enzyme capable of oxidizing proline residues present in the lantibiotic molecule.Or alternatively, ORF2 mlb can be inactivated in the producing strain, resulting in the formation of derivatives 107891 lacking an oxygen atom in amino acid 14.

The mlb cluster also includes many regulatory genes responsible for the direct or indirect activation of biosynthesis and resistance gene expression during production of 107891. These genes include ORF 3 and 5 mlb (SEQ ID NO: 4 and 6): ORF3 mlb (SEQ ID NO: 4 ) is a separate quorum-sensing peptide, partially responsible for the regulation of production of 107891; and ORF5 mlb (SEQ ID NO: 6) is closely related to Sigma-70, which is an extra-cytoplasmic functional family of sigma factors of RNA polymerase that act as a positive transcriptional regulator. ORF 3 and 5 mlb are new genetic elements that are absent in other clusters of lantibiotics. Two ORF genes 3 and 5 mlb can be cloned and expressed individually or in any combination of them in other strains producing lantibiotic to increase the yield of the resulting product.

Host strains include, but are not limited to, Actinomycetales strains, the families Streptosporangiaceae, Micromonosporaceae, Pseudonocardiaceae and Streptomycetaceae, the genera Microbispora, Actinoplanes, Planomonospora, Streptomyces and the like. Alternatively, these genes may be overexpressed individually or in any combination thereof in a strain producing 107891 to increase the yield of 107891.

The mlb cluster also includes many genes responsible for exporting lantibiotic intermediates or end products from the cytoplasm and for conferring resistance to the producer cell. These genes include ORF 1, 10-11, 13-14, and 17 mlb (SEQ ID NOS: 2, 11-12, 14-15, and 18). ORF mlb 1, 10-11, 13-14 encode ABC class transporters responsible for ATP-dependent excretion of 107891 or its intermediates, mlb ORF17 encodes Na / K antiporter ion, responsible for exporting 107891 or its intermediates against the proton gradient. These genes can be cloned and expressed either individually or in any combination of them in another lantibiotic producer strain to increase the yield of the resulting product. Host strains include, but are not limited to, Actinomycetales strains, the families Streptosporangiaceae, Micromonosporaceae, Pseudonocardiaceae and Streptomycetaceae, the Microbispora genus, Actinoplanes, Planomonospora, Streptomyces, and the like. Alternatively, any combination thereof, in a strain producing 107891, to increase the yield of 107891.

B. APPLICATIONS OF mlb CLUSTER

The present invention also provides nucleic acids for expressing the full-sized molecule 107891, any of its precursors or a derivative thereof. Such nucleic acids include an isolated (e) gene (s) cluster (s) containing (e) ORFs encoding polypeptides sufficient to drive assembly 107891. In one example, a complete mlb cluster (SEQ ID NO: 1) can be introduced into suitable vector and used to transform the desired host producer. In another aspect, the mlb cluster is cloned into two separate segments in two different vectors that may be compatible in the desired producer host. In yet another aspect, the mlb cluster can be divided into three segments, each of which is cloned in a separate compatible vector. Examples of the application of one-, two- or three-vector systems are described in the literature.

Once the mlb cluster has been suitably cloned into one or more vectors, it can be introduced into various suitable producer hosts in which the production of lantibiotics can be performed more efficiently than in the native host. Preferred host cells are cells of species or strains that can efficiently express actinomycete genes. Such a host includes, but is not limited to, Actinomycetales, Streptosporangiaceae, Micromonosporaceae, Pseudonocardiaceae and Streptomycetaceae, Microbispora, Actinoplanes, Planomonospora and Streptomyces and the like. Alternatively, a second copy of the mlb cluster cloned into one or more suitable vectors can be introduced into a strain producing 107891, where a second copy of the mlb genes increases the yield of 107891.

Transferring the producing ability to a well characterized host can significantly improve several stages of the optimization and development process: the titer of the natural product in the producing strain can be increased more efficiently; purification of the natural product can be carried out with a known background of possible interacting activities; the composition of the complex can be more effectively controlled; altered derivatives of a natural product can be more efficiently produced by manipulating fermentation conditions or by constructing a path.

Alternatively, a cluster of biosynthetic genes can be modified, inserted into a host cell and used to synthesize or chemically modify a wide range of metabolites: for example, open reading frames can be reordered, modified and combined with another cluster of lantibiotic biosynthesis genes.

Using the information provided in this application, the cloning and expression of 107891 nucleic acids can be carried out using standard and well-known methods.

In another possible application, selected ORFs from the mlb gene cluster are isolated and inactivated using standard molecular biology techniques. A mutant ORF cloned in a suitable vector containing DNA segments that flank the indicated ORF on the PTA-5024 Microbispora sp. Chromosome is introduced into said Microbispora strain, where two double cross homologous recombination events result in the inactivation of said ORF in the producer strain. This technique is useful for producing 107891 precursors or derivatives in an efficient manner.

In yet another possible application, selected ORFs from the mlb gene cluster are isolated and placed under the control of the desired promoter. The engineered ORF cloned in a suitable vector is then introduced into Microbispora sp. PTA-5024, by replacing the original ORF, as described above, or as an additional copy of the specified ORF. This technique is useful for increasing or decreasing the expression level of ORFs, which are critical for the production of the 107891 molecule, its precursors or derivatives.

EXPERIMENTAL SECTION

The following examples serve to illustrate the principles and methods by which the 107891 gene cluster is identified, and the principles and methods by which all mlb genes are identified and analyzed. These examples serve to illustrate the principles and methods of the present invention, but not to limit its scope.

General methods

Unless otherwise indicated, bacterial strains and cloning vectors can all be obtained from public collections or commercial sources. For molecular biology, standard methods are used. Microbispora was grown on NT agar and V6 medium (20 g / l glucose, 5 g / l yeast extract, 3 g / l casein hydrolyzate, 5 g / l meat extract, 5 g / l peptone, 1.5 g / l NaCl , 0.5% glycerol). Lantibiotics are isolated according to published methods. Sequence analyzes are performed using standard programs. Database searches are performed using Blast or Fasta programs on public websites.

Example 1 - Isolation of biosynthesis genes 107891

The genomic library is prepared using DNA from Microbispora sp. PTA-5024 in the Supercos 3 conjugate cosmic vector. It is constructed by inserting the aaclV-oriT-intFS9 cassette from pSET152 into Supercos 1 (Stratagene, La Jolla, CA 92037) in accordance with the following: the aaclV-oriT-intFSC31 cassette was obtained by PCR ( polymerase chain reaction) in the form of a 3.8 kb Nrul fragment from the pSET152 vector and inserted into the same supercosl site. Total DNA from Microbispora sp. PTA-5024 is partially digested with Sau3AI to optimize fragment sizes in the range of 40 kb. The partially digested DNA is treated with alkaline phosphatase and ligated into Supercos3 previously digested with BamHI. The ligation mixture is packaged in vitro and used to transfect E. coli XL1Blue cells. The resulting cosmid library was screened by hybridization with a 5'-GTSACSWSSTGGWSSYTSWSSACSGGSCCSTGCACSWSSCCSGGSGGSWSSAACWSSWSSTCCWSSTG-3 'oligonucleotide probe (SEQ ID NO: 19). The oligonucleotide is constructed based on the amino acid sequence deduced from structure 107891. Two cosmids positive for this probe are isolated and physically mapped with restrictase enzymes. Based on such experiments, the cosmids shown in FIG. 1 are identified. A segment thus identified from the genome of Microbispora sp. PTA-5024, contains the mlb gene cluster, responsible for the synthesis of antibiotic 107891.

The above example serves to illustrate the principle and methodologies by which the mlb cluster can be isolated. It will be apparent to those skilled in the art that the mlb cluster can be cloned in various vectors. However, it will be understood by those skilled in the art that having an mlb cluster of 20 kbp, preferred vectors are vectors capable of carrying large inserts such as lambda, cosmid and BAC vectors. Those skilled in the art will appreciate that other probes may be used to identify the mlb cluster from such a library. From the sequence shown in SEQ ID NO: 1, any fragment can be amplified by PCR from DNA of Microbispora sp. PTA-5024 and used for screening a library derived from such DNA. One or more clones can be identified from this library, which include any segment covered by SEQ ID NO: 1. In addition, the mlb cluster can also be identified by using heterologous probes, such as probes obtained from another lan cluster using the information provided in Table 1. Alternatively, other gene clusters that direct the synthesis of secondary metabolites contain genes closely related to the mlb genes to allow heterologous hybridization. All of these variations are within the scope of the present invention.

Example 2 - Analysis of the sequence of the gene cluster 107891

The mlb cluster, identified as described in Example 1, is sequenced using the shotgun method. The mlb cluster sequence in this application is given as SEQ ID NO: 1. The resulting DNA sequence is analyzed to identify the most likely coding sequences that are compared against other lan clusters or searched in GenBank. The exact start codon for each ORF is determined by multiple alignment of related sequences or by searching for the ribosome binding site located higher in the sequence. A total of 17 ORFs identified by ORF1 - ORF17 mlb were identified. The results of these analyzes are summarized in Table 1 and are listed in this application in the sequence listing as SEQ ID NO: 2 - SEQ ID NO: 18. Details are given below.

2A. Synthesis of Prepropeptide 107891

ORF 6 mlb is responsible for the synthesis of the prepropeptide. The prepropeptide contains 49 a.k. leader sequence and 24-a. propeptide (SEQ ID NO: 7), which undergo post-translational modification with the formation of a mature lantibiotic. Two general properties of the lantibiotic leader peptides are preserved in 107891: the conserved sequence of type A lantibiotics (for example, the FD / NLD / E motif) and the proline residue at position - 2. The C-terminal portion of the prepropeptide (SEQ ID NO: 7) is consistent with the published primary structure 107891 and its putative propeptide sequence.

2B. Post-translational modification of the propeptide 107891

Four proteins encoded by ORF 7-9 mlb (SEQ ID NO: 8-10) and ORF 17 mlb (SEQ ID NO: 18) are involved in the post-translational modification of prepropeptide 107891. Homologs of these gene products have been found in many lantibiotic clusters. Based on the sequence identities with dehydratases and cyclases found in other clusters of lantibiotics and their roles, the following assumptions can be made. The ORF7 mlb polypeptide is responsible for the dehydration of serine residues at positions 3, 5, 13, 18 and 21 and the threonine residue at positions 2 and 8 of propeptide 107891 to form the corresponding dehydrated residues. The ORF8 mlb polypeptide catalyzes the regio- and stereospecific addition of the conjugate of cysteine residues present in the 107891 propeptide to four dehydroalanine and one dehydrobutirin residues to form the corresponding five thioesters. Specifically, the ORF8 mlb polypeptide is involved in the formation of 3-7, 13-20, 18-23 and 21-24 lanthionins and 8-11 methylanthionines.

Based on the sequence identities found with decarboxylases encoded by the epidermine and mersacidin clusters, ORF9 mlb encodes the enzyme responsible for decarboxylation of the 21-24 lanthionine group. Based on the sequence identities found with flavin reductase encoded by other antibiotic clusters, ORF16 mlb encodes a flavoprotein reductase. Given the roles expected for oxidative decarboxylation during the formation of epidermis and mersacidin, ORF polypeptides of 9 and 16 mlb catalyze the formation of the S - [(Z) -2-aminovinyl] -D-cysteine residue present in 107891 (Formula I).

2B. Formation of β-hydroxyproline and chlorination of tryptophan

Two proteins encoded by ORF 2 and 15 mlb (SEQ ID NO: 3 and 16) are involved in the addition of one or two β-hydroxyl groups to the proline residue at position 14 and in the chlorination of the tryptophan residue at position 4 of propeptide 107891. The ORF2 mlb polypeptide shows significant identity with P450 monoxygenases (Table 1) and is involved in the hydroxylation of the proline residue. No ORF2 mlb homologs were found in other lantibiotic clusters, so this gene is a unique example of the P450 monoxygenase involved in the hydroxylation of the lantibiotic molecule. Additionally, based on the level of identity with other halogenases, the ORF15 mlb polypeptide is involved in the tryptophan chlorination and is a unique example of a halogenase involved in the modification of the lantibiotic molecule.

2G. Export and Sustainability

Five proteins encoded by ORF 1, 10, 11, 13, 14 and 17 (SEQ ID NO: 2, 11, 12, 14, 15 and 18) are involved in exporting 107891 or its precursor beyond the cytoplasm and in conferring resistance to the producing strain . Their alleged roles are as follows.

Homologs of ORF1 (SEQ ID NO: 2) were not found in other lantibiotic clusters. This gene encodes additional ABC-type transporters (Table 1), and is thus involved in imparting resistance to 107891 producing strain of Microbispora sp. PTA. Homologs of ORF 10, 13-14 and 17 (SEQ ID NO: 11, 14-15 and 18) are presented in other clusters of lantibiotics (Table 1). They encode ABC-type transmembrane transporters and ion-dependent transmembrane transporters, respectively. Thus, they are involved in the export and / or compartmentalization of 107891 or its predecessors. ORF17 mlb encodes a Na / K ionic antiporter responsible for exporting 107891 or its intermediates against the proton gradient.

2d. Regulation

Two proteins encoded by ORF 3 and 5 (SEQ ID NO: 4 and 6) are involved in the regulation of the expression of one or more mlb genes. The ORF5 mlb polypeptide (SEQ ID NO: 6) is a new genetic element whose homologues were not found in other clusters of lantibiotics. This protein belongs to the family of sigma factors that have extracitoplasmic function and act as positive transcriptional regulators. The ORF3 polypeptide (SEQ ID NO: 4) belongs to the LuxR-type transcription regulatory family. Thus, ORF 3 (SEQ ID NO: 4) is probably required for the expression of one or more mlb genes.

2E. Additional functions

Two additional ORFs are present in the mlb cluster: ORF4 (SEQ ID NO: 5) and ORF12 (SEQ ID NO: 13). Both ORFs are related to proteins with unknown function present in Salinispora tropica and Streptomyces ambofaciens, respectively (Table 1). However, their exact role in the biosynthesis of 107891 is still impossible to determine.

Example 3 - Manipulation of 107891 by Gene Replacement

Using the information given in Example 2, a deletion within the reading frame in ORF 2 is constructed as follows. Fragment A is obtained by amplification with 5'-AAGCTTGCATCTGCGTGGGCGTCCTGC-3 'oligonucleotides (SEQ ID NO: 20) and 5'-TCTAGACGGTCCGAAGATCATGGCCGCGG-3' (SEQ ID NO: 21); and fragment B is obtained by amplification with oligonucleotides 5'-TCTAGATCCATGTGAACCGGCGGGTGGCCG-3 '(SEQ ID NO: 22) and 5'-GAATTCCGGTCGCTCTCCTCGTCCTTTTGCC-3' (SEQ ID NO: 23).

Then fragment A was digested with EcoRI and Xbal, fragment B with Xbal and HindIII, and both fragments were ligated into pSET152, previously digested with EcoRI and HindIII. After transformation of E. coli DH5α, the resulting plasmid, designated pDM1, was recognized by the presence of fragments of 4 kb in length. and 1.5 kb after cleavage with EcoRI and HindIII. An aliquot of pDM1 was transferred to E. coli ET12567 cells (pUB307) to obtain strain DM1. Then, approximately 10 ⁸ CFU (colony forming units) of DM1 cells from overnight culture in LB are mixed with approximately 10 ⁷ CFU Microbispora PTA5024 grown in Rare3 medium for approximately 80 hours. The resulting mixture is applied to NT plates, which are then incubated at 28 ° C. for approximately 20 hours. After removing the excess E. coli cells by careful washing with water, the plates are coated with 3 ml of soft agar containing 200 μg of nalidixic acid and 15 μg / ml of apramycin. After additional incubation at 28 ° C for 3-5 weeks, Microbispora exconjugants are applied in strips to fresh medium containing apramycin. One such exconjugant called the Mb-DM1 strain is further processed. The Mb-DM1 strain is then grown in several passages in NT medium without apramycin, and the appropriate dilutions are applied to NT agar without apramycin. Individual colonies are then analyzed by PCR using 5'-CGCGCTGCTCGGGGGCCAAC-3 'oligonucleotides (SEQ ID NO: 24) and 5'-AGGAAACGGCCAGCCCGTGG-3' (SEQ ID NO: 25).

Colonies containing the deleted ORF2 allele are recognized by the presence of a 1.5 kb band. One of these colonies, designated Mb-DM2, is grown in NT medium, and the formation of dehydroxyl-107891 is confirmed by comparison with a reliable standard.

The above example serves to illustrate the principle and methods by which an ORF selected from any of the defined SEQ ID NOs: 2-18 can be replaced by a mutant copy in a strain of Microbispora sp. PTA5024 producing 107891. It will be apparent to those skilled in the art that ORF2 (SEQ ID NO: 3) is just an example of methods for constructing intra-frame deletions in a cluster as defined by SEQ ID NO: 1.

Those skilled in the art will also understand that intra-frame deletions are just one way to create mutations, and that other methods, including but not limited to reading frame mutations, insertions, and site-directed mutations, can also be used to produce null mutants in any of the ORFs defined by SEQ ID NO: 2-18.

It will also be understood by those skilled in the art that having a generally accepted method for creating mutations in any of the ORFs defined by SEQ ID NOS: 1, these methods can be used to alter the expression levels of the same ORFs. Examples of how this can be achieved include, but are not limited to, integrating multiple copies of these ORFs anywhere in the genome of Microbispora sp. PTA 5024, modifying promoters that control the expression of these ORFs, removing antisense RNAs, or transcribing terminators that affect their expression.

Finally, variations of the vectors used to embed mutant alleles in Microbispora sp. PTA 5024, under the conditions of conjugation and cultivation of a donor and recipient strain, in a method for the selection and screening of exconjugants and their derivatives, all fall within the scope of the present invention.

Example 4 - Halogenation of the in vitro lantibiotic

Using the information given in Example 2, ORF15 mlb (SEQ ID NO: 16) is overexpressed in E. coli as follows. A 1.6 kb fragment obtained by amplification with 5'-TTTTTCATATGGGTGGGAGTGATCGGCGGCG-3 'oligonucleotides (SEQ ID NO: 26) and 5'-TTTTTTGTCGACCTACTGCTGGCCGCGGTCCGGACT-3' (SEQ ID NO: 26) Ncol and Sall and ligated into pET22b previously cleaved with Ncol and Xhol. After transformation of E. coli DH5α cells, the resulting pHAL plasmid was recognized by the presence of 5.5 kb fragments. and 1.6 kb after cleavage with Ndel and Xhol, BL21 (DE3) is introduced into E. coli cells. Cell cultures of E. coli BL21 (DE3) bearing pHAL were grown at 20 ° C. in LB to OD600 0.6. Then IPTG (isopropyl-beta-D-1-thiogalactopyranoside) was added up to 1 mM and the cells were grown for another 6 hours. Cells were harvested, sonicated and the His-terminal sequences bearing the ORF15 polypeptide were removed from the Ni-agarose column. In vitro halogenation of substrates such as lacticin 481 or 97518 is carried out, and the formation of a chlorinated derivative of the lantibiotic is confirmed by MS (mass spectrometry) analysis.

The above example serves to illustrate the principle and methods by which an ORF selected from any of the ORF designated by SEQ ID NO: 2-18 can be overexpressed in a convenient host, the resulting enzyme is overproduced and used to transform the natural lantibiotic product into another compound. Those skilled in the art will understand that ORF15 (SEQ ID NO: 16) is used as an example of methods for overproducing any polypeptide encoded by SEQ ID NO: 1. Those skilled in the art will also understand that other methods for overproducing proteins, including, but not limited to, the use of various affinity terminal sequences, the use of various vectors, various host strains and induction methods can also be used to overproduce polypeptides defined by ORF 1-17 (SEQ ID NO: 2-18).

Those skilled in the art will recognize that other lantibiotic substrates with or without a tryptophan residue can be used to add chlorine atoms to ORF15 (SEQ ID NO: 16). Those skilled in the art will also appreciate that other ORF polypeptides defined by the mlb cluster (SEQ ID NO: 1) can be used for post-translational modification of other lantibiotic prepropeptides. ORF polypeptides that can be used for this task include, but are not limited to, ORF2 (SEQ ID NO 3). Specifically, ORFs 7 and 8 (SEQ ID NOs 8 and 9) can be used to dehydrate and incorporate thioether bridges into other lantibiotic prepropeptides; ORFs 9 and 16 (SEQ ID NOs 10 and 17) can be used to decarboxylate another lantibiotic containing a C-terminal Cys residue; and ORF 2 (SEQ ID NO 3) can be used to hydroxylate another lantibiotic containing proline.

Example 5 - Expression of the mlb cluster in a heterologous host

Using the information given in Examples 1 and 2, cosmid 1G6 (FIG. 1) containing an entire mlb cluster (SEQ ID NO: 1) was introduced into Streptomyces albus by conjugation as described by Kieser et al., 2000. Apramycin-resistant exconjugants grown under suitable conditions and 107891 purified as described.

It will also be understood by those skilled in the art that S. albus is just one of the producer strains, and that other strains can also be used to introduce the whole mlb cluster (SEQ ID NO: 1) and to produce 107891. Preferred cells are hosts are host cells of species or strains that can efficiently express actinomycete genes. Such a host includes Actinomycetales, Streptosporangiaceae, Micromonosporaceae, Pseudonocardiaceae and Streptomycetaceae, Microbispora, Actinoplanes, Planomonospora and Streptomyces and the like, and the like. Those of skill in the art will understand that, provided that suitable promoters are placed in front of mlb operons, the producing hosts need not be limited to cells that can efficiently express actinomycete genes. Suitable host producers in this latter category can be found among producer hosts that are easy to manipulate genetically, or among producer hosts that naturally produce other lantibiotics. Examples of such producer hosts include Escherichia coli and related species, Bacillus spp., Streptococcus spp., Lactobacillus spp., Staphylococcus spp. and the like, but are not limited to this.

It will also be understood by those skilled in the art that using the methods described in this example, the mlb cluster can be introduced as a second copy into the original producer strain 107891 Microbispora sp. PTA 5024 was the second copy of mlb genes, which increases the yield of 107891.

It will also be understood by those skilled in the art that various vectors, various methods of introducing an mlb cluster, various methods of selecting recombinant clones bearing an mlb cluster, various methods and conditions for growing said recombinant clones, and various methods for detecting production of 107891 can be effective for expressing mlb cluster in heterologous host.

Example 6 - Formation of a library of options 107891

Using the information given in Examples 2, 3 and 5, ORF 6 mlb (SEQ ID NO: 7) is modified for the production of variants 107891 as follows. Firstly, a vector carrying an intra-frame deletion of ORF 6 (SEQ ID NO: 7) is constructed according to the methods described in Example 3 using 5'-GCAGCCAGGCTCGCACCGGC-3 'oligonucleotides and CGCCCGTAACGAGCGA (SEQ ID NO 28 and 29) for of fragment A and 5'-GCAGCTTCTGCTGCTGA-3 'and 5'-TCCCGGCCAGCCACTT-3' oligonucleotides (SEQ ID NOS 30 and 31) for fragment B for amplification of ORF 6. The resulting construct was used to replace ORF 6 in S. albus strain obtained as described in Example 5, according to the methods of Example 3 and the preparation of strain SA-D6. Then the ORF 6 mlb prepeptide region (SEQ ID NO: 7), obtained by amplification with 5'-CCGGAAAGGAGCGAGCATATG-3 'oligonucleotides (SEQ ID NO: 32) and 5'-CAGATCTGCCAATACAGT-3' (SEQ ID NO: 33), is cleaved using Ndel and BgIII and ligated into pIJ8600 (Kieser et al., 2000), previously cleaved with Ndel and BgIII, to obtain plasmid pPRE1. In a parallel experiment A oligonucleotides 5'-CGGTGTCGAGGAGATCACCGCCGGGCCGGCGNNNNNNAGCNNNNNNNNNTGCACCNNNNNNTGCNNNAGCNNNNNNNNNNNNAGCNNNTGCAGCNNNTGCTGCTGAAGATCT-3 'and oligonucleotide 5'-TTCAGCAGCANNNGCTGCANNNGCTNNNNNNNNNNNNGCTNNNGCANNNNNNGGTGCANNNNNNNNNGCTNNNNNNCGCCGGCCCGGCGGTGATCTCCTCGACACCGATCGA-3' (SEQ ID 34 and 35) were denatured and annealed to form a mixture of DNA segments (SEQ ID NO: 36) which encode polypeptides with all possible changes in the amino acid sequence of the propeptide region of the ORF 6 polypeptide, with the exception of those involved in the formation of a thioether (specifically, amino acid 3, 7, 8, 11, 13, 18, 20, 21, 23 and 24). This mixture of DNA segments is ligated with plasmid pPRE1 previously cleaved with BSA O1 and BgIII to form a library of plasmids bearing all possible variant forms (except for (methyl) lanthionine bridges) of the ORF 6 segment encoding the propeptide fused to the ORF 6 segment, encoding a leader peptide. This plasmid library was introduced into strain SA-D6, and the resulting exconjugants grown in the presence of μg / ml thiostrepton were screened for the production of variants 107891 using biological assays or HPLC (high performance liquid chromatography) analysis. The variants of interest are further characterized by sequencing the propeptide region of the ORF 6 variant and identifying the structure.

Those skilled in the art will recognize that the leader peptide region of the ORF 6 polypeptide can also be modified so that enzymes involved in post-translational modifications (SEQ ID NOs: 8 and 9) can recognize these various leader peptides. Variations of the leader peptide region of the ORF 6 polypeptide are within the scope of the invention.

Those skilled in the art will understand that another method can be used to construct a library of ORF 6 variants, including, but not limited to, the use of various oligonucleotides, vectors, inducing systems, and host strains. In addition, it will be understood by those skilled in the art that methylanthionine residues can be substituted with lanthionine residues and vice versa to form additional ORF 6 variants. It will also be understood by those skilled in the art that site-directed mutagenesis can be used to form selected ORF 6 variants. resulting in the production of certain derivatives of 107891.

Claims (34)

1. The selected nucleic acid encoding the polypeptides involved in the biosynthesis of lantibiotic 107891 and its homologues, containing a nucleotide sequence selected from the group consisting of:
a) mlb gene cluster (SEQ ID NO: 1); or
b) the nucleotide sequence corresponding to sequence (a) due to the degeneracy of the genetic code and encoding the same polypeptides encoded by the mlb gene cluster (SEQ ID NO: 1), or
c) any of the ORF nucleotide sequences (open reading frame) of 1-17 mlb encoding polypeptides having the amino acid sequences of SEQ ID NO: 2-18; or
d) a nucleotide sequence corresponding to any of the sequences (c) due to the degeneracy of the genetic code and encoding the same polypeptides (SEQ ID NO: 2-18) encoded by any of the ORF 1-17 mlb;
or combinations thereof. 2. The selected nucleic acid encoding the polypeptides involved in the biosynthesis of lantibiotic 107891 and its homologues, containing a nucleotide sequence selected from the group consisting of:
the nucleotide sequence encoding a polypeptide whose amino acid sequence is at least 65%, at least 86%, at least 90%, or at least 95% or more identical to the amino acid sequence of the polypeptide (SEQ ID NO: 3, 7-10, 16-17) encoded by any of ORF 2, 6-9, 15-16 mlb, or a combination thereof. 3. The selected nucleic acid according to claim 1 or 2, containing a combination of nucleotide sequences that encode the polypeptides necessary for the synthesis of the lantibiotic prepropeptide 107891 (SEQ ID NO: 7) encoded by ORF 6 mlb, or nucleotide sequences corresponding to the indicated ORF due to degeneracy genetic code and encoding the same polypeptides. 4. The isolated nucleic acid according to claim 1 or 2, containing a combination of nucleotide sequences consisting of ORFs of 9 and 16 mlb that encode the polypeptides (SEQ ID NO: 10 and 17) necessary for the oxidative decarboxylation of the C-terminal Cys residue present in lantibiotic 107891, or nucleotide sequences corresponding to the indicated ORF due to the degeneracy of the genetic code and encoding the same polypeptides. 5. The isolated nucleic acid according to claim 1 or 2, comprising a combination of nucleotide sequences consisting of ORFs of 7 and 8 mlb that encode the polypeptides (SEQ ID NO: 8 and 9) necessary for the dehydration and formation of the (methyl) lanthionine propeptide portion of the lantibiotic 107891, or nucleotide sequences corresponding to the indicated ORF due to the degeneracy of the genetic code and encoding the same polypeptides. 6. The isolated nucleic acid according to claim 1 or 2, containing a nucleotide sequence consisting of ORF 15 mlb, which encodes a polypeptide (SEQ ID NO: 16), necessary for the chlorination of aromatic amino acid residues at position 4 of the lantibiotic 107891, or nucleotide sequences corresponding to indicated ORF due to the degeneracy of the genetic code and encoding the same polypeptide. 7. The isolated nucleic acid according to claim 1 or 2, containing a nucleotide sequence consisting of ORF 2 mlb, which encodes a polypeptide (SEQ ID NO: 3), necessary for hydroxylation of the proline residue of the amino acid at position 14 of the lantibiotic 107891, or nucleotide sequences corresponding to indicated ORF due to the degeneracy of the genetic code and encoding the same polypeptide. 8. The selected nucleic acid according to claim 1 or 2, containing a combination of nucleotide sequences consisting of ORFs 1, 10, 11, 13, 14 and 17 mlb that encode polypeptides (SEQ ID NO: 2, 11, 14, 15 and 18 ), necessary for exporting the lantibiotic 107891 or some of its precursors beyond the cytoplasm and to give the producer strain resistance to the lantibiotic 107891, or nucleotide sequences corresponding to the indicated ORF due to the degeneracy of the genetic code and encoding the same polypeptides. 9. The selected nucleic acid according to claim 1 or 2, containing a combination of nucleotide sequences consisting of ORF 3 and 5 mlb that encode the polypeptides (SEQ ID NO: 4 and 6) necessary for regulating the expression of one or more than one gene of the gene cluster mlb, or nucleotide sequences corresponding to the indicated ORF due to the degeneracy of the genetic code and encoding the same polypeptides. 10. The isolated nucleic acid according to claim 1 or 2, containing the nucleotide sequence consisting of the mlb gene cluster encoding the polypeptide necessary for the synthesis of the lantibiotic 107891, in which is introduced into the nucleotide sequence encoding the polypeptides necessary for the hydroxylation of the proline residue in the lantibiotic 107891 intra-frame deletion. 11. The selected nucleic acid sequence according to claim 1 or 2, containing a nucleotide sequence carrying at least one extracopy of at least one of the ORF 1-17 mlb encoding the polypeptides (SEQ ID NO: 2-18), or the nucleotide sequence, corresponding to the indicated ORF mlb due to the degeneracy of the genetic code and encoding the same polypeptides. 12. The selected nucleic acid according to claim 1 or 2, where the nucleotide sequence is a DNA sequence. 13. Recombinant DNA vector for expression of the lantibiotic 107891 or its homologues or precursors, which contains the DNA sequence, as defined in clause 12. 14. A host cell transformed with the vector of claim 13, capable of producing a lantibiotic 107891 or its homologs or precursors. 15. The transformed host cell according to 14, which refers to the order of Actinomycetales. 16. The transformed host cell according to clause 15, which belongs to a family selected from Streptosporangiaceae, Micromonosporaceae, Pseudonocardiaceae and Streptomycetaceae. 17. The transformed host cell according to clause 16, which refers to the genera selected from Microbispora, Actinoplanes, Planomonospora, Streptomyces and the like. 18. The transformed host cell according to 14, which relates to the order of Bacillales. 19. The transformed host cell according to claim 18, which refers to a family selected from Bacillaceae, Lactobacillaceae, Streptococcaceae or Staphylococcaceae. 20. The transformed host cell according to claim 19, which refers to the genera selected from Bacillus, Lactococcus, Streptococcus, Staphylococcus and the like. 21. The transformed host cell according to 14, which refers to species of Escherichia coli. 22. A method of increasing the production of lantibiotic 107891 or its homologs using a microorganism capable of producing lantibiotic 107891 or its precursor in a biosynthetic manner, including:
- transformation of the recombinant DNA vector according to item 13 of the microorganism that produces lantibiotic 107891 or its predecessor in a biosynthetic manner, where the specified DNA vector encodes an expression of activity that is speed-limiting in the specified path;
- culturing said microorganism transformed with said vector under conditions suitable for cell growth, expression of said gene and production of said lantibiotic or lantibiotic precursor. 23. A transformed microorganism producing a lantibiotic 107891 or its homologs, or its predecessor or derivative, where the biosynthesis genes of the lantibiotic 107891 in its genome are modified by inserting the nucleotide sequence of claim 10. 24. A method for producing a lantibiotic 107891 or a precursor or derivative thereof, comprising culturing the transformed microorganism producing 107891 according to claim 23 under conditions suitable for cell growth, expression of said gene and production of said lantibiotic or its precursor or derivative. 25. A transformed microorganism producing 107891 having lantibiotic biosynthesis genes 107891 in its genome, where at least one of the lantibiotic biosynthesis genes 107891 selected from ORF 1-17 mlb (SEQ ID NO: 2-18) is damaged. 26. The transformed microorganism of claim 25, wherein the biosynthesis gene that is damaged is a gene involved in the addition of a chlorine residue. 27. A method of producing a precursor or derivative of a lantibiotic 107891, comprising culturing a transformed microorganism producing 107891, according to claim 25 under conditions suitable for cell growth, expression of said gene and production of said lantibiotic or its predecessor or derivative. 28. A method of producing a derivative of the lantibiotic 107891, including:
- transformation with a recombinant DNA vector of a microorganism that produces a lantibiotic 107891 or its precursor in a biosynthetic manner, where the specified vector or its portion contains the nucleotide sequence according to any one of claims 1 to 11, which encodes the expression of enzymatic activity that modifies the specified lantibiotic or precursor; and
- culturing the specified microorganism, transformed with the specified vector, under conditions suitable for cell growth, expression of the specified gene and production of the specified derivative of the lantibiotic. 29. The selected polypeptide involved in the biosynthesis of lantibiotic 107891 and its homologues, selected from:
- the ORF polypeptide (SEQ ID NO: 2-18) encoded by any of the ORF 1-17 mlb; and
a polypeptide selected from the group consisting of at least 65% polypeptide, at least 90% polypeptide, and at least 95% or more polypeptide identical in amino acid sequence to the ORF polypeptide (SEQ ID NO: 2-18) encoded by any of ORF 1-17 mlb. 30. The selected polypeptide according to clause 29, which is any of the polypeptides (SEQ ID NO: 3, 7-10, 16-17) encoded by ORF 2, 6-9, 15-16 mlb. 31. The selected polypeptide according to clause 29, containing the ORF mlb polypeptide (SEQ ID NO: 3, 7-10, 16-17) encoded by any of ORF 2, 6-9, 15-16 mlb, or a polypeptide selected from the group consisting of a polypeptide that is at least 65%, a polypeptide that is at least 90%, and a polypeptide that is at least 95% or more identical in amino acid sequence to the polypeptide encoded by any of these ORF mlb. 32. The selected polypeptide according to clause 29, containing the ORF mlb polypeptide (SEQ ID NO: 2, 11-12, 14-15 and 18), encoded by any of the ORF 1,10-11, 13-14 and 17 mlb, or a polypeptide selected from the group consisting of at least 65% polypeptide, at least 90% polypeptide, and at least 95% or more polypeptide identical in amino acid sequence to the polypeptide encoded by any of these ORF mlb. 33. The selected polypeptide according to clause 29, containing the ORF mlb polypeptide (SEQ ID NO: 4 and 6) encoded by any of ORF 3 and 5 mlb, or a polypeptide selected from the group consisting of a polypeptide that is at least 65% , a polypeptide that is at least 90%, and a polypeptide that is at least 95% or more identical in amino acid sequence to the polypeptide encoded by any of these ORF mlb. 34. The selected polypeptide containing the polypeptide involved in the biosynthesis pathway of the lantibiotic 107891 selected from polypeptides encoded by any of the nucleic acids according to any one of claims 1 to 12.

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