# Patent

## Title
Polymyxin synthetase and gene cluster thereof

## Abstract

The present invention relates to a polymyxin synthetase isolated from Gram-positive Paenibacillus sp. and a gene cluster encoding the same, more precisely a polymyxin synthetase isolated from Paenibacillus polymyxa E681, a gene cluster encoding thereof and a preparation method of polymyxin or its derivatives using the gene cluster. The polymyxin synthetase of the present invention can be effectively used for the increase of productivity of polymyxin and the development of a novel antibiotic.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
This application is the National Stage filed under 35 U.S.C. 371 of International Application No. PCT/KR2006/0044665, filed Nov. 8, 2006, which claims priority to KR 10-2005-120878, filed Dec. 9, 2005.
TECHNICAL FIELD
The present invention relates to a polymyxin synthetase isolated from Gram-positive Paenibacillus sp. and a gene cluster encoding thereof, more precisely a polymyxin synthetase isolated from Paenibacillus polymyxa E681, a gene cluster encoding thereof and a preparation method of polymyxin or its derivatives using the gene cluster.
BACKGROUND ART
Non-ribosomal peptide synthetase (referred as ‘NRPS’ hereinafter) is organized by at least one ORF (open reading frame) forming NRPS complex, and each NRPS or NRPS subunit comprises one or more modules. A module is defined as the catalystic unit that incorporates one building block (for example, one amino acid) into the growing chain. Order and specificity of the modules within the NRPS determine the sequence and structure of the peptide product. Thus, NRPS which is not involved in ribosomal RNA translation used to be carried out by genetic code can produce peptides of wider structural diversity than those peptides translated from RNA template by ribosome. The peptides produced by NRPS can be further modified by the connection between hydroxyl acid and D- and L-amino acid, mutation and oxidation in main peptide chain forming linear, cyclic or branched cyclic structure, acylation, glycosylation, N-methylation and heterocyclic ring formation.
Polymyxin synthetase, one of NRPSs, stepwisely combines each amino acid monomer forming polymyxin and if necessary transforms the amino acid to complete the entire amino acid chain and to form a ring structure in order to synthesize a peptide antibiotic. Each module of NRPS is organized by at least three domains, which are A, C, and T domains. A domain (adenylation domain) plays a role in the selection and activation of an amino acid monomer, C domain (condensation domain) catalyzes peptide bond formation and T domains (thiolation domain, also called PCP) is involved in rotating phosphopantheteine group to incorporate an amino acid monomer into the growing peptide chain.
Recently, the tertiary structure of A domain recognizing phenylalanine of gramicidin biosynthesis gene has been identified, in which a specific amino acid binding site contains 8 amino acid residues (Conti E. et al., 1997. EMBO J. 16: 4174-4183). The amino acid sequence of this A domain was compared with that of the conventional A domain, as a result this A domain had high homology in 8 amino acid residues with the conventional A domain. Thus, analyzing the 8 amino acid residues may lead to the understanding of the association of a specific A domain with an amino acid (Challis G. L. et al., 2000. Chem. Biol. 7: 211-224).
In addition to these major domains, there are E domain (epimerization domain) playing a role in conversion of L-amino acid into D-amino acid and TE domain (termination domain), which are characterized by a specific amino acid motif.
A novel enzyme characterized by specificity can be designed by the modification of numbers and locations of modules at DNA level by genetic engineering and in vivo recombination techniques. For example, a domain originated from heterologous NRPS is substituted by using a recombinant technique (Schneider et al., Mol. Gen. Genet., 257, pp. 308-318, 1998) or a module can be designed to have specificity by changing residues forming the substrate binding pocket of A domain (Cane et al., Chem. Biol. vol. 6, p. 319-325, 1999).
Unlike other general peptides ribosomally translated, polymyxin is an antibiotic isolated from Bacillus sp. or Paenibacillus sp., which is produced by non-ribosomal peptide synthetase (Marahiel M. A. et al., 1997, Chem. Rev. 97, 2651-2673; Doekel S. et al., 2001, Metab. Eng. 6, 64-77).
The molecular weight of polymyxin is approximately 1200 Da (1.2 kDa) (Storm D. R. et al., Ann Rev. Biochem. 1977; 46:723-763). The basic structure of polymyxin is polyketidic peptide ring comprising 8 ̃10 amino acids and 2,4-diaminobutyric acid (Dab) at high concentration. Fatty acid is also attached on the peptide ring, which is preferably 6-methyoctanoic acid or 6-methylheptanoic acid (see FIG. 4). This structure favors solubility of polymyxin, suggesting that polymyxin is soluble in both water and an organic solvent.
Polymyxin is an antibiotic that is able to induce apoptosis by changing permeability of cell membrane and is functioning according to the following mechanisms.
First, polyketidic peptide ring of polymyxin changes the bridge between magnesium and calcium that stabilizes lipopolysaccharide of cell membrane to be bound to the cell. Then, lipopolysaccharide of cell membrane is reacted with fatty acid residue of polymyxin to make the linkage between polymyxin and cell membrane strong and tight. At last, polymyxin is incorporated into the outer membrane of cell, resulting in the destruction of the cell membrane (Hermsen E. D. et al., 2003, Infect. Dis. Clin. N. Am. 17: 545-562).
Polymyxin B was first isolated from Paenibacillus polymyxa in 1947 and since then 15 polymyxins have been reported (Storm D. R., et al., 1977, Annu. Rev. Biochem., 46: 723-763; Silaev, A. B. et al., 1975, Zh. Obshch. Khim. 45: 2331-2337; Martin N. I. et al., 2003, J. Biol. Chem. 278: 13124-13132). The polymyxin based antibiotic ‘polymyxin B sulfate’ killed 88% of Pseudomonas aeruginosa at the concentration of 0.01 μg/ml. Polymyxin E showed lethal effect at the concentration of 0.1 μg/ml. Polymyxin B and polymyxin E exhibited lethal effect on most Escherichia coli strains and Pseudomonas aeruginosa at the concentration under 2 μg/ml, in addition to on every Enterobacter, Salmonella, Shigella, Pasteurella, Brucella and Bordetella. However, both polymyxin B and E showed no lethal effect on Proteus, Serratia, Providencia and Edwardsiella even at the higher concentrations than 200 μg/ml. They had no effect on gram-positive bacteria, fungi and anaerobic bacteria, either (Nord N. M. et al., 1964, N. Engl. J. Med. 270: p. 1030-1035).
Thus, polymyxin had been used as a therapeutic agent for many diseases caused by pathogenic microorganisms until early 1970. But, it carried serious side effects such as fever, eruption and pain and induced severe neurotoxicity and hepatotoxicity (Pedersen M. F. et al., 1971, Invest. Urol. 9: p. 234-237). So, it has been replaced with other antibiotics with improved stability and most recently it is only being applied on local wounds as a form of ointment.
According to the increased use of antibiotics, pathogenic microorganisms having resistance to those antibiotics have been frequently noticed. In the midst, polymyxin draws our attention since it has excellent bactericidal effect on Gram-negative bacteria, in particular Pseudomonas aeruginosa and Acinetobacter baumannii exhibiting resistance against β-lactam, aminoglycoside and fluoroquinolone antibiotics.
Levin, et al reported that colistin (polymyxin E) was intravenously injected to 60 patients infected with Pseudomonas aeruginosa and Acinetobacter baumannii exhibiting resistance against the conventional antibiotics and as a result 58% of the patients were improved (Levin A. S. et al. 1999. Clin. Infect. Dis. 28:1008-1011). And there is another report by Stein, et al. saying that 3 osteomyelitis patients infected with Pseudomonas aeruginosa having resistance against almost all antibiotics were improved by the treatment of colistin (Stein A. et al., 2002, Clin. Infect. Dis. 35: p. 901-902). In another report, meningitis caused by Acinetobacter having resistance against antibiotics was also successfully treated by colistin (Jimenez-Mejias M. E. et al., 2002. Eur. J. Clin. Microbiol. Infect. Dis. 21: p. 212-214). Another report says that ventriculis caused by antibiotics-resistant Klebsiella pneumoniae was successfully treated by polymyxin B (Segal-Maurer S. et al., 1999, Clin. Infect. Dis. 28: p. 1134-1138).
As described hereinbefore, polymyxin seems to have therapeutic effect on Gram-negative bacteria having resistance against the conventional antibiotics, so that it is in increasing demand.
It had been tried to introduce an antibiotic biosynthesis gene into an industrially mass-productive strain in order to increase antibiotic productivity (Eppelmann K. et al., 2001. J. Biol. Chem. 276: p. 34824-34831; Pfeifer B. A. et al., 2001, Microbiol. Mol. Biol. Rev. 65: 106-118) and in fact it was confirmed that the substitution of a promoter of the antibiotic biosynthesis gene with a stronger one resulted in the increase of productivity (Tsuge K. et al., 2001. J. Bacteriol. 183: p. 6265-6273). There is an attempt to develop a novel antibiotic by re-constructing modules or domains of an antibiotic biosynthesis gene (Mootz H. D. et al., 2000. Proc. Natl. Acad. Sci. USA 97: p. 5848-5853; Ferra F. D. et al., 1997. J. Biol. Chem. 272: p. 25304-25309) or substituting a specific amino acid of the domains (Eppelmann K. et al., Biochemistry 41: p. 9718-9726). However, no polymyxin biosynthesis gene has been identified so far, therefore it had hardly been tried to increase productivity or develop a novel antibiotic based on the above mentioned techniques.
Therefore, it is important to identify a polymyxin biosynthesis gene and secure the information on the gene to increase production of polymyxin or develop polymyxin with less side effects and polymyxin based novel antibiotics.
The present inventors isolated, purified and analyzed polymyxin from Paenibacillus polymyxa E681. And the inventors confirmed that the strain produced polymyxin and found out and isolated a gene cluster encoding NRPS by sequencing the entire nucleotide sequence. The present inventors finally completed this invention by confirming with the domain analysis that the gene cluster was polymyxin biosynthesis gene.
DISCLOSURE
Technical Problem
It is an object of the present invention to provide a polymyxin synthetase isolated from Paenibacillus polymyxa E681, a gene cluster encoding the enzyme, and a preparation method for polymyxin and its derivatives using the gene cluster.
Technical Solution
The present invention provides a gene cluster encoding one or more polypeptides involved in polymyxin synthesis.
The present invention also provides a polymyxin synthetase encoded by the gene cluster.
The present invention further provides a recombinant vector containing the gene cluster.
The present invention also provides a host cell transformed by the above vector.
The present invention also provides an amino acid additional module of the polymyxin synthetase, in which A-T-E, C-A-T, C-A-T-C, C-A-T-E, C-A-T-TE or A-T-TE domains are combined stepwisely.
The present invention also provides a gene encoding each amino acid additional module.
The present invention also provides a polymyxin synthetase produced by the combination of the amino acid additional modules.
And, the present invention provides a preparation method of polymyxin or its derivatives comprising the following steps:
1) Inserting a gene cluster encoding the polymyxin synthetase into an expression vector;
2) Transforming a host cell with the expression vector containing the gene cluster of step 1);
3) Culturing the transformant of step 2); and
4) Isolating and purifying polymyxin or its derivatives from the culture product of step 3).
The descriptions for the terms used in the present invention are given hereinafter.
Non-ribosomal peptide synthetase (NRPS): composed of one or more ORFs (open reading frame) forming NRPS complex. Each NRPS or NRPS subunit contains one or more modules.
Module: a catalytic unit that incorporates a building block (ex: an amino acid) into the growing peptide chain. NRPSs produced peptides of enormous structural diversity, compared with ribosomally synthesized peptides.
Polymyxin: an antibiotic isolated from Bacillus sp or Paenibacillus sp, which is generated by NRPS not by ribosomal synthesis after being encoded.
Polymyxin synthetase: one of NRPSs, which combines each amino acid monomer forming polymyxin stepwisely and modifies the amino acid to complete the entire amino acid chain and to form a ring structure to produce a peptide antibiotic.
NRPS module: composed of A, C and T domains and additional E and TE domains.
A domain (adenylation domain) plays a role in selection and activation of an amino acid monomer and C domain (condensation domain) catalyzes a peptide bond formation, while T domain (thiolation domain, PCP) is involved in rotating phophopantheteine group to incorporate the amino acid monomer into growing polypeptide chain, E domain (epimerization) plays a role in conversion of L-amino acid into D-amino acid, and TE domain (termination domain) terminates the addition reaction of amino acids.
Hereinafter, the present invention is described in detail.
The present invention provides a gene cluster encoding one or more polypeptides involved in polymyxin synthesis. The present invention also provides a polymyxin synthetase encoded by the gene cluster.
The gene cluster is preferably isolated from Paenibacillus polymyxa E681.
The polymyxin herein is preferably polymyxin B, E or M and more preferably polymyxin M having polyketidic ring structure characterized by the stepwise binding of L-DAB (diaminobutyric acid), L-Thr (threonine), L-DAB and L-DAB to the amino group of 6-MOA (methyloctanoyl acid) and the location of D-Phe (phenylalanine) or D-Leu (leucine) on the 6th and the location of L-Leu, L-Thr, L-Ile (isoleucine) or L-Phe on the 7th site from the above amino group (see FIG. 1).
Polymyxin synthetase is one of NRPSs composed of one or more polypeptides, which is encoded by a gene cluster. The “gene cluster” herein is defined as the genome segment of a microorganism containing every gene necessary for the synthesis of secondary metabolites.
Particularly, the polymyxin synthetase consists of the polypeptide Pmx A represented by SEQ. ID. NO: 4, the polypeptide Pmx B represented by SEQ. ID. NO: 5, and the polypeptide Pmx E represented by SEQ. ID. NO: 6, but not always limited thereto. The variants of the above polypeptides, which are polypeptides having functional identity even though there are addition, deletion or substitution of one or more modules, domains and/or amino acids, can also be included. That is, every genes encoding Pmx A, Pmx B, Pmx E and their variants are included in the criteria of the invention, and particularly those nucleotide sequences represented by SEQ. ID. NO: 1, NO: 2 and NO: 3 are preferred.
The present inventors sequenced the nucleotide sequence of Paenibacillus polymyxa E681 genome by using whole-genome shotgun sequencing strategy. As a result, it was confirmed that Paenibacillus polymyxa E681 genome is approximately 5.4 Mbps in length and has a single circular chromosome. The present inventors also identified a polymyxin biosynthetic gene cluster from the genome above.
Approximately 4800 genes encoding proteins have been identified from the nucleotide sequence of E681 genome by using Critica (Badger J. H. and Olsen G. J., 1999, Mol. Biol. Evol. 16, 512), glimmer (Delcher A. L. et al., 1999, Nucleic Acids Res. 27, 4636) and zcurve (Guo F.-B. et al., 2003, Nucleic Acids Res. 31, 1780) programs. To investigate the functions of each gene product, the genes were translated into amino acid sequences and compared with sequences in the protein sequence database (Altschul S. F. et al., 1997, Nucleic Acids Res. 25: 3389-3402). Next, domain and protein family analysis (Bateman A. et al., 2004, Nucleic Acids Res. 32 (Database issue):D138-141; Haft D. H. et al., 2003, Nucleic Acids Res. 31:371-373), motif and pattern screening (Hulo N. et al., 2004, Nucleic Acids Res. 32 (Database issue):D134-137) and protein site prediction analysis (Gardy J. L. et al., 2003, Nucleic Acids Res. 31:3613-3617) were performed.
From the above screening, at least 4 NRPS gene clusters encoding 4 different antibiotic synthetases have been identified.
The substrate specificity of adenylation (A) domain of each gene cluster was compared with the chart showing active amino acids associated with A domain substrate specificity prepared by Challis et al (Challis G. L. et al., 2000, Chem. Biol. 7: p. 211-224). As a result, one of the gene clusters was identified as the gene cluster encoding polymyxin synthetase (see FIG. 2).
Each polypeptide of the polymyxin synthetase of the present invention contains one or more modules and each module is preferably organized by at least 2 domains selected from a group consisting of A, C, T, E and TE domains.
In polymyxin synthetase organized with PmxA, PmxB and PmxE, the fifth module of PmxE, C domain joins in the first module ‘A-T-E’ of PMXA, and the last C domain of the forth module of PmxA joins A-T-TE of PmxB. PmxC and PmxD were proved not to be involved in polymyxin synthesis but expected to be involved in polymyxin secretion.
Polymyxin is synthesized by completing polyketidic peptide ring by the stepwise binding of L-DAB (diaminobutyric acid), L-Thr (threonine), L-DAB, L-DAB, and L-DAB to the amino group of 6-MOA (methyloctanoyl acid) by Pmx E polypeptide module (domain) and locating D-Phe (phenylalanine) or D-Leu (leucine) on the 6th site and then locating L-Leu, L-Thr, L-Ile (isoleucine) or L-Phe on the 7th site, followed by the stepwise binding of L-DAB and L-DAB, and at last linking L-Thr by Pmx B polypeptide module (domain) (see FIG. 1A).
The polymyxin synthetase predicted by the nucleotide sequence of the gene, as shown in FIG. 4, enabled the prediction of polymyxin B, polymyxin E or polymyxin M. Considering that polymyxin isolated in Example 1 was polymyxin M, the gene cluster of the invention was presumed to be polymyxin M synthetase.
A novel polymyxin synthetase having a different specificity can be derived from the gene cluster of the invention by genetic alteration such as changing number or position of a module or a domain in the gene cluster. For example, heterologous NRPS originated domain was substituted (Schneider et al., Mol. Gen. Genet., 257, p. 308-318, 1998) or a residue forming substrate binding pocket of A domain was replaced to design a novel substrate specificity (Cane and Walsh, Chem. Biol. vol. 6, p. 319-325, 1999), or structural modification was performed by addition, substitution or deletion of one or more modules, domains or amino acids or by the linkage between D- and L-amino acid and hydroxyl acid, mutation and oxidation of peptide chain, acylation, glycosylation, N-methylation and heterocyclic ring formation.
Therefore, the gene provided by the present invention can be effectively used for the development of polymyxin with minimized side effects and its derivatives or a novel antibiotic according to the above method.
The present invention also provides a recombinant vector containing the gene cluster of the invention and a host cell transformed with the vector.
The gene cluster encoding polymyxin synthetase of the invention can be cloned into such vectors as BAC, plasmid, and fosmid, and the vector can be introduced into a relevant host cell to produce a polymyxin antibiotic.
In the present invention, Paenibacillus polymyxa, E. coli, and Bacillus subtillis are preferably used as host cells. A recombinant vector can be introduced into such host cells by a conventional method well-known to those in the art including heat-shock method or electric-shock method. It is also well understood to those in the art that different strains can be used as host cells according to the purpose of expression or various vectors.
The present invention also provides an amino acid additional module of polymyxin synthetase A-T-E, C-A-T, C-A-T-C, C-A-T-E C-A-T-TE or A-T-TE and a gene encoding each of the amino acid additional module.
Each module forming a polypeptide is described herein. First, Pmx A polypeptide is organized by the following 4 modules,
The first module: A(adenylation)-T(thiolation)-E(epimerization) domain;
The second module and the third modules: C(condensation)-A-T domain; and
The forth module: C-A-T-C domain.
Pmx B polypeptide comprises one module which is A-T-TE(termination) domain,
And Pmx E polypeptide is organized by the following 5 modules,
The first and the second modules: C-A-T domain;
The third module: C-A-T-E domain;
The forth module: C-A-T domain; and
The fifth module: C-A-T-C domain (see FIG. 3).
Genes encoding each domain and module of Pmx A, Pmx B and Pmx E are those represented by SEQ. ID. NO: 7 ̃NO: 66, in which linker genes combining each domain are also included. The SEQ. ID. NO. of each domain is presented in Table 1.
The present invention also provides a polymyxin synthetase produced by the combination of the amino acid additional modules.
Each polymyxin synthetase is formed by the combination of modules arranged as A-T-E, C-A-T, C-A-T-C, C-A-T-E, C-A-T-TE or A-T-TE. Therefore, the construction of such recombinant expression vector that contains the combination of gene corresponding each module leads to the diversity of polymyxin synthetases.
The present invention also provides a preparation method of polymyxin or its derivatives comprising the following steps:
1) Inserting a gene cluster encoding the polymyxin synthetase into an expression vector;
2) Transforming a host cell with the expression vector containing the gene cluster of step 1);
3) Culturing the transformant of step 2); and
4) Isolating and purifying polymyxin or its derivatives from the culture product of step 3).



DESCRIPTION OF DRAWINGS
The application of the preferred embodiments of the present invention is best understood with reference to the accompanying drawings, wherein:
FIG. 1 illustrates the structure of polymyxin, FIG. 1(A) is a representative diagram of polymyxin and FIG. 1(B) illustrates the structure of polymyxin M isolated from Paenibacillus polymyxa E681,
FIG. 2 is a diagram illustrating the structure of a polymyxin biosynthetic gene cluster originated from Paenibacillus polymyxa E681,
FIG. 3 is a diagram illustrating the structure of the domain of a polymyxin synthetase gene originated from Paenibacillus polymyxa E681 genome,
A: Domain A (adenylation);
C: Domain C (condensation);
E: Domain E (epimerization);
T: Domain T (thiolation); and
TE: Domain TE (termination)
FIG. 4 is a diagram illustrating the structure of polymyxin predicted from the domain structure of polymyxin biosynthesis gene separated from Paenibacillus polymyxa E681 genome.



MODE FOR INVENTION
Practical and presently preferred embodiments of the present invention are illustrative as shown in the following Examples.
However, it will be appreciated that those skilled in the art, on consideration of this disclosure, may make modifications and improvements within the spirit and scope of the present invention.
Example 1
Isolation and Analysis of Polymyxin from Paenibacillus polymyxa 
<1-1> Culture of Paenibacillus polymyxa 
Paenibacillus polymyxa E681 was cultured in the medium designed by Paulus and Gray (Paulus H and Gray E. 1964, J. Biol. Chem. 239:865-871) under aerobic condition at 25° C. with 180 rpm for 3 days, followed by centrifugation (7000 rpm, 10 min) to separate supernatant.
<1-2> Identification of Polymyxin by LC/MS Analyzing System
The composition of the supernatant was analyzed by LC/MS system.
LC/MS was performed using high pressure liquid chromatography system provided by Thermo Electron Co. (USA) and ion spectrometer. The sample proceeded to reversed-phase column (YMC Hydrosphere C18 column) and analyzed in a mixed solvent of acetonitrile and water containing 0.1% formic acid (0.2 ml/min).
As a result, (M+H)+ ion peak was 1158, and the actual molecular weight was confirmed to be 1157, which is the same molecular weight of the conventional polymyxin M.
To confirm if the target compound was polymyxin M, acetylation was performed. Particularly, the target compound was treated with pyridine:acetic anhydride (1:1), followed by reaction for 12 hours at room temperature. The molecular weight of the target compound was measured by LC/MS. As a result, it was confirmed that the target compound had the molecular weight of 1493, which was the same weight as that Martinet al (Martin N I, et al., 2003. J. Biol. Chem. 278: 13124-13132) reported earlier (FIG. 1).
Example 2
Sequencing of Polymyxin Biosynthesis Gene
The nucleotide sequence of Paenibacillus polymyxa E681 genome was completely sequenced by whole-genome shotgun sequencing strategy and then the polymyxin biosynthetic gene cluster was identified.
<2-1> Library Construction
Paenibacillus polymyxa E681 was cultured by the same manner as described in Example 1, and chromosomal DNA was extracted by the method described in Genome Analysis, A laboratory manual Vol. III Cloning systems (CSHL Press, Cold Spring Harbor, N.Y., USA), and the DNA was fragmented to construct a shotgun library for sequencing.
The high molecular chromosomal DNA fragmentation was performed with VCX-500 ultrasonicator (Sonics, Newtown, Conn., USA) with 19% strength, 0.3/3 sec of pulse on/off time, 6 times. The DNA fragments of 2 kb, 5 kb, 8 kb and 10 kb in size were recovered and used to construct the library. pUC18, pUC19, pUC118 or pBCKS (Stratagene, La Jolla, Calif., USA), and pTrueBlue (Genomics One (Laval, Quebec, Canada) vectors were used. The DNAs of  ̃40 kb and  ̃100 kb in size were used to construct fosmid library and BAC library, which would be used for forming the contig structure.
The fosmid library was constructed by using a fosmid library production kit (CopyControlTM fosmid library production kit, Epicentre Biotechnologies, Madison, Wis., USA) and the BAC library was constructed by inserting the chromosomal DNA digested with HindIII into pindigo 536 vector (Peterson D. G. et al., 2000, J. Agric. Genomics, volume 5, see internet, ncrg.org/research/jag, Luo M. et al., 2001, Genome 44: p. 154-62).
The reactant for the plasmid library was inserted into E. coli DH10B by electroporation, which was smeared on a LB agar plate medium containing X-gal/IPTG/Amp (Ampicillin). White recombinant colony was inoculated to a 96 deep-well plate containing LB(Amp) liquid medium, followed by shaking-culture in a 37° C. incubator with 250 rpm for 48 hours. Cells were recovered and plasmid DNA was separated and purified according to the standard method.
<2-2> Nucleotide Sequence Analysis
DNA sequencing was performed by using BigDyeTM terminator cycle sequencing kit (Applied Biosystems, CA, USA) and the reactant was analyzed with ABI 3700 and 3730 DNA analyzer (Applied Biosystems, Foster City, Calif., USA). Files containing the results were analyzed with phred/phrap/consed program (on the worldwide web, phrap.org).All the result files were analyzed with phred to organize nucleotide sequences and relevant results were collected to mask the sequence of the vector. Sequence combining was carried out by phrap and contig confirmation and edition and primer design were carried out by consed.
Approximately 61,700 sequence fragments (6.7 times) were obtained from the termini of the plasmid and fosmid/BAC, followed by sequencing combining. As a result, approximately 800 contig sequences were obtained, followed by finishing.
Clones connecting contigs by the sequences of the both ends were screened and then a primer was designed to read the gap between sequences, followed by determination of the nucleotide sequence. Only those fosmids connecting a big part having the gap of at least 15 kb were selected, followed by limited shotgun sequencing. The incorrectly combined sequence by repetitive sequences such as rRNA gene or transferase gene was corrected by using consed program. To remove physical gaps, primers were designed based on the end of each contig, followed by recombinant PCR or RT-PCR to obtain the sequences of the unknown region. All the gaps were eliminated to prepare authentic circular chromosome sequence, and Phred was operated. PCR was performed again to amplify the uncertain region. The aim of the accuracy was >99.99% (up to 1 bp error per 10 kb).
The whole nucleotide sequence of the identified Paenibacillus polymyxa E681 genome was approximately 5.4 Mbps in total length and had the single circular chromosome structure (% G+C, 45.8).
<2-3> Prediction of a Protein from a Gene
Approximately 4800 protein encoding genes were identified from the genome by running Critica (Badger J. H. and Olsen G. J., 1999, Mol. Biol. Evol. 16, 512), glimmer (Delcher A. L. et al., 1999, Nucleic Acids Res. 27, 4636) and zcurve (Guo F.-B. et al., 2003, Nucleic Acids Res. 31, 1780). To investigate the functions of each gene product, those genes were translated into amino acid sequences, which were screened by blastp with the known protein sequence databases (Altschul S F, et al., 1997. Nucleic Acids Res. 25:3389-3402). At this time, the databases used were COG (Tatusov R. L. et al., 2003, BMC Bioinformatics. 4:41), UniProt Knowledgebase (Bairoch A. et al., 2005, Nucleic Acids Res. 33(Database issue):D154-159), NCBI-NR (on the internet, ncbi.nih.ogv/blast/db/nr.tar.gz) and KEGG-Genes (Kanehisa M. et al., 2004, Nucleic Acids Res. 32(Database issue):D277-280).
For the analysis of a domain and a protein family, Pfam (Bateman A. et al., 2004. Nucleic Acids Res. 32(Database issue):D138-141) and TIGRFAMs (Haft D H, et al., 2003. Nucleic Acids Res. 31:371-373) databases were used. For the investigation of a motif and a pattern, Prosite (Hulo N. et al., 2004, Nucleic Acids Res. 32(Database issue):D134-137) database was used.
Psort-B was used to predict the location of a protein (Gardy J. L. et al., 2003, Nucleic Acids Res., 31:3613-3617). The proteins were given hierarchical names considering liability of the screening results. The protein had no homologs having E-value of lower than 10−5 from UniProt screening was named hypothetical protein.
From the analysis of genome information, at least 4 NRPS gene clusters encoding 4 different antibiotic synthetases have been identified. The substrate specificity of A domain of each gene cluster was compared with the substrate specificity associated active amino acid chart made by Challis et al (Challis G. L. et al., 2000, Chem. Biol. 7: 211-224). As a result, one of them was identified as the gene cluster encoding polymyxin synthetase (FIG. 2).
Example 3
Prediction of Polymyxin Structure from the Nucleotide Sequence of Polymyxin Biosynthesis Gene
The polymyxin biosynthesis gene of the present invention was analyzed based on the chart showing the substrate specificity associated active amino acids summarized by Challis et al (Challis G. L. et al., 2000. Chem. Biol. 7: 211-224). As a result, the gene was confirmed to be organized by such domains as pmx A, pmx B and pmx E, as shown in FIG. 3. Each A domain recognized amino acids such as DAB, Leu and Thr. The amino acid sequence of each domain was determined and the SEQ. ID. NO. corresponding to each domain is shown in Table 1.












TABLE 1







Domain
pmx A
pmx B
pmx E









A
 8, 15, 20, 26
32
39, 44, 50, 57, 62



T
10, 17, 22, 28
34
41, 46, 52, 59, 64



C
14, 19, 24, 30

38, 43, 48, 56, 61, 66



E
12

54



TE

36










As shown in FIG. 4, the predicted polymyxin structure was confirmed to be that of polymyxin M, which was consistent with the result of Example 1.
INDUSTRIAL APPLICABILITY
As explained hereinbefore, the present inventors confirmed that polymyxin could be produced, separated and purified from Paenibacillus polymyxa E681 and then the whole nucleotide sequence of the genome and its domain were analyzed, by which the gene cluster was identified as polymyxin biosynthesis gene. The polymyxin synthetase of the invention can be effectively used for the development of a novel antibiotic and the increase of productivity of polymyxin.
[Sequence List Text]
SEQ. ID. NO: 1 is Pmx A DNA sequence, SEQ. ID. NO: 2 is Pmx B DNA sequence, SEQ. ID. NO: 3 is Pmx E DNA sequence, SEQ. ID. NO: 4 is Pmx A amino acid sequence, SEQ. ID. NO: 5 is Pmx B amino acid sequence, SEQ. ID. NO: 6 is Pmx E amino acid sequence, SEQ. ID. NO: 7 is N terminal (297 aa: 1-297) sequence, SEQ. ID. NO: 8 is A1 (505 aa: 298-802) sequence, SEQ. ID. NO: 9 is A1-T1 linker (18 aa: 803-820) sequence,
SEQ. ID. NO: 10 is T1 (64 aa: 821-884) sequence, SEQ. ID. NO: 11 is T1-E1 linker (17 aa: 885-901) sequence, SEQ. ID. NO: 12 is E1 (460 aa: 902-1361) sequence, SEQ. ID. NO: 13 is E1-C2 linker (9 aa: 1362-1370) sequence, SEQ. ID. NO: 14 is C2 (437 aa: 1371-1807) sequence, SEQ. ID. NO: 15 is A2 (530 aa: 1798-2327) sequence, SEQ. ID. NO: 16 is A2-T2 linker (20 aa: 2328-2347) sequence, SEQ. ID. NO: 17 is T2 (63 aa: 2348-2410) sequence, SEQ. ID. NO: 18 is T2-C3 linker (21 aa: 2411-2431) sequence, SEQ. ID. NO: 19 is C3 (425 aa: 2432-2856) sequence,
SEQ. ID. NO: 20 is A3 (557 aa: 2855-3411) sequence 2 aa overlapped with C3, SEQ. ID. NO: 21 is A3-T3 linker (18 aa: 3412-3429) sequence, SEQ. ID. NO: 22 is T3 (65 aa: 3430-3494) sequence, SEQ. ID. NO: 23 is T3-C4 linker (22 aa: 3495-3516) sequence, SEQ. ID. NO: 24 is C4 (424 aa: 3517-3940) sequence, SEQ. ID. NO: 25 is C4-A4 linker (19 aa: 3941-3959) sequence, SEQ. ID. NO: 26 is A4 (509 aa: 3960-4468) sequence, SEQ. ID. NO: 27 is A4-T4 linker (18 aa: 4469-4486) sequence, SEQ. ID. NO: 28 is T4 (65 aa: 4487-4551) sequence, SEQ. ID. NO: 29 is T4-C5 linker (21 aa: 4552-4572) sequence,
SEQ. ID. NO: 30 is C5 (381 aa: 4573-4953) sequence, SEQ. ID. NO: 31 is N terminal (300 aa: 1-300) sequence, SEQ. ID. NO: 32 is A (530 aa: 301-830) sequence, SEQ. ID. NO: 33 is A-T linker (14 aa: 831-844) sequence, SEQ. ID. NO: 34 is T (62 aa: 845-906) sequence, SEQ. ID. NO: 35 is T-TE linker (25 aa: 907-931) sequence, SEQ. ID. NO: 36 is TE (171 aa: 932-1102) sequence, SEQ. ID. NO: 37 is N terminal (70 aa: 1-70) sequence, SEQ. ID. NO: 38 is C1 (432 aa: 71-502) sequence, SEQ. ID. NO: 39 is A1 (535 aa: 498-1032) sequence 5 aa overlapped with C1,
SEQ. ID. NO: 40 is A1-T1 linker (15 aa: 1033-1047) sequence, SEQ. ID. NO: 41 is T1 (64 aa: 1048-1111) sequence, SEQ. ID. NO: 42 is T1-C2 linker (21 aa: 1112-1132) sequence, SEQ. ID. NO: 43 is C2 (427 aa: 1133-1559) sequence, SEQ. ID. NO: 44 is A2 (520 aa: 1558-2077) sequence 2 aa overlapped with C2, SEQ. ID. NO: 45 is A2-T2 linker (20 aa: 2078-2097) sequence, SEQ. ID. NO: 46 is T2 (63 aa: 2098-2160) sequence, SEQ. ID. NO: 47 is T2-C3 linker (21 aa: 2161-2181) sequence, SEQ. ID. NO: 48 is C3 (427 aa: 2182-2608) sequence, SEQ. ID. NO: 49 is C3-A3 linker (31 aa: 2609-2639) sequence,
SEQ. ID. NO: 50 is A3 (556 aa: 2640-3195) sequence, SEQ. ID. NO: 51 is A3-T3 linker (14 aa: 3196-3209) sequence, SEQ. ID. NO: 52 is T3 (61 aa: 3210-3270) sequence, SEQ. ID. NO: 53 is T3-E3 linker (20 aa: 3271-3290) sequence, SEQ. ID. NO: 54 is E3 (459 aa: 3291-3749) sequence, SEQ. ID. NO: 55 is E3-C4 linker (11 aa: 3750-3760) sequence, SEQ. ID. NO: 56 is C4 (437 aa: 3761-4197) sequence, SEQ. ID. NO: 57 is A4 (556 aa: 4195-4750) sequence 3 aa overlapped with C4, SEQ. ID. NO: 58 is A4-T4 linker (18 aa: 4751-4768) sequence, SEQ. ID. NO: 59 is T4 (65 aa: 4769-4833) sequence,
SEQ. ID. NO: 60 is T4-C5 linker (21 aa: 4834-4854) sequence, SEQ. ID. NO: 61 is C5 (425 aa: 4855-5279) sequence, SEQ. ID. NO: 62 is A5 (556 aa: 5277-5832) sequence 3 aa overlapped with C5, SEQ. ID. NO: 63 is A5-T5 linker (18 aa: 5833-5850) sequence, SEQ. ID. NO: 64 is T5 (64 aa: 5851-5914) sequence, SEQ. ID. NO: 65 is T5-C6 linker (21 aa: 5915-5935) sequence, SEQ. ID. NO: 66 is C6 (377 aa: 5936-6312) sequence.
Those skilled in the art will appreciate that the conceptions and specific embodiments disclosed in the foregoing description may be readily utilized as a basis for modifying or designing other embodiments for carrying out the same purposes of the present invention. Those skilled in the art will also appreciate that such equivalent embodiments do not depart from the spirit and scope of the invention as set forth in the appended claims.

## Claims

1. An isolated polymyxin synthetase comprising a Pmx A polypeptide subunit having the sequence set forth in SEQ ID NO:4, Pmx B polypeptide subunit having the amino acid sequence set forth in SEQ ID NO:5 and Pmx E polypeptide subunit having the amino acid sequence set forth in SEQ ID NO:6.

2. The polymyxin synthetase of claim 1, wherein the polypeptide subunits contain one or more modules and each module is organized by at least two domains selected from a group consisting of A(adenylation), C(condensation), T(thiolation), E(epimerization) and TE(termination) domains.

3. A gene cluster which encodes the polymyxin synthetase of claim 1, wherein the polymyxin synthesized by the polymyxin synthetase is polymyxin M.

4. The gene cluster of claim 3, comprising a gene which encodes the Pmx A polypeptide subunit, said gene comprising the sequence set forth in SEQ ID NO:1.

5. The gene cluster of claim 3, comprising a gene which encodes the Pmx B polypeptide subunit, said gene comprising the sequence set forth in SEQ ID NO:2.

6. The gene cluster of claim 3, comprising a gene which encodes the Pmx E polypeptide subunit, said gene comprising the sequence set forth in SEQ ID NO:3.

7. The gene cluster of claim 3, wherein the gene cluster is isolated from Paenibacillus polymyxa E681.

8. An expression vector comprising a gene encoding a polypeptide subunit selected from the gene cluster of claim 3.

9. A transformed host cell with the expression vector of claim 8.

10. A method for preparing polymyxin M comprising the following steps:
1) constructing a recombinant expression vector by inserting genes encoding polymyxin synthetase, said polymyxin synthetase comprising a Pmx B polypeptide subunit comprising the amino acid sequence set forth in SEQ ID NO:5, a Pmx A polypeptide subunit comprising the amino acid sequence set forth in SEQ ID NO:4, and a Pmx E polypeptide subunit comprising the amino acid sequence set forth in SEQ ID NO:6, into an expression vector;
2) transforming a host cell with the expression vector containing the gene cluster of step 1) to produce a transformant;
3) culturing the transformant of step 2); and
4) isolating and purifying polymyxin M from the culture product of the transformant of step 3).

11. The method of claim 10, wherein the gene encoding the Pmx A polypeptide subunit, said gene comprising the sequence set forth in SEQ ID NO:1.

12. The method of claim 10, comprising a gene encoding the Pmx B polypeptide subunit, said gene comprising the sequence set forth in SEQ ID NO:2.

13. The method of claim 10, comprising a gene encoding the Pmx E polypeptide subunit, said gene comprising the sequence set forth in SEQ ID NO:3.

14. The method of claim 10, wherein the gene cluster is isolated from Paenibacillus polymyxa E681.