WO2007086645A1 - Fusaricidin synthetase and gene thereof - Google Patents

Fusaricidin synthetase and gene thereof Download PDF

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WO2007086645A1
WO2007086645A1 PCT/KR2006/005126 KR2006005126W WO2007086645A1 WO 2007086645 A1 WO2007086645 A1 WO 2007086645A1 KR 2006005126 W KR2006005126 W KR 2006005126W WO 2007086645 A1 WO2007086645 A1 WO 2007086645A1
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fusaricidin
seq
domain
group
gene
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Seung-Hwan Park
Jihyun F Kim
Choonghwan Lee
Soo-Keun Choi
Heayoung Jeong
Seong-Bin Kim
Yon Kyoung Park
Rumi Kim
Choong-Min Ryu
Soo-Young Park
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Korea Research Institute Of Bioscience And Biotechnology
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    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F1/00—Treatment of water, waste water, or sewage
    • C02F1/50—Treatment of water, waste water, or sewage by addition or application of a germicide or by oligodynamic treatment
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/18—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing at least two hetero rings condensed among themselves or condensed with a common carbocyclic ring system, e.g. rifamycin
    • C12P17/188—Heterocyclic compound containing in the condensed system at least one hetero ring having nitrogen atoms and oxygen atoms as the only ring heteroatoms
    • C12P17/189—Heterocyclic compound containing in the condensed system at least one hetero ring having nitrogen atoms and oxygen atoms as the only ring heteroatoms containing the rifamycin nucleus
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01F—MIXING, e.g. DISSOLVING, EMULSIFYING OR DISPERSING
    • B01F23/00—Mixing according to the phases to be mixed, e.g. dispersing or emulsifying
    • B01F23/50—Mixing liquids with solids
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B08—CLEANING
    • B08B—CLEANING IN GENERAL; PREVENTION OF FOULING IN GENERAL
    • B08B1/00—Cleaning by methods involving the use of tools
    • B08B1/10—Cleaning by methods involving the use of tools characterised by the type of cleaning tool
    • B08B1/12—Brushes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/93—Ligases (6)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione

Definitions

  • the present invention relates to a fusaricidin synthetase isolated from Gram-positive Paenibacillus sp . and a gene encoding thereof, more precisely a fusaricidin synthetase isolated from Paenibacillus polymyxa E681, a gene encoding thereof and a preparation method of fusaricidin or its derivatives using the gene.
  • 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.
  • NRPS which is not involved in ribosomal RNA translation according to 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-atnino acid, mutation and oxidation in main peptide chain forming linear, cyclic or branched cyclic structure, acylation, glycosylation, N-methylation and heterocyclic ring formation.
  • Fusaricidin synthetase one of NRPSs, stepwisely combines each amino acid monomer forming fusaricidin 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
  • T domain thiolation domain, also called PCP
  • PCP thiolation domain
  • 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 . , MoI. Gen. Genet., 257:308-318, 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. 6:319-325, 1999) .
  • Fusaricidin is an antibiotic isolated from
  • Paenibacillus sp which has a ring structure composed of 6 amino acid residues in addition to 15-guanidino-3- hydroxypentadecanoic acid (see Fig. 3) .
  • Fusaricidins isolated from Paenibacillus polymyxa so far are LI-F03, LI- F04, LI-F05, LI-F07 and LI-F08 (Kurusu K, Ohba K, Arai T and Fukushima K. J " . Antibiotics 40:1506-1514, 1987) and additional fusaricidins A, B, C and D have been reported (Kajimura Y and Kaneda M. J " .
  • Fusaricidin has excellent germicidal activity to plant pathogenic fungi such as Fusarium oxysporum, Aspergillus niger, Aspergillus oryzae and Penicillum thomii, and particularly fusaricidin B has germicidal activity to Candida albicans and Saccharomyces cerevisiae. Fusaricidin also has excellent germicidal activity to Gram-positive bacteria including Staphylococcus aureus (Kajimura Y and Kaneda M. J. Antibiotics 49:129-135, 1996; Kajimura Y and Kaneda M. J " . Antibiotics 50:220-228, 1997).
  • fusaricidin has antifungal activity against Leptosphaeria maculans which causes black root rot of canola (Beatty PH and Jensen SE. Can. J. Microbiol. '48: 159-169, 2002). According to the recent reports on the excellent germicidal activity of fusaricidin against pathogenic Gram- positive bacteria and plant pathogenic fungi, fusaricidin seems to have great potential for industrial uses and thereby is in increasing demand. As a part of the effect to increase productivity of an antibiotic, an antibiotic biosynthesis gene was inserted into a host strain to industrially mass-produce the antibiotic (Eppelmann K, Doekel S and Marahiel MA. J " . Biol. Chem.
  • the present inventors isolated, purified and analyzed fusaricidin from Paenibacillus polymyxa E681. And the inventors confirmed that the strain produced fusaricidin and found out and isolated a gene encoding NRPS by sequencing of the entire nucleotide sequence. The present inventors finally completed this invention by confirming with the domain analysis that the gene was a fusaricidin biosynthesis gene.
  • the present invention provides a polypeptide involved in fusaricidin synthesis.
  • the present invention also provides a gene encoding the polypeptide.
  • the present invention further provides a recombinant vector containing the gene.
  • 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 fusaricidin synthetase, in which C-A-T, C-A-T-E or C-A-T-TE domains are combined in order.
  • the present invention also provides a gene encoding each amino acid additional module.
  • the present invention also provides a fusaricidin synthetase produced by the combination of the amino acid additional modules.
  • the present invention provides a preparation method of fusaricidin or its derivatives comprising the following steps:
  • step 2) Transforming a host cell with the expression vector containing the gene of step 1) ; 3) Culturing the transforman'c of step 2) ; and 4) Isolating and purifying fusaricidin or its derivatives from the culture product of step 3) .
  • Non-ribosomal peptide synthetase composed of one or more ORFs (open reading frame) forming NRPS complex.
  • Each NRPS or NRPS subunit contains one or more modules.
  • NRPS a catalytic unit that incorporates a building block (ex: an amino acid) into the growing peptide chain.
  • NRPSs produce peptides of enormous structural diversity, compared with ribosomally synthesized peptides.
  • Fusaricidin an antibiotic isolated from Bacillus sp or Paenibacillus sp, which is generated by NRPS not by ribosomal synthesis after being encoded.
  • Fusaricidin synthetase one of NRPSs, which stepwisely combines each amino acid monomer forming fusaricidin in order 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, T domains and additional E, TE domains.
  • a 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 the growing polypeptide chain,
  • E domain plays a role in conversion of L- amino acid into D-amino acid
  • TE domain termination domain
  • the present invention provides a polypeptide involved in fusaricidin synthesis and a gene encoding the same.
  • the polypeptide involved in fusaricidin synthesis of the invention is represented by SEQ. ID. NO: 2 and its variants, that is, other polypeptides which are equally functioning but modified by addition, deletion and substitution of one or more modules, domains and/or amino acids are also included in the criteria of the invention. All genes encoding the polypeptides and their variants above are also included in the criteria of the invention and the one represented by SEQ. ID. NO: 1 is preferred.
  • the fusaricidin of the invention has the polyketidic ring structure organized by the stepwise binding of L-Thr
  • the fusaricidins possibly isolated from Paen.ibacillus polymyxa are fusaricidin A, fusaricidin B, fusaricidin C, fusaricidin D and L1-F03, LI-F04, LI-F07 and LI-F08, etc, but not always limited thereto.
  • 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 fusaricidin biosynthesis gene from the genome above.
  • a domain substrate specificity prepared by Challis et al (Challis G. L. et al . , Chem. Biol. 7:211-224, 2000).
  • Challis et al Challis G. L. et al . , Chem. Biol. 7:211-224, 2000.
  • one of the genes was identified as the gene encoding fusaricidin synthetase (see Fig. 2) .
  • Each polypeptide involved in fusaricidin biosynthesis 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 .
  • Each A domain of module composing fusaricidin synthetase recognized such amino acids as Thr, Leu/lle/Val, Tyr, Thr, Asn and VaI, as shown in Fig. 2.
  • fusaricidins A and B were isolated from E681 strain and fusaricidin A included L-Thr, D-VaI, L-VaI, D-allo-Thr, D-Asn, D-AIa, etc
  • fusaricidin B included L-Thr, D-VaI, L-VaI, D-allo-Thr, D-GIn, D-AIa, etc. From the whole genome assay of E681 was confirmed that only one gene was involved in fusaricidin synthesis.
  • the third A domain recognized Tyr or VaI, the fifth A domain recognized Asn or Gin and the sixth A domain recognized VaI or Ala was proved to be the' polyketidic peptide ring organized by the stepwise binding of L-Thr (threonine) D- VaI (valine), L-Tyr (tyrosine) or L-VaI, D-allo-Thr, D-Asn or D-GIu and lastly D-VaI or D-AIa (see Fig. 3) . So, the gene of the present invention was confirmed to be the fusaricidin synthetase and be able to produce fusaricidin of diversity (see Fig. 3) .
  • 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.
  • genetic alteration such as changing number or position of a module or a domain in the gene cluster.
  • heterologous NRPS originated domain was substituted (Schneider et al . , MoI. Gen. Genet., 257:308- 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.
  • the gene provided by the present invention can be effectively used for the development of fusaricidin derivatives or a novel antibiotic according to the above method.
  • the present invention also provides a recombinant vector containing the gene of the invention and a host cell transformed with the vector.
  • the gene encoding fusaricidin 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 fusaricidin antibiotic.
  • BAC BAC
  • plasmid plasmid
  • fosmid a relevant host cell to produce a fusaricidin antibiotic.
  • Paenibacillus polymyxa, E. coli, and Bacillus subtillis are preferably used as host cells.
  • a recombinant vector can be introduced into such host cells by one of the conventional methods 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 fusaricidin synthetase C-A-T, C-A-T-E or C-A-T-TE and a gene encoding each amino acid additional module.
  • the first module C (condensation) -A (adenylation) - T (thiolation) domain;
  • the second module C-A-T-E (epimerization) domain
  • the third module C-A-T domain
  • the forth module and the fifth module C-A-T-E domain;
  • the sixth module C-A-T-TE (termination) domain (see Fig. 2) .
  • the genes encoding the domain and module of fusaricidin synthetase are represented by SEQ. ID. NO: 3 ⁇
  • the present invention also provides a fusaricidin synthetase produced by the combination of the amino acid additional modules.
  • Each fusaricidin synthetase is formed by the combination of modules arranged as C-A-T, C-A-T-E or C-A-T- TE. Therefore, the construction of such recombinant expression vector that contains the combination of genes corresponding to each module leads to the diversity of fusaricidin synthetases.
  • the present invention provides a preparation method of fusaricidin or its derivatives comprising the following steps: 1) Inserting a gene encoding the polypeptide involved in fusaricidin synthesis into an expression vector;
  • step 3 Culturing the transformant of step 2); and 4) Isolating and purifying fusaricidin or its derivatives from the culture product of step 3) .
  • Fig. 1 is a diagram illustrating the structure of fusaricidin isolated from Paenibacillus polymyxa E681,
  • Fig. 2 is a diagram illustrating the structure of the domain of a fusaricidin synthetase gene originated from Paenibacillus polymyxa E681 genome,
  • A A (adenylation) domain
  • C C (condensation) domain
  • E E (epimerization) domain
  • T T (thiolation) domain
  • TE TE (termination) domain
  • Fig. 3 is a diagram illustrating the structure of fusaricidin predicted from the domain structure of fusaricidin biosynthesis gene isolated from Paenibacillus polymyxa E681 genome.
  • Example 1 Isolation and analysis of fusaricidin from Paenibacillus polymyxa
  • Paenibacillus polymyxa E681 (KCTC 8801P) was cultured in the medium designed by Paulus and Gray (Paulus H and
  • the supernatant was analyzed by LC/MS (Thermo electron Co., USA) using a mixed solvent of water and acetonitrile containing 0.1% formic acid under the condition of 0.2 M/min. (M+H) + ion peaks was 883, 897 and 911, which were the molecular weights of respectively fusaricidin A, fusaricidin B and LI-F05b.
  • LI-F05b is a member of fusaricidin series having only a difference in amino acid residues in the whole structure (Kurusu K, Ohba K, Arai T and Fukushima K. J " . Antibiotics 40:1506-1514, 1987) .
  • the nucleotide sequence of Paenibacillus polymyxa E681 genome was completely sequenced by whole-genome shotgun sequencing strategy and then the fusaricidin biosynthesis gene was identified.
  • 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. Ill Cloning systems (CSHL Press, Cold Spring Harbor, NY, 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, CT, USA) with 19% strength, 0.3/3 sec of pulse on/off time, 6 times.
  • the DNA fragments of 2 kb, 5kb, 8kb and 10 kb in size were recovered and used to construct the library.
  • pUC18, pUC19, pUC118 or pBCKS (Stratagene, La Jolla, CA, 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, WI, USA) and the BAC library was constructed by inserting the chromosomal DNA digested with HindiII into plndigo 536 vector (Peterson D. G. et al . , J. Agric. Genomics, 5, 2000; www.ncgr.org/research/jag / Luo M. et al . , Genome 44:154-62, 2001) .
  • the reactant for the plasmid library was inserted into E.
  • coli DHlOB by electroporation, which was smeared on a LB agar plate medium containing X-gal/lPTG/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.
  • 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, CA, USA) . Files containing the results were analyzed with phred/phrap/consed program (http : //www. 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.
  • the whole nucleotide sequence of the identified Paenibacillus polymyxa E681 genome was approximately 5.4 Mbps in total length and had single circular chromosome structure (%G+C: 45.8) .
  • Psort-B was used to predict the location of a protein (Gardy J. L. et al . , Nucleic Acids Res., 31:3613-3617, 2003).
  • 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.
  • Example 3 Prediction of fusaricidin structure from the nucleotide sequence of fusaricidin biosynthesis gene NRPS is a high molecular protein, which is selectively binding to a specific amino acid to activate it and then assemble the amino acids stepwisely. The recognition and activation of an amino acid happen in A domain. Recently, three-dimensional structure of A domain recognizing phenylalanine of gramicidin biosynthesis gene has been identified and a specific amino acid binding site therein was associated with 8 amino acid residues (Conti E, Stachelhaus T, Marahiel MA and Brick P. EMBO J. 16:4174- 4183, 1997).
  • the fusaricidin 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 . As a result, as illustrated in Fig. 2, each A domain recognized such amino acids as Thr, Leu/Ile/Val, Tyr, Thr, Asn and VaI.
  • the fusaricidin structure was as shown in Fig. 3.
  • fusaricidins A and B were isolated from E681 strain and fusaricidin A included L-Thr, D-VaI, L-VaI, D- allo-Thr, D-Asn, D-AIa, etc, and fusaricidin B included L- Thr, D-VaI, L-VaI, D-allo-Thr, D-GIn, D-AIa, etc. From the whole genome assay of E681 was confirmed that only one gene was involved in fusaricidin synthesis. The third A domain recognized Tyr or VaI, the fifth A domain recognized Asn or GIn and the sixth A domain recognized VaI or Ala. This result indicates that fusaricidin biosynthesis gene isolated from E681 strain can produce various fusaricidins.
  • the present inventors completed this invention by confirming that Paenibacillus polymyxa E681 produces fusaricidin, identifying fusaricidin biosynthesis gene by whole genome analysis, and confirming by domain analysis that the gene of the invention generates fusaricidin.
  • fusaricidin 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 was identified as fusaricidin biosynthesis gene.
  • the fusaricidin synthetase of the invention can be effectively used for the development of a novel antibiotic and the increase of productivity of fusaricidin.
  • SEQ. ID. NO: 1 is the sequence of fusaricidin biosynthesis gene
  • SEQ. ID. NO: 2 is the amino acid sequence of fusaricidin synthetase
  • SEQ. ID. NO: 3 is N terminal (88 aa : 1-88)
  • SEQ. ID. NO: 4 is Cl (435 aa : 89-523)
  • SEQ. ID. NO: 5 is Al (513 aa : 523-1035)
  • SEQ. ID. NO: 6 is Al-Tl linker (18 aa : 1036- 1053)
  • SEQ. ID. NO: 7 is Tl (64 aa : 1054-1117)
  • SEQ. ID. NO: 8 is T1-C2 linker (21 aa: 1118-1138)
  • SEQ. ID. NO: 9 is C2 (424 aa: 1139-1562)
  • SEQ. ID. NO: 10 is A2 (509 aa : 1560-2068) ,
  • SEQ. ID. NO: 11 is A2-T2 linker (18 aa: 2069-2086), SEQ. ID. NO: 12 is T2 (60 aa : 2087-2146), SEQ. ID. NO: 13 is T2-E2 linker (21 aa: 2147-2167), SEQ. ID. NO: 14 is E2 (458 aa: 2168-2625), SEQ. ID. NO: 15 is E2-C3 linker (11 aa: 2626-2636), SEQ. ID. NO: 16 is C3 (436 aa : 2637-3072), SEQ. ID. NO: 17 is A3 (505 aa : 3068-3572), SEQ. ID.
  • SEQ. ID. NO: 18 is A3-T3 linker (20 aa : 3573-3592), SEQ. ID. NO: 19 is T3 (64 aa: 3593-3656), SEQ. ID. NO: 20 is T3-C4 linker (21 aa : 3657-3677) ,
  • SEQ. ID. NO: 21 is C4 (426 aa : 3678-4103), SEQ. ID.
  • SEQ. ID. NO: 22 is C4-A4 linker (11 aa: 4104-4114), SEQ. ID. NO: 23 is A4 (502 aa: 4115-4616), SEQ. ID. NO: 24 is A4-T4 linker
  • SEQ. ID. NO: 25 is T4 (62 aa : 4636- 4697 )
  • SEQ . ID . NO : 26 is T4 -E4 linker ( 17 aa : 4698 -4714 )
  • SEQ. ID. NO: 27 is E4 (457 aa : 4715-5171)
  • SEQ. ID. NO: 28 is E4-C5 linker (11 aa: 5172-5182)
  • SEQ. ID. NO: 29 is C5 (438 aa: 5183-5620)
  • SEQ. ID. NO: 30 is A5 (515 aa: 5617- 6131)
  • SEQ. ID. NO: 31 is A5-T5 linker (15 aa : 6132-6146)
  • SEQ. ID. NO: 32 is T5 (63 aa : 6147-6209)
  • SEQ. ID. NO: 33 is T5-E5 linker (17 aa: 6210-6226)
  • SEQ. ID. NO: 34 is E5 (459 aa: 6227-6685)
  • SEQ. ID. NO: 35 is E5-C6 linker (10 aa: 6686-6695)
  • SEQ. ID. NO: 36 is C6 (439 aa : 6696-7134)
  • SEQ. ID. NO: 37 is A6 (520 aa : 7129-7648)
  • SEQ. ID. NO: 38 is A6-T6 linker (19 aa : 7649-7667), SEQ. ID.
  • SEQ. ID. NO: 39 is T6 (63 aa: 7668-7730), SEQ. ID. NO: 40 is T6-TE linker (26 aa : 7731-7756), SEQ. ID. NO: 41 is TE (152 aa : 7757-7908).

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Abstract

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

Description

[DESCRIPTION] [invention Title]
FUSARICIDIN SYNTHETASE AND GENE THEREOF
[Technical Field]
The present invention relates to a fusaricidin synthetase isolated from Gram-positive Paenibacillus sp . and a gene encoding thereof, more precisely a fusaricidin synthetase isolated from Paenibacillus polymyxa E681, a gene encoding thereof and a preparation method of fusaricidin or its derivatives using the gene.
[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 according to 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-atnino acid, mutation and oxidation in main peptide chain forming linear, cyclic or branched cyclic structure, acylation, glycosylation, N-methylation and heterocyclic ring formation.
Fusaricidin synthetase, one of NRPSs, stepwisely combines each amino acid monomer forming fusaricidin 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 domain (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., EMBO J. 16:4174-4183, 1997). 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 led to the understanding of linkage of a specific A domain to an amino acid (Challis G. L. et al., Chem. Biol. 7:211-224, 2000). 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 . , MoI. Gen. Genet., 257:308-318, 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. 6:319-325, 1999) .
Fusaricidin is an antibiotic isolated from
Paenibacillus sp, which has a ring structure composed of 6 amino acid residues in addition to 15-guanidino-3- hydroxypentadecanoic acid (see Fig. 3) . Fusaricidins isolated from Paenibacillus polymyxa so far are LI-F03, LI- F04, LI-F05, LI-F07 and LI-F08 (Kurusu K, Ohba K, Arai T and Fukushima K. J". Antibiotics 40:1506-1514, 1987) and additional fusaricidins A, B, C and D have been reported (Kajimura Y and Kaneda M. J". Antibiotics 49:129-135, 1996; Kajimura Y and Kaneda M. J. Antibiotics 50:220-228, 1997). The amino acid chain of fusaricidin is not ribosomally generated by being encoded like any other general polypeptide but generated by non-ribosomal peptide synthetase (Marahiel MA, Stachelhaus T and Mootz HD. Chem. Rev. 97:2651-2673, 1997; Doekel S and Marahiel MA. Metab. Eng. 6:64-77, 2001) .
Fusaricidin has excellent germicidal activity to plant pathogenic fungi such as Fusarium oxysporum, Aspergillus niger, Aspergillus oryzae and Penicillum thomii, and particularly fusaricidin B has germicidal activity to Candida albicans and Saccharomyces cerevisiae. Fusaricidin also has excellent germicidal activity to Gram-positive bacteria including Staphylococcus aureus (Kajimura Y and Kaneda M. J. Antibiotics 49:129-135, 1996; Kajimura Y and Kaneda M. J". Antibiotics 50:220-228, 1997). In addition, fusaricidin has antifungal activity against Leptosphaeria maculans which causes black root rot of canola (Beatty PH and Jensen SE. Can. J. Microbiol. '48: 159-169, 2002). According to the recent reports on the excellent germicidal activity of fusaricidin against pathogenic Gram- positive bacteria and plant pathogenic fungi, fusaricidin seems to have great potential for industrial uses and thereby is in increasing demand. As a part of the effect to increase productivity of an antibiotic, an antibiotic biosynthesis gene was inserted into a host strain to industrially mass-produce the antibiotic (Eppelmann K, Doekel S and Marahiel MA. J". Biol. Chem. 276:34824-34831, 2001; Pfeifer BA and Khosla C. Microbiol. MoI. Biol. Rev. 65:106-118, 2001). It was also tried to substitute a promoter of the antibiotic biosynthesis gene with a stronger promoter to increase productivity (Tsuge K, Akiyama T and Shoda M. J". Bacteriol. 183:6265-6273, 2001). However, any fusaricidin biosynthesis gene has not been identified, yet, and thus the reported methods seem not to be effective to increase productivity of fusaricidin. There has been an attempt to develop a novel antibiotic by re-constructing modules or domains of an antibiotic biosynthesis gene (Mootz HD, Schwarzer D and Marahiel MA. Proc. Natl. Acad. Sci . USA 97:5848-5853, 2000; Ferra FD, Rodriguez F, Tortora O. Tosi C and Grandi G. J". Biol. Chem. 272:25304-25309, 1997) or replacing a certain amino acid in a domain (Eppelmann K, Stachelhaus T and Marahiel MA. Biochemistry 41:9718-9726, 2000), which is expected to contribute to the development of a novel antibiotic having excellent activity. However, to do so, the fusaricidin biosynthesis gene has to be first identified. Therefore, it is important to identify a fusaricidin biosynthesis gene and secure the information on the gene to increase production of fusaricidin or develop fusaricidin based novel antibiotics.
The present inventors isolated, purified and analyzed fusaricidin from Paenibacillus polymyxa E681. And the inventors confirmed that the strain produced fusaricidin and found out and isolated a gene encoding NRPS by sequencing of the entire nucleotide sequence. The present inventors finally completed this invention by confirming with the domain analysis that the gene was a fusaricidin biosynthesis gene.
[Disclosure] [Technical Problem]
It is an object of the present invention to provide a fusaricidin synthetase isolated from Paenibacillus polymyxa E681, a gene encoding the enzyme, and a preparation method of fusaricidin or its derivatives using the gene. [Technical Solution]
The present invention provides a polypeptide involved in fusaricidin synthesis.
The present invention also provides a gene encoding the polypeptide.
The present invention further provides a recombinant vector containing the gene.
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 fusaricidin synthetase, in which C-A-T, C-A-T-E or C-A-T-TE domains are combined in order.
The present invention also provides a gene encoding each amino acid additional module. The present invention also provides a fusaricidin synthetase produced by the combination of the amino acid additional modules.
And, the present invention provides a preparation method of fusaricidin or its derivatives comprising the following steps:
1) Inserting a gene encoding the polypeptide involved in fusaricidin synthesis into an expression vector;
2) Transforming a host cell with the expression vector containing the gene of step 1) ; 3) Culturing the transforman'c of step 2) ; and 4) Isolating and purifying fusaricidin 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 produce peptides of enormous structural diversity, compared with ribosomally synthesized peptides.
Fusaricidin: an antibiotic isolated from Bacillus sp or Paenibacillus sp, which is generated by NRPS not by ribosomal synthesis after being encoded.
Fusaricidin synthetase: one of NRPSs, which stepwisely combines each amino acid monomer forming fusaricidin in order 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, T domains and additional E, 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 the 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 polypeptide involved in fusaricidin synthesis and a gene encoding the same.
The polypeptide involved in fusaricidin synthesis of the invention is represented by SEQ. ID. NO: 2 and its variants, that is, other polypeptides which are equally functioning but modified by addition, deletion and substitution of one or more modules, domains and/or amino acids are also included in the criteria of the invention. All genes encoding the polypeptides and their variants above are also included in the criteria of the invention and the one represented by SEQ. ID. NO: 1 is preferred. The fusaricidin of the invention has the polyketidic ring structure organized by the stepwise binding of L-Thr
(threonine) , D-VaI (valine) , L-VaI or L-Tyr (tyrosine) , D- allo-Thr, D-Asn or D-GIn, and D-AIa (alanine) or D-VaI to the amino group of GHPD (15-guanidino-3- hydroxypentadecanoic acid) (see Fig. 1) . The fusaricidins possibly isolated from Paen.ibacillus polymyxa are fusaricidin A, fusaricidin B, fusaricidin C, fusaricidin D and L1-F03, LI-F04, LI-F07 and LI-F08, etc, but not always limited thereto.
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 fusaricidin biosynthesis gene 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., MoI. Biol. Evol. 16:512, 1999), glimmer (Delcher A. L. et al . , Nucleic Acids Res. 27:4636, 1999) and zcurve (Guo F. -B. et al . , Nucleic Acids Res. 31:1780, 2003) 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., Nucleic Acids Res. 25:3389-3402, 1997). Next, domain and protein family analysis (Bateman A. et al . , Nucleic Acids Res. 32 (Database issue) :D138-141, 2004; Haft D. H. et al., Nucleic Acids Res. 31:371-373, 2003), motif and pattern screening (HuIo N. et al . , Nucleic Acids Res. 32 (Database issue) :D134-137 , 2004) and protein site prediction analysis (Gardy J. L. et al . , Nucleic Acids Res. 31:3613-3617, 2003) were performed.
From the above screening, at least 4 NRPS genes encoding 4 different antibiotic synthetases have been identified.
The substrate specificity of adenylation (A) domain of each gene 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 . , Chem. Biol. 7:211-224, 2000). As a result, one of the genes was identified as the gene encoding fusaricidin synthetase (see Fig. 2) .
Each polypeptide involved in fusaricidin biosynthesis 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 .
Each A domain of module composing fusaricidin synthetase recognized such amino acids as Thr, Leu/lle/Val, Tyr, Thr, Asn and VaI, as shown in Fig. 2. However, as explained in Example 1, fusaricidins A and B were isolated from E681 strain and fusaricidin A included L-Thr, D-VaI, L-VaI, D-allo-Thr, D-Asn, D-AIa, etc, and fusaricidin B included L-Thr, D-VaI, L-VaI, D-allo-Thr, D-GIn, D-AIa, etc. From the whole genome assay of E681 was confirmed that only one gene was involved in fusaricidin synthesis. The third A domain recognized Tyr or VaI, the fifth A domain recognized Asn or Gin and the sixth A domain recognized VaI or Ala. Thus, the predicted structure of fusaricidin was proved to be the' polyketidic peptide ring organized by the stepwise binding of L-Thr (threonine) D- VaI (valine), L-Tyr (tyrosine) or L-VaI, D-allo-Thr, D-Asn or D-GIu and lastly D-VaI or D-AIa (see Fig. 3) . So, the gene of the present invention was confirmed to be the fusaricidin synthetase and be able to produce fusaricidin of diversity (see Fig. 3) .
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 . , MoI. Gen. Genet., 257:308- 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 fusaricidin derivatives or a novel antibiotic according to the above method.
The present invention also provides a recombinant vector containing the gene of the invention and a host cell transformed with the vector.
The gene encoding fusaricidin 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 fusaricidin 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 one of the conventional methods 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 fusaricidin synthetase C-A-T, C-A-T-E or C-A-T-TE and a gene encoding each amino acid additional module.
Each module forming a polypeptide is described herein. First, a polypeptide is organized by the following 6 modules,
The first module: C (condensation) -A (adenylation) - T (thiolation) domain;
The second module: C-A-T-E (epimerization) domain; The third module: C-A-T domain;
The forth module and the fifth module: C-A-T-E domain; and
The sixth module: C-A-T-TE (termination) domain (see Fig. 2) . The genes encoding the domain and module of fusaricidin synthetase are represented by SEQ. ID. NO: 3 ~
NO: 41, in which linker genes combining each domain are also included. The SEQ. ID. NOs. of each domain are presented in Table 1.
The present invention also provides a fusaricidin synthetase produced by the combination of the amino acid additional modules. Each fusaricidin synthetase is formed by the combination of modules arranged as C-A-T, C-A-T-E or C-A-T- TE. Therefore, the construction of such recombinant expression vector that contains the combination of genes corresponding to each module leads to the diversity of fusaricidin synthetases.
And, the present invention provides a preparation method of fusaricidin or its derivatives comprising the following steps: 1) Inserting a gene encoding the polypeptide involved in fusaricidin synthesis into an expression vector;
2) Transforming a host cell with the expression vector containing the gene of step 1) ;
3) Culturing the transformant of step 2); and 4) Isolating and purifying fusaricidin 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 is a diagram illustrating the structure of fusaricidin isolated from Paenibacillus polymyxa E681,
Fig. 2 is a diagram illustrating the structure of the domain of a fusaricidin synthetase gene originated from Paenibacillus polymyxa E681 genome,
A: A (adenylation) domain, C: C (condensation) domain, E: E (epimerization) domain, T: T (thiolation) domain, TE: TE (termination) domain
Fig. 3 is a diagram illustrating the structure of fusaricidin predicted from the domain structure of fusaricidin biosynthesis gene isolated 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 fusaricidin from Paenibacillus polymyxa
<!-!> Culture of Paenibacillus polymyxa
Paenibacillus polymyxa E681 (KCTC 8801P) was cultured in the medium designed by Paulus and Gray (Paulus H and
Gray E. J. Biol. Chem. 239:865-871, 1964) under aerobic condition at 25 "C with 180 rpm for 3 days, followed by centrifugation (7000 rpm, 10 min) to obtain supernatant.
<l-2> Identification of fusaricidin by LC/MS analyzing system The composition of the supernatant was analyzed by LC/MS system. To separate and purify the supernatant, the culture solution was centrifuged at 8,000 rpm for 20 minutes to eliminate cells and the supernatant was extracted with butanol, concentrated under the reduced pressure in a rotary evaporator to eliminate butanol and proceeded to silica gel column containing chloroform:methanol (4:1 ~ 2:1) to give active fractions. The fractions were concentrated again under the reduced pressure and proceeded to Sephadex LH-20 using methanol as a solvent, followed by HPLC, resulting in the pure active fusaricidin A (20 mg) and fusaricidin B (8 mg) .
The supernatant was analyzed by LC/MS (Thermo electron Co., USA) using a mixed solvent of water and acetonitrile containing 0.1% formic acid under the condition of 0.2 M/min. (M+H) + ion peaks was 883, 897 and 911, which were the molecular weights of respectively fusaricidin A, fusaricidin B and LI-F05b. LI-F05b is a member of fusaricidin series having only a difference in amino acid residues in the whole structure (Kurusu K, Ohba K, Arai T and Fukushima K. J". Antibiotics 40:1506-1514, 1987) .
Example 2 : Sequencing of fusaricidin biosynthesis gene
The nucleotide sequence of Paenibacillus polymyxa E681 genome was completely sequenced by whole-genome shotgun sequencing strategy and then the fusaricidin biosynthesis gene was identified.
<2-l> 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. Ill Cloning systems (CSHL Press, Cold Spring Harbor, NY, 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, CT, USA) with 19% strength, 0.3/3 sec of pulse on/off time, 6 times. The DNA fragments of 2 kb, 5kb, 8kb and 10 kb in size were recovered and used to construct the library. pUC18, pUC19, pUC118 or pBCKS (Stratagene, La Jolla, CA, 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 (CopyControl™ fosmid library production kit, Epicentre Biotechnologies, Madison, WI, USA) and the BAC library was constructed by inserting the chromosomal DNA digested with HindiII into plndigo 536 vector (Peterson D. G. et al . , J. Agric. Genomics, 5, 2000; www.ncgr.org/research/jag/Luo M. et al . , Genome 44:154-62, 2001) . The reactant for the plasmid library was inserted into E. coli DHlOB by electroporation, which was smeared on a LB agar plate medium containing X-gal/lPTG/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 BigDye™ terminator cycle sequencing kit (Applied Biosystems, CA, USA) and the reactant was analyzed with ABI 3700 and 3730 DNA analyzer (Applied Biosystems, Foster City, CA, USA) . Files containing the results were analyzed with phred/phrap/consed program (http : //www. 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 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., MoI. Biol. Evol . 16:512, 1999), glimmer (Delcher A. L. et al . , Nucleic Acids Res. 27: 4636, 1999) and zcurve (Guo F. -B. et al . , Nucleic Acids Res. 31:1780, 2003) . 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 SF, et al :, Nucleic Acids Res. 25:3389-3402, 1997). At this time, the databases used were COG (Tatusov R. L. et al . , BMC Bioinformatics . 4:41, 2003), UniProt Knowledgebase (Bairoch A. et al . , Nucleic Acids Res. 33 (Database issue) :D154-159, 2005), NCBI-NR (ftp://ftp.ncbi.nih.gov/blast/db/nr.tar.gz) and KEGG-Genes (Kanehisa M. et al . , Nucleic Acids Res. 32 (Database issue) :D277-280, 2004) . For the analysis of domain and protein family, Pfam (Bateman A. et al . , Nucleic Acids Res. 32 (Database issue) :D138-141, 2004) and TIGRFAMs (Haft DH, et al . , Nucleic Acids Res. 31:371-373, 2003) databases were used. For the investigation of motif and pattern, Prosite (HuIo N. et al., Nucleic Acids Res. 32 (Database issue) :D134-137, 2004) database was used.
Psort-B was used to predict the location of a protein (Gardy J. L. et al . , Nucleic Acids Res., 31:3613-3617, 2003). 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 NPRS genes encoding 4 different antibiotic synthetases were 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 . , Chem. Biol. 7:211-224, 2000). As a result, one of them was identified as the gene encoding fusaricidin synthetase (Fig. 2) .
Example 3 : Prediction of fusaricidin structure from the nucleotide sequence of fusaricidin biosynthesis gene NRPS is a high molecular protein, which is selectively binding to a specific amino acid to activate it and then assemble the amino acids stepwisely. The recognition and activation of an amino acid happen in A domain. Recently, three-dimensional structure of A domain recognizing phenylalanine of gramicidin biosynthesis gene has been identified and a specific amino acid binding site therein was associated with 8 amino acid residues (Conti E, Stachelhaus T, Marahiel MA and Brick P. EMBO J. 16:4174- 4183, 1997). The amino acid sequence of this A domain was compared with that of the conventional A domain and high homology in 8 amino acid residues was confirmed. 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 GL, Ravel J and Townsend CA. Chem. Biol. 7:211-224, 2000).
The fusaricidin 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 . As a result, as illustrated in Fig. 2, each A domain recognized such amino acids as Thr, Leu/Ile/Val, Tyr, Thr, Asn and VaI. The fusaricidin structure was as shown in Fig. 3. However, as explained in Example 1, fusaricidins A and B were isolated from E681 strain and fusaricidin A included L-Thr, D-VaI, L-VaI, D- allo-Thr, D-Asn, D-AIa, etc, and fusaricidin B included L- Thr, D-VaI, L-VaI, D-allo-Thr, D-GIn, D-AIa, etc. From the whole genome assay of E681 was confirmed that only one gene was involved in fusaricidin synthesis. The third A domain recognized Tyr or VaI, the fifth A domain recognized Asn or GIn and the sixth A domain recognized VaI or Ala. This result indicates that fusaricidin biosynthesis gene isolated from E681 strain can produce various fusaricidins.
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 ]
Figure imgf000027_0001
The present inventors completed this invention by confirming that Paenibacillus polymyxa E681 produces fusaricidin, identifying fusaricidin biosynthesis gene by whole genome analysis, and confirming by domain analysis that the gene of the invention generates fusaricidin.
[industrial Applicability] As explained hereinbefore, the present inventors confirmed that fusaricidin 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 was identified as fusaricidin biosynthesis gene. The fusaricidin synthetase of the invention can be effectively used for the development of a novel antibiotic and the increase of productivity of fusaricidin.
[Sequence List Text] SEQ. ID. NO: 1 is the sequence of fusaricidin biosynthesis gene,
SEQ. ID. NO: 2 is the amino acid sequence of fusaricidin synthetase, SEQ. ID. NO: 3 is N terminal (88 aa : 1-88), SEQ. ID. NO: 4 is Cl (435 aa : 89-523), SEQ. ID. NO: 5 is Al (513 aa : 523-1035), SEQ. ID. NO: 6 is Al-Tl linker (18 aa : 1036- 1053), SEQ. ID. NO: 7 is Tl (64 aa : 1054-1117), SEQ. ID. NO: 8 is T1-C2 linker (21 aa: 1118-1138) , SEQ. ID. NO: 9 is C2 (424 aa: 1139-1562), SEQ. ID. NO: 10 is A2 (509 aa : 1560-2068) ,
SEQ. ID. NO: 11 is A2-T2 linker (18 aa: 2069-2086), SEQ. ID. NO: 12 is T2 (60 aa : 2087-2146), SEQ. ID. NO: 13 is T2-E2 linker (21 aa: 2147-2167), SEQ. ID. NO: 14 is E2 (458 aa: 2168-2625), SEQ. ID. NO: 15 is E2-C3 linker (11 aa: 2626-2636), SEQ. ID. NO: 16 is C3 (436 aa : 2637-3072), SEQ. ID. NO: 17 is A3 (505 aa : 3068-3572), SEQ. ID. NO: 18 is A3-T3 linker (20 aa : 3573-3592), SEQ. ID. NO: 19 is T3 (64 aa: 3593-3656), SEQ. ID. NO: 20 is T3-C4 linker (21 aa : 3657-3677) ,
SEQ. ID. NO: 21 is C4 (426 aa : 3678-4103), SEQ. ID.
NO: 22 is C4-A4 linker (11 aa: 4104-4114), SEQ. ID. NO: 23 is A4 (502 aa: 4115-4616), SEQ. ID. NO: 24 is A4-T4 linker
(19 aa: 4617-4635), SEQ. ID. NO: 25 is T4 (62 aa : 4636- 4697 ) , SEQ . ID . NO : 26 is T4 -E4 linker ( 17 aa : 4698 -4714 ) , SEQ. ID. NO: 27 is E4 (457 aa : 4715-5171), SEQ. ID. NO: 28 is E4-C5 linker (11 aa: 5172-5182) , SEQ. ID. NO: 29 is C5 (438 aa: 5183-5620), SEQ. ID. NO: 30 is A5 (515 aa: 5617- 6131) , SEQ. ID. NO: 31 is A5-T5 linker (15 aa : 6132-6146),
SEQ. ID. NO: 32 is T5 (63 aa : 6147-6209), SEQ. ID. NO: 33 is T5-E5 linker (17 aa: 6210-6226), SEQ. ID. NO: 34 is E5 (459 aa: 6227-6685), SEQ. ID. NO: 35 is E5-C6 linker (10 aa: 6686-6695), SEQ. ID. NO: 36 is C6 (439 aa : 6696-7134), SEQ. ID. NO: 37 is A6 (520 aa : 7129-7648), SEQ. ID. NO: 38 is A6-T6 linker (19 aa : 7649-7667), SEQ. ID. NO: 39 is T6 (63 aa: 7668-7730), SEQ. ID. NO: 40 is T6-TE linker (26 aa : 7731-7756), SEQ. ID. NO: 41 is TE (152 aa : 7757-7908).
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

[CLAIMS]
[Claim l]
A polypeptide involved in fusaricidin synthesis or a variant thereof.
[Claim 2]
The polypeptide according to claim 1, which is represented by SEQ. ID. NO: 2.
[Claim 3]
The polypeptide according to claim 1, wherein the fusaricidin is selected from a group consisting of fusaricidin A, fusaricidin B, fusaricidin C, fusaricidin D, LI-F03, LI-F04, LI-F05, LI-F07 and LI-F08.
[Claim 4]
The polypeptide according to claim 1, wherein the polypeptide contains one or more modules and each module contains at least two domains selected from a group consisting of A, C, T, E and TE domains.
[Claim 5]
The polypeptide according to claim 4, wherein the structure has been modified by the addition, deletion or substitution of one or more modules, domains or amino acids, or the linkage between hydroxyl acid and D- and L-amino acid, mutation and oxidation of peptide chain, acylation, glycosylation, N-methylation and heterocyclic ring formation.
[Claim 6]
A gene encoding the polypeptide of claim 1 or claim 5.
[Claim 7]
The gene according to claim 6, which is represented by SEQ. ID. NO: 1.
[Claim 8] A recombinant vector containing the gene of claim 6.
[Claim 9]
A host cell transformed with the vector of claim 8.
[Claimio]
An amino acid additional module of the fusaricidin synthetase, which is characterized by the stepwise binding of C (condensation) domain selected from a group consisting of sequences represented by SEQ. ID. NOs: 4, 9, 16, 21, 29 and 36, A (adenylation) domain selected from a group consisting of sequences represented by SEQ. ID. NOs: 5, 10, 17, 23, 30 and 37, and T (thiolation) domain selected from a group consisting of sequences represented by SEQ. ID. NOs: 7, 12, 19, 25, 32 and 39.
[Claim ll]
A gene encoding the module of claim 10.
[Claiml2] An amino acid additional module of the fusaricidin synthetase, which is characterized by the stepwise binding of C (condensation) domain selected from a group consisting of sequences represented by SEQ. ID. NOs: 4, 9, 16, 21, 29 and 36, A (adenylation) domain selected from a group consisting of sequences represented by SEQ. ID. NOs: 5, 10, 17, 23, 30 and 37, T (thiolation) domain selected from a group consisting of sequences represented by SEQ. ID. NOs: 7, 12, 19, 25, 32 and 39, and E (epimerization) domain selected from a group consisting of sequences represented by SEQ. ID. NOs: 14, 17 and 34/
[Claim 13]
A gene encoding the module of claim 12.
[Claiml4] An amino acid additional module of the fusaricidin synthetase, which is characterized by the stepwise binding of C (condensation) domain selected from a group consisting of sequences represented by SEQ. ID. NOs: 4, 9, 16, 21, 29 and 36, A (adenylation) domain selected from a group consisting of sequences represented by SEQ. ID. NOs: 5, 10,
17, 23, 30 and 37, T (thiolation) domain selected from a group consisting of sequences represented by SEQ. ID. NOs:
7, 12, 19, 25, 32 and 39, and TE (termination) domain represented by SEQ. ID. NO: 41.
[Claim 15]
A gene encoding the module of claim 14.
[Claim 16]
A fusaricidin synthetase generated by the combination of modules of claim 10, claim 12 or claim 14.
[Claim 17] A preparation method of fusaricidin or its derivatives comprising the following steps:
1) Constructing a recombinant expression vector by inserting a gene encoding the fusaricidin synthetases of claim 6 into an expression vector; 2) Transforming a host cell with the expression vector containing the gene of step 1) ;
3) Culturing the transformant of step 2); and
4) Isolating and purifying fusaricidin or its derivatives from the culture product of step 3) .
PCT/KR2006/005126 2006-01-24 2006-11-30 Fusaricidin synthetase and gene thereof WO2007086645A1 (en)

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