# Patent

## Title
Biosynthetic gene cluster for chejuenolide of marine microorganism Hahella chejuensis

## Abstract

The present invention relates to a biosynthetic gene cluster for a chejuenolide of the marine microorganism Hahella chejuensis, and to the function of an enzyme involving the biosynthetic pathway of a chejuenolide encoded by the genes. Since the present invention can be applied to the development of a novel material as a mechanism for biosynthesis by combining of biosynthetic genes for chejuenolide, the present invention can suggest a new direction in the study of polyketide antibiotics, and is thus very useful.

## Background
TECHNICAL FIELD
The present invention relates to a biosynthetic gene cluster for chejuenolide that is a polyketide macrolide-based natural substance. More particularly, the present invention relates to isolated DNAs encoding enzymes associated with a biosynthetic pathway for a chejuenolide, and the function of a gene product associated with the production of chejuenolide. Also, the present invention relates to a mutant strain in which genes and enzymes associated with the biosynthesis of chejuenolide are disrupted, a linear polyketide that is a chejuenolide precursor for biosynthesizing the mutant strain, and a method of preparing the same.
BACKGROUND ART
Marine organisms have attracted attention as an important source for development of new medicines. In particular, marine microorganisms have been known to produce secondary metabolites having various structures and activities. The marine microorganisms often produce natural bioactive substances different from the species found in land environments as the biochemical metabolisms in the body are adapted to marine environments (salinity, a pressure, a high-concentration halogen, etc.) which are completely different from the land environments (Fenical and Jensen 2006; Marris 2006).
New 17-membered carbocyclic tetraene natural substances, chejuenolides A, B and C, have been found from a culture extract of a marine microorganism Hahella chejuensis MB-1084 isolated from a marine sediment within the intertidal region in coastal waters of Geojedo Island (Choi, Sohn et al. 2008; Seo and Oh 2009). Only an antibiotic, lankacidin, isoliated from Streptomyces greseofuscus, S. violaceoniger, and S. rochei var. volubilis was previously reported as the antibiotic containing a 17-membered carbocyclic ring (Uramoto, Otake et al. 1969; Higashide, Fugono et al. 1971). Lankacidin is a very powerful antibacterial agent that is currently used to treat an infection of Serpulina (Treponema) hyodysenteriae that is a type of bacteria called a spirochete causing diseases in animals and also known to have an anticancer activity and an immunosuppressive effect (Oostu, Matsumoto et al. 1975; Hayashi, Suenaga et al. 1988). Lankacidin have various kinds of analogs (some analogs are also referred to as T-2636 or bundlins). In this case, lankacidin C and chejuenolide highly resemble each other in a structural aspect (Harada and Kishi 1974; Harada 1975; Nakahama, Harada et al. 1975). However, the lankacidin is different from the chejuenolide in that the lankacidin has a 6-membered-δ-lactone ring structure in a 17-membered macrocyclic ring and a 2-hydroxy-propanamide chain at a 3rd carbon atom (Harada and Kishi 1974; Harada 1975; Choi, Sohn et al. 2008). It was reported that δ-lactone rings are disrupted as the metabolites, lankacyclinol and lankacyclinol A (12-O-acetyl lankacyclinol), obtained after administration of lankacidin C into rats undergo a decarboxylation action, but a macrolide-based polyketide natural substance having a 17-membered carbocyclic tetraene residue without a 6-membered-δ-lactone ring (deficient in ester bonds) is only a chejuenolide (Harada, Tanayama et al. 1973; Choi, Sohn et al. 2008).
The polyketide-based natural substance is one of the secondary metabolites from the microorganisms which are of medicinal importance, and belong to the antibiotics (tetracycline, erythromycin A, rifamycin S, etc.), the anticancer drugs (daunorubicin, epothilone, etc.), the cholesterol lowering agents (lovastatin, etc.), the anthelminthics (Avermectin, etc.), the antimicrobial drugs (Amphotericin B, etc.), the pesticides (Spinosy A, etc.), and the immunosuppressive drugs (rapamycin, FK506, etc.) (Staunton and Weissman 2001; Shen 2003).
A polyketide is biosynthesized by several simple condensation reactions of carboxylic acid by a polyketide synthase (PKS), a pathway of which is similar to the biosynthesis pathway of fatty acid (Shen 2003; Muller 2004). Type I polyketide is biosynthesized by type I PKS that is a huge multifunctional enzyme, one protein of which shows various enzymatic activities. In this case, such a PKS complex is composed of several modules associated with consecutive condensation reactions (Gokhale, Tsuji et al. 1999; Muller 2004). Each of the modules is composed of acyl transferase (AT), acyl carrier protein (ACP) and β-ketoacyl synthase (KS) domains, which are directly associated with the condensation reaction of carboxylic acid, and ketone reductase (KR), dehydratase (DH) and enoyl reductase (ER) domains which are associated with the reduction of β-ketone groups produced as a result of the condensation reaction (Kennedy, Auclair et al. 1999). The type of carboxylic acid that is an extension unit used in each condensation reaction is widely determined according to the characteristics of the AT domains of each module, a level of reduction of β-ketone groups produced as a result of the condensation reaction varies according to the presence of the reduction domains (KR, DH, and ER) in each module, and the length of the polyketide chain is determined according to the total number of the modules (Donadio and Katz 1992; MacNeil, Occi et al. 1992; Aparicio, Molnar et al. 1996). That is, polyketides having various structures may be biosynthesized according to the combinations of the respective modules and domains. In this aspect, PKS has become an important study subject for combinatorial biosynthesis in addition to a non-ribosomal peptide synthetase (NRPS; a peptide synthetase that is not synthesized in the ribosomes) (Cortes, Wiesmann et al. 1995; Hutchinson and Fujii 1995; Oliynyk, Brown et al. 1996; Jacobsen, Hutchinson et al. 1997; Katz 1997; McDaniel, Thamchaipenet et al. 1999; Menzella, Reid et al. 2005).
The marine microorganism H. chejuensis biosynthesizes a polyketide chejuenolide having a peculiar 17-membered macrocyclic structure. When enzymes contributing to the biosynthesis pathway of chejuenolide, and genes encoding the enzymes are elucidated and then applied to development of novel materials as a mechanism for combinatorial biosynthesis, there is a high probability of addressing the new directionality to studies of polyketide-based antibiotics. As the prior-art technique of the present invention, Korean Registered Patent No. 10-0661175 (Dec. 22, 2006) discloses an algicidal preparation including prodigiosin and a prodigiosin biosynthetic gene cluster, and Japanese Unexamined Patent Laid-open Publication No. JP P2006-296419A (Nov. 2, 2006) discloses a method for producing an antibiotic-producing microorganism by gene disruption and the resultant antibiotic-producing microorganism and a method for producing an antibiotic metabolic intermediate (Wnashi Y. and Arakawa K. (2006).). Also, U.S. Pat. No. 4,914,206 (Apr. 3, 1900) discloses lankacidin derivatives and a production method thereof (Minamida. I. and Hashimoto N. (1993) Lankacidin derivatives and production thereof; JP62240687 (Oct. 21, 1987), U.S. Pat. No. 4,914,206 (Apr. 3, 1990), CN1014050 (Jun. 20, 1993), and EP00226896 (Sep. 15, 1993)). However, the isolated gene cluster required to biosynthesize a chejuenolide, and the functions of enzymes encoded by the gene cluster remains to be elucidated.
PRIOR-ART DOCUMENTS
Patent Documents


    Korean Registered Patent No. 10-0661175 (Dec. 22, 2006)
    Japanese Unexamined Patent Laid-open Publication No. JP P2006-296419A (Nov. 2, 2006)
    U.S. Registered Patent No. 04914206 (Apr. 3, 1900)


Non-patent Documents


    Arakawa, K., F. Sugino, et al. (2005). “Cyclization mechanism for the synthesis of macrocyclic antibiotic lankacidin in Streptomyces rochei.” Chem. Biol. 12(2): 249-256.
    Choi, Y. H., J. H. Sohn, et al. (2008). “Chejuenolides A and B, new macrocyclic tetraenes from the marine bacterium Hahella chejuensis.” Tetrahedron Lett. 49 (50): 7128-7131.
    Dickschat, J. S., O. Vergnolle, et al. (2011). “An Additional Dehydratase-Like Activity is Required for Lankacidin Antibiotic Biosynthesis.” ChemBioChem 12 (16): 2408-2412.
    Seo, C. and H. Oh (2009). “Chejuenolide C: A New Macrocyclic Metabolite from the Marine Bacterium Hahella chejuensis.” Bull. Korean Chem. Soc. 30(5): 1181-1183.
    Tatsuno, S., K. Arakawa, et al. (2007). “Analysis of modular-iterative mixed biosynthesis of lankacidin by heterologous expression and gene fusion.” J. Antibiot. 60(11): 700-708.
    Tatsuno, S., K. Arakawa, et al. (2009). “Extensive mutational analysis of modular-iterative mixed polyketide biosynthesis of lankacidin in Streptomyces rochei.” Biosci. Biotechnol. Biochem. 73(12): 2712-2719.


DISCLOSURE
Technical Problem
The present inventors cloned a chejuenolide biosynthetic gene from a marine microorganism Hahella chejuensis and sequenced the chejuenolide biosynthetic gene. Therefore, the present invention has been completed based on these facts.
Therefore, an object of the present invention provides DNA sequences of genes encoding respective proteins associated with the biosynthesis of a chejuenolide from H. chejuensis. 
Another object of the present invention provides a method of preparing a chejuenolide mutant using a mutant strain in which one or more of the genes having the DNA sequences are deleted or inactivated.
However, the other objects and advantages of the present invention will become more apparent to those of ordinary skill in the art by describing in detail exemplary embodiments thereof.
Technical Solution
According to one aspect of the present invention, there is provided a biosynthetic gene cluster for an isolated chejuenolide having a DNA sequence set forth in SEQ ID NO: 1.
According to another aspect of the present invention, there is provided a protein having amino acid sequences set forth in SEQ ID NOS: 2 to 27 encoded by the biosynthetic gene cluster.
According to still another aspect of the present invention, there is provided an isolated gene encoding the protein having amino acid sequences set forth in SEQ ID NOS: 2 to 27.
According to still another aspect of the present invention, there is provided a gene encoding a protein having at least one amino acid sequence selected from the group consisting of the followings:
(a) a polyketide synthesis module protein having an amino acid sequence set forth in at least one selected from the group consisting of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 13;
(b) an amine oxidase having an amino acid sequence set forth in SEQ ID NO: 9;
(c) an ABC transporter membrane permease, a transmembrane protein, an ABC transporter periplasmic protein, or an ABC transporter ATPase having an amino acid sequence set forth in at least one selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, and SEQ ID NO: 23;
(d) a transcriptional regulatory enzyme having an amino acid sequence set forth in SEQ ID NO: 14 or SEQ ID NO: 26; and
(e) an isochorismatase, a haloacid dehalogenase, an aminopeptidase, or a NADPH-quinone reductase having an amino acid sequence set forth in at least one selected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 15, SEQ ID NO: 24 and SEQ ID NO: 25.
According to still another aspect of the present invention, there is provided a vector containing at least one of the genes.
According to still another aspect of the present invention, there is provided a microorganism transformed with the vector.
According to still another aspect of the present invention, there is provided a method of preparing a chejuenolide, which includes culturing the microorganism.
According to still another aspect of the present invention, there is provided a transformant microorganism for producing a chejuenolide or a precursor thereof in which one or more of genes encoding proteins having amino acid sequences set forth in SEQ ID NOS: 2 to 27 are disrupted, wherein the transformant microorganism includes a chejuenolide biosynthetic gene set forth in SEQ ID NO: 1 in the genome thereof.
According to still another aspect of the present invention, there is provided a chejuenolide precursor O3P2 represented by the following Formula 1




According to yet another aspect of the present invention, there is provided a method of preparing a precursor of chejuenolide. Here, the method includes culturing a H. chejuensis mutant strain in which a 20,900th to 19,482nd region (orf3) encoding the amine oxidase in the DNA sequence set forth in SEQ ID NO: 1 is deleted or inactivated.
Hereinafter, the present invention will be described in detail.
The present invention provides a set of isolated nucleic acid molecules required to biosynthesize a chejuenolide. According to one exemplary embodiment of the present invention, the nucleic acid molecules represent a cluster of biosynthetic genes for a chejuenolide isolated from H. chejuensis sp. MB-1084, and are selected from an adjacent DNA sequence (SEQ ID NO: 1) consisting of 26 open reading frames (ORFs) encoding enzymes required for the chejuenolide formation (see FIG. 1). Amino acid sequences of the enzymes encoded by the 26 ORFs are set forth in SEQ ID NOS: 2 to 27, respectively.
Therefore, according to one aspect of the present invention, there is provided a biosynthetic gene cluster for an isolated chejuenolide having a DNA sequence set forth in SEQ ID NO: 1.
According to another aspect of the present invention, there is provided a protein having amino acid sequences set forth in SEQ ID NOS: 2 to 27 encoded by the biosynthetic gene cluster.
According to still another aspect of the present invention, there is provided an isolated gene encoding the protein having amino acid sequences set forth in SEQ ID NOS: 2 to 27.
According to still another aspect of the present invention, there is provided a gene encoding a protein having at least one amino acid sequence selected from the group consisting of the followings:
(a) a polyketide synthesis module protein having an amino acid sequence set forth in at least one selected from the group consisting of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 13;
(b) an amine oxidase having an amino acid sequence set forth in SEQ ID NO: 9;
(c) an ABC transporter membrane permease, a transmembrane protein, an ABC transporter periplasmic protein, or an ABC transporter ATPase having an amino acid sequence set forth in at least one selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, and SEQ ID NO: 23;
(d) a transcriptional regulatory enzyme having an amino acid sequence set forth in SEQ ID NO: 14 or SEQ ID NO: 26; and
(e) an isochorismatase, a haloacid dehalogenase, an aminopeptidase, or a NADPH-quinone reductase having an amino acid sequence set forth in at least one selected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 15, SEQ ID NO: 24 and SEQ ID NO: 25.
According to one exemplary embodiment, the gene encoding the protein in group (a) may have DNA sequences set forth in a 11,776th to 8,576th region (orfA) of SEQ ID NO: 1, a 18,803rd to 11,766th region (orf1) of SEQ ID NO: 1, a 21,800th to 20,919th region (orf4) of SEQ ID NO: 1, a 27,598th to 21,863rd region (orf5) of SEQ ID NO: 1, a 28,462nd to 27,605th region (orf6) of SEQ ID NO: 1, and a 33,473rd to 28,431st region (orf7) of SEQ ID NO: 1.
The gene encoding the protein in group (b) may have a DNA sequence set forth in a 20,900th to 19,482nd region (orf3) of SEQ ID NO: 1.
The gene encoding the protein in group (c) may have DNA sequences set forth in a 8,507th to 7,212th region (orfB) of SEQ ID NO: 1, a 6,300th to 2,431st region (orfC) of SEQ ID NO: 1, a 2,082nd to 1,327th region (orfD) of SEQ ID NO: 1, a 1,071st to 307th region (orfE) of SEQ ID NO: 1, a 39,788th to 41,404th region (orf13) of SEQ ID NO: 1, a 41,488th to 42,408th region (orf14) of SEQ ID NO: 1, a 42,517th to 43,350th region (orf15) of SEQ ID NO: 1, a 43,445th to 44,410th region (orf16) of SEQ ID NO: 1, and a 44,435th to 45,430th region (orf17) of SEQ ID NO: 1.
The gene encoding the protein in group (d) may have DNA sequences set forth in a 34,641st to 35,405th region (orf8) of SEQ ID NO: 1, and a 48,357th to 49,250th region (orf20) of SEQ ID NO: 1.
The gene encoding the protein in group (e) may have DNA sequences set forth in a 19,469th to 18,915th region (orf2) of SEQ ID NO: 1, a 36,205th to 35,576th region (orf9) of SEQ ID NO: 1, a 45,557th to 47,344th region (orf18) of SEQ ID NO: 1, and a 48,233rd to 47,682nd region (orf19) of SEQ ID NO: 1.
According to one exemplary embodiment, each of the isolated nucleic acid molecules according to one exemplary embodiment of the present invention may include a combination of the ORFs selected from orfA to orfE and orf1 to orf21 (SEQ ID NOS: 2 to 27, respectively), which are required to biosynthesize the polyketide chain of the chejuenolide.
According to another exemplary embodiment, each of the nucleic acid molecules according to one exemplary embodiment of the present invention may include a combination of the ORFs selected from orfA to orfE and orf1 to orf21 (SEQ ID NOS: 2 to 27, respectively), which encode enzymes required to extend the polyketide chain of the chejuenolide.
According to still another exemplary embodiment, each of the nucleic acid molecules according to one exemplary embodiment of the present invention may include a combination of the ORFs selected from orfA to orfE and orf1 to orf21 (SEQ ID NOS: 2 to 27, respectively), which encode enzymes required to terminate the extension of the polyketide chain of the chejuenolide and cyclize the polyketide chain of the chejuenolide.
According to still another exemplary embodiment, each of the nucleic acid molecules according to one exemplary embodiment of the present invention may include a combination of the ORFs selected from orfA to orfE and orf1 to orf21 (SEQ ID NOS: 2 to 27, respectively), which encode enzymes required to transport the chejuenolide to the outside and have a tolerance to the chejuenolide.
According to still another exemplary embodiment, each of the nucleic acid molecules according to one exemplary embodiment of the present invention may include a combination of the ORFs selected from orfA to orfE and orf1 to orf21 (SEQ ID NOS: 2 to 27, respectively), which encode enzymes required to regulate expression of the enzymes for biosynthesizing a chejuenolide.
According to yet another exemplary embodiment, each of the nucleic acid molecules including at least one DNA segment selected from SEQ ID NO: 1, which promote an expression level of the ORFs selected from orfA to orfE and orf1 to orf21 (SEQ ID NOS: 2 to 27, respectively) are provided.
The present inventors appreciate that the present invention providing the nucleic acid sequences encoding the enzymes associated with the chejuenolide biosynthesis also provides nucleic acid bases encoding fragments derived from such enzymes. In addition, the present inventors appreciate that the same enzymes set forth in SEQ ID NOS: 2 to 27 may be encoded by nucleic acid sequences rather than the nucleic acid sequences of natural or artificial variants of the orfA to orfE and the orf1 to orf21, that is, the nucleic acid sequences of the genomes, which encode the same enzymes but are set forth in the orfA to orfE and the orf1 to orf21, due to the degeneracy of the genetic code. Also, the present inventors appreciate that the variants having the same function(s) as the original enzymes but including additions, deletions or substitutions of amino acids which are not essential to the folding or functions, or conservative substitutions of essential amino acids may occur as the naturally occurring or artificially constructed variants of the enzymes set forth in SEQ ID NOS: 2 to 27.
Also, the present inventors appreciate that the present invention providing the entire nucleic acid sequence of the cluster required to biosynthesize a chejuenolide also provide a nucleic acid sequence required for expression of the genes present in the cluster. A non-limiting example of such a regulatory sequence includes a promoter and enhancer sequence, an antisense sequence, and a transcription terminator and semi-terminator sequence. Such sequences are useful in regulating the expression of the genes present in the chejuenolide gene cluster. Cells having the nucleotide sequence alone or fused with another nucleotide sequence also fall within the scope of the present invention.
Further, the present invention provides orfA (SEQ ID NO: 6) encoding a polyketide synthesis module including ketosynthase-acyl carrier protein-thioesterase (KS-ACP-TE) domains that extend a polyketide chain by means of a condensation reaction and release the extended polyketide chain by means of a hydrolysis or cyclization procedure, orf1 (SEQ ID NO: 7) encoding a polyketide synthesis module having two pairs of the ketone reductase-ketosynthase-acyl carrier protein (KR-KS-ACP) domains that extend a polyketide chain by means of a condensation reaction and reduce a β-ketone residue of the extended polyketide chain, or orf2 (SEQ ID NO: 8) encoding an enzyme, isochorismatase, that catalyze hydrolysis of isochorismate into 2,3-dihydroxy-2,3-dihydrobenzoate. Also, the present invention provides orf3 (SEQ ID NO: 9) encoding an amine oxidase that oxidize an amine group. In addition, the present invention provides orf4 (SEQ ID NO: 10) encoding a polyketide synthesis module having one acyl transferase (AT) domain providing a malonyl-co-enzyme A (malonyl-CoA) required to extend a polyketide, orf5 (SEQ ID NO: 11) encoding a polyketide synthesis module having ketone reductase-transmethylase-acyl carrier protein1-acyl carrier protein2-ketosynthase (KR-MT-ACP1-ACP2-KS) domains that condense a polyketide chain, reduce the condensed polyketide chain and add one methyl molecule derived from methionine to the polyketide chain, orf6 (SEQ ID NO: 12) encoding a polyketide synthesis module having one dehydratase (DH) domain useful in removing one molecule of water from a β-hydroxyl group during extension of the polyketide to form a double bond between α- and β-carbon atoms, or orf7 (SEQ ID NO: 13) encoding an NRPS-PKS hybrid module coding for condensase-adenylase-peptidyl carrier protein-ketosynthase (C-A-PCP-KS) domains that condense one molecule of glycine with one molecule of malonyl-co-enzyme A during polyketide biosynthesis, or an isolated nucleic acid molecule having a nucleic acid sequence encoding a naturally occurring variant or derivative of the enzyme.
Further, the present invention provides a nucleic acid molecule having a nucleic acid sequence encoding an enzyme encoded by at least one selected from the group consisting of orfB to orfE and orf13 to orf17 (SEQ ID NOS: 2 to 5 and SEQ ID NOS:19 to 23, respectively), which are useful in transporting a chejuenolide or a chejuenolide precursor from cells to the outside and giving tolerance to the chejuenolide or chejuenolide precursor, an enzyme encoded by at least one selected from the group consisting of orf8 and orf20 (SEQ ID NOS: 14 and 26, respectively), which directly or indirectly permit or activate biosynthesis and expression of tolerance-related genes during preparation of the chejuenolide, an enzyme encoded by at least one selected from the group consisting of orf9, orf18 and orf19 (SEQ ID NOS: 15, 24 and 25, respectively), which are considered to indirectly participate in biosynthesis pathway of the chejuenolide, or a naturally occurring or artificially constructed variant or derivative of the enzyme.
According to still another aspect of the present invention, there are provided a vector including the gene, a microorganism transformed with the vector, and a method of preparing chejuenolide, which includes culturing the microorganism.
According to still another aspect of the present invention, there is provided a transformant microorganism for producing a chejuenolide or a precursor thereof which includes a chejuenolide biosynthetic gene set forth in SEQ ID NO: 1 in the genome thereof and in which one or more of genes encoding proteins having amino acid sequences set forth in SEQ ID NOS: 2 to 27 are disrupted. Preferably, the microorganism is a mutant strain H. chejuensis O3KO (International Accession No.: KCTC 12315BP; and Korean Accession No. KACC91712P) in which a gene encoding an amine oxidase in the DNA sequence set forth in SEQ ID NO: 1 is disrupted.
According to one exemplary embodiment, there may be provided a chejuenolide-producing strain having extra copies of the nucleic acid sequence specifying one ORF selected from the group consisting of orfA to orfE and orf1 to orf21 (SEQ ID NOS: 2 to 27, respective).
According to preferred exemplary embodiments, such a chejuenolide-producing strain is any strain belonging to the Oceanospirillales order. According to other preferred exemplary embodiments, such a chejuenolide-producing strain is a family of the Hahella genus. According to one additional aspect of the present invention, there is provided a Hahella strain having one or more modifications in the nucleic acid sequence set forth in SEQ ID NO: 1, which results in increased or decreased expression of one or more of orfA to orfE and orf1 to orf21 (SEQ ID NOS: 2 to 27, respectively).
According to one preferred exemplary embodiment, the present invention provides a nucleic acid molecule having a nucleic acid sequence set forth in SEQ ID NO: 1 or a partial region thereof, which contains at least one vector, wherein the nucleic acid molecule is useful in preparing at least one of a chejuenolide and a precursor thereof, or a derivative of the chejuenolide in other cells. According to preferred exemplary embodiments, the nucleic acid sequence or partial region thereof is included in a single vector. According to other preferred exemplary embodiments, such a vector is a bacterial artificial chromosome (BAC).
Also, the present invention provides a knockout cassette and a knockout vector, both of which includes at least one selected from the group consisting of a gene orfA having a thioesterase domain positioned at the end of the polyketide synthase cluster among the 26 ORFs, a gene orfB encoding a membrane permease, genes orfC and orfD downstream of the orfB, genes orf8, orf9 and orf19 upstream of a gene orf7 encoding a polyketide/peptide hybrid synthesis module, and genes orf2 and orf3 accompanied in a PKS-related gene cluster forming the backbone of a chejuenolide.
Specifically, the present invention provides various pairs of DNA knockout cassettes in which at least one selected from a thioesterase domain in the polyketide synthesis module designated as the orfA positioned at the end of the polyketide synthase cluster, a membrane permease designated as the orfB positioned downstream of the polyketide synthase cluster, a transmembrane protein designated as the orfC, an isochorismatase designated as the orf2 positioned inside the polyketide synthase cluster, an amine oxidase designated as the orf3, a transcriptional regulatory enzyme designated as the orf8 positioned upstream of the polyketide synthase cluster, a haloacid dehalogenase designated as the orf9, and a NADPH-quinone reductase designated as the orf19 is substituted with an antibiotic apramycin-resistant gene (see Table 2). PCR primers having a sequence of 36 nucleic acid bases tailored immediately upstream and downstream of the initiation codon and the stop codon of each of the above-described genes are constructed to amplify an apramycin-resistant gene cassette consisting of an antibiotic-resistant gene and an origin of transfer (OriT) gene (FIG. 2). The amplified DNA fragment serves as a knockout cassette. Then, the knockout cassette may be used to construct a knockout vector in which the antibiotic-resistant gene and ORF are optionally substituted (FIG. 2). For this purpose, a PCR targeting approach (Gust, Challis et al. 2003) is used, but various approaches may be applied.
The present invention provides a knockout cassette including at least one selected from the group consisting of the thioesterase domain, the membrane permease, the transmembrane protein, the isochorismatase, the amine oxidase, the transcriptional regulator, and the dehalogenasor NADPH-quinone reductase gene.
According to specific exemplary embodiments of the present invention, there are provided a pBG6E11-derved knockout vector in which each of the transcriptional regulator, dehalogenase, NADPH-quinone reductase, isochorismatase and amine oxidase genes constructed as the knockout cassette are substituted with the antibiotic-resistant gene, and a pBG19A6-derived knockout vector derived in which the thioesterase, membrane permease or transmembrane protein gene is substituted with the antibiotic-resistant gene.
In addition, the present invention provide gene mutant strains in which at least one selected from the group consisting of the thioesterase domain, the membrane permease, the transmembrane protein, the isochorismatase, the amine oxidase, the transcriptional regulator, and the dehalogenasor NADPH-quinone reductase genes is disrupted, wherein the gene mutant strains are transformed by means of conjugation between Escherichia coli and a heterogeneous bacterial strain using the knockout vectors (FIG. 2).
When H. chejuensis is transformed using the knockout vector according to one exemplary embodiment of the present invention, the DNA sequences upstream and downstream of the ORF substituted with the antibiotic-resistant gene in the knockout vector have the same DNA sequence as chromosomal DNA, resulting in induction of a homologous recombination event. A mutant strain in which the ORF is optionally disrupted by means of the homologous recombination is prepared (FIG. 2).
The present inventors extracted secondary metabolites from the H. chejuensis mutant strain using a liquid-liquid chromatography technique, and finally examined an effect of the disrupted genes and enzymes on biosynthesis of a chejuenolide using high-performance liquid chromatography (HPLC) and mass spectrometry (MS).
Among the mutant strains constructed through a series of procedures, a H. chejuensis mutant strain O3KO in which the amine oxidase is disrupted was deposited (International Accession No.: KCTC 12315BP; and Korean Accession No. KACC91712P). In this case, a compound produced by the mutant strain O3KO is a linear polyketide O3P2 (Formula: C24H35NO6) that is a chejuenolide precursor represented by the following Formula 1.




Furthermore, the present invention provides a method of preparing the gene-disrupted mutant strain, a method of producing a linear polyketide, and a linear polyketide produced using the method.
More particularly, the present invention provides a method of preparing a precursor of a chejuenolide, which includes culturing a H. chejuensis mutant strain in which a 20,900th to 19,482nd region (orf3) encoding the amine oxidase in the DNA sequence set forth in SEQ ID NO: 1 is deleted or inactivated (Gust, B., G. L. Challis, et al. (2003). “PCR-targeted Streptomyces gene replacement identifies a protein domain needed for biosynthesis of the sesquiterpene soil odor geosmin.” Proceedings of the National Academy of Sciences 100 (4): 1541.)
A. Chejuenolide Biosynthetic Gene from H. chejuensis 
A chejuenolide is a polyketide macrolide-based natural metabolite produced by a marine microorganism H. chejuensis MB-1084. The present invention provides a nucleic acid sequence of a gene cluster for biosynthesis of a chejuenolide, and enzymes. A physical hierarchy of the chejuenolide gene cluster is shown together with an adjacent DNA sequence in FIG. 1. FIG. 1 shows a physical map of a nucleic acid molecule having a length of 51.4 kb from the chromosomal DNA of H. chejuensis, and a set of fosmids specifying such a segment. A genetic hierarchy of the DNA segments governing the chejuenolide biosynthesis is shown in FIG. 1, and a nucleic acid sequence of the genetic construct is set forth in SEQ ID NO: 1.
An exact boundary of the cluster may be constructed through comparison with another biosynthetic gene cluster for a polyketide, particularly comparison with another biosynthetic gene cluster for a 17-membered carbocyclic tetraene antibiotic lankacidin, and the functions of a gene product of the another biosynthetic gene cluster. Therefore, as shown in the left end of FIG. 1, the chejuenolide cluster is demarked by a gene orfE (SEQ ID NO: 2) encoding an amino acid ABC transporter periplasmic protein having a width spanning from a 1,071st residue to a 307th residue of the nucleic acid sequence set forth in SEQ ID NO: 1. As shown in the right end of FIG. 1, the chejuenolide cluster is demarked by the remainder of ORF whose functions are not known and which has a width spanning from a 49,530th residue to a 50,609th residue of the nucleic acid sequence set forth in SEQ ID NO: 1. The chejuenolide cluster includes 26 ORFs which have a width of approximately 51,400 base pairs and are set forth in orfA to orfE and orf1 to orf21, respectively.
The nucleic acid sequence set forth in SEQ ID NO: 1 encodes 26 enzymes set forth in SEQ ID NOS: 2 to 27, respectively. The orfE (SEQ ID NO: 2) represents a sequence of 254 amino acid residues derived by translating a 1,071st to 307th nucleic acid sequence on the complementary double helix. The orfD (SEQ ID NO: 3) represents a sequence of 251 amino acid residues derived by translating a 2,082nd to 1,327th nucleic acid sequence on the complementary double helix. The orfC (SEQ ID NO: 4) represents a sequence of 1,269 amino acid residues derived by translating a 6,300th to 2,431st nucleic acid sequence on the complementary double helix. The orfB (SEQ ID NO: 5) represents a sequence of 431 amino acid residues derived by translating a 8,507th to 7,212th nucleic acid sequence on the complementary double helix. The orfA (SEQ ID NO: 6) represents a sequence of 1,066 amino acid residues derived by translating a 11,776th to 8,576th nucleic acid sequence on the complementary double helix. The orf1 (SEQ ID NO: 7) represents a sequence of 2,345 amino acid residues derived by translating a 18,803rd to 11,766th nucleic acid sequence on the complementary double helix. The orf2 (SEQ ID NO: 8) represents a sequence of 184 amino acid residues derived by translating a 19,469th to 18,915th nucleic acid sequence on the complementary double helix. The orf3 (SEQ ID NO: 9) represents a sequence of 472 amino acid residues derived by translating a 20,900th to 19,482nd nucleic acid sequence on the complementary double helix. The orf4 (SEQ ID NO: 10) represents a sequence of 293 amino acid residues derived by translating a 21,800th to 20,919th nucleic acid sequence on the complementary double helix. The orf5 (SEQ ID NO: 11) represents a sequence of 1,911 amino acid residues derived by translating a 27,598th to 21,863rd nucleic acid sequence on the complementary double helix. The orf6 (SEQ ID NO: 12) represents a sequence of 285 amino acid residues derived by translating a 28.462nd to 27,605th nucleic acid sequence on the complementary double helix. The orf7 (SEQ ID NO: 13) represents a sequence of 1,680 amino acid residues derived by translating a 33,473rd to 28,431st nucleic acid sequence on the complementary double helix. The orf8 (SEQ ID NO: 14) represents a sequence of 254 amino acid residues derived by translating a 34,641st to 35,405th nucleic acid sequence on the complementary double helix. The orf9 (SEQ ID NO: 15) represents a sequence of 209 amino acid residues derived by translating a 36,205th to 35,576th nucleic acid sequence on the complementary double helix. The orf10 (SEQ ID NO: 16) represents a sequence of 147 amino acid residues derived by translating a 37,230th to 36,787th nucleic acid sequence on the complementary double helix. The orf11 (SEQ ID NO: 17) represents a sequence of 158 amino acid residues derived by translating a 38,496th to 38,020th nucleic acid sequence on the complementary double helix. The orf12 (SEQ ID NO: 18) represents a sequence of 126 amino acid residues derived by translating a 38,963rd to 38,583rd nucleic acid sequence on the complementary double helix. The orf13 (SEQ ID NO: 19) represents a sequence of 538 amino acid residues derived by translating a 39,788th to 41,404th nucleic acid sequence on the complementary double helix. The orf14 (SEQ ID NO: 20) represents a sequence of 306 amino acid residues derived by translating a 41,488th to 42,408th nucleic acid sequence on the complementary double helix. The orf15 (SEQ ID NO: 21) represents a sequence of 277 amino acid residues derived by translating a 42,517th to 43,350th nucleic acid sequence on the complementary double helix. The orf16 (SEQ ID NO: 22) represents a sequence of 321 amino acid residues derived by translating a 43,445th to 44,410th nucleic acid sequence on the complementary double helix. The orf17 (SEQ ID NO: 23) represents a sequence of 331 amino acid residues derived by translating a 44,435th to 45,430th nucleic acid sequence on the complementary double helix. The orf18 (SEQ ID NO: 24) represents a sequence of 595 amino acid residues derived by translating a 45,557th to 47,344th nucleic acid sequence on the complementary double helix. The orf19 (SEQ ID NO: 25) represents a sequence of 183 amino acid residues derived by translating a 48,233rd to 47,682nd nucleic acid sequence on the complementary double helix. The orf20 (SEQ ID NO: 26) represents a sequence of 297 amino acid residues derived by translating a 48,357th to 49,250th nucleic acid sequence on the complementary double helix. The orf21 (SEQ ID NO: 27) represents a sequence of 359 amino acid residues derived by translating a 49,530th to 50,609th nucleic acid sequence on the complementary double helix.
The biosynthetic gene cluster for the chejuenolide is summarized in the following Table 1.









TABLE 1







Chejuenolide cluser
Lke clustera
Gene bankb
















DNA sequence
Size

Volume




















ORFc
Start
End
(bp)
Gene
matchd
Entrye
Errorf
Proposed functiong
CDh



















orfB
1071
307
765


YP_434645.1
3e-170
Amino acid ABC transporter 
Bacterial periplasmic 










periplasmic protein
substrate binding protein


orfD
2082
1927
756


YP_434646.1
2e-174
Amino acid ABC transporter 
Bacterial periplasmic 










periplasmic protein
substrate binding protein


orfC
6300
2431
3870


ZP_1696941.1
0.0
Transmembrane protein
G8 domain-VCBS repeat


orfB
8507
7212
1295
orf20
53%
YP_434654.1
0.0
Membrane permease
MFS carrier


orfA
11776
8576
3201
LkeG
51%
YP_434655.1
0.0
Polyketide synthesis module
KS-ACP-TB


orf1
18803
11766
7038
LkeF
49%
YP_434656.1
0.0
Polyketide synthesis module
KR1-ACP1-KS1-KR2-ACP2-KS2


orf2
19469
18915
555
LkeH
51%
YP_434657.1
6e-131
Nicotinamidase-like enzyme
Isochorismatase


orf3
20900
19482
1419
LkeE
63%
YP_434658.1
0.0
Amine oxidase
Amine oxidase


orf4
21800
20919
882
LkeD
54%
YP_434659.1
0.0
Acyl transferase
AT


orf5
27598
21863
5735
LkeC
54%
YP_434660.1
0.0
Polyketide synthesis module
KR-MT-ACP1-ACP2-Ks


orf6
28452
27605
858
LkeB
50%
YP_434661.1
0.0
Dehydratase
DH


orf7
33473
28431
5043
LkeA
49%
YP_434662.1
0.0
Polyketide/peptide hybrid 
C-A-PCP-KS










synthesis module



orf8
34641
35405
755


ZP_8991191.1
0.0
Transcriptional regulatory 











enzyme



orf9
36205
35576
630


YP_3807586.1
2e-70
Haloacid dehalogenase
Haloacid dehalogenase-like 











enzyme


orf10
37230
36787
444


YP_1518378.1
0.001
Unknown



orf11
38496
38020
477


YP_434319.1
3e-103
Unknown



orf12
38953
38569
381


YP_434318.1
2e-77
Unknown



orf13
39733
41404
1617


YP_434316.1
0.0
Oligopeptide ABC transporter 
Oligopeptide ABC transporter 










periplasmic protein
periplasmic protein


orf14
41499
42408
921


YP_434315.1
0.0
Dipeptide/oligopeptide/Ni ABC 
Dipeptide/oligopeptide/Ni ABC 










transporter membrane permease
transporter membrane permease


orf15
42517
48350
834


YP_434314.1
0.0
Dipeptide/oligopeptide/Ni ABC 
Dipeptide/oligopeptide/Ni ABC 










transporter membrane permease
transporter membrane permease


orf16
43445
44410
966


YP_434313.1
0.0
Dipeptide/oligopeptide/Ni ABC 
Dipeptide/oligopeptide/Ni ABC 










transporter ATPase
transporter ATPase


orf17
44435
45430
996


YP_434312.1
0.0
Oligopeptide ABC transporter 
Oligopeptide ABC transporter 










ATPase
ATPase


orf18
45557
47344
1788


YP_434311.1
0.0
Aminopeptidase
Xaa-Pro aminopeptidase


orf19
48233
47682
552


YP_434310.1
0.0
NADPH-quinone reductase
NADPH-quinone reductase


orf20
48357
49250
894


YP_434309.1
0.0
Transcriptional regulatory 
Transcriptional regulatory 










enzyme
enzyme


orf21
49530
50609
1083


YP_390605.1
0.0
Unknown
YaaC-like protein





aLankacidin Biosynthetic gene cluster


bBLAST search results from GenBank


cOpen reading frame having a DNA sequence translated as an amino acid sequence. i.e., a DNA sequence spanning from initiation to a stop codons


dThis column reports the sequence homology (%) of the optimum matches from lankacidin biosynthetic gene cluster


eAccession Number GenBank Entry with the highest score


fError probability score obtained from the BLAST research


gProposed functions of lankacidin cluster and chejuenolide gene cluster based on the combined results from the BLAST reserach


hConservative domains reported by the BLAST research






In fact, a gene encoding a polyketide synthase (PKS) associated with polymerization of a polyketide chain was constructed into a region separated by a 24.9-kb segment to be transcribed (FIG. 1). This indicated that the PKS gene modules were concentrated in a compact region, which coincided with a common opinion indicating that most of the genes required for PKS or NRPS biosynthesis form a cluster.
Unlike another known type I polyketide biosynthesis enzyme, all the PKS modules in the chejuenolide cluster coded for an acyl transferase (AT) domain and a dehydratase (DH) domain under separate ORFs of orf4 (SEQ ID NO: 10) and orf6 (SEQ ID NO: 12). Also, a PKS module encoding orf7 (SEQ ID NO: 13) is hybridized with a condensation domain (C), an adenylation domain (A) and a peptide carrier peptide (PCP) of NPRS.
Such characteristics are also examined in another known 17-membered carbocyclic tetraene biosynthesis cluster. Representatively, the biosynthetic gene cluster for lankacidin is similar to the biosynthetic gene cluster for the chejuenolide in that the biosynthetic gene cluster for lankacidin includes the PKS modules similar to those of the biosynthetic gene cluster for the chejuenolide, the configuration and arrangement of the domains constituting the PKS modules are similar to each other, and both the amino acid sequences constituting the PKS gene modules have a similarity of 49 to 63%. However, the biosynthetic gene cluster for lankacidin is different from the biosynthetic gene cluster for the chejuenolide in that a position of the orf2 (SEQ ID NO: 8) encoding the isochorismatase of the chejuenolide on the biosynthetic gene cluster is different from that of LkcH encoding an isochorismatase of the lankacidin cluster unlike the lankacidin. Also, the biosynthetic gene cluster for lankacidin is highly different from the biosynthetic gene cluster for the chejuenolide in a structural aspect in that the substantially biosynthesized lankacidin and chejuenolide are deficient in 6-membered-5-lactone rings linked via an ester bond formed by a thioesterase, and the biosynthetic gene cluster for lankacidin has an acetyl amide chain at the 3rd carbon atom instead of the 2-hydroxypropane amide chain (Arakawa et al. 2007; Choi, Sohn et al. 2008).
In conclusion, the genetic hierarchy of the chejuenolide cluster disclosed herein is distinguished from the hierarchies of other clusters participating in the synthesis of other polyketide macrolides, and are substantially different from that of the lankacidin cluster having a structure similar to the chejuenolide cluster in aspect of the configuration and contexts. Therefore, the gene cluster according to one exemplary embodiment of the present invention presents the first example of the cluster having a new genetic hierarchy.
B. Role of Chejuenolide Biosynthetic Gene
In particular, the present invention discloses a DNA sequence encoding PKS resulting in synthesis of a 17-membered carbocyclic tetraene polyketide precursor of H. chejuensis MB-1084. The PKS of the chejuenolide consists of six enzymes, each of which includes one and two modules. The six enzymes are represented by orfA, orf1, orf4, orf5, orf6, and orf7 (SEQ ID NOS: 6, 7, 10, 11, 12, and 13, respectively). The synthesis of the polyketide by the PKS is performed by a modular system in which a series of extension modules proceed after a loading module.
In general, the PKS loading module is composed of an acyl transferase (AT) and an acyl carrier protein (ACP). Each module consists of a ketosynthase (KS), AT, ACP, and enzyme domains serving to change β-ketone groups in an extending polyketide chain, that is, a ketone reductase (KR), a dehydratase (DH) and a methyl transferase (MT). The final module includes a thioesterase (TE) domain serving to terminate the extension by separating the mature polyketide chain from the PKS.
The PKS of the chejuenolide is composed of six modules, each of which consists of a total of 5 KS domains, 5 ACP domains, 3 KR domains, one DH domain, one AT domain, one MT domain, one C domain, one A domain, and one PCP domain. Specifically, the orf7 (SEQ ID NO: 13) specifies a sequence of C-A-PCP-KS as an NRPS-PKS hybrid module, and is a loading module of the chejuenolide that provides a starting material as one molecule of amino acid; the orf6 (SEQ ID NO: 12) is a module consisting of one DH domain in which an unsaturated bond is formed between α-carbon and β-carbon by removing one hydroxyl group of the β-carbon in the form of a water molecule in an extension procedure; the orf5 (SEQ ID NO: 11) specifies KR-MT-ACP1-ACP2-KS as a PKS extension module, and participates in rarely and repeatedly polymerizing a polyketide chain as type I PKS; the orf4 (SEQ ID NO: 10) supplies malonate required for an extension procedure as a module consisting of one AT domain; the orf3 (SEQ ID NO: 9) coats an amine oxidase, and participates in macrocyclization of a chain extending in a post PKS procedure; the orf2 (SEQ ID NO: 8) coats an isochorismatase, and is also assumed to play a role in the post PKS procedure; the orf1 (SEQ ID NO: 7) specifies KR1-ACP-KS1-KR2-ACP2-KS2 as a PKS extension module, and polymerizes a polyketide chain in which ketone groups of condensed β-carbons are reduced into hydroxyl groups; and the orfA (SEQ ID NO: 6) is a PKS extension termination module, and separates an extended chain from the module by means of hydrolysis.
Other genes present in the chejuenolide cluster represent genetic factors useful in synthesizing novel metabolites. Among theses, the orf3 (SEQ ID NO: 9) coats an amine oxidase that is an enzyme participating in oxidation of amines, and plays an important role in macrocyclization of a chejuenolide. This gene is closed and expressed in a heterologous host, and thus may be used to cyclize different types of polyketide chains having characteristics similar to the chejuenolide precursor. Optionally, the orf3 may be inactivated in a producing strain to produce a linear polyketide chain that is the chejuenolide precursor. Such a linear polyketide may be obtained by chemical approaches, but it is desirable to produce the linear polyketide chain using a single fermentation process without any chemical intervention.
Also, the chejuenolide cluster includes many genes transporting chejuenolide intermediates or final products from the cytoplasm to the outside or resulting in endowment of tolerance to producer cells. These genes include orfB to orfE and orf13 to orf17 (SEQ ID NOS: 2 to 5 and SEQ ID NOS: 19 to 23, respectively). The orfB, orf14 and orf15 coat an ABC transporter membrane permease, the orfC coats a transmembrane protein, the orfD, orfE and orf13 coat an ABC transporter periplasmic protein, and the orf16 and orf17 coat an ABC transporter ATPase. The genes may be cloned alone or in combination, and expressed in another polyketide producer strain, thereby causing an increase in yield of the formed products. Alternatively, the genes may be overexpressed alone or in combination in a chejuenolide-producing strain, thereby increasing the yield of the chejuenolide.
Further, the chejuenolide cluster many regulatory genes that directly or indirectly permit or activate biosynthesis and expression of resistant genes during production of the chejuenolide. These genes also include orf8 and orf20 (SEQ ID NOS: 14 and 26, respectively). The three genes may be cloned alone or in combination, and expressed in another polyketide-producing strain, thereby increasing the yield of the formed products. Alternatively, the genes may be overexpressed alone or in combination in a chejuenolide-producing strain, thereby increasing the yield of the chejuenolide.
Further, the chejuenolide cluster includes genes encoding enzymes indirectly contributing to biosynthesis of a chejuenolide. The genes also include orf2, orf9, orf18 and orf19 (SEQ ID NOS: 8, 15, 24, and 25, respectively). The orf2 coats an enzyme, isochorismatase, that catalyzes hydrolysis of isochorismate into 2,3-dihydroxy-2,3-dihydrobenzoate, the orf9 coats a haloacid dehalogenase that dehalogenates haloacid by means of hydrolysis, the orf18 coats an aminopeptidase that is an N-terminal peptide hydrolase, and the orf19 coats an NADPH-quinone reductase that converts quinone into hydroquinone. The four genes may be cloned alone or in combination, and expressed in another polyketide-producing strain, thereby causing an increase in yield of the formed products. Alternatively, the genes may be overexpressed alone or in combination in a chejuenolide-producing strain, thereby increasing the yield of the chejuenolide.
C. Use of Chejuenolide Biosynthetic Gene Cluster
Also, the present invention provides a nucleic acid molecule for expressing any one of a full-length chejuenolide molecule, and a precursor or derivative thereof. Such a nucleic acid molecule includes an isolated gene cluster(s) containing ORF, which coat a polyketide sufficient to direct the assembly of a chejuenolide. By way of example, the full-length chejuenolide cluster (SEQ ID NO: 1) may be introduced into a proper vector, and used to transform a desired producing host. In an aspect, this DNA segment is introduced into a proper vector in which a larger DNA segment may be handled. Non-limiting examples of such a vector include bacterial artificial chromosome (BAC) vectors or differentiated derivatives. In another aspect, the two separate segments are cloned into two distinct vectors with which the chejuenolide cluster may have compatibility in a desired producing host. In still another aspect, the chejuenolide cluster may be sub-cleaved into three segments, which may be cloned into separate compatible vectors. Use examples of 1-, 2- or 3-vector systems are disclosed in Tatsuno, Arakawa et al. 2007; Tatsuno, Arakawa et al. 2009; and Dickschat, Vergnolle et al. 2011. Once the chejuenolide cluster is cloned into at least one proper vector, the vector may be introduced into many proper producing hosts that can produce a chejuenolide more effectively than the natural hosts. Desirable host cells are host cells from species and strains that can effectively express these genes. Alternatively, the second copy of the chejuenolide cluster cloned into at least one proper vector may be introduced into a chejuenolide-producing strain, and the second copies of the chejuenolide biosynthetic genes may be expressed in the chejuenolide-producing strain to increase the yield of the chejuenolide.
The endowment of well-specified host with production capability may substantially improve various parts of processes causing optimization and development. That is, the optimum concentration of natural products in the producing strain may be effectively enhanced; the natural products may be purified under the known background of purifiable interference activities; compositions of a complex may be adjusted more effectively; and modified derivatives of the natural products may be more effectively produced through handling of fermentation conditions or by pathway engineering treatment.
Alternatively, the biosynthetic gene cluster may be modified, inserted into host cells, and widely used to synthesize various metabolites or chemically modify the metabolites. That is, for example, the open reading frames may be re-arranged, modified, and combined with another antibiotic biosynthetic gene cluster. By using information provided herein, the cloning and expression of a chejuenolide nucleic acid molecule may be achieved using conventional methods known in the related art.
As another possible use of the chejuenolide gene cluster, the ORFs selected from the chejuenolide gene cluster may be isolated and inactivated using conventional molecular biological techniques. An ORF substituted with an antibiotic marker cloned into a proper vector containing a DNA segment flanking the ORF in the MB-1804 chromosome is introduced into the producer strain, and two double crossing-over events of homogenous combinations result in inactivation of the ORF in the producer strain. This procedure is useful in producing the precursors or derivatives of the chejuenolide in an effective manner.
As still another possible use of the chejuenolide gene cluster, the ORFs selected from the chejuenolide gene cluster may be isolated, and brought under control of a desirable promoter. Then, the engineered ORF cloned into a proper vector is replaced with an original ORF as described above, or is introduced into a H. chejuensis strain as an additional copy of the ORF. This procedure is useful in increasing or decreasing an expression level of the ORF which is important to produce a chejuenolide molecule, or a precursor or a derivative thereof.
D. Construction of Mutant Strain and Production of Precursor
According to the present invention, a method of preparing a gene mutant strain in which an amine oxidase gene is disrupted, and a method of producing a novel linear polyketide from the gene mutant strain includes:
(1) constructing a chromosomal DNA library of H. chejuensis using a fosmid vector and obtaining a fosmid clone including a chejuenolide biosynthetic gene cluster (operation 1);
(2) constructing a knockout cassette (an antibiotic-resistant gene cassette) including DNA sequence upstream and downstream of the ORF used to encode an amine oxidase in the chejuenolide biosynthetic gene cluster (operation 2);
(3) constructing a knockout vector by replacing an amine oxidase gene with an antibiotic-resistant gene in a fosmid including the chejuenolide biosynthetic gene cluster using the knockout cassette (operation 3);
(4) introducing the knockout vector into H. chejuensis by means of an E. coli-Hahella fusion (operation 4);
(5) screening a H. chejuensis mutant strain, in which the amine oxidase gene is replaced with the antibiotic-resistant gene to be disrupted by means of homologous recombination, using an antibiotic (operation 5);
(6) culturing the mutant strain in which the screened amine oxidase is disrupted in a ZoBell liquid medium to obtain a culture broth (operation 6); and
(7) separating and purifying a novel linear polyketide from the culture broth through a series of chromatographic processes (operation 7).
Advantageous Effects
As described above, according to the exemplary embodiments of the present invention, a nucleic acid molecule and a DNA sequence of a gene cluster biosynthesizing a natural polyketide-based chejuenolide having a peculiar 17-membered macrocyclic tetraene structure, which is produced by a marine microorganism H. chejuensis MB-1084, are provided, and the functions of gene products (enzymes) encoded by the nucleic acid molecule are disclosed.
Specifically, the genes of the chejuenolide cluster is selectively disrupted using a molecular biological method, and an effect of an enzyme encoded by the selected gene on the chejuenolide biosynthesis is disclosed, a knockout cassette and a knockout vector in which the gene is disrupted are provided, and a mutant strain prepared using the knockout vector, and a chejuenolide precursor produced by the mutant strain are also disclosed.
Furthermore, the chejuenolide biosynthetic gene(s) is applicable to development of novel materials as a mechanism for combinatorial biosynthesis, and is very useful in offering new guidance for studies of polyketide-based antibiotics.



DESCRIPTION OF DRAWINGS
FIG. 1 shows a genetic map for isolated DNA segments and a chejuenolide biosynthesis cluster derived from the chromosome of H. chejuensis MB-1804 according to one exemplary embodiment of the present invention. A thick line represents a segment set forth in SEQ ID NO: 1, and fosmids carrying the isolated DNA segment are represented by pBG6E11 and pBG19A6. Each ORF is indicated by arrow, and numbered in Table 1. The direction of the genes is shown in FIG. 1.
FIG. 2 is a schematic diagram showing a knockout system using homologous recombination according to one exemplary embodiment of the present invention. An orf3 knockout cassette encoding an amine oxidase is constructed using PCR, and a recombinant vector in which the orf3 in a fosmid pBG6E11 is substituted with an apramycin-resistant gene cassette (AprR+OriT) is prepared using homologous recombination, and introduced into a chejuenolide-producing strain by means of an E. coli-heterologous strain fusion to screen a homologously recombined knockout mutant strain.
FIG. 3 is a diagram showing the results obtained by analyzing the molecular weight of a chejuenolide precursor. The ions of the precursor O3P2 are detected to be m./z 434 [M+H]+ and 867 [2M+H]+ in an ESI-MS+ mode, and detected to be m/z 432 [M−H]- and 865 [2M−H]− in an ESI-MS− mode. Therefore, the molecular weight of the chejuenolide precursor O3P2 is 433 Da.
FIG. 4 shows a chemical structure of the chejuenolide precursor O3P2. The chemical structures of the chejuenolide precursor O3P2, chejuenolide A and lankacidin C are shown for comparison in the order from the left.



BEST MODE
Hereinafter, preferred embodiments of the present invention will be described in further detail with reference to the accompanying drawings. It should be understood that the description proposed herein is merely a preferable example for the purpose of illustration only, and not intended to limit the scope of the invention, as apparent to those skilled in the art.
Example 1
Detection and Characterization of Chejuenolide Biosynthetic Gene
The present inventors constructed a chromosomal DNA library of H. chejuensis, and screened clones including a biosynthetic gene cluster for a polyketide synthase (PKS). DNA information of the two clones was combined to construct one contig, and the functions of the protein encoded by DNA were characterized using database BLASTP (a basic local alignment search tool for protein).
More particularly, a chromosomal DNA library was constructed from a chejuenolide-producing strain, H. chejuensis MB-1084, using a fosmid vector pCC2FOS (Epicentre, USA), and clones having a nucleic acid molecule encoding a ketosynthase was screened from the library using a PCR-based screening method.
To determine whether the fosmid vector was included in the screened clones, a ketosynthase was disrupted using a knockout system. As a result, it was confirmed through HPLC and MS that no chejuenolide extracted from a culture broth of H. chejuensis was produced.
Two fosmids having a group of the biosynthetic genes were screened from the library, and designated as ‘pBG6E11’ and ‘pBG19A6,’ respectively. The shotgun whole genome sequencing and primer walking of the two fosmids were requested to Macrogen Inc. to analyze a full-length base sequence of the chejuenolide biosynthetic gene cluster (SEQ ID NO: 1).
Next, the full-length 54.9 kb-long DNA sequence was divided into a total of 26 open reading frames (ORF) based on the BLASTp database from the National Center for Biotechnology Information (NCBI) (Table 1).
Therefore, the present invention provides one contig containing the chejuenolide biosynthetic gene cluster, and two fosmids including the contig. The contig set forth in SEQ ID NO: 1 had a full-length 54.9 kb-long DNA sequence, and consisted of a total of 26 ORFs (see FIG. 1). In addition to the genes directly associated with biosynthesis of the polyketide, an isochorismatase, an amine oxidase, a dehalogenase, various reductases, a transporter, and transcriptional regulatory enzymes were found in the gene cluster. A clone including the two fosmids designated respectively as pBG6E11 (International Accession No.: KCTC 12316BP; and Korean Accession No. KACC91713P) and pBG19A6 (International Accession No.: KCTC 12317BP; and Korean Accession No. KACC91714P) was deposited in the International Depositary Authorities, Korean Collection for Type Cultures (KCTC) and Korean Agricultural Culture Collection (KACC).
Example 2
Construction of Knockout Cassette for Amine Oxidase Gene Orf3 that is One of Chejuenolide Biosynthetic Genes and Method of Constructing a Knockout Fosmid Vector in which an Amine Oxidase Gene is Substituted with an Antibiotic-Resistant Gene
To remove an amine oxidase from the fosmid clone (pBG6E11) obtained in Example 1, a knockout cassette was constructed using an antibiotic-resistant gene cassette. To knock out an amine oxidase gene (FIG. 2), a PCR targeting approach (Gust, Challis et al. 2003) using an apramycin antibiotic-resistant gene and a gene OriT associated with a fusion between Actinomyces sp. and E. coli was used.
For this purpose, a knockout cassette, which had an apramycin antibiotic-resistant gene and an OriT gene positioned therein and in which DNA sequences having a length of 36 nucleotides upstream and downstream of the initiation codon and the stop codon of the amine oxidase gene were included at the 5′ terminus and 3′ terminus of the gene, was constructed and used (Table 2).
To replace the amine oxidase gene in the fosmid clone pBG6E11 carrying a group of chejuenolide biosynthetic genes using an apramycin-resistant gene-OriT cassette, first of all, a pBG6E11 vector was transformed into E. coli BW25113/pHS8 using electrophoration, and the knockout cassette obtained by a PCR method was then transfected into BW25113/pHS8/pBG6E11 using electrophoration. Thereafter, the transfected cell line was induced so that the knockout cassette was recombined with pBG6E11 using a Lambda Red recombination vector pHS8, and cultured in a Luria-Bertani (LB) agar medium supplemented with 50 μg/ml of ampicillin, 50 μg/ml of chloramphenicol and 50 μg/ml of apramycin to screen transformants in which the amine oxidase gene was recombined with the knockout cassette.
To determine whether the amine oxidase gene was replaced with the knockout cassette in the E. coli strain (BW25113/pHS8/pBG6E11::Δorf3) screened thus, primers for confirming the knockout from the nucleic acid sequence spaced at approximately 100 bp from both of the primers used in the knockout were constructed to re-determine whether the amine oxidase gene was replaced with the apramycin-OriT cassette. The recombinant fosmid constructed thus was used to transform H. chejuensis with an amine oxidase knockout vector.
To introduced the constructed amine oxidase knockout vector pBG6E11::Δorf3 into H. chejuensis, the knockout vector was isolated from E. coli BW25113 and electroporated into DNA methylase-deficient E. coli (dam−/dcm− E. coli) ET12567/pUZ8002 in order to avoid a potent methyl-specific restriction system of a heterogeneous strain, and a transformed strain (ET12567/pUZ8002/pBG6E11::Δorf3) was screened on an LB agar medium supplemented with 50 μg/ml of apramycin and 50 μg/ml of kanamycin. A list of primers for constructing a knockout cassette is summarized in Table 2 below.








TABLE 2





Primers
DNA Sequence (5′-3′)







CheORF2_F
CACAAATTCATCAAGGACTTAATGGTTTAAAGGAGTCGAATGattccggggatccgtcgacc



(SEQ ID NO: 28)


 


CheORF2 R
TGCTCCGGGAGGGATTGCGCCTGCGCTCAGACAGGGTCAtgtaggctggagctgcttc



(SEQ ID NO: 29)


 


CheORF3_F3
CACGCACAGGCTTAACCCATAGAGGTAATCAAAATGAAAATGattccggggatccgtcgacc



(SEQ ID NO: 30)


 


CheORF3_R3
CGCTTTTTCTTTTTGTAGCAACATTGTTCGACTCCTTTATCAtgtaggctggagctgcttc



(SEQ ID NO: 31)


 


CheORF8 F
AGCATCCTAACAACAAAAATCACGGAGTCTGGCGAGATGattccggggatccgtcgacc



(SEQ ID NO: 32)


 


CheORF8_R
TCCCTGCACGATCACGCCAAATCATCCCCACTAAGCCTTTCAtgtaggctggagctgcttc



(SEQ ID NO: 33)


 


CheORF9 F
GGGGAGAGGACGTGCATCTAGTCATGTTTGACATAGACATGattccggggatccgtcgacc



(SEQ ID NO: 34)


 


CheORF9_R
TTATTTTAATCCAATAAACCTAAAGGCCTTATCAGATTCAtgtaggctggagctgcttc



(SEQ ID NO: 35)


 


CheORF19 F
ACGCACTGAAAACCGAACTGAAAAGGACCGCCCGACATGattccggggatccgtcgacc



(SEQ ID NO: 36)


 


CheORF19_R
GATAACGCTCACAGGCGATATCCAGTTCCGCATCCATCAtgtaggctggagctgcttc



(SEQ ID NO: 37)


 


CheORFA_F
TGTTGCTGTGTCTGGGCGGTCTGGCGATGAGCTATGTCTATGattccggggatccgtcgacc



(SEQ ID NO: 38)


 


CheORFA_R
ACCGCAAACGGGACAGCGTTGTTTATTTTGCTAATCCGCTCAtgtaggctggagctgcttc



(SEQ ID NO: 39)


 


CheORFB_F
TGTTGCTGTGTCTGGGCGGTCTGGCGATGAGCTATGTCTATGattccggggatccgtcgacc



(SEQ ID NO: 40)


 


CheORFB R
ACCGCAAACGGGACAGCGTTGTTTATTTTGCTAATCCGCTCAtgtaggctggagctgcttc



(SEQ ID NO: 41)


 


CheORFC_F
TGCACTGTGGAAGACTATAAAATGAACTTGGGTGAAACGATGattccggggatccgtcgacc



(SEQ ID NO: 42)


 


CheORFC_R
TCCGGCGCATAAGAAAGACTAGCGGTCGCTAATCAGATATCAtgtaggctggagctgcttc



(SEQ ID NO: 43)









Example 3
Construction of H. chejuensis Mutant Strain in which a Chejuenolide Amine Oxidase Gene is Disrupted
To obtain a mutant strain in which an amine oxidase gene was disrupted in the chejuenolide biosynthetic gene cluster from H. chejuensis according to one exemplary embodiment of the present invention, the amine oxidase knockout vector constructed in Example 2 was introduced by means of a fusion between E. coli ET12567/pUZ8002/pBG6E11::Δorf3 and H. chejuensis. 
For this purpose, the ET12567/pUZ8002/pBG6E11::Δorf3 strain was pre-cultured overnight in 5 ml of an LB liquid medium supplemented with 50 μg/ml of apramycin and 50 μg/ml of kanamycin, pre-cultured in 10 ml of an LB liquid medium supplemented with the same concentrations of the antibiotics, and then cultured at 37° C. for approximately 4 to 5 hours until an optical density of 600 nm (OD600) reached 0.4. The culture broth was centrifuged to obtain a pellet. Then, the pellet was washed twice with the same volume of LB to remove the antibiotics completely, and suspended in 0.5 ml of an LB medium.
As the same time, the H. chejuensis was cultured overnight at 28° C. while stirring in 5 ml of a Zobell medium (5 g of peptone, 1 g of yeast extract, 10 mg of FePO4, 30 g of seawater salt, 15 g of agar, and 1 L of distilled water), and centrifuged to obtain a pellet. Then, the pellet was washed twice with the same volume of an LB medium, and suspended in 0.5 ml of a fresh LB medium.
Next, the cell suspensions of the E. coli strain ET12567/pUZ8002/pBG6E11::Δorf3 and H. chejuensis were added into a 1.5 ml microcentrifuge tube (or an ependorf tube) at an amount of 0.5 ml, mixed, centrifuged at 13,000 rpm for 30 seconds. Thereafter, the resulting suspension remaining after discarding most of the supernatant was plated on a Zobell agar medium supplemented with 10 mM MgCl2, and cultured at 28° C. for 16 to 20 hours. Subsequently, 0 ml of sterilized water including 0.5 mg of nalidixic acid and 1.25 mg of apramycin was added, and evenly plated again.
Clones were formed when the cells were cultured at 28° C. for approximately 2 to 3 days. The clones were transferred to a Zobell agar medium supplemented with 25 μg/ml of nalidixic acid and 50 μg/ml of apramycin. The formed mutant strain was subjected to PCR to re-determine whether the amine oxidase gene was replaced with the knockout cassette. The mutant strain from which the amine oxidase gene was removed was designated as H. chejuensis O3KO, and deposited in the KACC from the National Institute of Agricultural Botany (NIAB) (International Accession No.: KCTC 12315BP; Korean Accession No. KACC91712P).
Example 4
Purification of Novel Linear Polyketide Produced in Mutant Strain O3KO in which a Chejuenolide Amine Oxidase Gene is Disrupted
200 ml of a Zobell liquid medium was put into a 1 L shaking flask, and a seed of fresh clones from the amine oxidase mutant strain was inoculated in the liquid medium, and pre-cultured at 28° C. and 200 rpm for 1 to 2 days while stirring. The pre-culture broth was inoculated at a concentration of 1% in 1 L of a Zobell medium prepared in the same conditions as the pre-culturing conditions, and then cultured at 28° C. and 200 rpm for one day while stirring.
The culture broth obtained thus was centrifuged at 10,000 g for 30 minutes to obtain a pellet.
The obtained pellet was suspended in 500 ml of methanol, and then extracted for 2 hours using an ultrasonicator.
The methanol extract was evaporated under a reduced pressure, and finally recovered with a small amount of methanol.
The methanol extract was fractionated in a 0-to-100% aqueous methanol solution using Diaion HP-20 column chromatography (Mitsubishi Corporation, Japan).
The resulting fractions of 40% aqueous methanol solution were concentrated under a reduced pressure, and recovered with a small amount of methanol. Then, the methanol extract was
Peaks appearing when observed under a UV light of 254 nm while allowing a 20%-to-50% aqueous acetonitrile solution including 0.1% formic acid to flow at a flow rate of 2 ml/min through HPLC were analyzed using electrospray ionization mass spectrometry (ESI/MS) to obtain 2 mg of a metabolite having a molecular weight of 433 Da (FIG. 3).
The purified metabolite was dissolved in CD3OD (Cambridge Isotope Laboratories, USA), and subjected to 1H-, 13C- and 2D-NMR (COSY, HMBC, and HSQC) experiments using a 500 MHz nuclear magnetic resonance (NMR) spectrometer. As a result, it was confirmed that the general spectrum pattern resembled the NMR data for the chejuenolide, indicating that the purified metabolite was an intermediate produced in the chejuenolide biosynthesis pathway. It was confirmed that a new δC 169.5 ppm carbon signal which was not found in the NMR data for the chejuenolide was observed for the compound. In this case, the carbon signal corresponded to a carbon atom in a carbonyl group of a lactone ring, which corresponded to carbonyl carbon in a 6-membered d-lactone ring structure of the lankacidin.
Also, it was revealed that a hydrogen atom appearing as a triplet corresponding to one hydrogen atom in 5H 4.98 ppm linked to a 3rd carbon atom of the chejuenolide moved upfield, and appeared as a doublet corresponding to two hydrogen atoms in δH 3.90 ppm. These results could be obtained when a bond between 2nd and 3rd carbon atoms disappeared. Also, the formation of a new double bond between 1st and 2rd carbon atoms was confirmed from the fact that a 18th methyl residue of δH 1.72 ppm appeared as a singlet, a 2nd carbon atom of δC 49.9 ppm moved downfield toward δC 96.0 ppm, and a 3rd carbon atom of the carbonyl group appeared in δC 205.6 ppm moved upfiled toward δC 169.5 ppm. It was confirmed a secondary metabolite having a molecular weight of 433 Da biosynthesized in H. chejuensis in which the amine oxidase gene was disrupted was a linear polyketide accumulated as a chejuenolide biosynthetic precursor, which was a novel compound whose structure was not reported so far (see Table 3 and FIG. 4).











TABLE 3





Carbon No.
δH
δC
COSY
HMBC



















1

169.5




2

96.0


3

171.2


4
2.48(1H, m)
37.0
20
3, 20


5
4.15(1H, m)
78.0
6a, 6b
 7


6a
1.77(1H, m)
40.6
5, 6b, 7
4, 5


6b
2.08(1H, m)

5, 6a, 7
4, 5, 7 ,8


7
4.29(1H, q, 7.0)
69.4
6a, 6b, 8
5, 6, 7, 8


8
5.57(1H, dd, 15.5, 7.0)
128.3
7, 9
7, 10


9
6.30(1H, d, 15.5)
136.0
 8
7, 10, 11, 21


10

134.5


11
5.55(1H, t, 7.3)
128.2
12a
10, 12, 13, 21


12a
2.39(1H, m)
36.2
11
10, 11, 13, 14


12b
2.43(1H, m)

13
10, 11, 13, 14


13
4.17(1H, m)
71.9
12b, 14
11, 12, 14, 15


14
5.70(1H, dd, 6.7, 15.7)
130.9
13, 15
13, 16


15
6.23(1H, d, 15.5J)
133.8
14
13, 16, 17, 22


16

135.5


17
5.46(1H, t, 6.8)
126.9
18
15, 18, 22


18
3.9(2H, d, 7.0)
37.0
17
16, 17, 23


19
1.72(3H, s)
7.2

1, 2, 3


20
1.25(3H, d, 7.5)
15.2
 4
3, 4, 5


21
1.77(3H, s)
11.2

9, 10, 11


22
1.80(3H, s)
11.2

15, 16, 17


23

171.6


24
1.94(3H, s)
21.0

23


N—H
8.45(1H, br s)

## Claims

1. A transformant microorganism transformed from a microorganism having a genetic sequence of SEQ ID NO: 1 in its genome, wherein one or more genes encoding amino acid sequences of SEQ ID NOS: 2 to 27 that are included the SEQ ID: 1 are disrupted in the transformant microorganism.

2. The transformant microorganism according to claim 1, wherein the microorganism is a mutant strain Hahella chejuensis O3KO (International Accession No.: KCTC 12315BP) in which a gene encoding an amine oxidase in the DNA sequence of SEQ ID NO: 1 is disrupted.

3. A method of preparing a precursor of chejuenolide, comprising:
(1) culturing a mutant strain in which one or more of genes encoding proteins having amino acid sequences set forth in SEQ ID NOS: 2 to 27 are disrupted by knockout system, wherein the mutant strain includes a chejuenolide biosynthetic gene set forth in SEQ ID NO: 1 in the genome thereof; and
(2) obtaining the precursor of chejuenolide from the culture broth.

4. The method according to claim 3, wherein the mutant strain is disrupted at a 20,900th to 19,482nd region (orf3) encoding an amine oxidase having an amino acid sequence set forth in SEQ ID NO: 9 by knockout system.

5. The method according to claim 4, wherein the mutant strain is a Hahella chejuensis O3KO (International Accession No.: KCTC 12315BP).

6. The method according to claim 3, wherein the precursor is a chejuenolide precursor O3P2 represented by Formula 1.