WO2011068341A2 - Gene and protein for biosynthesis of tricyclocompounds - Google Patents

Gene and protein for biosynthesis of tricyclocompounds Download PDF

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WO2011068341A2
WO2011068341A2 PCT/KR2010/008496 KR2010008496W WO2011068341A2 WO 2011068341 A2 WO2011068341 A2 WO 2011068341A2 KR 2010008496 W KR2010008496 W KR 2010008496W WO 2011068341 A2 WO2011068341 A2 WO 2011068341A2
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tacrolimus
kctc
gene
ascomycin
sequence
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WO2011068341A3 (en
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김재종
임시규
김동환
이금순
유정현
이미옥
임상면
이보미
김상준
차선호
윤여준
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(주)제노텍
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    • 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
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    • 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
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
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    • C12P1/00—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes
    • C12P1/04—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes by using bacteria
    • 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

Definitions

  • the present invention relates to polyketides, in particular tricyclocompounds and more particularly to nucleic acid sequences of genes directly related to the biosynthesis of tacrolimus and its analogs used as immunosuppressants It relates to a protein and a method for producing an active substance using the same.
  • Tricyclocompounds are secondary microbial metabolites that exhibit antifungal and immunosuppressive activity mainly produced by microorganisms, particularly actinomycetes. In general, these substances are very toxic and have high pharmacological activity, and thus have been developed as immunosuppressants, anticancer agents, etc., and are sold as expensive drugs such as Prograf TM and Rapamune TM .
  • Tacrolimus FK506
  • ascomycin FK520
  • rapamycin sirolimus
  • meridamycin meridamycin
  • Tacrolimus, rapamycin, ascomycin and their structural analogs have similar structural sites that inhibit the activation of immune cells, T cells, in vivo and in vitro.
  • These compounds have a pyranose-pipecolinyl region (C1-C15 in FIG. 1), which is similar in structure to leucine-proline peptides, and has a peptidyl proyl cis / trans It has been reported that the binding of isomerase (peptidyl prolyl cis / trans isomerase) shows various physiological activities (Rosen MK et al., 1990).
  • Ascomycin is a 23-membered macrolide compound formed of 23 carbons and an ethyl analog of tacrolimus (see FIG. 1) (Hatanaka H. et al., 1998). And dihydrotacrolimus is a propyl analog of the tacrolimus C21 position, FK523 is a methyl analog of the tacrolimus C21 position, and FK525 is a prolyl analog of tacrolimus ( prolyl analog).
  • Tacrolimus Streptomyces tsukubaensis 9993 (US Pat. No. 4,894,366), Streptomyces sp. ATCC 55098, Streptomyces sp. ATCC 53770, Streptomyces claus It has been reported to be produced in strains such as S treptomyces clavuligerus CKD1119 (Korean Patent 10-0485877), Streptomyces kanamyceticus KCC S-043 (KCTC 9225) (Muramatsu H. et al.
  • FK506 and structurally similar chain FK520 also Streptomyces high-gloss nose kusu subgenus ascorbyl Mai Shetty kusu (Streptomyces hygroscopicus subsp. ascomyceticus) ATCC 14891, Streptomyces high-gloss nose kusu subgenus Yakushima N-Sys (Streptomyces hygroscopicus subsp. yakusimaensis Produced by 7238 and Strepptomyces tsukubaensis 993 Reported.
  • rapamycin has been reported to produce high-gloss Streptomyces nose kusu subgenus high-gloss nose kusu (Streptomyces hygroscopicus subsp. Hygroscopicus) ATCC 29253.
  • Tricyclocompounds belong to a group of polyketide and nonribosomal peptide hybrids. Their chemical structure is a polyketide formed by condensation of two carbon units and a cyclohexyl structure and a pipeusic acid (unusual amino acid) of pipecolic acid [FK525] Proline] is a macrolide compound formed by linking. These compounds are typically very difficult to synthesize chemically, and the amounts produced by wild-type producing strains are very small. Therefore, the biosynthetic mechanism of these compounds can be used as a means for generating new materials and increasing productivity by changing the structure of these compounds.
  • the biosynthetic mechanism of macrolide-producing microorganisms is a megasynthase, which is composed of multiple large proteins complex.
  • polyketide site biosynthesis is achieved by the claisen condensation of acyl-CoA, and enzymes encoded by genes responsible for biosynthesis of these substances are in the form of modules. It is known to be configured and act sequentially. This is classified as a modular type I polyketide synthase (PKS).
  • PKS modular type I polyketide synthase
  • Module type 1 polyketide synthase (modular type I PKS) consists of a series of sets of modules in which the catalytic domains elongate and modify each chain length. As a module progresses by one cycle, the carbon constituting the skeleton grows by two.
  • Typical module type 1 polyketide synthase is a multienzyme system, which is typically composed of a loading module, multiple extender modules and a releasing module.
  • acyl-CoA acylcoenzyme-A
  • the expansion module basically consists of three enzymatic domains: Ketosynthase (KS), Acyltransferase (AT), and Acyl carrier protein (ACP).
  • KS Ketosynthase
  • AT Acyltransferase
  • ACP Acyl carrier protein
  • enzymes involved in the modification of beta-carbon, ie ketoductase (KR), dehydroatase (DH), and enolyl reductase (ER) May be added to form a module.
  • One action of the module adds an acyl coei residue, which transfers the acyl moiety to the corresponding acyl group transport protein (ACP) to generate acyl-ACP and to synthesize keto groups.
  • the enzyme (KS) gradually increases the carbon number by allowing the acyl group of the acyl-ACP generated in the molecule added by the preceding module to perform carbon-carbon bonds through claisen condensation. That is, once these sets work, one acyl residue may be added to increase the carbon number of the backbone carbon chain by two.
  • keto-reductase, dehydrogenase, and enol reductase act in turn to transform the keto group into an alcohol group, an alcohol group into a double bond, and a double bond into a saturated single bond.
  • the biosynthetic genetic machinery of a compound such as polyketide is generally configured to reflect the structure of a substance in charge.
  • Biosynthetic devices are composed of continuous process systems, and modifications of continuous process systems such as module modifications often lead to the application of materials of a different structure than the ones in charge. Therefore, intentional modification of modular assembly lines can be an important way to create new structures of material.
  • the modification of the module which is a part line of the biosynthetic gene, means the creation of a new structure of substance.
  • combinatorial biosynthesis is a technique that attempts to create a variety of new materials by systematically and efficiently modifying, deleting, or replacing genes in charge of specific modules. In order to apply this technique, biosynthetic genes must be obtained.
  • the first rapamycin biosynthetic gene and its protein have been cloned from Streptomyces high-gloss high-gloss nose kusu subgenus nose kusu (Streptomyces hygroscopicus subsp. Hygroscopicus), ATCC 29253 (T. Schwecke et al., 1995).
  • tacrolimus biosynthesis genes were cloned from Streptomyces sp. MA6548 (Motamedi H. et al., 1998 and Motamedi H. et al., 1997). It is said gene is a three poly Kane Tide synthase (PKS) gene that is fkbA, fkbB, fkbC
  • PKS poly Kane Tide synthase
  • ascomycin-producing strain of Streptomyces high-gloss nose kusu subgenus ascorbyl Mai Shetty kusu were from ATCC 14891 cloning the ascomycin biosynthetic gene cluster (Wu, KL et al., 2000), a total of about 80kb Analyzes the linker gene sequence to suggest fkbA, B, C, P, D, M, O, L, N, Q, S, K, J, I, H, G, F, E that are believed to be involved in ascomycin biosynthesis 22 genes of R1, R2, U, and W were identified.
  • tacrolimus biosynthesis gene and ascomycin biosynthesis gene were very similar.
  • the domain composition constituting the polyketide synthase is different from the report that the dehydrogenase (DH) domains present in modules 3 and 8 of ascomycin biosynthetic polyketide synthase do not exist in the tacrolimus biosynthetic gene.
  • DH dehydrogenase
  • Tacrolimus biosynthesis was commonly found in FkbO (oxidase), peptide synthase (FkbP), p450 hydroxylase (FkbD), O-methyl transferase (FkbM), and lysine cyclodeaminase (FkbL).
  • tacrolimus biosynthetic gene family contains Fkb G, H, I, J, K, which are believed to be responsible for the synthesis of methoxymalonyl ACP present in ascomycin (FK520). Therefore, the presence or absence of a gene responsible for a specific transcription regulator FkbN is not known.
  • the composition of the tacrolimus producing strain biosynthesis gene is different from that of the ascomycin producing strain biosynthesis gene. That is, in the case of tacrolimus producing strain, another gene responsible for the C21 position biosynthesis gene is likely to exist in addition to the ascomycin biosynthesis gene. In addition, another gene involved in regulating the biosynthesis of tricyclic compounds such as tacrolimus and ascomycin may be used to increase the production of tacrolimus and other tricyclic compounds or to generate various derivatives.
  • the present inventors fully secured biosynthetic genes from tacrolimus producing strains, identified the function of the genes, and confirmed the availability.
  • the present invention further expands the tacrolimus biosynthetic gene group, which is in charge of the previously incompletely identified tacrolimus biosynthesis apparatus, to secure genes that do not exist in the ascomycin biosynthetic gene but exist only in the tacrolimus biosynthetic gene. That is, the nucleic acid sequence was revealed by securing a gene group in charge of a complete tacrolimus biosynthesis apparatus including FkbA, B, C and FkbO, P, D, M, L found in the existing tacrolimus biosynthesis gene group. The biosynthetic association between the gene containing the nucleic acid sequence, the polypeptide produced from the gene, and the tricyclic compounds including tacrolimus and its analogs was identified and its industrial applicability was confirmed.
  • the present invention provides a tacrolimus biosynthetic gene cluster represented by a sequence that is the same as or greater than or equal to 85% identity with SEQ ID NO: 1, 54 or 93, or a sequence complementary to the sequence.
  • the present invention provides a polyketide synthase gene represented by a sequence that is identical to, or complementary to, SEQ ID NO: 3, 56, or 95 or more than 80% identity.
  • the present invention provides a polyketide synthetase represented by a sequence that is identical to or exhibits 80% or more identity to SEQ ID NO: 2, 55 or 94.
  • the present invention provides a polyketide synthase gene represented by a sequence showing the same or 80% or more identity to SEQ ID NO: 5, 58 or 97 or a sequence complementary to the sequence.
  • the present invention provides a polyketide synthetase represented by a sequence which is identical to SEQ ID NO: 4, 57 or 96 or which exhibits at least 80% identity.
  • the present invention provides a polyketide synthase gene represented by a sequence that is identical to, or complementary to, SEQ ID NO: 7, 60, or 99, or at least 90% identity.
  • the present invention provides a polyketide synthetase represented by a sequence that is identical to or exhibits 90% or more identity to SEQ ID NO: 6, 59, or 98.
  • the present invention provides a polyketide synthase gene represented by a sequence that is identical to, or complementary to, SEQ ID NO: 9, 62, or 101 or 90% identity.
  • the present invention provides a polyketide synthetase represented by a sequence that is identical to or exhibits 90% or more identity to SEQ ID NO: 8, 61 or 100.
  • the present invention provides a 9-dioxo-31-O-desmethyl-tacrolimus, 9-dioxo- by culturing a mutant in which the fkbD gene coding region of the tacrolimus biosynthetic gene cluster of claim 1 is deleted or inactivated.
  • the present invention is cultured mutant in which the fkbM gene coding region of the tacrolimus biosynthetic gene cluster of claim 1 is deleted or inactivated 31-O- desmethyl- tacrolimus, 31-O- desmethyl- ascomycin and A method of producing at least one selected from 31-O-desmethyl-36,37-dihydro-tacrolimus is provided.
  • the tacrolimus biosynthetic gene group includes three types of tacrolimus producing strain Streptomyces sp. ATCC 55098, Streptomyces kanamyceticus KCTC 9225, Streptomyces sp .) Obtained from KCTC 11604BP.
  • 19 genes commonly found in the tacrolimus biosynthetic gene group (SEQ ID NOs: 3,5,7,9,21,23,25,27,29,31,33,35,37,39,41, 43,45,47,49,56,58,60,62,64,66,68,70,72,74,76,78,80,82,84,86,88,90,92,95,97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131 were confirmed (Fig. 2).
  • the inventors of the present invention have shown that the genes of tcsA, B, C, D and the polypeptides in which these genes are responsible (SEQ ID NOs: 2,3,4,5,6,7,8,9,55,56,57, 58,59, 60,61,62,94,95,96, 97,98,99,100,101 are involved in the synthesis of tacrolimus and the side chain (alyl-malonyl derivative) at position C-21 of the analogue. Said. Therefore, these genes can be used for economic production through the production of new materials or by increasing the productivity of tacrolimus and / or its related materials and reducing impurities.
  • the present inventors have used 9-dioxo-31-O-desmethyl-tacrolimus (ascomycin) ⁇ 9-deoxo-31-O by a mutation method of fkbD and fkbM (SEQ ID NOs: 42,43,44,45 ).
  • -desmethyl-tacrolimus (ascomycin) ⁇ and 31-O-desmethyl-tacrolimus (ascomycin) ⁇ 31-O-desmethyl-tacrolimus (ascomycin) ⁇ was found to be capable of high production.
  • Tacrolimus FK506, Ascomycin (FK520), Dihydrotacrolimus, Prolyl tacrolimus (FK525), FK523 (L-683795), L-687819, 9-dioxo-31-O- Desmethyl-tacrolimus, 9-dioxo-31-O-desmethyl-ascomycin, 9-dioxo-tacrolimus, 9-dioxo-ascomycin, 9-hydroxy-9-dioxo-ta Crowlimus, 9-dioxo-36,37-dihydro-tacrolimus, 31-O-desmethyl-tacrolimus, 31-O-desmethyl-ascomycin, 31-O-desmethyl-36,37 -Dihydro-tacrolimus, 9-dioxo-31-O-desmethyl-36,37-dihydro-tacrolimus, prolyl-dihydro-tacrolimus,
  • Mutants that can be reasonably made by the method of constructing the fkbM , fkbD mutants presented in the examples of the present invention are 9-dioxo-31-O-dimethyl-tacrolimus, 9-dioxo-31-O-dimethyl -Ascomycin, 9-dioxo-31-O-dimethyl-36,37-dihydro-tacrolimus, 31-O-dimethyl-tacrolimus, 31-O-dimethyl-ascomycin and 31-O-dimethyl
  • productivity of -36,37-dihydro-tacrolimus can be increased, and thus it can be applied to high production of the tacrolimus modified material produced in small amounts in the wild type.
  • Tcs A, Tcs B, Tcs C, and Tcs D genes obtained by the present invention and the proteins encoded by the genes are directly expressed or mutated in the production or modification of the side branches (allyl-malonyl derivatives) at tacrolimus 21. It can be used as a mechanism and can be applied to structural modification or reduction of impurities of the entire polyketide material using the same.
  • 1 is a structure (formula) of a three-membered ring compound.
  • FIG. 2 shows the tacrolimus biosynthesis gene group.
  • A Streptomyces sp. KCTC 11604BP, respectively;
  • B Streptomyces kanamyceticus KCTC 9225;
  • C Streptomyces sp. was obtained from the ATCC 55098 strain.
  • Figure 3 shows the modules (Modules) and domain composition and putative biosynthesis process of the biosynthetic gene group.
  • the upper domain is from Streptomyces sp. KCTC 11604BP and the lower is from Streptomyces kanamyceticus KCTC 9225.
  • FIG. 6 is a diagram illustrating the synthesis process of tacrolimus C-21 side chain.
  • KS ketosynthase
  • AT acyltransferase
  • ACP acyl carrier protein
  • aDH acyl-ACP (CoA) dehydrogenase ⁇ acyl-ACP (CoA) dehydrogenase ⁇
  • R / C reductase / carboxylase
  • biosynthetic genes were obtained from strains producing tacrolimus.
  • Actinomycetes used in the present invention are Streptomyces kanamyceticus KCTC 9225 (Muramatsu H. et al., 2005) and Streptomyces sp. KCTC 11604BP and Streptomyces reported production of tacrolimus. Streptomyces sp. ATCC 55098.
  • Streptomyces strains were prepared in 30 ml GT-TA-1 medium [1% aqueous starch, 0.7% glycerol, 0.3% bactopeptone, 0.3% yeast extract, 0.5% soyton peptone, 0.05 in a 500 ml Erlenmeyer Erlenmeyer flask.
  • % AZ-20R (antifoam), pH 6.8] after activating for one day at 28 °C, 240rpm conditions, the activated species culture strain 150ml GT-TA-2 medium contained in 3L Elenmeyer Erlenmeyer flask [1% oxidized starch, 1% glycerol, 2% soybean mill, 0.2% CaCO 3 , 0.5% CSL, 0.05% AZ-20R (antifoam), pH 6.5] and incubated for one day under the same conditions.
  • Culture cultured in GT-TA-2 medium was carried out in 2.7 L GT-TA-4 medium [7% oxidized starch, 1.7% yeast powder, 0.5% soybean mill, 0.1% (NH 4 ) 2 in a 5 L fermenter.
  • Metabolites were extracted with 75% acetone from cultures or HP-20 resins recovered from fermented cultures. Extracted metabolites were analyzed under HPLC conditions as shown in Table 1.
  • LC-MS / MS was performed to identify tacrolimus and its analog.
  • Samples were partially purified using HLB cartridges for LC-MS / MS as follows. Acetonitrile was mixed in the same volume to the culture and extracted for 30 minutes, and then water was added to finally give 25% of acetonitrile. This was passed through an HLB cartridge (OASIS HLC cartridge, 1 ml) and then the unadsorbed material was washed off with twice the capacity of 30% acetonitrile. It was then eluted in 1-fold with 40, 50, 60, 70% acetonitrile. Mass spectrometry was performed under the LC-MS conditions as shown in Table 2 using 50% acetonitrile eluent as the main fraction in the eluent stepwise.
  • Condition item Condition content column YMC Ultra C18 (5 * 2mm, 2 ⁇ m) Mobile phase 50 -100% gradient of acetonitrile Flow rate 1ml / min device Shimadzu LCMS-IT-TOF Ionization ESI, positive Fragmentation 100 V Scan range 200-1000 m / z Sample injection amount 1 to 5 ⁇ l
  • Tacrolimus (FK506) with HR-MS (M + Na) + m / z 826.4730 at approximately 19.9 min under conditions of C18 HPLC in the fermentation medium of Streptomyces kanamyceticus KCTC 9225 and Streptomyces KCTC 11604BP. Mr. 803.4820), several analogs existed before and after the tacrolimus peak.
  • Aspergillus niger ATCC 9642 was used as the test for antifungal activity measurement.
  • Potato Dextrose Agar medium (0.4% potato starch, 2% dextrose, 1.5% agar) was sterilized and cooled to 40 ⁇ 50 °C, and then inoculated with the specimen to prepare a specimen medium.
  • the target extract for the antimicrobial activity obtained from the cultures of wild and mutant strains was buried in paper disks (ADVANTEC, Toyo Roshi Kaisha, Ltd.) and placed on a pre-prepared test medium and incubated at 28 ° C. for two days. The antimicrobial activity was confirmed by measuring the size of the clear (clear zone).
  • Biosynthetic genes were obtained from three strains specified in Example 1 above.
  • the fosmid library of each strain was prepared, and the phosmid library was used by separating chromosomes using the method suggested by Kieser et al. (Kieser T. et al., 2000).
  • the kit was purchased and manufactured according to the manufacturer's instructions.
  • About 1,500 phosphide ends prepared from each strain were sequenced with an accuracy of 0.02 / 10 kb or more using an ABI-3730xl autosequencer using Sanger's didioxy methods to obtain sequencing information.
  • phosphide clones containing polyketase synthase gene information were found, and they were sequenced using shotgun sequencing.
  • KCTC 11604BP of Streptomyces genus analyzed the Fos1004F01 (1-40366), Fos1005D02 (39116-80661), Fos1006D05 (58172-97743) to obtain a total of about 97kb (SEQ ID NO: 1).
  • Biosynthetic genes derived from Streptomyces kanamyceticus KCTC 9225 were analyzed by Fos1006G02 (1-35521), Fos1012A09 (31026-67758), Fos1004E04 (41430-85253), and Fos1010E10 (76978-111990). (SEQ ID NO 54) was obtained. Biosynthetic genes derived from Streptomyces sp. Connection information (SEQ ID NO: 93) was obtained. Previously known tacrolimus biosynthesis genes are secured and analyzed based on polyketase synthase and only a few genes are insufficient for understanding and industrial utilization of the entire composition of tacrolimus biosynthesis. Enough genes of sufficient size were obtained, which could be systematically analyzed (FIG. 2).
  • the sequence obtained from each strain was predicted by predicting orf (open reading frame) by Glimmer program and comparing each function with BLAST-2.2.21.
  • Streptomyces KCTC 11604BP-derived genes a total of about 44 orf were predicted (Table 3), and Streptomyces kanamyceticus About 70 orf were predicted for KCTC 9225 and about 56 orf for ATCC 55098 in Streptomyces.
  • genes encoding three major polyketase synthase fkbA , B , C ), methoxymalonyl ACP synthesis genes ( fkbG, H, I, J, K ), pipepelic acid (pipecolate) biosynthetic gene ( fkbL ), modification related putative genes ( fkbM, D, O ), regulatory genes ( fkbN ), nonribosomal peptide synthase ( fkbP ), and type II thioesterase (TE) gene ( fkbQ ) has been reported in the ascomycin biosynthetic gene family.
  • fkbA , B , C genes encoding three major polyketase synthase ( fkbA , B , C ), methoxymalonyl ACP synthesis genes ( fkbG, H, I, J, K ), pipepelic acid (pipecolate) biosynthetic gene ( fkbL
  • tcsA, B, C, D Four new genes ( tcsA, B, C, D ) and their polypeptides are believed to be specific for tacrolimus biosynthesis and are the first genes and products identified in accordance with the present invention (SEQ ID NOs: 2,3,4). , 5,6,7,8,9,55,56,57,58,59,60,61,62,94,95,96, 97,98,99,100,101).
  • ACP -Acyl carrier protein
  • Serine residues essential for 4'-phosphopantetheine attachment to ACP and TcsA's ACP domains and up to FkbJ in 10 modules on polyketide synthase are well conserved (Florova G. et al. 2002).
  • KS domains on polyketide synthase are well conserved for cysteine, histidine and lysine amino acids essential for decarboxylation and acyl transfer (Dreier J. and Khosla C., 2000).
  • the 10 AT domains on the polyketide synthase preserve well the amino acid residues that make up the nucleophile elbow of Gx (H) S xG, as well as the other amino acids required for the catalyst (Keatinge-Clay AT et al., 2003).
  • KR domains There are eight KR domains, one for each of modules 1,2,3,5,6,7,8,9.
  • the parts necessary for NADPH or NADH binding and tyrosine (Tyr), which play a central role in the formation of the Rosaman fold, are conserved (Reid, R. et al. 2003), and the LDD motifs are well conserved (Caffrey, P., 2003). This is consistent with the B-type KRs having an alcohol stereochemical structure.
  • DH domains From modules 1 to 9 there were DH domains. In addition to the DH domain analogues present in Modules 3 and 8, the seven DH domains are well conserved for the HxxxGxxxP motif, an enzyme activity site, and histidine and proline, which are essential for enzyme activity (Joshi, AK, and Smith, S., 1993). ).
  • DH module 8 the active site is completely lost, and the DH domain of module 3 derived from Streptomyces kanamyceticus KCTC 9225 is changed to serine because the amino acid residue constituting the active site is changed to serine. Is considered difficult.
  • the DH of module 2 and module 4 is not required due to the module configuration, but the amino acid residues necessary for activity are well preserved.
  • the ER domain contains the ER domain in the loading module and modules 6, 7, and 9, and LxHxxxGVG, which constitutes the NADPH binding site, which is important for ER function, is well conserved (Witkowski, A. et al., 1991, Amy). CM et al., 1989).
  • FkbQ has conserved Ser, His, Asp and GxSxG constituting the nucleophile elbow constituting the catalytic triad (Bruner, S. D. et al., 2002).
  • the ATCC 55098 gene of Streptomyces genus was missing some modules of polyketide synthase and was not in perfect form. From this, Streptomyces kanamyceticus Unlike the biosynthetic genes derived from KCTC 9225 and Streptomyces KCTC 11604BP strains, biosynthetic genes derived from Streptomyces ATCC 55098 were estimated to be insufficient for biosynthesis of tacrolimus and its structural analogues. It is estimated that there was (FIG. 2).
  • FkbM is 31-O- methyl transferase FkbM-sk (sk means Streptomyces kanamyceticus KCTC 9225 derived) by enzyme-FkbM asco (asco refers to the Streptomyces hygroscopicus subsp. Ascomyceticus ATCC 14891-derived) and 82%, and 71% RapI Similarity is shown. It is assumed that the enzyme catalyzes the O-methylation of 4,5-DHCHC (dihydroxylcyclohex-1-enecarboxylic acid) starter unit derived from Shikimate.
  • 4,5-DHCHC dihydroxylcyclohex-1-enecarboxylic acid
  • FkbD is C9-p450 hydroxylase.
  • FkbD-sk showed 88% similarity with FkbD-asco, 76% with RapJ, and 35% with MerE. This gene is believed to be responsible for the hydroxylation of C9 Oxo FK506 to produce C9-OH FK506.
  • a recent study (Moss SJ et al., 2004) shows that C9 keto form pre-rapamycin (pre-) by RapJ directly from C9-oxo pre-rapamycin. rapamycin) can be synthesized. This suggests that FkbD, an analog of RapK, is also responsible for producing C9-ketoform tacrolimus directly from C9-oxoform.
  • FkbO is an oxidase.
  • FkbO-sk showed 83% similarity to FkbO-asco and 71% with RapK.
  • the function of FkbO is unclear. It is assumed that the C9 position is involved in the oxidation of the hydroxyl group, but both rapamycin and FK506 / FK520 require an oxidation step of the C9-OH group to form a C9 keto group.
  • C9-OH tacrolimus was found to be a by-product of fermentation and can be assumed to act as a C9-OH oxidase.
  • RapK an analogue of FkbO in rapamycin biosynthetic genetic machinery
  • it may play a role in biosynthesis or regulation of DHCHC, the initiating unit, rather than as a C-9 oxidase (Gregory MA et al., 2005). ).
  • the regulatory gene found in all ascomyceticus ATCC 14891 was an fkbN analogue encoding the LuxR family transcriptional regulator.
  • fkbN analogs was the gene is placed in the same orientation as was present between the fkbQ fkbN fkbM both analog and is the reverse, and, fkbQ analogs.
  • the polyketide moiety is the most commonly used precursor of malonyl-CoA and methylmalonyl-CoA.
  • Tacrolimus and ascomycin use unique precursors, which are methoxylmalonyl-ACP. It is estimated that five genes , fkbG, H, I, J, K, are involved so far for synthesis (Wantanabe K. et al., 2003).
  • S. kanamyceticus KCTC 9225 comes with S. hygroscopicus subsp.
  • FkbG from ascomyceticus ATCC 14891 showed 86%
  • FkbH showed 83% similarity
  • 85% FkbI, 77% FkbJ, 87% FkbK was divided into two.
  • Tacrolimus, ascomycin, rapamycin and meridamycin are composed of a special toric amino acid, L-pipecolate, bound to a polyketide.
  • L-Pipecholate is derived from L-lysine and L-lysine is produced through cyclodeamination reaction, and the enzyme responsible for this is known as FkbL and cyclodeaminase.
  • FkbL-sk showed 89% similarity with the analogue FkbL-asco, 76% with RapL, and 43% with TubZ.
  • Enzymes that bind L-pipecolate to polyketides are FkbP and non ribosomal peptide synthetase (NRPS).
  • FkbP-sk shows a similarity of 58% with RapP, 95% with FkbP-MA5648, 84% with FkbP-Asco, and 47% with MerP.
  • FkbP consists of a condensation domain, an adenylation domain, a PCP domain, and again a condensation domain. However, it is not known exactly how each domain performs the binding of L-pipepelate and polyketide.
  • the L-pipecolate synthesized by FkbL is charged to the PCP of FkbP by the adenylation domain, which in turn is condensed with the PK structure by the condensation domain of FkbP, resulting in final formation into the framework of complete tacrolimus and its analogous material. It is estimated.
  • TcsA-sk (SEQ ID NO: 55) had 88% similarity with TcsA-55098 (SEQ ID NO: 94) and 79% with TcsA-11604BP (SEQ ID NO: 2), and these TcsA analogs had AT domains.
  • TcsB-sk showed 86% similarity to TcsB-55098 and 79% to TcsB-11604BP, and TcsB contained the KS domain found in polyketide synthase.
  • the N-terminus of TcsB has a well-conserved KS domain, and all three analogs are also well conserved at the C-terminal position, which plays an important role in the formation of propylmalonyl CoA (ACP) or its analogs. It is estimated.
  • ACP propylmalonyl CoA
  • TcsC is an analog of SalG (ABP73651) included in the salinosporamide A biosynthetic apparatus of Salinispora tropica CNB-476.
  • TcsC-11604BP, TcsC-sk, and TcsC-55098 show high concordance with SalG of 50%, 51% and 51%, respectively.
  • Some of these TcsC analogues exist as FkbS in the ascomycin biosynthetic gene group, but only 152 amino acids in the C terminus of FkbS have been reported, and their function is not known exactly.
  • TcsC analogues found in many macrolide biosynthetic genes are thought to play a role in supplying side branches, such as the ethylmalonyl site of macrolides, but the exact mechanism is unknown. Only recently has SalG been reported to act as a reductive carboxylase.
  • TcsD is a dehydrogenase that is more specific for the synthesis of propylmalonyl ACP (CoA) rather than the general ACAD, and is known to form allyl (propylenyl) malonyl ACP (CoA) through reduction of propylmalonyl ACP (CoA). It is assumed to be involved.
  • the biosynthetic gene loaded on the phosphide vector was moved to a cosmid vector having a high copy number.
  • SuperCos1 vector (Staratagene, USA) was digested with restriction enzyme Bam HI / Nhe I and repaired with T4 DNA polymerase (Fill-in) to construct SuperCos1-1 with one extra coarse site removed. Cut Fos1006G02 with Spe I and Xba I to cut about 38 kb, Fos-1004F01 with Xba I to cut about 37 kb, and Fos-1006D05 with Spe 1 and Xba 1 to cut about 41 kb.
  • Cos-1006G02, Cos-1004F01, and Cos-1006D05 were constructed by ligation to the SuperCos1-1 vectors digested with Xba I, respectively.
  • Cosmids constructed using lambda packaging extracts (Epicentre, Madison, Wis., USA) were used to address the inefficiencies caused by the selection of low ligands due to large size inserts. The method of packaging and introducing into E. coli DH5 ⁇ was used.
  • the introduction of the carrier vector for expression and mutation of the gene into the actinomycetes was performed by an exconjugation method between E. coli / Streptomyces (Kieser, T. et al., 2000). Non-methylated to give (non-methylating donor) into dam -, dc m - of the E. coli ET12567 / pUZ8002 expression vector or a cosmid vector in the actinomycetes This allows the next junction (conjugation) introduced into the electroosmotic (electrophoration) method Introduced.
  • mutants used in the present invention were carried out according to the method reported by Gust B. et al. (2003).
  • the initiators used for constructing the mutants are shown in Table 5.
  • the obtained fragments were introduced into strains introduced into E. coli BW25113 / pIJ790 strains transformed with Cos-1004F01, Cos-1006G02, and Cos-1006D05 by electroshock method, thereby producing mutant cosmid vectors as follows.
  • Cos-1006D05 ( ⁇ tcsD -sk :: aac (3) IV-oriT ) was constructed.
  • the constructed mutant cosmids were transferred to E. coli ET12567 / pUZ8002, respectively, and then transformed into Streptomyces KCTC 11604BP and Streptomyces kanamyceticus KCTC 9225 through the exconjugation of Example 9. Likewise, mutants of each gene could be obtained.
  • S. sp . KCTC 11604BP ( ⁇ tcsA ) , S. sp . KCTC 11604BP ( ⁇ tcsB ), S. sp . KCTC 11604BP ( ⁇ tcsC ), S. sp . KCTC 11604BP ( ⁇ tcsD ), S. sp . KCTC 11604BP ( ⁇ tcs1 ), S. sp . KCTC 11604BP ( ⁇ tcs2 ), S. sp . KCTC 11604BP ( ⁇ tcs3 ), S. sp . KCTC 11604BP ( ⁇ tcs4 ), S.
  • KCTC 11604BP ( ⁇ tcs5 ), S. sp . KCTC 11604BP ( ⁇ tcs7 ), S. sp . KCTC 11604BP ( ⁇ fkbA ), S. sp . KCTC 11604BP ( ⁇ fkbD ), S. sp. KCTC 11604BP ( ⁇ fkbM ), S. sp . KCTC 11604BP ( ⁇ fkbN ), S. sp . KCTC 11604BP ( ⁇ fkbQ ), S. sp . KCTC 11604BP ( ⁇ fkbR ), S.
  • kanamyceticus KCTC 9225 ( ⁇ tcsA ), S. kanamyceticus KCTC 9225 ( ⁇ tcsB ), S. kanamyceticus KCTC 9225 ( ⁇ tcsC ), S. kanamyceticus KCTC 9225 ( ⁇ tcsD )
  • the mutants were Southern cross and PCR confirmed double cross mutants.
  • Fkb A-deficient mutants were prepared and analyzed.
  • FkbA is a tacrolimus polyketide synthetase, contains four modules, and is an essential enzyme for biosynthesis of polyketide backbone. No antifungal activity was shown in the culture of the fkbA- deficient variant, Streptomyces KCTC 11604BP ( ⁇ fkbA ), constructed in Example 10.
  • tacrolimus analogs such as tacrolimus and ascomycin, DH-tacrolimus, FK525 could be confirmed by HPLC and LC-MS of Example 2 (FIG. 4).
  • the gene group secured in the present invention is essential for the formation of biosynthetic machinery of the analogs including tacrolimus and ascomycin. From this fact, it was confirmed that the synthesis of tacrolimus and ascomycin is performed on the same basis, unlike the conventional ascomycin and tacrolimus biosynthesis mechanisms expected to be different from each other. That is, the gene obtained in the present invention means that tacrolimus can be used for the synthesis and modification of ascomycin and its structural analogues.
  • the regulatory factor fkbN mutant Streptomyces KCTC 11604BP ( ⁇ fkbM ) constructed in Example 10 also lost antifungal activity in the same manner as Streptomyces KCTC 11604BP ( ⁇ fkbA ), by HPLC analysis of Example 2 No production of any tacrolimus derivatives could be confirmed. In other words, it was confirmed that FkbN is a very specific positive regulator of tacrolimus biosynthesis gene.
  • KCTC 11604BP ( ⁇ tcsB ), S. sp. KCTC 11604BP ( ⁇ tcsC ), S. sp. Tacrolimus production was not detected by HPLC for KCTC 11604BP ( ⁇ tcsD ) (FIG. 4).
  • the fkbQ and Tcs1,2,3,4,5,7 mutants still produced tacrolimus and ascomycin (FIG. 4).
  • essential tacrolimus biosynthesis genes could be estimated from fkbG to FkbN , including tcsA, B, C, D.
  • these genes are all genes commonly identified in the tacrolimus biosynthetic gene group (FIG. 2).
  • Example 10 As constructed in Example 10 and analyzed in Example 11, the tcsA, B, C, D mutants were unable to confirm tacrolimus production by HPLC.
  • S. kanamyceticus KCTC 9225 ( ⁇ tcsA ), S. kanamyceticus KCTC 9225 ( ⁇ tcsB ), S. kanamyceticus KCTC 9225 ( ⁇ tcsC ), and S. kanamyceticus KCTC 9225 ( ⁇ tcsD ) constructed in Example 10. Production could not be detected by HPLC. However, these strains were wild type S. sp. Lower than KCTC 11604BP but still high antifungal activity was confirmed.
  • KCTC 11604BP ( ⁇ tcsC ) it was confirmed that ascomycin (ethyl form of tacrolimus) and FK523 (methyl form of tacrolimus) are still produced, S. sp.
  • KCTC 11604BP ( ⁇ tcsD ) it was confirmed that a tacrolimus derivative of 37, 38-dihydrofoam was produced.
  • tcsA, B, C, D is a gene that plays a very important role in the side chain formation of tacrolimus position 21.
  • S. sp KCTC 11604BP ( ⁇ tcsB ), S. sp .
  • KCTC 11604BP ( ⁇ tcsC ) produces very small amounts of tacrolimus
  • analogs that partially replace ketosynthase and reductive carboxylase, so that some weakly functioning or complex media
  • allyl (propylenyl) malonic acid or its analogues eg, 4-fethenoic acid, valeric acid or propyl malonyl acid
  • Tacrolimus was not detected at all in KCTC 11604BP ( ⁇ tcsA ), indicating that the AT and ACP domains of TcsA play an absolute role in delivering the allyl-malonyl moiety to the main PKS (ACP or AT of Module 4). It will be. Also S. sp. KCTC 11604BP ( ⁇ tcsD ) was found only in the saturated form of the side chains of all compounds. This may be due to the loss of its role as acyl-CoA dehydrogenase (ACAD). Inferred from the above results, TcsA, B, C, D is a mechanism estimated as shown in Figure 6 in the formation of tacrolimus side chain will play an important role in the synthesis of tacrolimus and its derivatives.
  • ACAD acyl-CoA dehydrogenase
  • TcsA, B, C, D and their gene mutants Utilization of the TcsA, B, C, D and their gene mutants, or the expression of proteins through the introduction of individual or population of genes, the production of new substances, tacrolimus and its analogs, or other that can be newly produced It is thought to be very useful for increasing the production of C. limousin flexible substances or for removing important impurities of tacrolimus such as ascomycin.
  • S. sp . KCTC 11604BP ( ⁇ fkbD ), S. sp .
  • the production of tacrolimus and its analogs of KCTC 11604BP ( ⁇ fkbM ) was confirmed. Both variants lost tacrolimus production capacity.
  • S. sp . KCTC 11604BP ( ⁇ fkbD ) is a 9-dioxo-31-O-desmethyl- ascomycin , including 9-dioxo-31-O-desmethyl-tacrolimus, a deoxo derivative of tacrolimus.
  • 9-dioxo-31-O-desmethyl-tacrolimus and 31-O-desmethyl-tacrolimus are S. sp .
  • high production was possible at 80-150 mg / L, which can be compared with the production of tacrolimus produced in each wild-type strain.
  • Tacrolimus FK506, Ascomycin (FK520), Dihydrotacrolimus, Prolyl tacrolimus (FK525), FK523 (L-683795), L-687819, 9-dioxo-31-O- Desmethyl-tacrolimus, 9-dioxo-31-O-desmethyl-ascomycin, 9-dioxo-tacrolimus, 9-dioxo-ascomycin, 9-hydroxy-9-dioxo-ta Crowlimus, 9-dioxo-36,37-dihydro-tacrolimus, 31-O-desmethyl-tacrolimus, 31-O-desmethyl-ascomycin, 31-O-desmethyl-36,37 -Dihydro-tacrolimus, 9-dioxo-31-O-desmethyl-36,37-dihydro-tacrolimus, prolyl-dihydro-tacrolimus,
  • Mutants that can be reasonably made by the method of constructing the fkbM , fkbD mutants presented in the examples of the present invention are 9-dioxo-31-O-dimethyl-tacrolimus, 9-dioxo-31-O-dimethyl -Ascomycin, 9-dioxo-31-O-dimethyl-36,37-dihydro-tacrolimus, 31-O-dimethyl-tacrolimus, 31-O-dimethyl-ascomycin and 31-O-dimethyl
  • productivity of -36,37-dihydro-tacrolimus can be increased, and thus it can be applied to high production of the tacrolimus modified material produced in small amounts in the wild type.
  • Tcs A, Tcs B, Tcs C, and Tcs D genes obtained by the present invention and the proteins encoded by the genes are directly expressed or mutated in the production or modification of the side branches (allyl-malonyl derivatives) at tacrolimus 21. It can be used as a mechanism and can be applied to structural modification or reduction of impurities of the entire polyketide material using the same.
  • Sequences described in the Sequence Listing of the present invention are gene and protein sequences of enzymes involved in polyketide synthesis, including tacrolimus.

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Abstract

The present invention relates to a gene constituting biosynthetic loci used in biosynthesis of tricyclocompounds produced by microorganisms, particularly the Actinomycete sp., tacrolimus (FK506) and dihydrotacrolimus, which is a structural analog thereof, ascomycin (FK520), prolyl-tacrolimus (FK525), FK523 and the like, and a protein which is encoded by the gene. The gene and the protein can be used to produce and chemically modify tacrolimus, ascomycin and materials having the chemical structure related thereto and to reduce impurities.

Description

삼원환 화합물의 생합성을 담당하는 유전자와 단백질Genes and proteins responsible for the biosynthesis of three-membered ring compounds
본 발명은 폴리케타이드류(polyketides), 특히 삼원환 화합물(tricyclocompounds) 더욱 특히, 면역억제제로 사용되고 있는 타크롤리무스와 그 유연물질의 생합성에 직접적으로 관계하는 유전자의 핵산 서열과 그 서열로부터 생산되는 단백질 및 이를 이용한 활성물질의 생산방법에 관한 것이다.The present invention relates to polyketides, in particular tricyclocompounds and more particularly to nucleic acid sequences of genes directly related to the biosynthesis of tacrolimus and its analogs used as immunosuppressants It relates to a protein and a method for producing an active substance using the same.
삼원환 화합물(Tricyclocompounds)은 주로 미생물, 특히 방선균 속이 생산하는 항진균 활성 및 면역억제 활성을 보이는 미생물 이차 대사 산물이다. 이들 물질은 일반적으로 독성이 매우 약하고 약리활성이 매우 높아 면역억제제, 항암제 등으로 개발되었으며, 프로그랍(PrografTM), 라파뮨(RapamuneTM) 등과 같은 고가의 의약품으로 판매되고 있다. Tricyclocompounds are secondary microbial metabolites that exhibit antifungal and immunosuppressive activity mainly produced by microorganisms, particularly actinomycetes. In general, these substances are very toxic and have high pharmacological activity, and thus have been developed as immunosuppressants, anticancer agents, etc., and are sold as expensive drugs such as Prograf TM and Rapamune TM .
대표적인 삼원환 화합물로서 타크롤리무스(tacrolimus, FK506), 아스코마이신(ascomycin, FK520), 라파마이신(rapamycin, sirolimus), 메리다마이신(meridamycin) 등이 알려져 있다.Tacrolimus (FK506), ascomycin (FK520), rapamycin (sirolimus), meridamycin and the like are known as representative three-membered ring compounds.
타크롤리무스, 라파마이신, 아스코마이신과 그 구조 유사체들은 유사 구조 부위를 가지고 있어 인비보(in vivo)와 인비트로(in vitro)에서 면역세포인 T 세포의 활성화를 억제한다. 이들 화합물은 피라노스-피페콜린 영역(pyranose-pipecolinyl region)(도 1의 C1~C15)을 가지고 있는데 이는 루이신-프롤린 펩타이드(leucine-proline peptide)와 유사한 구조로서, 펩티딜 프로릴 시스/트랜스 이성화 효소 (peptidyl prolyl cis/trans isomerase)의 결합으로 다양한 생리적 활성을 나타낸다고 보고되었다 (Rosen M. K. 등, 1990). Tacrolimus, rapamycin, ascomycin and their structural analogs have similar structural sites that inhibit the activation of immune cells, T cells, in vivo and in vitro. These compounds have a pyranose-pipecolinyl region (C1-C15 in FIG. 1), which is similar in structure to leucine-proline peptides, and has a peptidyl proyl cis / trans It has been reported that the binding of isomerase (peptidyl prolyl cis / trans isomerase) shows various physiological activities (Rosen MK et al., 1990).
아스코마이신은 23개의 탄소로 형성되는 마크롤라이드(23-membered macrolide) 구조의 화합물이며, 타크롤리무스의 에틸 유사체(ethyl analog)이다 (도 1 참조)(Hatanaka H. 등, 1998). 그리고 다이하이드로타크롤리무스 (dihydrotacrolimus)는 타크롤리무스 C21 위치의 프로필 유사체 (propyl analog)이고, FK523은 타크롤리무스 C21 위치의 메틸 유사체 (methyl analog)이며, FK525는 타크롤리무스의 프롤릴 유사체 (prolyl analog)이다. Ascomycin is a 23-membered macrolide compound formed of 23 carbons and an ethyl analog of tacrolimus (see FIG. 1) (Hatanaka H. et al., 1998). And dihydrotacrolimus is a propyl analog of the tacrolimus C21 position, FK523 is a methyl analog of the tacrolimus C21 position, and FK525 is a prolyl analog of tacrolimus ( prolyl analog).
타크롤리무스는 스트렙토마이세스 츠쿠바엔시스(Streptomyces tsukubaensis) 9993(미국 특허 제4,894,366호), 스트렙토마이세스속 (Streptomyces sp.) ATCC 55098, 스트렙토마이세스속(Streptomyces sp.) ATCC 53770, 스트렙토마이세스 클라불리제루스(Streptomyces clavuligerus) CKD1119 (대한민국 특허 10-0485877), 스트렙토마이세스 카나마이세티쿠스(Streptomyces kanamyceticus) KCC S-043 (KCTC 9225) 등의 균주에서 생산되는 것으로 보고되었으며(Muramatsu H.등, 2005), FK506과 구조적으로 유사체인 FK520 또한 스트렙토마이세스 하이그로스코피쿠스 아속 아스코마이세티쿠스(Streptomyces hygroscopicus subsp. ascomyceticus) ATCC 14891, 스트렙토마이세스 하이그로스코피쿠스 아속 야쿠시마엔시스(Streptomyces hygroscopicus subsp. yakusimaensis) 7238과 스트렙토마이세스 츠쿠바엔시스(Streptomyces tsukubaensis) 993 등이 생산하는 것으로 보고되었다. 또한, 라파마이신은 스트렙토마이세스 하이그로스코피쿠스 아속 하이그로스코피쿠스 (Streptomyces hygroscopicus subsp. hygroscopicus) ATCC 29253이 생산하는 것으로 보고되었다. Tacrolimus Streptomyces tsukubaensis 9993 (US Pat. No. 4,894,366), Streptomyces sp. ATCC 55098, Streptomyces sp. ATCC 53770, Streptomyces claus It has been reported to be produced in strains such as S treptomyces clavuligerus CKD1119 (Korean Patent 10-0485877), Streptomyces kanamyceticus KCC S-043 (KCTC 9225) (Muramatsu H. et al. , 2005), FK506 and structurally similar chain FK520 also Streptomyces high-gloss nose kusu subgenus ascorbyl Mai Shetty kusu (Streptomyces hygroscopicus subsp. ascomyceticus) ATCC 14891, Streptomyces high-gloss nose kusu subgenus Yakushima N-Sys (Streptomyces hygroscopicus subsp. yakusimaensis Produced by 7238 and Strepptomyces tsukubaensis 993 Reported. In addition, rapamycin has been reported to produce high-gloss Streptomyces nose kusu subgenus high-gloss nose kusu (Streptomyces hygroscopicus subsp. Hygroscopicus) ATCC 29253.
삼원환 화합물(Tricyclocompounds)은 폴리케타이드(polyketide)와 비 리보좀유래 펩타이드 (nonribosomal peptide) 혼합형(hybrid) 화합물군에 속한다. 이들의 화학구조는 2개의 탄소유닛(carbon unit)이 축합되어 형성되는 폴리케타이드에 사이크로헥실(cyclohexyl) 구조와 비 단백질성 아미노산 (unusual amino acid)인 피페콜린산(pipecolic acid) [FK525의 경우 프롤린(proline)]이 연결되어 형성된 마크롤라이드(macrolide)계 화합물이다. 이들 화합물은 통상적으로 화학적으로 합성이 매우 힘들고, 야생형 생산균주에 의해 생산되는 양은 매우 적다. 그러므로, 이들 화합물의 생합성 기구는 이들 화합물의 구조 변경에 의한 신규물질 창출과 생산력 증대를 위한 수단으로 이용될 수 있다.Tricyclocompounds belong to a group of polyketide and nonribosomal peptide hybrids. Their chemical structure is a polyketide formed by condensation of two carbon units and a cyclohexyl structure and a pipeusic acid (unusual amino acid) of pipecolic acid [FK525] Proline] is a macrolide compound formed by linking. These compounds are typically very difficult to synthesize chemically, and the amounts produced by wild-type producing strains are very small. Therefore, the biosynthetic mechanism of these compounds can be used as a means for generating new materials and increasing productivity by changing the structure of these compounds.
미생물이 생산하는 마크롤라이드계 물질의 생합성 기구는 다수의 큰 단백질들의 복합체 (multiple large proteins complex)로 구성되어 있는 거대효소(megasynthase)이다. 특히 폴리케타이드 부위 생합성은 아실코에이(acyl-CoA)의 반복적 축합(claisen condensation)에 의해 이루어지며, 이들 물질의 생합성을 담당하는 유전자에 의해 암호화되어 있는 효소는 모듈 (module) 형태의 단위로 구성되어 순차적으로 작용하는 것으로 알려져 있다. 이를 소위 모듈 제1형 폴리케타이드 합성효소[modular type I polyketide synthase(PKS)]로 분류한다. The biosynthetic mechanism of macrolide-producing microorganisms is a megasynthase, which is composed of multiple large proteins complex. In particular, polyketide site biosynthesis is achieved by the claisen condensation of acyl-CoA, and enzymes encoded by genes responsible for biosynthesis of these substances are in the form of modules. It is known to be configured and act sequentially. This is classified as a modular type I polyketide synthase (PKS).
모듈 제1형 폴리케타이드 합성효소(modular type I PKS)는 각 사슬 길이를 연장(elongation)하고 변형(modification)하는 촉매부위(catalytic domain)가 모듈이라는 일련의 세트로 구성되어 있다. 하나의 모듈이 한 사이클씩 진행함에 따라 골격을 구성하는 탄소가 두개씩 커지게 된다. 전형적인 모듈 제1형 폴리케타이드 합성효소는 효소활성복합체 (multienzyme system)이며, 시작 모듈 (loading module), 다수의 확장 모듈 (multiple extender modules) 그리고 해리 모듈 (releasing module)로 구성된 것이 전형적인 모습이다. Module type 1 polyketide synthase (modular type I PKS) consists of a series of sets of modules in which the catalytic domains elongate and modify each chain length. As a module progresses by one cycle, the carbon constituting the skeleton grows by two. Typical module type 1 polyketide synthase is a multienzyme system, which is typically composed of a loading module, multiple extender modules and a releasing module.
시작 모듈은 일반적으로 아세틸(acetyl)혹은 프로피오닐(propionyl)기를 아실코엔자임-A (acyl-CoA)를 기질로 사용하지만 독특한 화학구조체를 사용하는 경우도 빈번하다. 확장 모듈은 기본적으로 케토기 합성효소(Ketosynthase; KS), 아실기 전이효소(Acyltransferase; AT), 아실기 운반 단백질(Acyl carrier protein; ACP)의 세 개의 효소도메인(enzymatic domains) 으로 구성되어 있다. 여기에 부가적으로 베타탄소 (beta-carbon)의 변형(modification)에 관여하는 효소들 즉, 케토환원효소(ketoreductase; KR), 탈수소효소(dehydratase; DH), 에놀기 환원효소(enoyl reductase; ER) 등이 첨가되어 모듈이 구성되기도 한다. 한 번의 모듈의 작용으로 아실 코에이 잔기가 첨가되는데 아실기 전이효소(AT)는 아실 잔기(acyl moiety)를 해당하는 아실기 운반 단백질(ACP)로 전달하여 아실-ACP를 생성하게 하고 케토기 합성효소(KS)는 앞의 모듈에 의해 첨가된 분자에 생성된 아실-ACP의 아실기를 클라이젠 축합(claisen condensation)을 통해 탄소-탄소 결합을 하도록 하여 점차적으로 탄소 수를 증가시킨다. 즉 이들 세트가 한 번 작용하면 하나의 아실 잔기가 첨가되어 골격 탄소사슬의 탄소수가 2개씩 증가될 수 있다. 이 과정에서 케토환원효소, 탈수소효소, 그리고 에놀기 환원효소가 차례로 작용하여 베타 탄소 위치의 케토기가 알콜기로, 알콜기가 이중결합으로, 이중결합이 포화 단일결합(saturated single bond)으로 변형되기도 한다. Starting modules generally use acetyl or propionyl groups as acylcoenzyme-A (acyl-CoA) as substrates but often use unique chemical structures. The expansion module basically consists of three enzymatic domains: Ketosynthase (KS), Acyltransferase (AT), and Acyl carrier protein (ACP). In addition, enzymes involved in the modification of beta-carbon, ie ketoductase (KR), dehydroatase (DH), and enolyl reductase (ER) ) May be added to form a module. One action of the module adds an acyl coei residue, which transfers the acyl moiety to the corresponding acyl group transport protein (ACP) to generate acyl-ACP and to synthesize keto groups. The enzyme (KS) gradually increases the carbon number by allowing the acyl group of the acyl-ACP generated in the molecule added by the preceding module to perform carbon-carbon bonds through claisen condensation. That is, once these sets work, one acyl residue may be added to increase the carbon number of the backbone carbon chain by two. In this process, keto-reductase, dehydrogenase, and enol reductase act in turn to transform the keto group into an alcohol group, an alcohol group into a double bond, and a double bond into a saturated single bond. .
상기와 같이 폴리케타이드와 같은 화합물의 생합성 유전자 기구는 일반적으로 담당하고 있는 물질의 구조를 반영하여 구성되어 있다. 생합성기구는 연속공정 시스템으로 구성되어 있으며, 모듈 변형과 같은 연속공정 시스템의 변형으로 종종 원래 담당하고 있는 물질과 다른 구조의 물질이 생산에 응용되기도 한다. 그러므로, 모듈로 구성된 조립라인의 의도적인 변형은 새로운 구조의 물질을 창출할 수 있는 중요한 방편이 될 수 있다. 즉, 생합성 유전자의 부품라인이라고 할 수 있는 모듈의 변형은 바로 새로운 구조의 물질 창출을 의미하게 된다. 이러한 원리에 착안하여, 특정 모듈을 담당하고 있는 유전자를 체계적, 효율적으로 변형, 결실 또는 치환하여 신규의 다양한 물질의 창출을 시도하고자 하는 기술이 조합생합성(combinatorial biosynthesis)이다. 이러한 기술을 적용하기 위해 근본적으로 생합성 유전자를 확보하여야 한다. As described above, the biosynthetic genetic machinery of a compound such as polyketide is generally configured to reflect the structure of a substance in charge. Biosynthetic devices are composed of continuous process systems, and modifications of continuous process systems such as module modifications often lead to the application of materials of a different structure than the ones in charge. Therefore, intentional modification of modular assembly lines can be an important way to create new structures of material. In other words, the modification of the module, which is a part line of the biosynthetic gene, means the creation of a new structure of substance. With this principle in mind, combinatorial biosynthesis is a technique that attempts to create a variety of new materials by systematically and efficiently modifying, deleting, or replacing genes in charge of specific modules. In order to apply this technique, biosynthetic genes must be obtained.
최근에는 몇 가지 삼원환화합물의 생합성을 담당하는 유전자군이 밝혀졌다. Recently, gene families responsible for the biosynthesis of several three-membered cyclic compounds have been identified.
가장 먼저 라파마이신 생합성 유전자와 그 단백질은 스트렙토마이세스 하이그로스코피쿠스 아속 하이그로스코피쿠스 (Streptomyces hygroscopicus subsp. hygroscopicus) ATCC 29253으로부터 클로닝되었다(Schwecke T. 등, 1995).The first rapamycin biosynthetic gene and its protein have been cloned from Streptomyces high-gloss high-gloss nose kusu subgenus nose kusu (Streptomyces hygroscopicus subsp. Hygroscopicus), ATCC 29253 (T. Schwecke et al., 1995).
또한, 스트렙토마이세스 속(Streptomyces sp.) MA6548로부터 타크롤리무스 생합성 유전자가 클로닝되었다(Motamedi H.등, 1998 와 Motamedi H.등, 1997). 이들이 밝힌 유전자는 3개의 폴리케타이드 합성효소(PKS) 유전자 즉 fkbA, fkbB, fkbC이며 이와 더불어, fkbA와 동일한 방향으로 그 상류(upstream)에 fkbO, fkbP가 존재하며 fkbC의 상류에 fkbL이 존재하고, fkbA 의 하류(down stream)에 fkbD 와 fkbM이 전체 약 63kb에 걸쳐 존재하고 있음을 밝혔다. In addition, tacrolimus biosynthesis genes were cloned from Streptomyces sp. MA6548 (Motamedi H. et al., 1998 and Motamedi H. et al., 1997). It is said gene is a three poly Kane Tide synthase (PKS) gene that is fkbA, fkbB, fkbC In addition, in the same direction as the fkbA in the upstream (upstream) fkbO, fkbP exists and the fkbL is present upstream of fkbC , it said that the downstream fkbD and the fkbM (down stream) of fkbA exists throughout the approximately 63kb.
또한, 아스코마이신 생산균주인 스트렙토마이세스 하이그로스코피쿠스 아속 아스코마이세티쿠스(Streptomyces hygroscopicus subsp. ascomyceticus) ATCC 14891로부터 아스코마이신 생합성 유전자군이 클로닝되었고(Wu, K. L. 등, 2000), 전체 약 80kb의 연결 유전자 서열을 분석하여 아스코마이신 생합성에 관련될 것으로 추정되는 fkbA,B,C,P,D,M,O,L,N,Q,S,K,J,I,H,G,F,E,R1,R2,U,W 22개의 유전자 정보를 확인하였다. Further, ascomycin-producing strain of Streptomyces high-gloss nose kusu subgenus ascorbyl Mai Shetty kusu (Streptomyces hygroscopicus subsp. Ascomyceticus) were from ATCC 14891 cloning the ascomycin biosynthetic gene cluster (Wu, KL et al., 2000), a total of about 80kb Analyzes the linker gene sequence to suggest fkbA, B, C, P, D, M, O, L, N, Q, S, K, J, I, H, G, F, E that are believed to be involved in ascomycin biosynthesis 22 genes of R1, R2, U, and W were identified.
타크롤리무스 생합성 유전자와 아스코마이신 생합성 유전자의 구성은 매우 유사하였다. 특히, 폴리케타이드 합성효소를 구성하고 있는 도메인 구성은 아스코마이신 생합성 폴리케타이드 합성효소의 모듈 3과 모듈 8에 존재하는 탈수소효소(DH) 도메인이 타크롤리무스 생합성 유전자에는 존재하지 아니한다는 보고 이외에는 동일하였다. 타크롤리무스 생합성 기구는 그 외 FkbO(oxidase) FkbP(peptide synthase), FkbD(p450 hydroxylase), FkbM(O-methyl transferase) 그리고 FkbL(lysine cyclodeaminase)이 공통적으로 발견되었다. 그러나, 타크롤리무스 생합성 유전자군의 불완전한 확보로 인해 아스코마이신(FK520)에 존재하는 메톡시말로닐 ACP(methoxymalonyl ACP)의 합성을 담당할 것으로 추정되는 FkbG,H,I,J,K를 포함하여 특이적인 전사조절인자인 FkbN 을 담당하는 유전자 등의 존재 유무는 밝혀지지 않았다. The composition of tacrolimus biosynthesis gene and ascomycin biosynthesis gene were very similar. In particular, the domain composition constituting the polyketide synthase is different from the report that the dehydrogenase (DH) domains present in modules 3 and 8 of ascomycin biosynthetic polyketide synthase do not exist in the tacrolimus biosynthetic gene. Same. Tacrolimus biosynthesis was commonly found in FkbO (oxidase), peptide synthase (FkbP), p450 hydroxylase (FkbD), O-methyl transferase (FkbM), and lysine cyclodeaminase (FkbL). However, due to incomplete acquisition of the tacrolimus biosynthetic gene family, it contains Fkb G, H, I, J, K, which are believed to be responsible for the synthesis of methoxymalonyl ACP present in ascomycin (FK520). Therefore, the presence or absence of a gene responsible for a specific transcription regulator FkbN is not known.
아스코마이신 생산균주인 스트렙토마이세스 하이그로스코피쿠스 아속 아스코마이세티쿠스(Streptomyces hygroscopicus subsp. ascomyceticus) ATCC 14891의 발효배양체에서 타크롤리무스, 다이하이드로타크롤리무스 등의 C21 위치에 탄소가 4개 부착된 유도체가 발견된 보고가 없다. 그러나, 타크롤리무스 생산균주인 스트렙토마이세스 츠쿠바엔시스(Streptomyces tsukubaensis) 993의 경우 아스코마이신 및 다이하이드로타크롤리무스, FK523 등 C21 위치의 유사체가 생산되는 것으로 보고되었다(미국 특허 4,894,366). 이러한 유사체 발효산물 의 존재로부터 타크롤리무스 생산균주 생합성 유전자의 구성이 아스코마이신 생산균주 생합성 유전자 구성과는 차이가 있음을 유추해 볼 수 있다. 즉, 타크롤리무스 생산균주의 경우 C21 위치 생합성 유전자를 담당하는 또 다른 유전자가 아스코마이신 생합성 유전자에 부가하여 존재할 가능성이 높은 것이다. 또한 타크롤리무스와 아스코마이신 등의 삼원환 화합물 생합성 조절에 관여하는 또 다른 유전자는 타크롤리무스 및 기타 삼원환 화합물의 생산 증대 혹은 다양한 유도체 창출이 가능하도록 이용할 수 있을 것이다.Ascomycin producing strain Streptomyces hygroscopicus asoke ascomyceticusStreptomyces hygroscopicus subsp.ascomyceticusIn the fermentation broth of ATCC 14891, there have been no reports of derivatives having four carbons attached to the C21 positions such as tacrolimus and dihydrotacrolimus. However, tacrolimus producing strain Streptomyces Tsukubaensis (Streptomyces tsukubaensis) In the case of 993, it has been reported that analogues of C21 positions, such as ascomycin and dihydrotacrolimus, FK523, are produced (US Pat. No. 4,894,366). From the presence of the analogue fermentation product, it can be inferred that the composition of the tacrolimus producing strain biosynthesis gene is different from that of the ascomycin producing strain biosynthesis gene. That is, in the case of tacrolimus producing strain, another gene responsible for the C21 position biosynthesis gene is likely to exist in addition to the ascomycin biosynthesis gene. In addition, another gene involved in regulating the biosynthesis of tricyclic compounds such as tacrolimus and ascomycin may be used to increase the production of tacrolimus and other tricyclic compounds or to generate various derivatives.
본 발명의 목적은 면역억제제로 사용되고 있는 타크롤리무스와 그 유연물질의 생합성에 직접적으로 관계하는 핵산 서열과 그 서열에 담겨 있는 폴리펩타이드 서열 정보를 제공하는 것이다.It is an object of the present invention to provide nucleic acid sequences directly related to the biosynthesis of tacrolimus and its analogs used as immunosuppressive agents and polypeptide sequence information contained in the sequences.
본 발명자들은 타크롤리무스 생산균주들로부터 생합성 유전자를 완전하게 확보하고 유전자의 기능을 규명하고 이용성을 확인하였다. 본 발명은 기존에 불완전하게 규명된 타크롤리무스 생합성 기구를 담당하고 있는 타크롤리무스 생합성 유전자군을 더욱 확대하여 아스코마이신 생합성 유전자에는 존재하지 않으며 타크롤리무스 생합성 유전자에만 존재하는 유전자를 확보하였다. 즉, 기존 타크롤리무스 생합성 유전자군에서 밝혀진 FkbA,B,C 및 FkbO,P,D,M,L을 포함하는 완전한 타크롤리무스 생합성기구를 담당하는 유전자군을 확보하여 핵산서열을 밝혔으며, 그 핵산 서열이 담고 있는 유전자와 그 유전자로부터 생산되는 폴리펩타이드와 타크롤리무스 및 그 유사체를 포함하는 삼원환 화합물들의 생합성 연관성을 규명하고 그 산업적 응용성을 확인하였다. The present inventors fully secured biosynthetic genes from tacrolimus producing strains, identified the function of the genes, and confirmed the availability. The present invention further expands the tacrolimus biosynthetic gene group, which is in charge of the previously incompletely identified tacrolimus biosynthesis apparatus, to secure genes that do not exist in the ascomycin biosynthetic gene but exist only in the tacrolimus biosynthetic gene. That is, the nucleic acid sequence was revealed by securing a gene group in charge of a complete tacrolimus biosynthesis apparatus including FkbA, B, C and FkbO, P, D, M, L found in the existing tacrolimus biosynthesis gene group. The biosynthetic association between the gene containing the nucleic acid sequence, the polypeptide produced from the gene, and the tricyclic compounds including tacrolimus and its analogs was identified and its industrial applicability was confirmed.
본 발명은 서열번호 1, 54 또는 93과 동일하거나 85% 이상의 동일성을 나타내는 서열 또는 상기 서열에 상보적인 서열로 표시되는 타크롤리무스 생합성 유전자 클러스터를 제공한다.The present invention provides a tacrolimus biosynthetic gene cluster represented by a sequence that is the same as or greater than or equal to 85% identity with SEQ ID NO: 1, 54 or 93, or a sequence complementary to the sequence.
본 발명은 서열번호 3, 56 또는 95와 동일하거나 80% 이상의 동일성을 나타내는 서열 또는 상기 서열에 상보적인 서열로 표시되는 폴리케타이드 합성효소 유전자를 제공한다.The present invention provides a polyketide synthase gene represented by a sequence that is identical to, or complementary to, SEQ ID NO: 3, 56, or 95 or more than 80% identity.
본 발명은 서열번호 2, 55 또는 94와 동일하거나 80% 이상의 동일성을 나타내는 서열로 표시되는 폴리케타이드 합성효소를 제공한다.The present invention provides a polyketide synthetase represented by a sequence that is identical to or exhibits 80% or more identity to SEQ ID NO: 2, 55 or 94.
본 발명은 서열번호 5, 58 또는 97과 동일하거나 80% 이상의 동일성을 나타내는 서열 또는 상기 서열에 상보적인 서열로 표시되는 폴리케타이드 합성효소 유전자를 제공한다.The present invention provides a polyketide synthase gene represented by a sequence showing the same or 80% or more identity to SEQ ID NO: 5, 58 or 97 or a sequence complementary to the sequence.
본 발명은 서열번호 4, 57 또는 96과 동일하거나 80% 이상의 동일성을 나타내는 서열로 표시되는 폴리케타이드 합성효소를 제공한다.The present invention provides a polyketide synthetase represented by a sequence which is identical to SEQ ID NO: 4, 57 or 96 or which exhibits at least 80% identity.
본 발명은 서열번호 7, 60 또는 99와 동일하거나 90% 이상의 동일성을 나타내는 서열 또는 상기 서열에 상보적인 서열로 표시되는 폴리케타이드 합성효소 유전자를 제공한다.The present invention provides a polyketide synthase gene represented by a sequence that is identical to, or complementary to, SEQ ID NO: 7, 60, or 99, or at least 90% identity.
본 발명은 서열번호 6, 59 또는 98과 동일하거나 90% 이상의 동일성을 나타내는 서열로 표시되는 폴리케타이드 합성효소를 제공한다.The present invention provides a polyketide synthetase represented by a sequence that is identical to or exhibits 90% or more identity to SEQ ID NO: 6, 59, or 98.
본 발명은 서열번호 9, 62 또는 101과 동일하거나 90% 이상의 동일성을 나타내는 서열 또는 상기 서열에 상보적인 서열로 표시되는 폴리케타이드 합성효소 유전자를 제공한다.The present invention provides a polyketide synthase gene represented by a sequence that is identical to, or complementary to, SEQ ID NO: 9, 62, or 101 or 90% identity.
본 발명은 서열번호 8, 61 또는 100과 동일하거나 90% 이상의 동일성을 나타내는 서열로 표시되는 폴리케타이드 합성효소를 제공한다.The present invention provides a polyketide synthetase represented by a sequence that is identical to or exhibits 90% or more identity to SEQ ID NO: 8, 61 or 100.
본 발명은 청구항 1의 타크롤리무스 생합성 유전자 클러스터 중 fkbD 유전자 코딩 영역을 결손시키거나 불활성화시킨 돌연변이체를 배양하여 9-디옥소-31-O-데스메틸-타크롤리무스, 9-디옥소-31-O-데스메틸-아스코마이신 및 9-디옥소-31-O-데스메틸-36,37-다이하이드로-타크롤리무스 중 선택된 1종 이상을 생산하는 방법을 제공한다.The present invention provides a 9-dioxo-31-O-desmethyl-tacrolimus, 9-dioxo- by culturing a mutant in which the fkbD gene coding region of the tacrolimus biosynthetic gene cluster of claim 1 is deleted or inactivated. Provided are methods for producing one or more selected from 31-O-desmethyl-ascomycin and 9-dioxo-31-O-desmethyl-36,37-dihydro-tacrolimus.
본 발명은 청구항 1의 타크롤리무스 생합성 유전자 클러스터 중 fkbM 유전자 코딩 영역을 결손시키거나 불활성화시킨 돌연변이체를 배양하여 31-O-데스메틸-타크롤리무스, 31-O-데스메틸-아스코마이신 및 31-O-데스메틸-36,37-다이하이드로-타크롤리무스 중 선택된 1종 이상을 생산하는 방법을 제공한다.The present invention is cultured mutant in which the fkbM gene coding region of the tacrolimus biosynthetic gene cluster of claim 1 is deleted or inactivated 31-O- desmethyl- tacrolimus, 31-O- desmethyl- ascomycin and A method of producing at least one selected from 31-O-desmethyl-36,37-dihydro-tacrolimus is provided.
본 발명에서 타크롤리무스 생합성 유전자군은 3가지 타크롤리무스 생산균주 스트렙토마이세스속(Streptomyces sp.) ATCC 55098, 스트렙토마이세스 카나마이세티쿠스(Streptomyces kanamyceticus) KCTC 9225, 스트렙토마이세스속(Streptomyces sp.) KCTC 11604BP로부터 확보하였다. 각 균주로부터 타크롤리무스 생합성 유전자군의 연속적인 핵산 서열을 확보하고(서열번호 1, 54, 93), 이들 연속적인 핵산서열로부터 유전자 (서열번호 3,5,7,9,11,13,15,17,19,21,23,25,27,29,31,33,35,37,39,41,43,45,47,49,51,53, 56,58,60,62,64,66,68,70,72,74,76,78,80,82,84,86,88,90,92,95,97,99,101,103,105,107,109,111,113,115,117,119,121,123,125,127,129,131)를 예측하고, 폴리펩타이드 서열(서열번호 2,4,6,8,10,12,14,16,18,20,22,24,26,28,30, 32,34,36,38,40,42,44,46,48,50,52,55,57,59,61,63,65,67,69,71,73,75,77,79,81,83,85,87,91,94,96,98,100,102,104,106,108,110,112,114,116,118,120,122,124,126,128,130)을 확인하였으며, 이들 유전자의 돌연변이체들을 작제하여 타크롤리무스 및 그 유사체 등의 생합성 연관관계를 규명하였다. 이러한 발명으로 타크롤리무스 생합성 유전자군에서 공통적으로 발견되는 유전자 19개(서열번호 3,5,7,9,21,23,25,27,29,31,33,35,37,39,41,43,45,47,49,56,58,60,62,64, 66,68,70,72,74,76,78,80,82,84,86,88,90,92,95,97,99,101,103,105,107,109,111,113,115,117,119,121,123,125,127,129,131)를 확인하였다(도 2). In the present invention, the tacrolimus biosynthetic gene group includes three types of tacrolimus producing strain Streptomyces sp. ATCC 55098, Streptomyces kanamyceticus KCTC 9225, Streptomyces sp .) Obtained from KCTC 11604BP. From each strain, a sequence of nucleic acid sequences of tacrolimus biosynthetic gene group was obtained (SEQ ID NOs: 1, 54, 93), and genes (SEQ ID NOs: 3, 5, 7, 9, 11, 13, 15) from these consecutive nucleic acid sequences , 17,19,21,23,25,27,29,31,33,35,37,39,41,43,45,47,49,51,53,56,58,60,62,64,66 , 68,70,72,74,76,78,80,82,84,86,88,90,92,95,97,99,101,103,105,107,109,111,113,115,117,119,121,123,125,127,129,131 , 10,12,14,16,18,20,22,24,26,28,30,32,34,36,38,40,42,44,46,48,50,52,55,57,59 , 61,63,65,67,69,71,73,75,77,79,81,83,85,87,91,94,96,98,100,102,104,106,108,110,112,114,116,118,120,122,124,126,128,130), and mutants of these genes were constructed The biosynthetic relationship between Crawlimus and its analogs was elucidated. In this invention, 19 genes commonly found in the tacrolimus biosynthetic gene group (SEQ ID NOs: 3,5,7,9,21,23,25,27,29,31,33,35,37,39,41, 43,45,47,49,56,58,60,62,64,66,68,70,72,74,76,78,80,82,84,86,88,90,92,95,97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131 were confirmed (Fig. 2).
본 발명자들은 특히 이들 유전자 중에 tcsA,B,C,D의 유전자 및 이들 유전자가 담당하고 있는 폴리펩타이드(서열번호 2,3,4,5,6,7,8,9,55,56,57,58,59, 60,61,62,94,95,96, 97,98,99,100,101)가 타크롤리무스와 그 유연물질의 C-21 위치의 곁사슬(알릴-말로닐 유도체)의 합성에 관여함을 밝혔다. 그러므로 이들 유전자가 신규한 물질의 창출 혹은 타크롤리무스 및/또는 그 유연물질의 생산성의 증대 및 불순물 감소를 통한 경제적인 생산에 활용될 수 있을 것이다. In particular, the inventors of the present invention have shown that the genes of tcsA, B, C, D and the polypeptides in which these genes are responsible (SEQ ID NOs: 2,3,4,5,6,7,8,9,55,56,57, 58,59, 60,61,62,94,95,96, 97,98,99,100,101 are involved in the synthesis of tacrolimus and the side chain (alyl-malonyl derivative) at position C-21 of the analogue. Said. Therefore, these genes can be used for economic production through the production of new materials or by increasing the productivity of tacrolimus and / or its related materials and reducing impurities.
또한, 본 발명자들은 fkbD와 fkbM (서열번호 42,43,44,45)의 돌연변이 방법으로 9-디옥소-31-O-데스메틸-타크롤리무스(아스코마이신){9-deoxo-31-O-desmethyl-tacrolimus(ascomycin)}와 31-O-데스메틸-타크롤리무스(아스코마이신){31-O-desmethyl-tacrolimus(ascomycin)}을 고생산할 수 있음을 확인하였다. In addition, the present inventors have used 9-dioxo-31-O-desmethyl-tacrolimus (ascomycin) {9-deoxo-31-O by a mutation method of fkbD and fkbM (SEQ ID NOs: 42,43,44,45 ). -desmethyl-tacrolimus (ascomycin)} and 31-O-desmethyl-tacrolimus (ascomycin) {31-O-desmethyl-tacrolimus (ascomycin)} was found to be capable of high production.
본 발명으로 타크롤리무스(FK506), 아스코마이신(FK520), 다이하이드로타크롤리무스, 프롤릴타크롤리무스(FK525), FK523(L-683795), L-687819, 9-디옥소-31-O-데스메틸-타크롤리무스, 9-디옥소-31-O-데스메틸-아스코마이신, 9-디옥소-타크롤리무스, 9-디옥소-아스코마이신, 9-하이드록시-9-디옥소-타크롤리무스, 9-디옥소-36,37-다이하이드로-타크롤리무스, 31-O-데스메틸-타크롤리무스, 31-O-데스메틸-아스코마이신, 31-O-데스메틸-36,37-다이하이드로-타크롤리무스, 9-디옥소-31-O-데스메틸-36,37-다이하이드로-타크롤리무스, 프롤릴-다이하이드로-타크롤리무스, 라파마이신, 메리다마이신 및 36-케토-메리다마이신 생합성에 필요한 유전자를 확보하였으며, 이들 물질의 산업적 생산 및 다른 신규한 구조의 화합물 창조를 위한 구조변형 등에 응용할 수 있다. Tacrolimus (FK506), Ascomycin (FK520), Dihydrotacrolimus, Prolyl tacrolimus (FK525), FK523 (L-683795), L-687819, 9-dioxo-31-O- Desmethyl-tacrolimus, 9-dioxo-31-O-desmethyl-ascomycin, 9-dioxo-tacrolimus, 9-dioxo-ascomycin, 9-hydroxy-9-dioxo-ta Crowlimus, 9-dioxo-36,37-dihydro-tacrolimus, 31-O-desmethyl-tacrolimus, 31-O-desmethyl-ascomycin, 31-O-desmethyl-36,37 -Dihydro-tacrolimus, 9-dioxo-31-O-desmethyl-36,37-dihydro-tacrolimus, prolyl-dihydro-tacrolimus, rapamycin, meridamycin and 36-keto The genes needed for the biosynthesis of meridamycin have been obtained, and they can be applied to the industrial production of these materials and structural modifications for the creation of other novel compounds.
본 발명의 예시에서 제시된 fkbM, fkbD 돌연변이체의 작제방법에 의해 합리적으로 만들어질 수 있는 돌연변이체는 9-디옥소-31-O-디메틸-타크롤리무스, 9-디옥소-31-O-디메틸-아스코마이신, 9-디옥소-31-O-디메틸-36,37-다이하이드로-타크롤리무스, 31-O-디메틸-타크롤리무스, 31-O-디메틸-아스코마이신 및 31-O-디메틸-36,37-다이하이드로-타크롤리무스의 생산성을 높일 수 있어 야생형에서 소량으로 생산되는 상기 타크롤리무스 변형물질의 고생산에 응용될 수 있다. Mutants that can be reasonably made by the method of constructing the fkbM , fkbD mutants presented in the examples of the present invention are 9-dioxo-31-O-dimethyl-tacrolimus, 9-dioxo-31-O-dimethyl -Ascomycin, 9-dioxo-31-O-dimethyl-36,37-dihydro-tacrolimus, 31-O-dimethyl-tacrolimus, 31-O-dimethyl-ascomycin and 31-O-dimethyl The productivity of -36,37-dihydro-tacrolimus can be increased, and thus it can be applied to high production of the tacrolimus modified material produced in small amounts in the wild type.
본 발명으로 확보한 TcsA,TcsB,TcsC,TcsD 유전자 및 그 유전자가 코딩하고 있는 단백질은 타크롤리무스 21 위치의 곁가지 (알릴-말로닐 유도체)의 생산 혹은 변형에 직접, 발현 혹은 돌연변이체 작제로 이용될 수 있으며 이를 이용한 전체 폴리케타이드 물질의 구조 변형 또는 불순물 감소에 응용될 수 있다.The Tcs A, Tcs B, Tcs C, and Tcs D genes obtained by the present invention and the proteins encoded by the genes are directly expressed or mutated in the production or modification of the side branches (allyl-malonyl derivatives) at tacrolimus 21. It can be used as a mechanism and can be applied to structural modification or reduction of impurities of the entire polyketide material using the same.
도 1은 삼원환 화합물의 구조(화학식)이다.1 is a structure (formula) of a three-membered ring compound.
도 2는 타크롤리무스 생합성 유전자군을 나타낸다. 각각 (A), 스트렙토마이세스속(Streptomyces sp.) KCTC 11604BP ; (B), 스트렙토마이세스 카나마이세티쿠스(Streptomyces kanamyceticus) KCTC 9225; (C), 스트렙토마이세스속(Streptomyces sp.) ATCC 55098 균주로부터 확보하였다. 2 shows the tacrolimus biosynthesis gene group. (A), Streptomyces sp. KCTC 11604BP, respectively; (B), Streptomyces kanamyceticus KCTC 9225; (C), Streptomyces sp. Was obtained from the ATCC 55098 strain.
도 3은 생합성 유전자군의 모듈(Modules)과 도메인 구성 및 추정 생합성 과정을 나타낸다. 위쪽 도메인 구성은 스트렙토마이세스속(Streptomyces sp.) KCTC 11604BP, 아래쪽은 스트렙토마이세스 카나마이세티쿠스(Streptomyces kanamyceticus) KCTC 9225 유래의 것이다. Figure 3 shows the modules (Modules) and domain composition and putative biosynthesis process of the biosynthetic gene group. The upper domain is from Streptomyces sp. KCTC 11604BP and the lower is from Streptomyces kanamyceticus KCTC 9225.
도 4는 생합성 유전자 돌연변이체의 HPLC 분석 결과이다.4 shows the results of HPLC analysis of biosynthetic gene mutants.
(a),(f), S. sp. KCTC 11604BP; (b), S. sp. KCTC 11604BP(ΔfkbA) d; (c), S. sp. KCTC 11604BP(ΔfkbN); (d), S. sp. KCTC 11604BP(Δtcs1); (e), S. sp. KCTC 11604BP(ΔfkbQ); (g), S. sp. KCTC 11604BP(ΔtcsA); (h), S. sp. KCTC 11604BP(ΔtcsB); (i), S. sp. KCTC 11604BP(ΔtcsC); (j), S. sp. KCTC 11604BP(ΔtcsD) (a), (f), S. sp. KCTC 11604BP; (b), S. sp. KCTC 11604BP (Δ fkbA ) d; (c), S. sp. KCTC 11604BP (Δ fkbN ); (d), S. sp. KCTC 11604BP (Δ tcs1 ); (e), S. sp. KCTC 11604BP (Δ fkbQ ); (g), S. sp. KCTC 11604BP (Δ tcsA ); (h), S. sp. KCTC 11604BP (Δ tcsB ); (i), S. sp. KCTC 11604BP (Δ tcsC ); (j), S. sp. KCTC 11604BP (Δ tcsD )
화합물 1, 타크롤리무스; 화합물 2, 아스코마이신; 화합물 3, 36,37-다이하이드로-타크롤리무스 Compound 1, tacrolimus; Compound 2, ascomycin; Compound 3, 36,37-dihydro-tacrolimus
(a)-(e), HP-20 레진의 첨가 없이 발효 배양하여 분석하였다. (a)-(e), analyzed by fermentation culture without addition of HP-20 resin.
(f)-(j), 5% HP-20 레진을 첨가하여 발효 배양하여 분석하였다.(f)-(j), 5% HP-20 resin was added and analyzed by fermentation culture.
도 5는 방선균 배양액의 총이온 질량스펙트럼 분석 결과이다.5 is a result of analyzing the total ion mass spectrum of the actinomycetes culture medium.
(A), S. sp. KCTC 11604BP; (B), S. sp. KCTC 11604BP(ΔtcsA); (C), S. sp. KCTC 11604BP(ΔtcsB); (D), S. sp. KCTC 11604BP(ΔtcsC); (E), S. sp. KCTC 11604BP(ΔtcsD)(A),S. sp. KCTC 11604BP; (B), S. sp. KCTC 11604BP (ΔtcsA); (C),S. sp. KCTC 11604BP (ΔtcsB); (D),S. sp. KCTC 11604BP (ΔtcsC);  (E),S. sp. KCTC 11604BP (ΔtcsD)
도 6은 타크롤리무스 C-21 측쇄의 합성과정 추정도이다.6 is a diagram illustrating the synthesis process of tacrolimus C-21 side chain.
약어: KS, 케토합성효소(ketosynthase); AT, 아실기 전이효소(acyltransferase); ACP, 아실기 운반 단백질(acyl carrier protein); aDH, 아실-ACP(CoA) 탈수소효소{acyl-ACP(CoA) dehydrogenase}; R/C, 환원효소/카복실라제(reductase/carboxylase)Abbreviations: KS, ketosynthase; AT, acyltransferase; ACP, acyl carrier protein; aDH, acyl-ACP (CoA) dehydrogenase {acyl-ACP (CoA) dehydrogenase}; R / C, reductase / carboxylase
도 7은 FkbD, FkbM 돌연변이체 배양액의 HPLC 분석 결과이다.7 shows the results of HPLC analysis of FkbD and FkbM mutant cultures.
(a), S. sp. KCTC 11604BP, (b), S. sp. KCTC 11604BP(ΔfkbD); (c), S. sp. KCTC 11604BP(ΔfkbM)(a), S. sp. KCTC 11604BP, (b), S. sp. KCTC 11604BP (Δ fkbD ); (c), S. sp. KCTC 11604BP (Δ fkbM )
피크: 1, 타크롤리무스; 2, 아스코마이신; 3,9-디옥시-31-디메틸-타크롤리무스; 4,9-디옥소-31-디메틸-아스코마이신; 5,9-디옥소-31-디메틸-36,37,-다이하이드로타크롤리무스; 6,31-디메틸-타크롤리무스; 7,31-디메틸아스코마이신; 8,31-디메틸-36,37-다이하이드로타크롤리무스.Peak: 1, tacrolimus; 2, ascomycin; 3,9-dioxy-31-dimethyl-tacrolimus; 4,9-dioxo-31-dimethyl-ascomycin; 5,9-dioxo-31-dimethyl-36,37, -dihydrotacrolimus; 6,31-dimethyl-tacrolimus; 7,31-dimethylascomycin; 8,31-dimethyl-36,37-dihydrotacrolimus.
배양은 레진 첨가 없이 진행하였으며, Sep-Pack(C18)으로 간단히 정제후 분석하였다.The cultivation proceeded without the addition of resin, followed by brief purification with Sep-Pack (C18).
이러한 발명의 구체적인 내용을 아래 실시예에서 상세히 설명하고자 하며, 하기 실시예에서 사용한 균주, 벡터, 프로모터, 개시체, PCR방법, 제한효소 및 제한효소 부위, 기타 핵산의 클로닝과 서열 분석에 사용한 효소, 시약 및 기기 등의 재료 및 방법, 돌연변이 방법은 본 발명의 실시방법을 예를 들어 상세히 설명하고자 하는 것이다. 그러나, 아래 실시예의 기재가 본 발명의 권리 범위를 구체적으로 제한하는 것은 아니다. Specific details of the present invention will be described in detail in the following examples, and the strains, vectors, promoters, initiators, PCR methods, restriction enzymes and restriction enzyme sites used in the following examples, enzymes used for cloning and sequencing of other nucleic acids, Materials and methods such as reagents and instruments, and mutant methods are intended to be described in detail by way of examples of the present invention. However, the description of the following examples does not specifically limit the scope of the present invention.
<실시예 1> 균주 및 발효 Example 1 Strains and Fermentations
본 발명에서는 타크롤리무스를 생산하는 균주들로부터 생합성 유전자를 확보하였다. 본 발명에 사용된 방선균은 타크롤리무스의 생산이 보고된 스트렙토마이세스 카나마이세티쿠스(Streptomyces kanamyceticus) KCTC 9225 (Muramatsu H. 등, 2005)와 스트렙토마이세스속(Streptomyces sp.) KCTC 11604BP 및 스트렙토마이세스속(Streptomyces sp.) ATCC 55098이다. In the present invention, biosynthetic genes were obtained from strains producing tacrolimus. Actinomycetes used in the present invention are Streptomyces kanamyceticus KCTC 9225 (Muramatsu H. et al., 2005) and Streptomyces sp. KCTC 11604BP and Streptomyces reported production of tacrolimus. Streptomyces sp. ATCC 55098.
상기 기록한 방선균속뿐만 아니라, 하기 본 발명에 기재된 상기 균주의 돌연변이체 및 형질전환체를 이용한 타크롤리무스 또는 그 유사체의 발효 배양은 아래 조건으로 수행하였다. In addition to the above described actinomycetes, the fermentation culture of tacrolimus or its analogs using the mutants and transformants of the strains described in the present invention was performed under the following conditions.
스트렙토마이세스속 균주를 500㎖ 엘렌마이어 삼각 플라스크에 들어있는 30㎖ GT-TA-1 배지[1% 수용성 전분, 0.7% 글리세롤, 0.3% 박토펩톤, 0.3% 효모추출물, 0.5% 소이톤펩톤, 0.05% AZ-20R(antifoam), pH 6.8]에 28℃, 240rpm의 조건에서 하루 동안 배양하여 활성화시킨 후, 활성화된 종배양 균주를 3ℓ 엘렌마이어 삼각플라스크에 들어있는 150㎖의 GT-TA-2 배지[1% 산화 전분, 1% 글리세롤, 2% 소이빈 밀, 0.2% CaCO3, 0.5% CSL, 0.05% AZ-20R(antifoam), pH 6.5]에 접종, 동일 조건에서 하루 동안 배양을 실시하였다. GT-TA-2 배지에서 배양된 배양액을 5ℓ 발효장치에 들어있는 2.7ℓ의 GT-TA-4 배지 [7% 산화 전분, 1.7% 효모 분말, 0.5% 소이빈 밀, 0.1% (NH4)2SO4, 0.1% CaCO3, 0.05% AZ-20R(antifoam), pH 8.5]에 최종 배양액 부피의 10%의 양을 접종하여 28℃의 온도에서 6일 동안 1.5vvm의 통기 조건에서 600~900rpm으로 배양을 실시하였다. 고농도로 배양할 필요가 있는 경우 5% HP-20 레진을 GT-TA-4 배지에 첨가하여 살균 후 사용하였다. Streptomyces strains were prepared in 30 ml GT-TA-1 medium [1% aqueous starch, 0.7% glycerol, 0.3% bactopeptone, 0.3% yeast extract, 0.5% soyton peptone, 0.05 in a 500 ml Erlenmeyer Erlenmeyer flask. % AZ-20R (antifoam), pH 6.8] after activating for one day at 28 ℃, 240rpm conditions, the activated species culture strain 150ml GT-TA-2 medium contained in 3L Elenmeyer Erlenmeyer flask [1% oxidized starch, 1% glycerol, 2% soybean mill, 0.2% CaCO 3 , 0.5% CSL, 0.05% AZ-20R (antifoam), pH 6.5] and incubated for one day under the same conditions. Culture cultured in GT-TA-2 medium was carried out in 2.7 L GT-TA-4 medium [7% oxidized starch, 1.7% yeast powder, 0.5% soybean mill, 0.1% (NH 4 ) 2 in a 5 L fermenter. SO 4 , 0.1% CaCO 3 , 0.05% AZ-20R (antifoam), pH 8.5] was inoculated with 10% of the final culture volume at 600-900 rpm for 6 days at a temperature of 1.5vvm at a temperature of 28 ° C. The culture was carried out. 5% HP-20 resin was added to GT-TA-4 medium and used after sterilization when it was necessary to culture at high concentration.
<실시예 2> 타크롤리무스 및 유연물질 분석Example 2 Tacrolimus and Lead Material Analysis
발효 배양된 배양액으로부터 회수된 배양액 혹은 HP-20 레진으로부터 75% 아세톤으로 대사물질을 추출하였다. 추출된 대사물질은 표 1과 같은 HPLC 조건 하에 분석하였다. Metabolites were extracted with 75% acetone from cultures or HP-20 resins recovered from fermented cultures. Extracted metabolites were analyzed under HPLC conditions as shown in Table 1.
표 1
항목 조건 내용
칼럼 Hypersil GOLD C18 analytical column(Thermo)
칼럼온도 55℃
흡수파장 210㎚
이동상 50% 아세토나이트릴(Acetonitrile)
유속 1㎖/min
시료 주입량 20㎕
Table 1
Item Condition content
column Hypersil GOLD C18 analytical column (Thermo)
Column temperature 55 ℃
Absorption wavelength 210 nm
Mobile phase 50% Acetonitrile
Flow rate 1ml / min
Sample injection volume 20 μl
타크롤리무스 및 그 유연물질을 동정하기 위해 LC-MS/MS를 수행하였다. LC-MS/MS를 위해 아래와 같이 HLB 카트리지를 이용하여 시료를 부분정제하였다. 배양액에 아세토니트릴을 동일 부피로 혼합하고 30분간 추출한 후 물을 부가하여 최종적으로 아세토니트릴이 25%가 되도록 하였다. 이를 HLB 카트리지(OASIS HLC cartridge, 1㎖)에 통과하고 이후에 2배 용량의 30% 아세토니트릴로 미흡착 물질을 씻어내었다. 그 후 40, 50, 60, 70% 아세토니트릴로 1배량으로 용리하였다. 단계별 용리액 중 50% 아세토니트릴 용리액을 주분획으로 사용하여 표 2와 같은 LC-MS 조건으로 질량분석을 진행하였다. LC-MS / MS was performed to identify tacrolimus and its analog. Samples were partially purified using HLB cartridges for LC-MS / MS as follows. Acetonitrile was mixed in the same volume to the culture and extracted for 30 minutes, and then water was added to finally give 25% of acetonitrile. This was passed through an HLB cartridge (OASIS HLC cartridge, 1 ml) and then the unadsorbed material was washed off with twice the capacity of 30% acetonitrile. It was then eluted in 1-fold with 40, 50, 60, 70% acetonitrile. Mass spectrometry was performed under the LC-MS conditions as shown in Table 2 using 50% acetonitrile eluent as the main fraction in the eluent stepwise.
표 2
조건 항목 조건 내용
칼럼 YMC Ultra C18(5*2㎜, 2㎛)
이동상 50 -100% gradient of acetonitrile
유속 1㎖/min
기기 Shimadzu LCMS-IT-TOF
이온화 ESI, positive
단편화세기 100V
스캔범위 200~1000 m/z
시료주입량 1~5㎕
TABLE 2
Condition item Condition content
column YMC Ultra C18 (5 * 2㎜, 2㎛)
Mobile phase 50 -100% gradient of acetonitrile
Flow rate 1ml / min
device Shimadzu LCMS-IT-TOF
Ionization ESI, positive
Fragmentation 100 V
Scan range 200-1000 m / z
Sample injection amount 1 to 5 μl
스트렙토마이세스 카나마이세티쿠스 KCTC 9225와 스트렙토마이세스속 KCTC 11604BP의 발효배양체에서 C18 HPLC 수행 조건에서 약 19.9분에 HR-MS (M+Na)+ m/z 826.4730으로 타크롤리무스(FK506)(Mr. 803.4820)가 확인되었으며, 타크롤리무스 피크 전후로 여러 유사체들이 존재하였다. 약 18.1분에 m/z 814.4661의 아스코마이신(FK520)(Mr. 791.4819), 16.7분에 m/z 812.455의 FK525(Mr. 789.4663), 28.1분에 m/z 818.4826의 다이하이드로타크롤리무스(dihydro tacrolimus; DH-FK506) (Mr. 805.4976)가 검출되었다. 그러나, 미국 종균협회(ATCC)에서 구입한 스트렙토마이세스속 ATCC 55098의 실시예 1의 방법으로 배양한 배양체에서는 타크롤리무스를 확인할 수 없었다. Tacrolimus (FK506) with HR-MS (M + Na) + m / z 826.4730 at approximately 19.9 min under conditions of C18 HPLC in the fermentation medium of Streptomyces kanamyceticus KCTC 9225 and Streptomyces KCTC 11604BP. Mr. 803.4820), several analogs existed before and after the tacrolimus peak. Ascomycin (FK520) in the m / z 814.4661 for about 18.1 minutes (Mr. 791.4819), of 16.7 minutes m / z 812.455 to FK525 (Mr. 789.4663), the m / z 818.4826 for 28.1 minutes dihydro tacrolimus (dihydro tacrolimus; DH-FK506) (Mr. 805.4976) was detected. However, tacrolimus could not be identified in the culture cultured by the method of Example 1 of the Streptomyces genus ATCC 55098 purchased from the American spawn association (ATCC).
하기 실시예에서 만들어진 여러 돌연변이체 및 형질전환체들도 실시예 1과 같이 배양하고, 표 1과 표 2에 준하여 HPLC 및 LC-MS 분석을 실시하였다. Several mutants and transformants made in the following examples were also cultured as in Example 1, and subjected to HPLC and LC-MS analysis according to Table 1 and Table 2.
<실시예 3> 항진균 활성 측정 Example 3 Antifungal Activity Measurement
항진균 활성 측정을 위해 아스퍼질러스 나이거(Aspergillus niger) ATCC 9642를 피검균으로 사용하였다. Potato Dextrose Agar 배지(0.4% 감자전분, 2% 덱스트로스, 1.5% 한천)을 멸균하여 40~50℃로 식힌 후, 피검균을 접종하여 피검균 배지를 준비하였다. 야생주 및 돌연변이주들의 배양액으로부터 확보한 항균활성 측정을 위한 대상 추출액을 페이퍼 디스크(ADVANTEC, Toyo Roshi Kaisha, Ltd.)에 묻힌 후 미리 준비한 피검균 배지에 올려 28℃에서 이틀 동안 배양하고, 저해활성환(clear zone)의 크기를 측정하여 항균력을 확인하였다. Aspergillus niger ATCC 9642 was used as the test for antifungal activity measurement. Potato Dextrose Agar medium (0.4% potato starch, 2% dextrose, 1.5% agar) was sterilized and cooled to 40 ~ 50 ℃, and then inoculated with the specimen to prepare a specimen medium. The target extract for the antimicrobial activity obtained from the cultures of wild and mutant strains was buried in paper disks (ADVANTEC, Toyo Roshi Kaisha, Ltd.) and placed on a pre-prepared test medium and incubated at 28 ° C. for two days. The antimicrobial activity was confirmed by measuring the size of the clear (clear zone).
<실시예 4> 생합성 유전자 확보Example 4 Biosynthesis
상기 실시예 1에 명시된 3종의 균주로부터 생합성 유전자를 확보하였다. 먼저 각 균주의 포스미드(fosmid) 라이브러리를 제작하고 포스미드 라이브러리는 Kieser 등이 제시한 방법(Kieser T. 등, 2000)을 이용하여 크로모좀을 분리하여 사용하였고, 포스미드 라이브러리 제작은 Epicentre사로부터 키트를 구입하여 제작사가 지시한 방법에 따라 제작하였다. 각 균주로부터 제작된 약 1,500개의 포스미드 말단을 Sanger의 다이디옥시방법(dideoxy methods)으로 ABI-3730xl autosequencer를 사용하여 0.02/10kb 이상의 정확도로 서열을 분석하여 염기서열정보를 확보하였다. 이렇게 구성된 정보를 이용하여 폴리케타이드 합성효소 유전자 정보가 함유된 포스미드 클론을 찾았으며, 이들을 숏건 서열분석(shotgun sequencing) 방법을 이용하여 염기서열 분석하였다. 각 분석된 reads 정보를 Phred-Phrap™(University of washington, Seatle, U.S.A) 프로그램을 이용하여 연결(assemble)하여 최종적으로 완전히 연결된 타크롤리무스(FK506) 생합성 유전자를 확보할 수 있었다. 스트렙토마이세스속 KCTC 11604BP는 Fos1004F01 (1-40366), Fos1005D02 (39116-80661), Fos1006D05 (58172-97743)을 분석하여 전체 약 97kb의 연결정보(서열번호 1)를 확보하였다. 스트렙토마이세스 카나마이세티쿠스 KCTC 9225 유래의 생합성 유전자는 Fos1006G02 (1-35521), Fos1012A09 (31026-67758), Fos1004E04 (41430-85253), Fos1010E10 (76978-111990)을 분석하여 전체 약 109kb의 연결정보(서열번호 54)를 확보하였다. 또, 스트렙토마이세스속 ATCC 55098 유래의 생합성 유전자는 Fos1011B11 (142-41780), Fos1010H09 (9845-44813), Fos1012B03 (27400-72808), FOS1001F05 (59902-95981)의 염기서열을 완전히 분석하여 전체 약 99kb의 연결정보(서열번호 93)를 확보하였다. 기존에 알려진 타크롤리무스 생합성 유전자는 폴리케타이드 합성효소를 중심으로 수개의 유전자만이 확보, 분석되어 완전한 타크롤리무스 생합성기구 전체 구성의 이해와 산업적 활용에 부족하였으나, 본 발명을 통해 전체 생합성 유전자가 포함된 충분한 크기의 유전자를 확보하고, 이를 체계적으로 분석할 수 있었다(도 2). Biosynthetic genes were obtained from three strains specified in Example 1 above. First, the fosmid library of each strain was prepared, and the phosmid library was used by separating chromosomes using the method suggested by Kieser et al. (Kieser T. et al., 2000). The kit was purchased and manufactured according to the manufacturer's instructions. About 1,500 phosphide ends prepared from each strain were sequenced with an accuracy of 0.02 / 10 kb or more using an ABI-3730xl autosequencer using Sanger's didioxy methods to obtain sequencing information. Using the information thus constructed, phosphide clones containing polyketase synthase gene information were found, and they were sequenced using shotgun sequencing. Each analyzed reads information was assembled using the Phred-Phrap ™ (University of washington, Seatle, U.S.A) program to finally obtain a fully linked tacrolimus (FK506) biosynthetic gene. KCTC 11604BP of Streptomyces genus analyzed the Fos1004F01 (1-40366), Fos1005D02 (39116-80661), Fos1006D05 (58172-97743) to obtain a total of about 97kb (SEQ ID NO: 1). Biosynthetic genes derived from Streptomyces kanamyceticus KCTC 9225 were analyzed by Fos1006G02 (1-35521), Fos1012A09 (31026-67758), Fos1004E04 (41430-85253), and Fos1010E10 (76978-111990). (SEQ ID NO 54) was obtained. Biosynthetic genes derived from Streptomyces sp. Connection information (SEQ ID NO: 93) was obtained. Previously known tacrolimus biosynthesis genes are secured and analyzed based on polyketase synthase and only a few genes are insufficient for understanding and industrial utilization of the entire composition of tacrolimus biosynthesis. Enough genes of sufficient size were obtained, which could be systematically analyzed (FIG. 2).
<실시예 5> 생합성 유전자 정보 분석Example 5 Biosynthesis Gene Information Analysis
각 균주로부터 확보된 염기서열을 Glimmer program으로 orf(open reading frame)를 예측하고 각 기능을 BLAST-2.2.21로 비교하여 예측하였다. 스트렙토마이세스속 KCTC 11604BP 유래 유전자의 경우 전체 약 44개의 orf들을 예측할 수 있었으며(표 3), 스트렙토마이세스 카나마이세티쿠스 KCTC 9225 유래의 경우 약 70개의 orf를 예측하였고, 스트렙토마이세스속 ATCC 55098의 경우 약 56개의 orf를 예측하였다. The sequence obtained from each strain was predicted by predicting orf (open reading frame) by Glimmer program and comparing each function with BLAST-2.2.21. In the case of Streptomyces KCTC 11604BP-derived genes, a total of about 44 orf were predicted (Table 3), and Streptomyces kanamyceticus About 70 orf were predicted for KCTC 9225 and about 56 orf for ATCC 55098 in Streptomyces.
이들 유전자에게서 공통적으로 발견되는 유전자는 모두 19개였다(서열번호 3,5,7,9,21,23,25,27,29,31,33,35,37,39,41,43,45,47,49,56,58,60,62,64,66, 68,70,72,74,76,78,80,82,84,86,88,90,92,95,97,99,101,103,105,107,109,111,113,115,117,119,121,123,125,127,129,131). 본 발명에서는 아스코마이신 생합성 유전자와 높은 유사도(유사도 70% 이상)를 나타내는 경우 Wu K.등(2000)에서 명명한 유전자명에 준하여 명명하였으며, 필요시 유전자의 유래를 표시하였다. 그 외 유전자들은 tcs (tacrolimus synthase)로 명명하였다. There were 19 genes commonly found in these genes (SEQ ID NOs: 3,5,7,9,21,23,25,27,29,31,33,35,37,39,41,43,45,47). , 49,56,58,60,62,64,66, 68,70,72,74,76,78,80,82,84,86,88,90,92,95,97,99,101,103,105,107,109,111,113,115,117,119,121,123,125,127,129,131). In the present invention, when showing high similarity (more than 70% similarity) with the ascomycin biosynthesis gene was named according to the gene name named in Wu K. et al. (2000), the origin of the gene is indicated if necessary. Other genes was named tcs (t a c rolimus ynthase s).
공통적으로 발견되는 19개 유전자 중 15개 유전자는 아스코마이신 생합성 유전자에서 발견되는 것과 유사하였다. 즉, 3개의 주요 폴리케타이드 합성효소를 코딩하고 있는 유전자들(fkbA,B,C), 메톡시말로닐(methoxymalonyl) ACP 합성관련 유전자들(fkbG,H,I,J,K), 피페콜산(pipecolate) 생합성 유전자(fkbL), 변형 관련 추정 유전자들(fkbM,D,O), 조절유전자(fkbN), 비 리보솜 펩타이드 합성효소 (fkbP), 그리고 타입 Ⅱ 티오에스터라제(thioesterase; TE) 유전자(fkbQ)는 아스코마이신 생합성 유전자군에서 보고되었다. Fifteen of the nineteen commonly found genes were similar to those found in ascomycin biosynthetic genes. That is, genes encoding three major polyketase synthase ( fkbA , B , C ), methoxymalonyl ACP synthesis genes ( fkbG, H, I, J, K ), pipepelic acid (pipecolate) biosynthetic gene ( fkbL ), modification related putative genes ( fkbM, D, O ), regulatory genes ( fkbN ), nonribosomal peptide synthase ( fkbP ), and type II thioesterase (TE) gene ( fkbQ ) has been reported in the ascomycin biosynthetic gene family.
새로운 4개의 유전자 (tcsA,B,C,D )와 그 폴리펩타이드는 타크롤리무스 생합성에 특이적인 것으로 추정되며, 본 발명에 이하여 처음으로 확인된 유전자 및 산물이다 (서열번호 2,3,4,5,6,7,8,9,55,56,57,58,59,60,61,62,94,95,96, 97,98,99,100,101). Four new genes ( tcsA, B, C, D ) and their polypeptides are believed to be specific for tacrolimus biosynthesis and are the first genes and products identified in accordance with the present invention (SEQ ID NOs: 2,3,4). , 5,6,7,8,9,55,56,57,58,59,60,61,62,94,95,96, 97,98,99,100,101).
<실시예 6> 주요 폴리케타이드 합성효소의 효소활성 도메인 분석 Example 6 Enzyme Activity Domain Analysis of Major Polyketide Synthetase
본 발명에서 확보한 생합성 유전자군이 타크롤리무스와 그 유연물질의 생합성을 온전히 담당할 수 있는지를 확인하기 위해 스트렙토마이세스속 KCTC 11604BP 와 스트렙토마이세스 카나마이세티쿠스 KCTC 9225의 생합성 유전자의 주요 폴리케타이드 합성효소(PKS)를 중심으로 효소활성에 필수적인 도메인 구성을 분석하였다 (도 3). In order to confirm whether the biosynthetic gene group obtained in the present invention is fully responsible for the biosynthesis of tacrolimus and its related substances, the major poly of the biosynthetic genes of Streptomyces KCTC 11604BP and Streptomyces kanamyceticus KCTC 9225 The domain configuration essential for enzymatic activity was analyzed around ketase synthase (PKS) (FIG. 3).
-ACP(Acyl carrier protein) 도메인-Acyl carrier protein (ACP) domain
폴리케타이드 합성효소 상의 10개 모듈 내의 ACP와 TcsA의 ACP 도메인, 그리고 FkbJ까지 모두 4'-포스포팬테티엔(4'-phosphopantetheine) 부착에 필수적인 세린 잔기가 잘 보존되어 있다(Florova G. 등, 2002).Serine residues essential for 4'-phosphopantetheine attachment to ACP and TcsA's ACP domains and up to FkbJ in 10 modules on polyketide synthase are well conserved (Florova G. et al. 2002).
-KS(Ketosynthase) 도메인-Ketosynthase (KS) domain
폴리케타이드 합성효소 상의 10개 KS 도메인에 탈카복시화(decarboxylation)와 아실기 전이에 필수적인 시스테인, 히스티딘, 라이신 아미노산이 잘 보존되어 있다 (Dreier J. 와 Khosla C., 2000). Ten KS domains on polyketide synthase are well conserved for cysteine, histidine and lysine amino acids essential for decarboxylation and acyl transfer (Dreier J. and Khosla C., 2000).
-AT(Acyltransferase) 도메인-Ac (transylase) domain
폴리케타이드 합성효소 상의 10개 AT 도메인에 Gx(H)SxG 의 호중 엘보(nucleophile elbow)를 구성하는 아미노산 잔기가 잘 보존되어 있으며, 또한 촉매에 필요한 기타 아미노산들이 잘 보존되어 있다 (Keatinge-Clay A. T. 등, 2003). The 10 AT domains on the polyketide synthase preserve well the amino acid residues that make up the nucleophile elbow of Gx (H) S xG, as well as the other amino acids required for the catalyst (Keatinge-Clay AT et al., 2003).
-KR(ketoreductase) 도메인Ketoreductase (KR) domain
모듈 1,2,3,5,6,7,8,9에 각 한 개씩 모두 8개의 KR 도메인이 존재하고 있다. NADPH 또는 NADH 결합에 필요한 부분과 로자만 폴드(Rossaman fold) 구성에 중심적인 역할을 하는 타이로신(Tyr)이 보존되어 있으며(Reid, R. 등 2003), 또한 LDD 모티프가 잘 보존되어 있다 (Caffrey, P., 2003). 이는 B-타입 KRs이 알콜 입체화학구조를 가지는 것과 부합하는 결과이다. There are eight KR domains, one for each of modules 1,2,3,5,6,7,8,9. The parts necessary for NADPH or NADH binding and tyrosine (Tyr), which play a central role in the formation of the Rosaman fold, are conserved (Reid, R. et al. 2003), and the LDD motifs are well conserved (Caffrey, P., 2003). This is consistent with the B-type KRs having an alcohol stereochemical structure.
-DH(Dehydrogenase) 도메인-DH (Dehydrogenase) domain
모듈 1에서 9까지 DH 도메인이 존재하였다. 모듈 3과 8에 존재하는 DH 도메인 유사체 이외의 7개의 DH 도메인은 효소활성부위인 HxxxGxxxP 모티프와 효소 활성에 필수적인 부위인 히스티딘과 프롤린이 잘 보존되어 있다(Joshi, A. K., and Smith, S., 1993).From modules 1 to 9 there were DH domains. In addition to the DH domain analogues present in Modules 3 and 8, the seven DH domains are well conserved for the HxxxGxxxP motif, an enzyme activity site, and histidine and proline, which are essential for enzyme activity (Joshi, AK, and Smith, S., 1993). ).
그러나, 모듈 8번 DH의 경우 활성부위가 완전히 소실되어 있으며, 스트렙토마이세스 카나마이세티쿠스 KCTC 9225 유래 모듈 3의 DH 도메인은 활성부위를 구성하는 아미노산 잔기인 프롤린이 세린으로 변경되어 있어 활성을 갖기는 어려운 것으로 판단된다. However, in the case of DH module 8, the active site is completely lost, and the DH domain of module 3 derived from Streptomyces kanamyceticus KCTC 9225 is changed to serine because the amino acid residue constituting the active site is changed to serine. Is considered difficult.
베타 케토 산화(Beta keto oxidation)에 관여하는 DH의 경우 모듈 구성상 모듈 2와 모듈 4의 DH는 요구되지 않으나 활성에 필요한 아미노산 잔기가 잘 보존되어 있다. In the case of DH involved in beta keto oxidation, the DH of module 2 and module 4 is not required due to the module configuration, but the amino acid residues necessary for activity are well preserved.
-ER(enoyl reductase) 도메인-ER (enoyl reductase) domain
ER 도메인은 시작 모듈(loading module)과 모듈 6,7,9에 ER 도메인이 존재하고 있으며 모두 ER 기능에 중요한 NADPH 결합 부위를 구성하는 LxHxxxGVG가 잘 보존되어 있다(Witkowski, A. 등, 1991,Amy C. M. 등, 1989). The ER domain contains the ER domain in the loading module and modules 6, 7, and 9, and LxHxxxGVG, which constitutes the NADPH binding site, which is important for ER function, is well conserved (Witkowski, A. et al., 1991, Amy). CM et al., 1989).
-TE(thioesterase) 도메인-TE (thioesterase) domain
FkbQ는 촉매 트리아드(catalytic triad)를 구성하는 Ser, His, Asp과 호중 엘보(nucleophile elbow)를 구성하는 GxSxG가 보존되어 있다(Bruner, S. D. 등, 2002). FkbQ has conserved Ser, His, Asp and GxSxG constituting the nucleophile elbow constituting the catalytic triad (Bruner, S. D. et al., 2002).
-스트렙토마이세스속 ATCC 55098 유래의 폴리케타이드 합성효소-Polyketide synthase from Streptomyces ATCC 55098
스토렙토마이세스속 ATCC 55098 유전자의 경우 폴리케타이드 합성효소의 일부 모듈이 소실되어 완전한 형태가 아니었다. 이로 미루어 스트렙토마이세스 카나마이세티쿠스 KCTC 9225 및 스토렙토마이세스속 KCTC 11604BP균주 유래의 생합성 유전자와 달리 스토렙토마이세스속 ATCC 55098 유래의 생합성 유전자는 타크롤리무스 및 그 구조 유사체의 생합성에 불충분할 것으로 추정되었으며, 균주의 보관 중 돌연변이가 있었을 것으로 추정된다(도 2). The ATCC 55098 gene of Streptomyces genus was missing some modules of polyketide synthase and was not in perfect form. From this, Streptomyces kanamyceticus Unlike the biosynthetic genes derived from KCTC 9225 and Streptomyces KCTC 11604BP strains, biosynthetic genes derived from Streptomyces ATCC 55098 were estimated to be insufficient for biosynthesis of tacrolimus and its structural analogues. It is estimated that there was (FIG. 2).
<실시예 7> 폴리케타이드 합성효소 이외의 유전자의 정보 분석 Example 7 Analysis of Genes Other Than Polyketide Synthetase
-전사후 변형 유전자 Post-transcriptional modified genes FkbFkb O, O, FkbFkb M, M, FkbFkb DD
FkbM은 31-O-메틸 전이효소로 FkbM-sk (sk는 Streptomyces kanamyceticus KCTC 9225 유래를 의미)는 FkbM-asco (asco는 Streptomyces hygroscopicus subsp. ascomyceticus ATCC 14891 유래를 의미)와 82%, RapI과 71%의 유사도를 보이고 있다. Shikimate에서 유래된 4,5-DHCHC(dihydroxylcyclohex-1-enecarboxylic acid) 개시 유닛(starter unit)의 O-메틸화를 촉매하는 효소로 추정된다. FkbM is 31-O- methyl transferase FkbM-sk (sk means Streptomyces kanamyceticus KCTC 9225 derived) by enzyme-FkbM asco (asco refers to the Streptomyces hygroscopicus subsp. Ascomyceticus ATCC 14891-derived) and 82%, and 71% RapI Similarity is shown. It is assumed that the enzyme catalyzes the O-methylation of 4,5-DHCHC (dihydroxylcyclohex-1-enecarboxylic acid) starter unit derived from Shikimate.
FkbD는 C9-p450 하이드록실레이즈(hydroxylase)이다. FkbD-sk는 FkbD-asco와 88%, RapJ와 76%, MerE와 35%의 유사도를 보였다. 이 유전자는 C9 Oxo FK506의 하이드록실화를 담당하여 C9-OH FK506을 생산하는 것으로 추정되고 있다. 그러나, 최근의 연구(Moss S. J. 등, 2004)를 볼 때 C9-옥소 프리-라파마이신(C9-oxo pre-rapamycin)으로부터 직접 RapJ에 의해 C9 케토폼(keto form)인 프리-라파마이신(pre-rapamycin)이 합성될 수 있음이 보고되었다. 이로 미루어 RapK의 유사체인 FkbD도 C9-케토폼인 타크롤리무스를 C9-옥소폼으로부터 직접 생성하는 것을 담당할 것으로 추정된다. FkbD is C9-p450 hydroxylase. FkbD-sk showed 88% similarity with FkbD-asco, 76% with RapJ, and 35% with MerE. This gene is believed to be responsible for the hydroxylation of C9 Oxo FK506 to produce C9-OH FK506. However, a recent study (Moss SJ et al., 2004) shows that C9 keto form pre-rapamycin (pre-) by RapJ directly from C9-oxo pre-rapamycin. rapamycin) can be synthesized. This suggests that FkbD, an analog of RapK, is also responsible for producing C9-ketoform tacrolimus directly from C9-oxoform.
FkbO는 산화효소이다. FkbO-sk는 FkbO-asco와 83%, RapK와 71%의 유사도를 보였다. FkbO의 기능은 불분명하다. C9 위치의 수산화기 산화에 관여할 것으로 추정하였으나, 라파마이신과 FK506/FK520 모두 C9 케토기를 형성하기 위해서는 C9-OH기의 산화 단계가 필요하다. C9-OH 타크롤리무스는 발효 부산물(by- product)로 발견되으로 C9-OH 산화효소 역할을 할 것으로 추정할 수 있다. 그러나, 라파마이신 생합성 유전자기구에 존재하는 FkbO의 유사체인 RapK의 연구결과는 C-9 산화효소로의 역할보다 개시 유닛인 DHCHC의 생합성 혹은 그의 조절 역할을 할 것으로 추정하기도 한다(Gregory M. A. 등, 2005). FkbO is an oxidase. FkbO-sk showed 83% similarity to FkbO-asco and 71% with RapK. The function of FkbO is unclear. It is assumed that the C9 position is involved in the oxidation of the hydroxyl group, but both rapamycin and FK506 / FK520 require an oxidation step of the C9-OH group to form a C9 keto group. C9-OH tacrolimus was found to be a by-product of fermentation and can be assumed to act as a C9-OH oxidase. However, the results of RapK, an analogue of FkbO in rapamycin biosynthetic genetic machinery, suggest that it may play a role in biosynthesis or regulation of DHCHC, the initiating unit, rather than as a C-9 oxidase (Gregory MA et al., 2005). ).
-전사조절 유전자, Transcriptional regulator genes, fkbNfkbN
S. kanamyceticus KCTC 9225, S. sp. ATCC 55098, S. sp. KCTC 11604BP, 그리고, S. hygroscopicus subsp. ascomyceticus ATCC 14891 모두에서 발견되는 조절 유전자는 LuxR 패밀리 전사조절인자를 코딩하는 fkbN 유사체였다. fkbN 유사체는 fkbQ 와 fkbN 사이에 존재하고 있었으며 모두 fkbM 유사체와는 역방향 그리고, fkbQ 유사체와는 동일한 방향으로 유전자가 배치되어 있었다. S. kanamyceticus KCTC 9225, S. sp. ATCC 55098, S. sp. KCTC 11604BP, and S. hygroscopicus subsp. The regulatory gene found in all ascomyceticus ATCC 14891 was an fkbN analogue encoding the LuxR family transcriptional regulator. fkbN analogs was the gene is placed in the same orientation as was present between the fkbQ fkbN fkbM both analog and is the reverse, and, fkbQ analogs.
-메톡시말로닐 ACP 합성 유전자-Methoxymalonyl ACP synthetic gene
일반적으로 폴리케타이드 부위는 말로닐-CoA와 메틸말로닐-CoA가 가장 흔히 사용되는 전구체이다. 그러나 타크롤리무스 및 아스코마이신은 독특한 전구체가 사용되는데 이는 메톡시말로닐(methoxylmalonyl)-ACP이다. 이의 합성을 위해 현재까지 5개의 유전자 즉 fkbG,H,I,J,K가 관여하는 것으로 추정하고 있다 (Wantanabe K.등, 2003). Generally, the polyketide moiety is the most commonly used precursor of malonyl-CoA and methylmalonyl-CoA. Tacrolimus and ascomycin, however, use unique precursors, which are methoxylmalonyl-ACP. It is estimated that five genes , fkbG, H, I, J, K, are involved so far for synthesis (Wantanabe K. et al., 2003).
S. kanamyceticus KCTC 9225유래와 S. hygroscopicus subsp. ascomyceticus ATCC 14891 유래 FkbG는 86%, FkbH는 83%의 상호 유사도를, FkbI는 85%, FkbJ는 77%, FkbK는 87%의 상호 유사도를 보였다. 그러나, S. sp. ATCC 55098의 FkbG는 두 개로 나누어져 있었다. S. kanamyceticus KCTC 9225 comes with S. hygroscopicus subsp. FkbG from ascomyceticus ATCC 14891 showed 86%, FkbH showed 83% similarity, 85% FkbI, 77% FkbJ, 87% FkbK. However, S. sp. The FkbG of ATCC 55098 was divided into two.
-피페콜레이트 첨가 관련 유전자, FkbP 및 FkbLGenes related to the addition of pipecolate, FkbP and FkbL
타크롤리무스, 아스코마이신, 라파마이신 및 메리다마이신(meridamycin)은 특별한 원환 아미노산인 L-피페콜레이트가 폴리케타이드에 결합하여 구성되어 있다. L-피페콜레이트는 L-라이신 유래로 L-라이신이 사이클로디아미네이션(cyclodeamination) 반응을 거쳐 생성되며 이를 담당하는 효소는 FkbL, 사이클로디아미네이즈(cyclodeaminase)로 알려져 있다. FkbL-sk는 유사체인 FkbL-asco와 89%, RapL과 76%, TubZ와 43%의 유사도를 나타낸다. L-피페콜레이트를 폴리케타이드에 결합하는 효소는 FkbP, NRPS(non ribosomal peptide synthetase)이다. FkbP-sk는 RapP와 58%, FkbP-MA5648과 95%, FkbP-Asco와 84%, MerP와 47%의 유사도를 나타낸다. FkbP는 축합 도메인, 아데닐화 도메인(adenylation domain), PCP 도메인, 그리고 다시 축합 도메인으로 구성되어 있다. 그러나 각 도메인이 어떻게 L-피페콜레이트와 폴리케타이드의 결합을 수행하는지에 대한 정확한 규명은 되어 있지 않다. FkbL에 의해 합성된 L-피페콜레이트는 아데닐화 도메인에 의해 FkbP의 PCP에 charge되고 이는 다시 FkbP의 축합 도메인에 의해 PK 구조체와 축합되어 완전한 타크롤리무스 및 그 유사구조 물질의 골격으로 최종 형성되게 할 것으로 추정된다. Tacrolimus, ascomycin, rapamycin and meridamycin are composed of a special toric amino acid, L-pipecolate, bound to a polyketide. L-Pipecholate is derived from L-lysine and L-lysine is produced through cyclodeamination reaction, and the enzyme responsible for this is known as FkbL and cyclodeaminase. FkbL-sk showed 89% similarity with the analogue FkbL-asco, 76% with RapL, and 43% with TubZ. Enzymes that bind L-pipecolate to polyketides are FkbP and non ribosomal peptide synthetase (NRPS). FkbP-sk shows a similarity of 58% with RapP, 95% with FkbP-MA5648, 84% with FkbP-Asco, and 47% with MerP. FkbP consists of a condensation domain, an adenylation domain, a PCP domain, and again a condensation domain. However, it is not known exactly how each domain performs the binding of L-pipepelate and polyketide. The L-pipecolate synthesized by FkbL is charged to the PCP of FkbP by the adenylation domain, which in turn is condensed with the PK structure by the condensation domain of FkbP, resulting in final formation into the framework of complete tacrolimus and its analogous material. It is estimated.
-DHCHC 합성 유전자-DHCHC synthetic gene
그외 Shikimate에서 유래된 DHCHC(4,5-dihydroxylcyclohex-1-enecarboxylic acid) 혹은 이와 유사한 구조체의 합성에 관련된 유전자군은 생합성 클러스터 내에 존재하지 않았으며, 이는 아마도 크로모좀 내의 다른 위치에 존재하는 것으로 보인다. 실제 ATCC 55098의 일부 게놈 분석 결과 이들 합성에 관계할 것으로 유추할 수 있는 일부 유전자군이 발견되었다. Other groups of genes involved in the synthesis of Shikimate-derived DHCHC (4,5-dihydroxylcyclohex-1-enecarboxylic acid) or similar constructs did not exist in the biosynthetic cluster, possibly in other locations within the chromosome. In fact, some genome analyzes of ATCC 55098 have identified some gene families that may be inferred to be involved in these synthesis.
-프로필말로닐-CoA(ACP) 합성-Propylmalonyl-CoA (ACP) synthesis
타크롤리무스의 합성 중에 대체적인 합성 기작은 상기에서 기술한 바와 같이 전체 생합성 기작을 논리적으로 유추할 수 있다. 그러나, 31 위치의 곁가지 합성에 대해서는 아직 밝혀지지 않은 상태이다. 본 발명에서는 아스코마이신과 라파마이신 생합성 유전자에서는 발견되지 않으나, 타크롤리무스를 생산하는 균종 즉, S. sp. KCTC 11604BP, S. kanamyceticus KCTC 9225 와 S. sp. ATCC 5508에서 공통적으로 발견되는 4개의 유전자 tcsA,B,C,D를 확보하였다 (서열번호 3,5,7,9,56,58,60,62,64,66,95,97,99,101). Alternative synthetic mechanisms during the synthesis of tacrolimus can logically infer the entire biosynthetic mechanism as described above. However, the synthesis of the side branch at position 31 is still unknown. In the present invention, although not found in ascomycin and rapamycin biosynthetic genes, the strains producing tacrolimus, ie, S. sp. KCTC 11604BP, S. kanamyceticus KCTC 9225 and S. sp. Four genes tcsA, B, C, D commonly found in ATCC 5508 were obtained (SEQ ID NOs: 3,5,7,9,56,58,60,62,64,66,95,97,99,101).
TcsA-sk(서열번호 55)는 TcsA-55098 (서열번호 94)과는 88%, TcsA-11604BP (서열번호 2)와는 79%의 유사도를 가지고 있었으며, 이들 TcsA 유사체는 AT 도메인을 가지고 있었으며, 아실기 전이효소 기능에 필요한 Gx(H)SxG의 호중 엘보(nucleophile elbow)를 구성하는 아미노산 잔기와 S. coelicolor 유래의 MAT(malonyl-CoA:ACP)와 S. hygroscopicus 유래의 RapAT2에서 촉매 작용에 관여하는 아르기닌, 히스티딘 등이 잘 보존되어 있다 (Keatinge-Clay A. T. 등, 2003).TcsA-sk (SEQ ID NO: 55) had 88% similarity with TcsA-55098 (SEQ ID NO: 94) and 79% with TcsA-11604BP (SEQ ID NO: 2), and these TcsA analogs had AT domains. Amino acid residues constituting the nucleophile elbow of Gx (H) S xG required for functional transferase function, and are involved in catalysis in S. coelicolor- derived MAT (malonyl-CoA: ACP) and S. hygroscopicus- derived RapAT2 Arginine, histidine and the like are well preserved (Keatinge-Clay AT et al., 2003).
TcsB-sk는 TcsB-55098 과 86%, TcsB-11604BP와 79%의 유사도를 나타내고 있었으며, TcsB는 폴리케타이드 합성효소에서 발견되는 KS 도메인을 함유하고 있었다. TcsB의 N 말단 쪽에 효소활성 부위가 잘 보존된 KS 도메인을 가지고 있으며, 이와 더불어 C 말단 위치에도 세 유사체 모두 잘 보존되어 있음으로 미루어 프로필말로닐 CoA(ACP) 혹은 그 유사체의 형성에 중요한 역할을 할 것으로 추정된다. TcsB-sk showed 86% similarity to TcsB-55098 and 79% to TcsB-11604BP, and TcsB contained the KS domain found in polyketide synthase. The N-terminus of TcsB has a well-conserved KS domain, and all three analogs are also well conserved at the C-terminal position, which plays an important role in the formation of propylmalonyl CoA (ACP) or its analogs. It is estimated.
TcsC는 살리니스포라 트로피카(Salinispora tropica) CNB-476의 살리노스포라마이드(salinosporamide) A 생합성기구에 포함된 SalG (ABP73651)의 유사체이다. TcsC-11604BP, TcsC-sk, TcsC-55098은 각각 SalG 와 50%, 51%, 51%의 높은 일치도를 나타낸다. 이러한 TcsC 유사체가 아스코마이신 생합성 유전자군에서도 FkbS로 일부 존재하나 FkbS는 C 말단쪽 152개의 아미노산만 보고되어 있으며, 그 기능 또한 정확히 알려져 있지 않고 있다. 여러 마크롤라이드 생합성 유전자에서 발견되는 TcsC 유사체는 마크롤라이드의 에틸말로닐 부위와 같은 곁가지의 공급에 역할을 할 것으로 추정하고 있으나 정확한 기작은 밝혀지지 않은 상태이다. 단지 최근에 SalG가 환원 카복실레이즈(reductive carboxylase) 역할을 한다는 것이 보고된 바 있다. TcsC is an analog of SalG (ABP73651) included in the salinosporamide A biosynthetic apparatus of Salinispora tropica CNB-476. TcsC-11604BP, TcsC-sk, and TcsC-55098 show high concordance with SalG of 50%, 51% and 51%, respectively. Some of these TcsC analogues exist as FkbS in the ascomycin biosynthetic gene group, but only 152 amino acids in the C terminus of FkbS have been reported, and their function is not known exactly. TcsC analogues found in many macrolide biosynthetic genes are thought to play a role in supplying side branches, such as the ethylmalonyl site of macrolides, but the exact mechanism is unknown. Only recently has SalG been reported to act as a reductive carboxylase.
TcsD-11604BP와 87%의 유사도를 보였으며, 플레시오시스티스 파시피카(Plesiocystis pacifica) SIR-1 유래의 ACAD (NZ_ABCS01000005.1)와 49%의 낮은 유사도를 보였다. 이러한 분석으로 보아 TcsD는 일반적인 ACAD보다는 프로필말로닐 ACP(CoA)의 합성에 매우 특이적인 탈수소효소로 프로필말로닐 ACP(CoA)의 환원을 통한 알릴(프로필레닐)말로닐 ACP(CoA)의 형성에 관여할 것으로 추정된다. It showed 87% similarity with TcsD-11604BP and 49% similar to ACAD (NZ_ABCS01000005.1) derived from Plesiocystis pacifica SIR-1. These analyzes suggest that TcsD is a dehydrogenase that is more specific for the synthesis of propylmalonyl ACP (CoA) rather than the general ACAD, and is known to form allyl (propylenyl) malonyl ACP (CoA) through reduction of propylmalonyl ACP (CoA). It is assumed to be involved.
표 3
start(bp) end(bp) Orf predicted function FK506 biosynthesis
192 1187 Orf(-11) ABC transporter not related
1184 1987 Orf(-10) ABC transporter
3453 2758 Orf(-9) RNA polymerase sigma factor
4319 3600 Orf(-8) hypothetical protein
5551 4685 Orf(-7) secreted cellulose-binding protein
5869 6012 Orf(-6) hypothetical protein
6016 6234 Orf(-5) predicted flavoprotein involved in k+ transport
6227 6796 Orf(-4) RNA polymerase sigma factor
6793 7428 Orf(-3) putative oxidoreductase
7550 7735 Orf(-2) Mscs Mechanosensitive ion channel
7732 8235 Orf(-1) Pilt protein-like
8724 10007 TcsA anyl transferase domain protein Putative tacrolimus biosythetic enzyme but not for ascomycin biosynthesis
tcsA-At(8733-9677), tcsA-PP(9747-9971)
10015 12405 TcsB polyketide synthase modules and related proteins
tcsB-KS(10060-11400), tcsB-KSb(11443-12141)
12402 13739 TcsC reductive carboxylase
tcsC-ER(12540-13637)
13736 14896 TcsD acyl-CoA dehydrogenase
tcsD-ACAD1(13742-14068), tcsD-ACAD2(14078-14236), tcsD-ACAD3(14387-14872)
16063 14909 Tcs1 methionine gamma lyase not realated
16123 16590 Tcs2 AsnC-family transcriptional regulatory protein
16694 17923 Tcs3 cytochrome P450
18108 19295 Tcs4 indole oxygenase
19308 20060 5Tcs short-chain dehydrogenase/reductase SDR
fkiA-KR(19326-19871)
20232 20900 FkbG O-methyl transferase precursor methoxymalonyl-ACP biosynthesis
21966 20878 FkbH predicted enzyme involved in methoxyalonyl-ACP biosynthesis
23077 21977 FkbI acyl CoA dehydrogenase
24205 23327 FkbK acyl CoA dehydrogenase
23330 23070 FkbJ acyl CoA dehydrogenase
25236 24199 FkbL lysine cyclodeaminase Precursor
36016 25377 FkbC polyketide synthase
M5-KS(36016-34785), M5-AT(34543-33705), M5-DH(33529-33045), M5-Kr(32197-31698), M5-PP(31423-31203) module 5
Ms-KS(31141-29901), M6-AT(29662-28824), M6-DH(28645-28152), M6-ER(27391-26529), M6-KR(26503-26016), M6-PP(25783-25563) module 6
59078 36138 FkbB FK506 polyketide synthase
Load-CAS(59003-57796), Load-ER(57092-56221), Load-PP(55718-55498) loading module
M1-KS(55454-54193), M1-AT(53954-53116), M1-DH(52937-52438), M1-KR(51668-51181), M1-PP(50942-50722) module 1
M2-KS(50639-49381), M2-AT(49019-4815), M2-DH(47993-47530), M2-KR(46697-46186), M2-pp(45941-45721) module 2
M3-KS(45662-44401), M3-AT(44165-43354), M3-DH(43202-42724) module 3
M3-KR(41954-41440), M3-PP(41183-40963), M4-KS(40904-39643), M4-AT(39368-38512), M4-DH(38279-37795), M4-PP(36530-36310) module 4
59326 60318 Fkb0 FK506 oxidase condensation
60315 64913 FkbP syntheaseFK506 peptide condensation & derailed
fkbP-CD(60480-61376), fkbP(61932-63137), fkbP-PP(63387-63605), fkbP-CD(63642-64496)
64916 84172 FkbA FK506 polyketide synthase
M7-ESD(64916-64996), M7-KS(65009-66229), M7-AT(66473-67249), M7-DH(67403-67897), M7-ER(68693-69577), M7-Kr(69605-70090), M7-PP(70364-70582) module 7
M8-KS(70646-71905), M8-AT(72167-72964), M8-DH(73100-73501), M8-KR(74276-74761), M8-PP(75047-75265) module 8
M9-KS(75323-76582), M9-AT(76826-77662), M9-DH(77858-78340), M9-ER(79115-79975), M9-KR(80003-80488), M9-PP(80789-81007) module 9
M10-Ks(81101-82390), M10-AT(82697-83572), M10-PP(83795-84013) module 10
84169 85355 FkbD C9 hydroxylase Post modification
85332 86114 FkbM 31-o-demethyl-FK506 methyltransferase Post modification
88900 86129 FkbN cholesterol oxidase regulator specific regulator
89743 88955 FkbQ thioesterase ⅠⅠ editing
fkbQ-TE(89677-89010)
89940 89731 Tcs6 Putative lipoprotein not related
90174 91598 Tcs7 LysR-famil transcriptional regulator not related
92059 91577 Orf(1) conserved hypothetical protein not related
92031 93020 Orf(2) PE-PGRS FAMILY PROTEIN
93068 93361 Orf(3) melanogenic peroxidase
93606 94610 Orf(4) putative lipoprotein
95839 96003 Orf(5) No hits found
95621 94725 Orf(6) PE-PGRS FAMILY PROTEIN
96124 97362 Orf(7) Aminotransferase, class Ⅰand ⅠⅠ
TABLE 3
start (bp) end (bp) Orf predicted function FK506 biosynthesis
192 1187 Orf (-11) ABC transporter not related
1184 1987 Orf (-10) ABC transporter
3453 2758 Orf (-9) RNA polymerase sigma factor
4319 3600 Orf (-8) hypothetical protein
5551 4685 Orf (-7) secreted cellulose-binding protein
5869 6012 Orf (-6) hypothetical protein
6016 6234 Orf (-5) predicted flavoprotein involved in k + transport
6227 6796 Orf (-4) RNA polymerase sigma factor
6793 7428 Orf (-3) putative oxidoreductase
7550 7735 Orf (-2) Mscs Mechanosensitive ion channel
7732 8235 Orf (-1) Pilt protein-like
8724 10007 TcsA anyl transferase domain protein Putative tacrolimus biosythetic enzyme but not for ascomycin biosynthesis
tcsA-At (8733-9677), tcsA-PP (9747-9971)
10015 12405 TcsB polyketide synthase modules and related proteins
tcsB-KS (10060-11400), tcsB-KSb (11443-12141)
12402 13739 TcsC reductive carboxylase
tcsC-ER (12540-13637)
13736 14896 TcsD acyl-CoA dehydrogenase
tcsD-ACAD1 (13742-14068), tcsD-ACAD2 (14078-14236), tcsD-ACAD3 (14387-14872)
16063 14909 Tcs1 methionine gamma lyase not realated
16123 16590 Tcs2 AsnC-family transcriptional regulatory protein
16694 17923 Tcs3 cytochrome P450
18108 19295 Tcs4 indole oxygenase
19308 20060 5Tcs short-chain dehydrogenase / reductase SDR
fkiA-KR (19326-19871)
20232 20900 FkbG O-methyl transferase precursor methoxymalonyl-ACP biosynthesis
21966 20878 FkbH predicted enzyme involved in methoxyalonyl-ACP biosynthesis
23077 21977 FkbI acyl CoA dehydrogenase
24205 23327 FkbK acyl CoA dehydrogenase
23330 23070 FkbJ acyl CoA dehydrogenase
25236 24199 FkbL lysine cyclodeaminase Precursor
36016 25377 FkbC polyketide synthase
M5-KS (36016-34785), M5-AT (34543-33705), M5-DH (33529-33045), M5-Kr (32197-31698), M5-PP (31423-31203) module 5
Ms-KS (31141-29901), M6-AT (29662-28824), M6-DH (28645-28152), M6-ER (27391-26529), M6-KR (26503-26016), M6-PP (25783 -25563) module 6
59078 36138 FkbB FK506 polyketide synthase
Load-CAS (59003-57796), Load-ER (57092-56221), Load-PP (55718-55498) loading module
M1-KS (55454-54193), M1-AT (53954-53116), M1-DH (52937-52438), M1-KR (51668-51181), M1-PP (50942-50722) module 1
M2-KS (50639-49381), M2-AT (49019-4815), M2-DH (47993-47530), M2-KR (46697-46186), M2-pp (45941-45721) module 2
M3-KS (45662-44401), M3-AT (44165-43354), M3-DH (43202-42724) module 3
M3-KR (41954-41440), M3-PP (41183-40963), M4-KS (40904-39643), M4-AT (39368-38512), M4-DH (38279-37795), M4-PP (36530 -36310) module 4
59326 60318 Fkb0 FK506 oxidase condensation
60315 64913 FkbP syntheaseFK506 peptide condensation & derailed
fkbP-CD (60480-61376), fkbP (61932-63137), fkbP-PP (63387-63605), fkbP-CD (63642-64496)
64916 84172 FkbA FK506 polyketide synthase
M7-ESD (64916-64996), M7-KS (65009-66229), M7-AT (66473-67249), M7-DH (67403-67897), M7-ER (68693-69577), M7-Kr (69605 -70090), M7-PP (70364-70582) module 7
M8-KS (70646-71905), M8-AT (72167-72964), M8-DH (73100-73501), M8-KR (74276-74761), M8-PP (75047-75265) module 8
M9-KS (75323-76582), M9-AT (76826-77662), M9-DH (77858-78340), M9-ER (79115-79975), M9-KR (80003-80488), M9-PP (80789 -81007) module 9
M10-Ks (81101-82390), M10-AT (82697-83572), M10-PP (83795-84013) module 10
84169 85355 FkbD C9 hydroxylase Post modification
85332 86114 FkbM 31-o-demethyl-FK506 methyltransferase Post modification
88900 86129 FkbN cholesterol oxidase regulator specific regulator
89743 88955 FkbQ thioesterase ⅠⅠ editing
fkbQ-TE (89677-89010)
89940 89731 Tcs6 Putative lipoprotein not related
90174 91598 Tcs7 LysR-famil transcriptional regulator not related
92059 91577 Orf (1) conserved hypothetical protein not related
92031 93020 Orf (2) PE-PGRS FAMILY PROTEIN
93068 93361 Orf (3) melanogenic peroxidase
93606 94610 Orf (4) putative lipoprotein
95839 96003 Orf (5) No hits found
95621 94725 Orf (6) PE-PGRS FAMILY PROTEIN
96124 97362 Orf (7) Aminotransferase, class Ⅰ and Ⅰ
표 4
Streptomyces sp. GT1005 S. kanamyceticus KCTC9225 S. sp. ATCC55098 S. hygroscopicus subsp. ascomyceticus ATCC 14891 S. sp. MA6548
ORF Length Length Identity/ Similarity Length Identity/ Similarity Length Identity/ Similarity Length Identity/ Similarity
TcsA 427 426 79/86 429 77/85 - - - -
TcsB 796 796 79/84 796 77/83 - - - -
TcsC 445 445 90/94 443 87/94 - - - -
TcsD 386 386 87/92 386 86/92 - - - -
Tcs1 384 - - - - - - - -
Tcs2 155 - - - - - - - -
Tcs3 409 - - - - - - -
Tcs4 395 - - - - - - - -
Tcs5 250 - - - - - - - -
FkbG 222 222 86/89 99 86/90 222 81/86 - -
FkbH 362 362 86/90 362 86/91 360 79/86 - -
FkbI 366 366 87/92 366 85/89 366 85/90 - -
FkbJ 86 86 85/89 86 80/88 86 73/84 - -
FkbK 292 283 88/90 282 86/89 282 83/89 - -
FkbL 345 345 89/94 345 88/92 345 86/93 - -
FkbC 3559 3597 81/86 - - 3591 77/84 - -
FkbB 7646 7591 83/87 - - 7525 78/84 7576 81/86
FkbO 330 332 85/90 333 84/88 344 81/86 332 86/90
FkbP 1532 1506 83/87 1592 77/81 1495 79/85 1504 82/87
FkbA 6418 6408 84/89 6445 82/87 6396 80/86 6420 81/87
FkbD 388 388 89/92 388 88/91 388 88/93 162 89/91
FkbM 260 212 82/90 260 85/91 260 83/89 - -
FkbN 923 934 82/89 934 82/89 913 71/82 - -
FkbQ 262 255 84/90 255 81/89 247 83/88 - -
Tcs6 69 - - - - - - - -
Tcs7 474 - - - - - - - -
Table 4
Streptomyces sp. GT1005 S. kanamyceticus KCTC9225 S. sp. ATCC55098 S. hygroscopicus subsp. ascomyceticus ATCC 14891 S. sp. MA6548
ORF Length Length Identity / Similarity Length Identity / Similarity Length Identity / Similarity Length Identity / Similarity
TcsA 427 426 79/86 429 77/85 - - - -
TcsB 796 796 79/84 796 77/83 - - - -
TcsC 445 445 90/94 443 87/94 - - - -
TcsD 386 386 87/92 386 86/92 - - - -
Tcs1 384 - - - - - - - -
Tcs2 155 - - - - - - - -
Tcs3 409 - - - - - - -
Tcs4 395 - - - - - - - -
Tcs5 250 - - - - - - - -
FkbG 222 222 86/89 99 86/90 222 81/86 - -
FkbH 362 362 86/90 362 86/91 360 79/86 - -
FkbI 366 366 87/92 366 85/89 366 85/90 - -
FkbJ 86 86 85/89 86 80/88 86 73/84 - -
FkbK 292 283 88/90 282 86/89 282 83/89 - -
FkbL 345 345 89/94 345 88/92 345 86/93 - -
FkbC 3559 3597 81/86 - - 3591 77/84 - -
FkbB 7646 7591 83/87 - - 7525 78/84 7576 81/86
FkbO 330 332 85/90 333 84/88 344 81/86 332 86/90
FkbP 1532 1506 83/87 1592 77/81 1495 79/85 1504 82/87
FkbA 6418 6408 84/89 6445 82/87 6396 80/86 6420 81/87
FkbD 388 388 89/92 388 88/91 388 88/93 162 89/91
FkbM 260 212 82/90 260 85/91 260 83/89 - -
FkbN 923 934 82/89 934 82/89 913 71/82 - -
FkbQ 262 255 84/90 255 81/89 247 83/88 - -
Tcs6 69 - - - - - - - -
Tcs7 474 - - - - - - - -
<실시예 8> 포스미드 클론을 코스미드 클론으로 변경<Example 8> Change of phosphide clone to cosmid clone
접합 효율을 높이기 위해 포스미드 벡터에 탑재되어 있는 생합성 유전자를 복제수가 높은 코스미드 벡터로 이동하고자 하였다. SuperCos1 벡터(Staratagene, USA)를 제한효소 BamHI/NheI으로 절단후 T4 DNA 중합효소로 수복(Fill-in)하여 여분의 코스 부위(cos site) 1개가 제거된 SuperCos1-1을 작제 하였다. Fos1006G02를 SpeI과 XbaI으로 절단하여 약 38kb의 단편을, Fos-1004F01를 XbaI으로 절단하여 약 37 kb의 단편을, Fos-1006D05를 Spe1과 Xba1으로 절단하여 약 41 kb의 단편을 얻어 XbaI으로 절단한 SuperCos1-1 벡터에 각각 연결(ligation)하여 Cos-1006G02와 Cos-1004F01, Cos-1006D05를 작제하였다. 큰 사이즈의 삽입물(insert)로 인한 낮은 연결체(ligate)의 선별에 의한 비효율성을 해결하기 위해 람다 패키징 추출물(lambda packaging extracts)(Epicentre, Madison, WI, USA)을 이용하여 작제된 코스미드를 패키지하여 이.콜리 DH5α에 도입하는 방법을 사용하였다. In order to increase the conjugation efficiency, the biosynthetic gene loaded on the phosphide vector was moved to a cosmid vector having a high copy number. SuperCos1 vector (Staratagene, USA) was digested with restriction enzyme Bam HI / Nhe I and repaired with T4 DNA polymerase (Fill-in) to construct SuperCos1-1 with one extra coarse site removed. Cut Fos1006G02 with Spe I and Xba I to cut about 38 kb, Fos-1004F01 with Xba I to cut about 37 kb, and Fos-1006D05 with Spe 1 and Xba 1 to cut about 41 kb. Cos-1006G02, Cos-1004F01, and Cos-1006D05 were constructed by ligation to the SuperCos1-1 vectors digested with Xba I, respectively. Cosmids constructed using lambda packaging extracts (Epicentre, Madison, Wis., USA) were used to address the inefficiencies caused by the selection of low ligands due to large size inserts. The method of packaging and introducing into E. coli DH5α was used.
<실시예 9> 접합에 의한 방선균의 형질 전환Example 9 Transformation of Actinomycetes by Conjugation
유전자의 발현 및 돌연변이를 위한 운반벡터의 방선균으로의 도입은 이.콜라이/스트렙토마이세스 간의 엑스콘쥬게이션(exconjugation) 방법(Kieser, T. 등, 2000)으로 수행하였다. 비 메틸화 주게(non-methylating donor)로 dam -, dcm- 인 E. coli ET12567/pUZ8002에 발현 벡터 또는 코스미드 벡터를 전기침투(electrophoration) 방법으로 도입한 다음 접합(conjugation)을 통해 이를 방선균에 도입하였다. The introduction of the carrier vector for expression and mutation of the gene into the actinomycetes was performed by an exconjugation method between E. coli / Streptomyces (Kieser, T. et al., 2000). Non-methylated to give (non-methylating donor) into dam -, dc m - of the E. coli ET12567 / pUZ8002 expression vector or a cosmid vector in the actinomycetes This allows the next junction (conjugation) introduced into the electroosmotic (electrophoration) method Introduced.
<실시예 10> 생합성 유전자 돌연변이체의 작제 방법 Example 10 Construction of a Biosynthetic Gene Mutant
본 발명에서 사용한 돌연변이체의 작제는 Gust B. 등이(2003) 보고한 방법에 준하여 실시하였다. 돌연변이체 작제에 사용한 개시체는 표 5와 같다. 작제를 위한 돌연변이 apr-OriT 카셋은 pIJ773을 주형으로 표 5에 제시된 개시체를 사용하여 PCR로 증폭하여 약 1.4kb 단편들을 확보하였다. 확보된 단편을 Cos-1004F01, Cos-1006G02와 Cos-1006D05가 형질전환된 이.콜리 BW25113/pIJ790 균주에 도입한 균주에 전기충격법으로 도입하여 하기와 같이 돌연변이 코스미드 벡터를 만들었다. The construction of the mutants used in the present invention was carried out according to the method reported by Gust B. et al. (2003). The initiators used for constructing the mutants are shown in Table 5. Mutant apr - OriT cassette for construction amplified pIJ773 by PCR using the initiators shown in Table 5 as templates to obtain approximately 1.4 kb fragments. The obtained fragments were introduced into strains introduced into E. coli BW25113 / pIJ790 strains transformed with Cos-1004F01, Cos-1006G02, and Cos-1006D05 by electroshock method, thereby producing mutant cosmid vectors as follows.
Cos-1004F01(ΔtcsA::aac(3)IV-oriT), Cos-1004F01 (Δ tcsA :: aac (3) IV-oriT ),
Cos-1004F01(ΔtcsB::aac(3)IV-oriT), Cos-1004F01 (Δ tcsB :: aac (3) IV-oriT ),
Cos-1004F01(ΔtcsC::aac(3)IV-oriT), Cos-1004F01 (Δ tcsC :: aac (3) IV-oriT ),
Cos-1004F01(ΔtcsD::aac(3)IV-oriT), Cos-1004F01 (Δ tcsD :: aac (3) IV-oriT ),
Cos-1004F01(Δtcs1::aac(3)IV-oriT), Cos-1004F01 (Δ tcs1 :: aac (3) IV-oriT ),
Cos-1004F01(Δtcs2::aac(3)IV-oriT), Cos-1004F01 (Δ tcs2 :: aac (3) IV-oriT ),
Cos-1004F01(Δtcs3::aac(3)IV-oriT), Cos-1004F01 (Δ tcs3 :: aac (3) IV-oriT ),
Cos-1004F01(Δtcs4::aac(3)IV-oriT), Cos-1004F01 (Δ tcs4 :: aac (3) IV-oriT ),
Cos-1004F01(Δtcs5::aac(3)IV-oriT), Cos-1004F01 (Δ tcs5 :: aac (3) IV-oriT ),
Cos-1006G02(ΔfkbA::aac(3)IV-oriT), Cos-1006G02 (Δ fkbA :: aac (3) IV-oriT ),
Cos-1006G02(ΔfkbD::aac(3)IV-oriT), Cos-1006G02 (Δ fkbD :: aac (3) IV-oriT ),
Cos-1006G02(ΔfkbM::aac(3)IV-oriT), Cos-1006G02 (Δ fkbM :: aac (3) IV-oriT ),
Cos-1006G02(ΔfkbN::aac(3)IV-oriT), Cos-1006G02 (Δ fkbN :: aac (3) IV-oriT ),
Cos-1006G02(ΔfkbQ::aac(3)IV-oriT), Cos-1006G02 (Δ fkbQ :: aac (3) IV-oriT ),
Cos-1006G02(ΔfkbR::aac(3)IV-oriT), Cos-1006G02 (Δ fkbR :: aac (3) IV-oriT ),
Cos-1006D05(ΔtcsA-sk(::aac(3)IV-oriT), Cos-1006D05 (Δ tcsA -sk (:: aac (3) IV-oriT ),
Cos-1006D05(ΔtcsB-sk::aac(3)IV-oriT), Cos-1006D05 (Δ tcsB -sk :: aac (3) IV-oriT ),
Cos-1006D05(ΔtcsC-sk::aac(3)IV-oriT), Cos-1006D05 (Δ tcsC -sk :: aac (3) IV-oriT ),
Cos-1006D05(ΔtcsD-sk::aac(3)IV-oriT)를 작제하였다. Cos-1006D05 (Δ tcsD -sk :: aac (3) IV-oriT ) was constructed.
상기 작제된 돌연변이 코스미드를 각각 E. coli ET12567/pUZ8002로 옮긴 후 실시예 9의 엑스콘쥬게이션을 통해 스트렙토마이세스속 KCTC 11604BP와 스트렙토마이세스 카나마이세티쿠스 KCTC 9225로 형질전환하였으며, 이를 통해 아래와 같이 각 유전자의 돌연변이체를 확보할 수 있었다. The constructed mutant cosmids were transferred to E. coli ET12567 / pUZ8002, respectively, and then transformed into Streptomyces KCTC 11604BP and Streptomyces kanamyceticus KCTC 9225 through the exconjugation of Example 9. Likewise, mutants of each gene could be obtained.
S. sp. KCTC 11604BP(ΔtcsA), S. sp. KCTC 11604BP(ΔtcsB), S. sp. KCTC 11604BP(ΔtcsC),S. sp. KCTC 11604BP(ΔtcsD), S. sp. KCTC 11604BP(Δtcs1), S. sp. KCTC 11604BP(Δtcs2), S. sp. KCTC 11604BP(Δtcs3), S. sp. KCTC 11604BP(Δtcs4),S. sp. KCTC 11604BP(Δtcs5), S. sp. KCTC 11604BP(Δtcs7), S. sp. KCTC 11604BP(ΔfkbA), S. sp. KCTC 11604BP(ΔfkbD), S. sp. KCTC 11604BP(ΔfkbM), S. sp. KCTC 11604BP(ΔfkbN), S. sp. KCTC 11604BP(ΔfkbQ), S. sp. KCTC 11604BP(ΔfkbR), S. kanamyceticus KCTC 9225(ΔtcsA), S. kanamyceticus KCTC 9225(ΔtcsB), S. kanamyceticus KCTC 9225(ΔtcsC), S. kanamyceticus KCTC 9225(ΔtcsD) S. sp . KCTC 11604BP (Δ tcsA ) , S. sp . KCTC 11604BP (Δ tcsB ), S. sp . KCTC 11604BP (Δ tcsC ), S. sp . KCTC 11604BP (Δ tcsD ), S. sp . KCTC 11604BP (Δ tcs1 ), S. sp . KCTC 11604BP (Δ tcs2 ), S. sp . KCTC 11604BP (Δ tcs3 ), S. sp . KCTC 11604BP (Δ tcs4 ), S. sp . KCTC 11604BP (Δ tcs5 ), S. sp . KCTC 11604BP (Δ tcs7 ), S. sp . KCTC 11604BP (Δ fkbA ), S. sp . KCTC 11604BP (Δ fkbD ), S. sp. KCTC 11604BP (Δ fkbM ), S. sp . KCTC 11604BP (Δ fkbN ), S. sp . KCTC 11604BP (Δ fkbQ ), S. sp . KCTC 11604BP (Δ fkbR ), S. kanamyceticus KCTC 9225 (Δ tcsA ), S. kanamyceticus KCTC 9225 (Δ tcsB ), S. kanamyceticus KCTC 9225 (Δ tcsC ), S. kanamyceticus KCTC 9225 (Δ tcsD )
돌연변이체는 서던 블랏 및 PCR로 이중 교차 돌연변이체(double cross over mutants)임을 확인하였다. The mutants were Southern cross and PCR confirmed double cross mutants.
표 5
Figure PCTKR2010008496-appb-T000001
 Table 5
Figure PCTKR2010008496-appb-T000001
표 6
strains Function Bioassay Metabolites
FK506 FK520 Others
Wild type +++ +++ + +
ΔtcsA allyl malonyl ACP biosynthesis + - + +
ΔtcsB allyl malonyl ACP biosynthesis + - + +
ΔtcsC allyl malonyl ACP biosynthesis + - + +
ΔtcsD allyl malonyl ACP biosynthesis + - + dihydro FK506
Δtcs1 Methionine gamma lyase +++ +++ + +
Δtcs2 AsnC-family transcriptional regulatory protein +++ +++ + +
Δtcs3 Cytochrome P450 +++ +++ + +
Δtcs4 Indole oxygenase +++ +++ + +
Δtcs5 Short-chain dehydrogenase +++ +++ + +
ΔfkbA FK506 polyketide synthase - - - -
ΔfkbD P450 9-deoxo-FK506 hydroxylase +++ - - +++
ΔfkbM 31-O -demethyl-FK506 methyltransferase +++ - - +++
Δ fkbN transcriptional regulator - - - -
ΔfkbQ type Ⅱ thioesterase +++ +++ + +
Δtcs7 Lys R-family transcriptional regulator +++ +++ + +
Table 6
strains Function Bioassay Metabolites
FK506 FK520 Other
Wild type +++ +++ + +
Δ tcsA allyl malonyl ACP biosynthesis + - + +
ΔtcsB allyl malonyl ACP biosynthesis + - + +
ΔtcsC allyl malonyl ACP biosynthesis + - + +
ΔtcsD allyl malonyl ACP biosynthesis + - + dihydro FK506
Δtcs1 Methionine gamma lyase +++ +++ + +
Δtcs2 AsnC-family transcriptional regulatory protein +++ +++ + +
Δtcs3 Cytochrome P450 +++ +++ + +
Δtcs4 Indole oxygenase +++ +++ + +
Δtcs5 Short-chain dehydrogenase +++ +++ + +
ΔfkbA FK506 polyketide synthase - - - -
ΔfkbD P450 9-deoxo-FK506 hydroxylase +++ - - +++
ΔfkbM 31-O- demethyl-FK506 methyltransferase +++ - - +++
Δ fkbN transcriptional regulator - - - -
ΔfkbQ type II thioesterase +++ +++ + +
Δtcs7 Lys R-family transcriptional regulator +++ +++ + +
<실시예 11> 삼원환 화합물 생합성 유전자 영역 결정Example 11 Determination of Three-membered Ring Compound Biosynthesis Gene Region
확보한 유전자가 타크롤리무스 생합성과 아스코마이신 등의 삼원환 화합물 생합성에 관여하는 생합성 유전자임을 증명하기 위해 FkbA 결손 돌연변이체를 제작하여 분석하였다. FkbA는 타크롤리무스 폴리케타이드 합성효소이며 4개의 모듈을 포함하고 있고, 폴리케타이드 골격의 생합성에 핵심적인 효소이다. 실시예 10에서 작제한 fkbA 결손 변이체, 스트렙토마이세스속 KCTC 11604BP(ΔfkbA)의 배양체에는 어떠한 항진균 활성도 나타내지 않았다. 또한 타크롤리무스 및 아스코마이신, DH-타크롤리무스, FK525 등 어떠한 타크롤리무스 유사체도 실시예 2의 HPLC와 LC-MS로 확인할 수 없었다(도 4). 이는 본 발명에서 확보한 유전자 군이 타크롤리무스와 아스코마이신을 포함하는 그 유사체의 생합성 기구 형성에 필수적임을 증명하는 것이다. 이러한 사실로부터 기존의 아스코마이신과 타크롤리무스 생합성 기구가 서로 상이할 것으로 예측한 것과는 달리 타크롤리무스와 아스코마이신 합성이 동일한 기반에서 이루어짐을 확인할 수 있었다. 즉, 본 발명에서 확보한 유전자는 타크롤리무스는 물론 아스코마이신과 그 구조 유사체의 합성 및 변형에 이용될 수 있음을 의미한다. In order to prove that the acquired gene is a biosynthetic gene involved in the trisynthetic compound biosynthesis such as tacrolimus biosynthesis and ascomycin, Fkb A-deficient mutants were prepared and analyzed. FkbA is a tacrolimus polyketide synthetase, contains four modules, and is an essential enzyme for biosynthesis of polyketide backbone. No antifungal activity was shown in the culture of the fkbA- deficient variant, Streptomyces KCTC 11604BP (Δ fkbA ), constructed in Example 10. In addition, no tacrolimus analogs such as tacrolimus and ascomycin, DH-tacrolimus, FK525 could be confirmed by HPLC and LC-MS of Example 2 (FIG. 4). This proves that the gene group secured in the present invention is essential for the formation of biosynthetic machinery of the analogs including tacrolimus and ascomycin. From this fact, it was confirmed that the synthesis of tacrolimus and ascomycin is performed on the same basis, unlike the conventional ascomycin and tacrolimus biosynthesis mechanisms expected to be different from each other. That is, the gene obtained in the present invention means that tacrolimus can be used for the synthesis and modification of ascomycin and its structural analogues.
또한, 실시예 10에서 작제한 조절인자 fkbN 돌연변이체인 스트렙토마이세스속 KCTC 11604BP(ΔfkbM)도 스트렙토마이세스속 KCTC 11604BP(ΔfkbA)와 동일하게 항진균 활성이 소실되었으며, 실시예 2의 HPLC분석으로 어떠한 타크롤리무스 유도체의 생성도 확인할 수 없었다. 즉 FkbN이 타크롤리무스 생합성 유전자의 매우 특이적인 양성 조절인자임을 확인하였다. In addition, the regulatory factor fkbN mutant Streptomyces KCTC 11604BP (Δ fkbM ) constructed in Example 10 also lost antifungal activity in the same manner as Streptomyces KCTC 11604BP (Δ fkbA ), by HPLC analysis of Example 2 No production of any tacrolimus derivatives could be confirmed. In other words, it was confirmed that FkbN is a very specific positive regulator of tacrolimus biosynthesis gene.
이렇게 확인된 타크롤리무스 생합성 유전자군에서 타크롤리무스 생합성에 직접적으로 관여하는 유전자를 확인하고자 하였다. 아스코마이신 생합성 유전자 로 알려진 fkbG와 fkbN를 벗어나는 유전자 영역이 타크롤리무스 생합성에 필요한지를 검증하였다. fkbG를 벗어나 존재하는 tcsA,B,C,D,1,2,3,4,5와 fkbN을 벗어난 fkbQ,7의 변이체를 실시예 10과 같이 작제하여 그 대사물질을 분석하였다. 그 결과, tcsA,B,C,D 돌연변이체인 S. sp. KCTC 11604BP(ΔtcsA), S. sp. KCTC 11604BP(ΔtcsB), S. sp. KCTC 11604BP(ΔtcsC), S. sp. KCTC 11604BP(ΔtcsD)의 경우 타크롤리무스 생산이 HPLC로 검출되지 않았다(도 4). 그러나, fkbQ 및 Tcs1,2,3,4,5,7 돌연변이체는 여전히 타크롤리무스 및 아스코마이신을 생산하였다(도 4). 이로 미루어 필수적인 타크롤리무스 생합성 유전자는 tcsA,B,C,D를 포함하여 fkbG에서 FkbN까지로 추정할 수 있었다. 또한 이들 유전자는 타크롤리무스 생합성 유전자군에서 모두 공통적으로 확인되는 유전자들이다(도 2). We tried to identify the genes directly involved in tacrolimus biosynthesis in the tacrolimus biosynthetic gene group thus identified. We tested whether fkbG and fkbN gene regions known as ascomycin biosynthesis genes are required for tacrolimus biosynthesis. Variants of tcsA, B, C, D, 1,2,3,4,5 and fkbN , which were out of fkbG and fkbN , out of fkbN were constructed as in Example 10 and analyzed for their metabolites. As a result, the tcsA, B, C, D mutant S. sp. KCTC 11604BP (Δ tcsA ), S. sp. KCTC 11604BP (Δ tcsB ), S. sp. KCTC 11604BP (Δ tcsC ), S. sp. Tacrolimus production was not detected by HPLC for KCTC 11604BP (Δ tcsD ) (FIG. 4). However, the fkbQ and Tcs1,2,3,4,5,7 mutants still produced tacrolimus and ascomycin (FIG. 4). Thus, essential tacrolimus biosynthesis genes could be estimated from fkbG to FkbN , including tcsA, B, C, D. In addition, these genes are all genes commonly identified in the tacrolimus biosynthetic gene group (FIG. 2).
<실시예 12> <Example 12> tcsA,B,C,DtcsA, B, C, D 돌연변이체의 LC-MS/MS 분석 및 타크롤리무스 생합성  LC-MS / MS Analysis and Tacrolimus Biosynthesis of Mutants
실시예 10에서 작제하고 실시예 11에서 분석한 것과 같이 tcsA,B,C,D 돌연변이체는 타크롤리무스 생산을 HPLC로 확인할 수는 없었다. 또한 실시예 10에서 작제한 S. kanamyceticus KCTC 9225(ΔtcsA), S. kanamyceticus KCTC 9225(ΔtcsB), S. kanamyceticus KCTC 9225(ΔtcsC), S. kanamyceticus KCTC 9225(ΔtcsD) 또한 타크롤리무스 생산을 HPLC로 검출할 수 없었다. 그러나 이들 균주들은 야생형인 S. sp. KCTC 11604BP보다는 낮지만 여전히 높은 항진균 활성이 확인되었다. 이로 미루어 항진균 활성을 가진 타크롤리무스나 아스코마이신 또는 그 유사체가 일부 생산될 수 있음이 추정되었다. 유도체의 존재를 확인하기 위해 S. sp KCTC 11604BP 균주와 그 돌연변이주들 배양체를 이용하여 LC-MS/MS로 대사체를 좀더 면밀하게 분석하였다. LC-MS/MS 결과 각 돌연변이체에서 확인된 물질은 표 7 및 도 5와 같다. 흥미롭게도 S. sp. KCTC 11604BP(ΔtcsA), S. sp. KCTC 11604BP(ΔtcsB), S. sp. KCTC 11604BP(ΔtcsC)의 경우 아스코마이신(타크롤리무스의 에틸 폼) 및 FK523(타크롤리무스의 메틸 폼)가 여전히 생산되고 있음을 확인할 수 있었으며, S. sp. KCTC 11604BP(ΔtcsD)에서는 37, 38-다이하이드로폼의 타크롤리무스 유도체가 생산되고 있음을 확인할 수 있었다. 이러한 결과로 보아 tcsA, B, C, D는 타크롤리무스 21번 위치의 곁사슬 형성에 매우 중요한 역할을 하는 유전자임을 확인할 수 있었다. S. sp. KCTC 11604BP(ΔtcsB), S. sp. KCTC 11604BP(ΔtcsC)에서는 매우 적은 양의 타크롤리무스가 생산되는 것으로 보아 케토합성효소(Ketosynthase)와 환원카복실레이즈(reductive carboxylase)를 일부 대체하는 유사체가 존재하여 일부 미약하게 기능하거나, 복합배지를 사용하는 발효조건에 의한 알릴 (프로필레닐) 말로닐산 혹은 그 유사체(예, 4-페테노익산, 발레릭산 또는 프로필 말로닐산)가 직접 TcsA-ACP에 도입(charging)되어 극미량 생성된 것으로 추정된다. 그러나, S. sp. KCTC 11604BP(ΔtcsA)에서는 타크롤리무스가 전혀 검출되지 않았으며, 이는 알릴-말로닐 부분을 주요 PKS (모듈 4의 ACP 혹은 AT)로 전달하는 데에 TcsA의 AT 및 ACP 도메인이 절대적인 역할을 하기 때문일 것이다. 또한 S. sp. KCTC 11604BP(ΔtcsD)는 모든 화합물의 21위치의 곁사슬이 포화 형태(saturated form)로만 발견되었다. 이는 아실-CoA 탈수소효소(ACAD)로의 역할의 소실로 인한 것으로 추정할 수 있다. 상기의 결과로부터 추론할 때, TcsA,B,C,D는 타크롤리무스 곁사슬(side chain) 형성에 도 6과 같이 추정되는 메카니즘으로 타크롤리무스 및 그 유도체의 합성에 중요한 역할을 할 것이다. As constructed in Example 10 and analyzed in Example 11, the tcsA, B, C, D mutants were unable to confirm tacrolimus production by HPLC. In addition, S. kanamyceticus KCTC 9225 (Δ tcsA ), S. kanamyceticus KCTC 9225 (Δ tcsB ), S. kanamyceticus KCTC 9225 (Δ tcsC ), and S. kanamyceticus KCTC 9225 (Δ tcsD ) constructed in Example 10. Production could not be detected by HPLC. However, these strains were wild type S. sp. Lower than KCTC 11604BP but still high antifungal activity was confirmed. It has been estimated from this that some production of tacrolimus, ascomycin or an analog thereof with antifungal activity may be produced. S to confirm the presence of derivatives. Metabolites were analyzed more closely by LC-MS / MS using sp KCTC 11604BP strain and its mutant cultures. LC-MS / MS results The substances identified in each mutant are shown in Table 7 and FIG. 5. Interestingly S. sp. KCTC 11604BP (Δ tcsA ), S. sp. KCTC 11604BP (Δ tcsB ), S. sp. For KCTC 11604BP (Δ tcsC ) it was confirmed that ascomycin (ethyl form of tacrolimus) and FK523 (methyl form of tacrolimus) are still produced, S. sp. In KCTC 11604BP (Δ tcsD ), it was confirmed that a tacrolimus derivative of 37, 38-dihydrofoam was produced. These results indicate that tcsA, B, C, D is a gene that plays a very important role in the side chain formation of tacrolimus position 21. S. sp . KCTC 11604BP (Δ tcsB ), S. sp . As KCTC 11604BP (Δ tcsC ) produces very small amounts of tacrolimus, there are analogs that partially replace ketosynthase and reductive carboxylase, so that some weakly functioning or complex media It is estimated that allyl (propylenyl) malonic acid or its analogues (eg, 4-fethenoic acid, valeric acid or propyl malonyl acid) produced by the fermentation conditions used are directly generated in the traces of TcsA-ACP. However, S. sp. Tacrolimus was not detected at all in KCTC 11604BP (Δ tcsA ), indicating that the AT and ACP domains of TcsA play an absolute role in delivering the allyl-malonyl moiety to the main PKS (ACP or AT of Module 4). It will be. Also S. sp. KCTC 11604BP (Δ tcsD ) was found only in the saturated form of the side chains of all compounds. This may be due to the loss of its role as acyl-CoA dehydrogenase (ACAD). Inferred from the above results, TcsA, B, C, D is a mechanism estimated as shown in Figure 6 in the formation of tacrolimus side chain will play an important role in the synthesis of tacrolimus and its derivatives.
상기 TcsA,B,C,D와 그들의 유전자 돌연변이체의 활용 또는 유전자의 개별 또는 집단 도입을 통한 단백질 발현 등의 활용은 신규 물질 생산, 타크롤리무스 및 그 유연물질, 또는 신규로 생산될 수 있는 타크롤리무스 유연물질의 생산 증대 또는 아스코마이신과 같은 타크롤리무스의 중요한 불순물의 제거를 도모하는데 매우 유용할 것으로 추정된다. Utilization of the TcsA, B, C, D and their gene mutants, or the expression of proteins through the introduction of individual or population of genes, the production of new substances, tacrolimus and its analogs, or other that can be newly produced It is thought to be very useful for increasing the production of C. limousin flexible substances or for removing important impurities of tacrolimus such as ascomycin.
표 7
Strains LC/MS 결과
WT(GT1005) 9-deoxo-31-O-demethyl-ascomycin: Mr 763.4870 (m/z 786.4[M+Na] + ) 9-deoxo-31-O-demethyl-tacrolimus Mr 775.4870 (m/z 798.4[M+Na] + ) 9-hydroxy-tacrolimus: Mr 805.4976 (m/z 828.49[M+Na] + ) 9-deoxo-31-O-demethyl-dihydro-tacrolimus: Mr 777.5027 (m/z 800.5[M+Na] + ) Prolinyl-tacrolimus (FK525): Mr 789.4663 (m/z 812.46[M+Na] + ) ascomycin (FK520): Mr 791.4819 (m/z 814.48[M+Na] + ) tacrolimus (FK506): Mr 803.48198 (m/z 826.48[M+Na] + ) Decarbonyl-tacrolimus(9-deoxo-FK506): Mr 789.5027 (m/z 812.5[M+Na] + ) dihydro-tacrolimus: Mr 805.49763 (m/z 828.48[M+Na] + )
ΔtcsA (acyltransferase) L-687819: Mr 763.45068(m/z 786.4301[M+Na] + ) (L-683795) FK523: Mr 777.4663(m/z 800.4587[M+Na] + ) ascomycinFK520: Mr 791.48198(m/z 814.46[M+Na] + )
ΔtcsB(ketosynthase) L-687819: Mr 763.45068(m/z 786.4301[M+Na]+)FK523: Mr 777.4663(m/z 800.4502[M+Na]+)ascomycin (FK520): Mr 791.48198(m/z 814.4682[M+Na]+)FK506: Mr 803.48198(m/z 826.4683[M+Na]+)(highly decreased)
ΔtcsC(crotonyl CoA reductase-reductive carboxylase) L-687819: Mr 763.45068 (m/z 786.4301[M+Na]+)FK523: Mr 777.4663 (m/z 800.4502[M+Na]+)ascomycin (FK520): Mr 791.48198 (m/z 814.4682[M+Na]+)tacrolimus (FK506): Mr 803.48198 (m/z 826.4683[M+Na]+(highly decreased)
ΔtcsD (acyl-CoA dehydrogenase) 31-O-demethyl-ascomycin: Mr 777.4663 (m/z 800.4655[M+Na] + ) 31-O-demethyl-DH-tacrolimus: Mr 791.48198 (m/z 814.4718[M+Na] + ) 9-deoxo-31-O-demethyl-DH-tacrolimus: Mr 777.50271 (m/z 800.4936[M+Na] + ) ascomycin (FK520): Mr 791.48198 (m/z 814.46[M+Na] + ) 9-deoxo-ascomycin: Mr 777.50271 (m/z 800.4928[M+Na] + ) Prolinyl-dihydro-tacrolimus: Mr 791.48198 (m/z 814.48[M+Na] + ) dihydro-tacrolimus: Mr 805.49763 (m/z 828.479[M+Na] + ) 9-deoxo-dihydro-tacrolimus: Mr 791.51836 (m/z 814.5[M+Na] + )
TABLE 7
Strains LC / MS Results
WT (GT1005) 9-deoxo-31-O-demethyl-ascomycin: Mr 763.4870 (m / z 786.4 [M + Na] + ) 9-deoxo-31-O-demethyl-tacrolimus Mr 775.4870 (m / z 798.4 [M + Na] + ) 9-hydroxy-tacrolimus: Mr 805.4976 (m / z 828.49 [M + Na] + ) 9-deoxo-31-O-demethyl-dihydro-tacrolimus: Mr 777.5027 (m / z 800.5 [M + Na] + ) Prolinyl -tacrolimus (FK525): Mr 789.4663 (m / z 812.46 [M + Na] + ) ascomycin (FK520): Mr 791.4819 (m / z 814.48 [M + Na] + ) tacrolimus (FK506): Mr 803.48198 (m / z 826.48 [M + Na] + ) Decarbonyl-tacrolimus (9-deoxo-FK506): Mr 789.5027 (m / z 812.5 [M + Na] + ) dihydro-tacrolimus: Mr 805.49763 (m / z 828.48 [M + Na] + )
ΔtcsA (acyltransferase) L-687819: Mr 763.45068 (m / z 786.4301 [M + Na] + ) (L-683795) FK523: Mr 777.4663 (m / z 800.4587 [M + Na] + ) ascomycin FK520: Mr 791.48198 (m / z 814.46 [M + Na] + )
ΔtcsB (ketosynthase) L-687819: Mr 763.45068 (m / z 786.4301 [M + Na] + ) FK523: Mr 777.4663 (m / z 800.4502 [M + Na] + ) ascomycin (FK520): Mr 791.48198 (m / z 814.4682 [M + Na] ] +) FK506: Mr 803.48198 ( m / z 826.4683 [m + Na] +) (highly decreased)
ΔtcsC (crotonyl CoA reductase-reductive carboxylase) L-687819: Mr 763.45068 (m / z 786.4301 [M + Na] + ) FK523: Mr 777.4663 (m / z 800.4502 [M + Na] + ) ascomycin (FK520): Mr 791.48198 (m / z 814.4682 [M + Na ] +) tacrolimus (FK506): Mr 803.48198 (m / z 826.4683 [m + Na] + (highly decreased)
ΔtcsD (acyl-CoA dehydrogenase) 31-O-demethyl-ascomycin: Mr 777.4663 (m / z 800.4655 [M + Na] + ) 31-O-demethyl-DH-tacrolimus: Mr 791.48198 (m / z 814.4718 [M + Na] + ) 9-deoxo- 31-O-demethyl-DH-tacrolimus: Mr 777.50271 (m / z 800.4936 [M + Na] + ) ascomycin (FK520): Mr 791.48198 (m / z 814.46 [M + Na] + ) 9-deoxo-ascomycin: Mr 777.50271 (m / z 800.4928 [M + Na] + ) Prolinyl-dihydro-tacrolimus: Mr 791.48198 (m / z 814.48 [M + Na] + ) dihydro-tacrolimus: Mr 805.49763 (m / z 828.479 [M + Na] + ) 9-deoxo-dihydro-tacrolimus: Mr 791.51836 (m / z 814.5 [M + Na] + )
<실시예 13> 디옥소- 및 디메틸레이티드형 타크롤리무스 유도체 고생산Example 13 High Production of Dioxo- and Dimethylated Tacrolimus Derivatives
실시예 10에서 작제된 돌연변이체들 즉, S. sp. KCTC 11604BP(ΔfkbD), S. sp. KCTC 11604BP(ΔfkbM)의 타크롤리무스 및 그 유사체의 생산을 확인하였다. 이 두 변이체는 타크롤리무스 생산 능력은 소실되었다. 그러나, S. sp. KCTC 11604BP(ΔfkbD)는 타크롤리무스의 디옥소(deoxo) 유도체인 9-디옥소-31-O-데스메틸-타크롤리무스를 포함하여 9-디옥소-31-O-데스메틸-아스코마이신, 9-디옥소-31-O-데스메틸-36,37-다이하이드로-타크롤리무스를 생산함을 확인하였다. 또한 S. sp. KCTC 11604BP(ΔfkbM)는 데스메틸 폼인 31-O-데스메틸-타크롤리무스, 31-O-데스메틸-아스코마이신, 31-O-데스메틸-36,37-다이하이드로-타크롤리무스를 생산함을 확인하였다. 특히 상기 물질은 fkbD와 fkbM 돌연변이체에서 야생주인 S. sp. KCTC 11604BP에서보다 매우 높은 생산이 가능함을 확인하였다. 특히, 9-디옥소-31-O-데스메틸-타크롤리무스와 31-O-데스메틸-타크롤리무스는 S. sp. KCTC 11604BP(ΔfkbD)와 S. sp. KCTC 11604BP(ΔfkbM)에서 각각 야생형 균주에서 생산되는 타크롤리무스 생산량에 비견될 수 있는 80~150㎎/ℓ로 고생산이 가능하였다. The mutants constructed in Example 10, ie, S. sp . KCTC 11604BP (Δ fkbD ), S. sp . The production of tacrolimus and its analogs of KCTC 11604BP (Δ fkbM ) was confirmed. Both variants lost tacrolimus production capacity. However, S. sp . KCTC 11604BP ( ΔfkbD ) is a 9-dioxo-31-O-desmethyl- ascomycin , including 9-dioxo-31-O-desmethyl-tacrolimus, a deoxo derivative of tacrolimus. , 9-dioxo-31-O-desmethyl-36,37-dihydro-tacrolimus. Also S. sp. KCTC 11604BP (Δ fkbM ) produced desmethyl foam 31-O-desmethyl-tacrolimus, 31-O-desmethyl-ascomycin, 31-O-desmethyl-36,37-dihydro-tacrolimus It was confirmed. In particular, the material S in the master wild fkbD and fkbM mutants. sp. It was confirmed that much higher production is possible than in KCTC 11604BP. In particular, 9-dioxo-31-O-desmethyl-tacrolimus and 31-O-desmethyl-tacrolimus are S. sp . KCTC 11604BP (Δ fkbD ) and S. sp . In KCTC 11604BP (Δ fkbM ), high production was possible at 80-150 mg / L, which can be compared with the production of tacrolimus produced in each wild-type strain.
본 발명으로 타크롤리무스(FK506), 아스코마이신(FK520), 다이하이드로타크롤리무스, 프롤릴타크롤리무스(FK525), FK523(L-683795), L-687819, 9-디옥소-31-O-데스메틸-타크롤리무스, 9-디옥소-31-O-데스메틸-아스코마이신, 9-디옥소-타크롤리무스, 9-디옥소-아스코마이신, 9-하이드록시-9-디옥소-타크롤리무스, 9-디옥소-36,37-다이하이드로-타크롤리무스, 31-O-데스메틸-타크롤리무스, 31-O-데스메틸-아스코마이신, 31-O-데스메틸-36,37-다이하이드로-타크롤리무스, 9-디옥소-31-O-데스메틸-36,37-다이하이드로-타크롤리무스, 프롤릴-다이하이드로-타크롤리무스, 라파마이신, 메리다마이신 및 36-케토-메리다마이신 생합성에 필요한 유전자를 확보하였으며, 이들 물질의 산업적 생산 및 다른 신규한 구조의 화합물 창조를 위한 구조변형 등에 응용할 수 있다. Tacrolimus (FK506), Ascomycin (FK520), Dihydrotacrolimus, Prolyl tacrolimus (FK525), FK523 (L-683795), L-687819, 9-dioxo-31-O- Desmethyl-tacrolimus, 9-dioxo-31-O-desmethyl-ascomycin, 9-dioxo-tacrolimus, 9-dioxo-ascomycin, 9-hydroxy-9-dioxo-ta Crowlimus, 9-dioxo-36,37-dihydro-tacrolimus, 31-O-desmethyl-tacrolimus, 31-O-desmethyl-ascomycin, 31-O-desmethyl-36,37 -Dihydro-tacrolimus, 9-dioxo-31-O-desmethyl-36,37-dihydro-tacrolimus, prolyl-dihydro-tacrolimus, rapamycin, meridamycin and 36-keto The genes needed for the biosynthesis of meridamycin have been obtained, and they can be applied to the industrial production of these materials and structural modifications for the creation of other novel compounds.
본 발명의 예시에서 제시된 fkbM, fkbD 돌연변이체의 작제방법에 의해 합리적으로 만들어질 수 있는 돌연변이체는 9-디옥소-31-O-디메틸-타크롤리무스, 9-디옥소-31-O-디메틸-아스코마이신, 9-디옥소-31-O-디메틸-36,37-다이하이드로-타크롤리무스, 31-O-디메틸-타크롤리무스, 31-O-디메틸-아스코마이신 및 31-O-디메틸-36,37-다이하이드로-타크롤리무스의 생산성을 높일 수 있어 야생형에서 소량으로 생산되는 상기 타크롤리무스 변형물질의 고생산에 응용될 수 있다. Mutants that can be reasonably made by the method of constructing the fkbM , fkbD mutants presented in the examples of the present invention are 9-dioxo-31-O-dimethyl-tacrolimus, 9-dioxo-31-O-dimethyl -Ascomycin, 9-dioxo-31-O-dimethyl-36,37-dihydro-tacrolimus, 31-O-dimethyl-tacrolimus, 31-O-dimethyl-ascomycin and 31-O-dimethyl The productivity of -36,37-dihydro-tacrolimus can be increased, and thus it can be applied to high production of the tacrolimus modified material produced in small amounts in the wild type.
본 발명으로 확보한 TcsA,TcsB,TcsC,TcsD 유전자 및 그 유전자가 코딩하고 있는 단백질은 타크롤리무스 21 위치의 곁가지 (알릴-말로닐 유도체)의 생산 혹은 변형에 직접, 발현 혹은 돌연변이체 작제로 이용될 수 있으며 이를 이용한 전체 폴리케타이드 물질의 구조 변형 또는 불순물 감소에 응용될 수 있다.The Tcs A, Tcs B, Tcs C, and Tcs D genes obtained by the present invention and the proteins encoded by the genes are directly expressed or mutated in the production or modification of the side branches (allyl-malonyl derivatives) at tacrolimus 21. It can be used as a mechanism and can be applied to structural modification or reduction of impurities of the entire polyketide material using the same.
본 발명의 서열목록에 기재된 서열은 타크롤리무스를 비롯한 폴리케타이드 합성에 관여하는 효소들의 유전자 및 단백질 서열이다.Sequences described in the Sequence Listing of the present invention are gene and protein sequences of enzymes involved in polyketide synthesis, including tacrolimus.

Claims (11)

  1. 서열번호 1, 54 또는 93과 동일하거나 85% 이상의 동일성을 나타내는 서열 또는 상기 서열에 상보적인 서열로 표시되는 타크롤리무스 생합성 유전자 클러스터Tacrolimus biosynthetic gene cluster represented by the sequence equal to or greater than 85% identity with SEQ ID NO: 1, 54, or 93, or a sequence complementary to the sequence
  2. 서열번호 3, 56 또는 95와 동일하거나 80% 이상의 동일성을 나타내는 서열 또는 상기 서열에 상보적인 서열로 표시되는 폴리케타이드 합성효소 유전자A polyketide synthase gene represented by a sequence that is the same as or greater than 80% identity with SEQ ID NO: 3, 56 or 95, or a sequence complementary to the sequence
  3. 서열번호 2, 55 또는 94와 동일하거나 80% 이상의 동일성을 나타내는 서열로 표시되는 폴리케타이드 합성효소A polyketide synthetase represented by a sequence that is identical to or exhibits 80% or more identity to SEQ ID NO: 2, 55, or 94
  4. 서열번호 5, 58 또는 97과 동일하거나 80% 이상의 동일성을 나타내는 서열 또는 상기 서열에 상보적인 서열로 표시되는 폴리케타이드 합성효소 유전자A polyketide synthase gene represented by a sequence that is identical to, or complementary to, SEQ ID NO: 5, 58, or 97;
  5. 서열번호 4, 57 또는 96과 동일하거나 80% 이상의 동일성을 나타내는 서열로 표시되는 폴리케타이드 합성효소A polyketide synthetase represented by a sequence that is identical to or exhibits at least 80% identity with SEQ ID NO: 4, 57, or 96
  6. 서열번호 7, 60 또는 99와 동일하거나 90% 이상의 동일성을 나타내는 서열 또는 상기 서열에 상보적인 서열로 표시되는 폴리케타이드 합성효소 유전자A polyketide synthase gene represented by a sequence that is identical to, or complementary to, SEQ ID NO: 7, 60, or 99, or at least 90% identity
  7. 서열번호 6, 59 또는 98과 동일하거나 90% 이상의 동일성을 나타내는 서열로 표시되는 폴리케타이드 합성효소A polyketide synthetase represented by a sequence that is identical to or exhibits 90% or more identity to SEQ ID NOs: 6, 59, or 98
  8. 서열번호 9, 62 또는 101과 동일하거나 90% 이상의 동일성을 나타내는 서열 또는 상기 서열에 상보적인 서열로 표시되는 폴리케타이드 합성효소 유전자A polyketide synthase gene represented by a sequence that is identical to or greater than 90% identity to SEQ ID NO: 9, 62, or 101, or a sequence complementary to the sequence
  9. 서열번호 8, 61 또는 100과 동일하거나 90% 이상의 동일성을 나타내는 서열로 표시되는 폴리케타이드 합성효소A polyketide synthetase represented by a sequence that is identical to or exhibits 90% or more identity to SEQ ID NOs: 8, 61, or 100
  10. 타크롤리무스 생합성 유전자 클러스터 중 fkbD 유전자를 결손시키거나 불활성화시킨 돌연변이체를 배양하여 9-디옥소-31-O-데스메틸-타크롤리무스, 9-디옥소-31-O-데스메틸-아스코마이신 및 9-디옥소-31-O-데스메틸-36,37-다이하이드로-타크롤리무스 중 선택된 1종 이상을 생산하는 방법9-dioxo-31-O-desmethyl-tacrolimus, 9-dioxo-31-O-desmethyl- asco , by culturing a mutant in which the fkbD gene was deleted or inactivated in the tacrolimus biosynthetic gene cluster Method for producing at least one selected from mycin and 9-dioxo-31-O-desmethyl-36,37-dihydro-tacrolimus
  11. 타크롤리무스 생합성 유전자 클러스터 중 fkbM 유전자를 결손시키거나 불활성화시킨 돌연변이체를 배양하여 31-O-데스메틸-타크롤리무스, 31-O-데스메틸-아스코마이신 및 31-O-데스메틸-36,37-다이하이드로-타크롤리무스 중 선택된 1종 이상을 생산하는 방법31-O-desmethyl-tacrolimus, 31-O-desmethyl-ascomycin, and 31-O-desmethyl-36 were cultured by culturing mutants that deleted or inactivated the fkbM gene in the tacrolimus biosynthetic gene cluster. Method for producing at least one selected from, 37-dihydro-tacrolimus
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