WO2011068341A2 - Gène et protéine pour la biosynthèse de composés tricycliques - Google Patents

Gène et protéine pour la biosynthèse de composés tricycliques 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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김재종
임시규
김동환
이금순
유정현
이미옥
임상면
이보미
김상준
차선호
윤여준
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(주)제노텍
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    • C12N15/09—Recombinant DNA-technology
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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
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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
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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
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    • 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

La présente invention concerne un gène constituant des locus biosynthétiques utilisés dans la biosynthèse de composés tricycliques produits par des micro-organismes, en particulier Actinomycete sp., tacrolimus (FK506) et dihydrotacrolimus, qui est un analogue structural de celui-ci, ascomycine (FK520), prolyl-tacrolimus (FK525), FK523 et similaire, et une protéine qui est codée par le gène. Le gène et la protéine peuvent être utilisés pour produire et modifier chimiquement le tacrolimus, l'ascomycine et des substances ayant la structure chimique associée à ceux-ci et pour réduire les impuretés.
PCT/KR2010/008496 2009-12-03 2010-11-29 Gène et protéine pour la biosynthèse de composés tricycliques WO2011068341A2 (fr)

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CN113939519A (zh) * 2018-12-11 2022-01-14 莫尔根生物有限公司 促进毛发生长的新化合物及包含它们的组合物
EP4085933A3 (fr) * 2016-04-12 2023-01-11 Ginkgo Bioworks, Inc. Compositions et procédés pour la production de composés

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EP4085933A3 (fr) * 2016-04-12 2023-01-11 Ginkgo Bioworks, Inc. Compositions et procédés pour la production de composés
KR102109168B1 (ko) * 2018-12-11 2020-05-12 인트론바이오테크놀로지 신규 화합물 및 이를 포함하는 진균감염 치료용 약학적 조성물
WO2020122607A1 (fr) * 2018-12-11 2020-06-18 주식회사 인트론바이오테크놀로지 Nouveau composé et composition pharmaceutique pour le traitement d'infections fongiques le comprenant
CN113939519A (zh) * 2018-12-11 2022-01-14 莫尔根生物有限公司 促进毛发生长的新化合物及包含它们的组合物
US12011497B2 (en) 2018-12-11 2024-06-18 Molgenbio Co. Ltd. Compounds, compositions containing same, and use thereof for promoting hair growth

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