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WO2000024775A1 - Groupe de genes intervenant dans la biosynthese de nogalmycine et son utilisation dans la production d'antibiotiques hybrides - Google Patents

Groupe de genes intervenant dans la biosynthese de nogalmycine et son utilisation dans la production d'antibiotiques hybrides Download PDF

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WO2000024775A1
WO2000024775A1 PCT/FI1999/000870 FI9900870W WO0024775A1 WO 2000024775 A1 WO2000024775 A1 WO 2000024775A1 FI 9900870 W FI9900870 W FI 9900870W WO 0024775 A1 WO0024775 A1 WO 0024775A1
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streptomyces
plasmid
genes
host
nogalamycin
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PCT/FI1999/000870
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WO2000024775A9 (fr
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Kristiina Ylihonko
Sirke Torkkell
Kaisa Palmu
Juha Hakala
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Galilaeus Oy
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Priority to EP99952669A priority Critical patent/EP1123310A1/fr
Priority to JP2000578345A priority patent/JP2002528068A/ja
Publication of WO2000024775A1 publication Critical patent/WO2000024775A1/fr
Publication of WO2000024775A9 publication Critical patent/WO2000024775A9/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes

Definitions

  • This invention relates to the gene cluster for nogalamycin biosynthesis derived from Streptomyces nogalater, and the use of the genes therein to obtain novel hybrid antibiotics for drug screening.
  • Anthracyclines are antitumor antibiotics, mainly produced by Streptomyces sp. Daunomycin family of anthracyclines is commercially most important, since almost all of the around ten anthracyclines currently in clinical use, or in late clinical trials for cytotoxic drugs, belong to this family. Despite the long history of anthracyclines, three decades or so, the studies on their biosynthesis are still going on, and there is further interest to obtain novel molecules for the development of cancer chemotherapeutics. A method currently used for finding novel molecules for drug screening is genetic engineering. Cloning the genes for anthracycline biosynthesis facilitates the production of hybrid anthracyclines, as well as their use in combinatorial biosynthesis to generate novel molecules.
  • Nogalamycin which was first described by Bhuyan and Dietz in 1965, is an anthracycline antibiotic produced by Streptomyces nogalater. It is highly active against tumor cells, whereas toxic properties of this compound have prevented its progress to clinical trials (Bhuyan and Smith, 1975).
  • menogaril (7-O-methylnogarol) is a semisynthetic derivative of nogalamycin, and its value in the treatment of cancer has been studied (e.g. Yoshida et al., 1996), the interest being now mainly in Japan.
  • Structurally nogalamycin (Fig. 1) differs from most other anthracyclines, as e.g.
  • the aglycone moiety is built from ten acetates; the neutral sugar, nogalose, is derived from glucose; and methyl groups of both of the sugars, nogalamine and nogalose, are transferred from methionine.
  • the origin of nogalamine was not clearly solved by Wiley, but most probably nogalamine is also derived from glucose.
  • the present invention concerns a gene cluster of Streptomyces nogalater, most of the genes of the cluster being derived from the deoxyhexose pathway for nogalamine and nogalose. Expressing a DNA fragment of the said region in S. galilaeus, which produces aclacinomycins, hybrid anthracyclines are obtained, wherein the aglycone moiety is derived from S. galilaeus, whereas the sugar moiety is characteristic neither to S. nogalater nor to S. galilaeus.
  • nogalamycinone is obtained, which is the aglycone of nogalamycin. Since the stereo- chemistry of nogalamycin differs from most other anthracyclines, using this gene enables the preparation of C-9 stereoisomers of the anthracycline molecules.
  • the present invention concerns particularly the gene cluster for nogalamycin biosynthesis (Sno5-cluster) causing the production of hybrid antibiotics with modifications in the sugar moiety.
  • the invention concerns in specific the use of the genes for nogalamine/nogalose biosynthesis to generate hybrid antibiotics modified in sugar moieties.
  • the invention also concerns the use of a specific cyclase gene included in the gene cluster of the invention, to generate the C-9 stereoisomers of typical anthracyclines.
  • the gene cluster according to the present invention is linked to the earlier reported clusters for nogalamycin biosynthesis.
  • the starting point of the present invention was the gene cluster for nogalamycin chromophor (International Patent Application WO 96/10581). Subsequently, we have found some genes for the deoxyhexose pathway of nogalamycin biosynthesis (Torkkell et al, 1997), and a part of the fragment comprising said genes was used to clone the genes for this invention.
  • the biosynthesis genes for nogalamycin can be isolated from Streptomyces sp., particularly from S. nogalater, which produces nogalamycin. Species which produce nogala- mycin-like anthracyclines can also be used, e.g. S. violaceochromogenes producing arugomycin (Kawai et al, 1987), or S. avidinii producing avidinorubicin (Aoki et al, 1991).
  • Genomic DNA of a Streptomyces strain carrying the genes for nogalamycin biosynthesis is used in preparing a genomic library.
  • Suitable gene fragments for cloning may be obtained by any frequently digesting restriction enzyme. Typically S ⁇ «3AI is used.
  • the isolated fragments could be inserted by Hgation in any Escherichia coli vector such as a plasmid, a phagemid, a phage, or a cosmid.
  • a cosmid vector is preferred since it enables the cloning of large DNA fragments.
  • a cosmid vector such as pFD666 (ATCC No. 77286) is suitable for this purpose, as it enables cloning of the fragments of about 40 kb.
  • the BamHl site of pFD666, giving sticky ends to the Sau3AI fragments may be used for cloning.
  • kits may be used to pack the DNA in phage particles.
  • Various E. coli strains can be used for the infection by the DNA packed.
  • An appropriate E. coli strain is, e.g. XLlBlue MRF', which is deficient in several restriction systems.
  • hybridization is an advantageous screening strategy.
  • the probe for hybridization may be any known fragment derived from the nogalamycin gene cluster, but a short fragment of about 1 kb derived from one end of the biosynthetic region previously cloned is preferred.
  • Colonies for the genomic library are transferred for filter hybridization to membranes, preferably to nylon membranes. Since the average size for a genomic DNA fragment is 40 kb, 2300 colonies gave 99.99% probability to find the expanded region for nogalamycin biosynthesis. Any method for hybridization may be used but, in particular, the DIG System (Boehringer Mannheim, GmbH, Germany) is useful.
  • the probe is homologous to the hybridized DNA, it is preferable to carry out the stringent washes of hybridization at 70°C in a low salt concentration according to Boehringer Mannheim's manual "DIG System User's Guide for Filter Hybridization". At least 80% homology is suggested to be needed for a DNA fragment to bind a probe in the conditions used for washes.
  • the positive clones may be digested with convenient restriction enzymes to demonstrate the physical linkage map of the DNA fragments.
  • the cosmid used for cloning was a shuttle cosmid replicating in both E. coli and Streptomyces sp.
  • S. lividans TK24 which is a typically used laboratory strain in cloning Streptomyces, resulted in deletions, and was omitted.
  • any plasmid being able to stably replicate in Streptomyces may be used for this purpose.
  • Two Bglll fragments of one of the clones were separately inserted into pIJ486 vectors, and the two plasmids obtained were transferred into a primary host, S. lividans TK24.
  • the recombinant plasmids obtained (pSY42 and pSY43), containing a 10 kb and a 7kb fragment from S. nogalater genomic DNA, respectively, were isolated from the primary host and further introduced into other Streptomyces strains by protoplast transformation.
  • the recombinant plasmid containing the 10 kb fragment caused the production of hybrid anthracyclines in the S.
  • galilaeus mutant strain H039 which endogenously produces aklavinone-rhodinose-rhodinose-rhodinose.
  • a few other S. galilaeus strains H075, H026, H063) mutated in deoxyhexose pathway for sugars in aclacinomycin were used in transformation, and new hybrid compounds were obtained. Since the structure of nogalamycin is almost unique among anthracyclines, the plasmids could be transferred to other anthracycline-producing strains, such as S. peucetius, which produces daunomycin, and S. purpurascens, which produces rhodomycins, to modify the structures of the characteristic antibiotics.
  • flanked Bglll fragments consisting of about 16000 bp revealed 15 complete ORFs.
  • the sequence analysis can be made by any computer based program, such as GCG (Madison, Wisconsin, USA) package.
  • GCG Garnier, Wisconsin, USA
  • the putative gene functions as deduced from the sequence homology of those available in the libraries are aminotransferase (snog ⁇ ), not completed
  • dTDP-glucose synthase (snogi) 2. aminomethyl transferase (snogA)
  • glycocyl transferase GTF (snogE)
  • g means that the gene involved in biosynthesis of the glycosidic proportion including glycosyl transferases, whereas a points out that the gene is needed for the formation of the aglycone moiety.
  • snoah responsible for the cyclization of the fourth ring of the aglycone moiety while determining the stereochemistry of the anthracyclinone
  • genes affecting the formation of nogalamine and nogalose snog], snogK, snogN, snogC, snogA
  • genes responsible for joining the sugar residues to the aglycone moiety snogD and snogE).
  • genes could be separately inserted in a vector using suitable restriction sites, or by amplifying the genes by PCR.
  • the fragments may contain an intrinsic promoter, or a promoter may be separately cloned. It is advantageous to use a vector carrying a promoter to allow expression of the genes in a Streptomyces strain.
  • the plasmid pIJE486 contains a promoter ermE for erythromycin resistance gene, allowing constitutive expression of the genes inserted in a correct orientation. Special attention is drawn to the gene encoding a cyclase for the aliphatic ring, but any gene of said cluster may be expressed in Streptomyces hosts.
  • the said cyclase converts the stereochemistry at C9 of auramycinone in TK24, if inserted into the plasmid possessing the other genes for auramycinone biosynthesis, except the cyclase responsible for the typical stereochemistry of anthracyclines.
  • Streptomyces strains, in particular S. galilaeus, carrying the recombinant plasmids are cultivated in media wherein antibiotics are produced.
  • the hybrid compounds are extracted with organic solvents from the culture broth, and the compounds are separated and purified using chromatographic techniques.
  • S. galilaeus H039 carrying the plasmid pSY42 and designated as H039/pSY42 produces aklavinone-4'-epi-2-deoxyfucose in El medium supplemented with thiostrepton to give selection pressure for the plasmid containing strains.
  • S. lividans TK24 carrying the plasmid pSY15c containing the genes for the nogalamycin chromophor and the genes for a cyclase (snoaU and a ketoreductase (snoaF), was cultivated in El medium supplemented with thiostrepton.
  • the compound 9-epi- auramycinone was produced, and this structure is now called nogalamycinone.
  • Any DNA fragment of the invention subcloned from a 17 kb nogalamycin biosynthesis region can be inserted in a vector replicating in Streptomyces, and the products may be produced by fermentation of the plasmid containing strains.
  • Fig. 1 shows the structures of nogalamycin, daunomycin and aclacinomycin.
  • Fig. 2 is a diagram of the gene cluster (Sno5) of the invention for nogalamycin biosynthesis.
  • Fig. 3 describes the proposed biosynthesis pathway for nogalamycin.
  • Fig. 4 shows a diagram of the plasmid pSY15c.
  • the genes snoah (aL) and snoaF (aF) shown black are inserted in the plasmid pSY15 to give pSY15c.
  • aL represents a cyclase snoah and aF is for C-7 ketoreductase snoaF.
  • WO 96/10581 generates the production of a tricyclic intermediate for nogalamycin biosynthesis in S. lividans.
  • the abbreviations al, a2 and a3 refer to the genes snoal, snoa2 and snoa3, respectively, for minimal PKS.
  • rA is the snoxA gene for an activator
  • aB is the snoaB gene for oxygenase
  • aC is the snoaC gene for methylase
  • aD is the snoaD gene for polyketide ketoreductase
  • aE is the snoaE for aromatase.
  • gF the snogF gene
  • gG the snogG gene
  • tsr is a thiostreptone resistance gene
  • Restriction enzymes used were purchased from Promega (Madison, Wisconsin, USA) or Boehringer Mannheim (Germany), and alkaline phosphatase from Boehringer Mannheim, and used according to the manufacturers' instructions.
  • Proteinase K was purchased from Promega and lysozyme from Sigma (St. Louis, USA).
  • HybondTM-N nylon membranes used in hybridization were purchased from Amersham (Buckinghamshire, England), DIG DNA Labelling Kit and DIG Luminescent Detection Kit from Boehringer Mannheim.
  • Qiaquick Gel Extraction Kit from Qiagen (Hilden, Germany) was used for isolating DNA from agarose.
  • the host strains to express the genes cloned were:
  • Streptomyces galilaeus H026 produces aclacinomycin N, ACMN, (aklavinone- rhodosamine-2-deoxyfucose-rhodinose) - Streptomyces galilaeus H063, produces aklavinone
  • H063 has not been described in the literature but it was obtained by NTG mutagenesis of S. galilaeus, and selected to be used as the host strain in the hybrid compound production, as it accumulates aklavinone without any sugar residues.
  • E. coli - Streptomyces shuttle cosmid pFD666 (ATCC 77286) was used for cloning the chromosomal DNA.
  • E. coli cloning vectors pSL1190 (Pharmacia) and pUC19 were used for preparing the subclones.
  • pIJ486 is a high copy plasmid vector provided by prof. Sir David Hopwood, John Innes Centre, UK (Ward et al, 1986)
  • pIJ ⁇ 486 is a vector containing ermE gene in the polylinker of pIJ486 (Bibb et al., 1985).
  • pSY15 is a pIJ486 based plasmid construct, wherein the genes of polyketide pathway for nogalamycin biosynthesis were cloned (Ylihonko et al, 1996a).
  • TSB medium For cultivation of S. nogalater for total DNA isolation TSB medium was used. Lysozyme solution (0.3 M sucrose, 25 mM Tris, pH 8 and 25 mM EDTA pH 8) was used in isolation of total DNA. TE buffer (10 mM Tris, pH 8.0 and ImM EDTA) was used to dissolve the DNA.
  • NMR data was collected with a JEOL JNM-GX 400 spectrometer at the ambient temperature. ! H and 13 C NMR samples were internally referenced to TMS.
  • the anthracycline metabolites were detected by HPLC (LaChrom, Merck Hitachi, pump L-7100, detector L-7400 and integrator D-7500) using a LiChroCART RP-18 column (4.6x250mm).
  • Acetonitrile ⁇ otassium hydrogen phosphate buffer 60 mM, pH 3.0 adjusted with citric acid was used as the mobile phase.
  • Gradient system starting from 65% to 30% of potassium dihydrogen phosphate buffer was used to separate the compounds. The flow rate was 1 ml/min and the detection was effected at 430 nm.
  • ISP4 plates supplemented with thiostrepton 50 ⁇ g/ml were used to maintain the plasmid carrying cultures.
  • Streptomyces nogalater ATCC 27451 was grown for three days in 50 ml of TSB medium supplemented with 0.5% of glycine. The cells were harvested by centrifuging for 15 min at 3900 x g in 12 ml Falcon tubes, and the cells were stored at -20°C. Cells from a 12 ml sample of the culture were used to isolate the DNA. 5 ml of lysozyme solution containing 5 mg of lysozyme/ml was added onto the cells, incubated for 20 min at 37°C.
  • the chromosomal DNA was partially digested with S ⁇ w3AI.
  • the DNA fragments were separated by agarose gel electrophoresis, and the fragments of 30 to 50 kb were cut from the 0.3% low gelling temperature SeaPlaque® agarose.
  • the DNA bands were isolated from the gel by heating to 65°C, extracting with equal volume of equilibrated phenol, and the phases were separated by centrifuging for 15 min at 2500 x g.
  • the phenol phase was extracted with TE buffer, centrifuged and the water phases were pooled.
  • the DNA was precipitated by adding 0.1 volumes of NaAc, pH 5.8 and 2 volumes of ethanol at -20°C for 30 min, centrifuged for 30 min at 15 000 rpm in Sorvall RC5C centrifuge using SS-34 rotor with adapters for 10 ml tubes. The pellet was air dried and dissolved in 20 ⁇ l of TE buffer. The isolated fragments were ligated to pFD666 cosmid vector digested with BamHl and dephosphorylated. The DNA was packed into phage particles, and infected to E. coli using Gigapack® III XL Packing Extract Kit according to the manufacturer's instructions.
  • the infected cells were grown on LB plates containing 50 ⁇ gl kanamycin and transferred to HybondTM-N nylon membranes (Amersham). The membranes were handled according to the protocol described in Boehringer Mannheim's manual "The DIG System User's Guide for Filter Hybridization".
  • the probe used to screen the colonies for an expanded nogalamycin gene cluster was a 1.07 kb Sacl fragment from the cluster described earlier (Torkkell et al, 1997).
  • the plasmid carrying the probe was digested with S ⁇ cl, and the fragment was separated from the vector by agarose gel electrophoresis and isolated from the gel using Qiaquick Gel Extraction Kit (Qiagen).
  • the probe was labelled by digoxygenin using random prime labelling system according to Boehringer Mannheim's manual "The DIG System User's Guide for Filter Hybrid- ization”. 5000 colonies were screened by hybridization at 70°C using the probe described. Positive colonies were detected using DIG Luminescent Detection Kit (Boehringer Mannheim). Seven colonies gave a positive signal. Cosmids from the positive clones were isolated from a 5ml culture by alkaline lysis method. Restriction analysis showed that the cloned fragments overlapped each other representing at least 60 kb of the continuous DNA. The positive clones obtained were designated as pFDSwol to pFDSn ⁇ 7.
  • pFDSno5 Clone No. 5, designated as pFDSno5
  • pFDSno5 was digested with Bglll, and for subcloning two fragments of about 10 kb and 7 kb were isolated and ligated to pSL1190 digested with Bglll and dephosphorylated.
  • the plasmids obtained were named as pSn42 and pSn43, respectively. These two fragments cover the DNA region flanked to the previously characterized area of nogalamycin biosynthesis cluster.
  • E. coli XL1 Blue MRF' cells were cultivated overnight at 37 °C in 5 ml of LB- medium supplemented with 50 ⁇ g/ml of ampicillin.
  • Wizard Plus Minipreps DNA Purification System kit of Promega, or Biometra silica spin plasmid miniprep kit of Biotechnikische Analytik Gmbh were used according to the manufacturers' instructions.
  • sequenced DNA fragment contained 15 complete open reading frames (ORFs), and the 5' end of other two ORFs in the both ends of the fragment according to the invention.
  • ORFs complete open reading frames
  • the functions of the genes were concluded by comparing the amino acid sequences translated from their base sequences to the known protein sequences in the data banks. The results are shown in Table 1. The positions given refer to the appended sequence listing. The amino acid sequences of the peptides are given in SEQ ID NO:2 to SEQ ID NO:18.
  • the 10 kb Bglll fragment from pFDS «o5 was cloned into the plasmid pIJ486 and the plasmid obtained was named as pSY42.
  • the 7 kb Bglll fragment from pFDSno5 was cloned into the plasmid pIJE486, and the plasmid pSY43 was obtained.
  • Plasmid pSY42 was introduced into S. lividans strain TK24 by protoplast transformation, isolated from it and further introduced into S. galilaeus mutant H039, and after propagation in H039, transferred to other S.
  • pSY42 contained a cyclase designated as NAMEC (nogalonic acid methyl ester cyclase), and in pSY43 a ketoreductase gene was identified.
  • Expression constructions were prepared which contained all the genes needed for the formation of nogalamycin aglycone.
  • a 1.4 kb BamHl-Sacl fragment from pSY42 (containing NAMEC) and a 1.1 kb Mlul-Kpnl fragment from pSY43 carrying the gene for a ketoreductase of C-7 keto group were Hgated to pSY15 linearized by Sacl, to form the plasmid pSY15c (Fig. 4).
  • Plasmid pSY15c was introduced into S. lividans TK24, and the strain TK24/pSY15c was cultivated in El medium supplemented with thiostrepton. An aglycone compound was produced, and this structure is now called nogalamycinone.
  • the seed culture 180 ml of El culture of the plasmid containing strain, H039/pSY42 or TK24/pSY15c, was obtained by cultivating the strain in three 250 ml Erlenmeyer flasks containing 60 ml of El medium supplemented with thiostrepton (5 ⁇ g/ml) for four days at 30°C, 330 rpm.
  • the combined culture broths 180 ml
  • the cells were harvested by centrifuging. 2.6 1 of methanol was used to break the bacterial cells and to extract anthracycline metabolites accumulated.
  • the anthracycline metabolites were extracted using 2 1 of dichloromethane at pH 6.
  • the organic layer was evaporated to dryness.
  • the viscous residue was flashed through a polyamide (11) column using wate ⁇ methanol from 1:9 to 0:10 as the eluent.
  • NMR analysis included NON, BMC, NOE, DEPT and HMBC techniques. Protons were assignated using NOESY and 2D pTOCSY techniques and carbons using DEPT and HMBC techniques.
  • microorganisms were deposited according to the Budapest Treaty at Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ), Mascheroder Weg lb, D-38124 Braunschweig, Germany.
  • Hybrid anthracycline antibiotics production of new anthracyclines by cloned genes from Streptomyces purpurascens in Streptomyces galilaeus. Microbiol. 140: 1351-1358.
  • Tork ell S., Ylihonko, K., Hakala, J., Skurnik, M., and Mantsala, P. 1997. Characterization of Streptomyces nogalater genes encoding enzymes involved in glycosylation steps in nogalamycin biosynthesis. Mol. Gen. Genet. 256: 203-209.

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Abstract

L'invention concerne le groupe de gène intervenant dans la biosynthèse de nogalamycine dérivée de Streptomyces nogalater, et l'utilisation des gènes s'y trouvant dans le but d'obtenir de nouveaux antibiotiques hybrides en vue du criblage des médicaments.
PCT/FI1999/000870 1998-10-23 1999-10-20 Groupe de genes intervenant dans la biosynthese de nogalmycine et son utilisation dans la production d'antibiotiques hybrides WO2000024775A1 (fr)

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Application Number Priority Date Filing Date Title
EP99952669A EP1123310A1 (fr) 1998-10-23 1999-10-20 Groupe de genes intervenant dans la biosynthese de nogalmycine et son utilisation dans la production d'antibiotiques hybrides
JP2000578345A JP2002528068A (ja) 1998-10-23 1999-10-20 ノガラマイシン生合成に関与する遺伝子クラスター、およびハイブリッド抗生物質の製造におけるその使用

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FI982295 1998-10-23
FI982295A FI107053B (fi) 1998-10-23 1998-10-23 Nogalamysiinin biosynteesiin liittyvä geeniryhmittymä ja sen käyttö hybridiantibioottien tuotossa

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002074800A1 (fr) * 2001-03-19 2002-09-26 Galilaeus Oy Batterie de genes pour la biosynthese de la rabelomycine, et leur utilisation pour obtenir des composes en vue du criblage de medicaments
EP2586791A1 (fr) * 2011-10-28 2013-05-01 Sanofi Groupes de gène pour la biosynthèse de griselimycine et de méthylgriselimycine
WO2013053857A3 (fr) * 2011-10-12 2013-08-01 Sanofi Grappe de gènes pour la biosynthèse de la grisélimycine et de la méthylgrisélimycine
CN110713992A (zh) * 2019-11-07 2020-01-21 凯莱英医药化学（阜新）技术有限公司 酮还原酶突变体及生产手性醇的方法

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1990405B1 (fr) 2007-05-08 2017-07-26 Provivo Oy Souches modifiées génétiquement produisant des métabolites anthracyclines utiles en tant que médicaments contre le cancer

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WO1996010581A1 (fr) * 1994-09-30 1996-04-11 Galilaeus Oy Procedes de production des anthracyclines et de leurs intermediaires

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Publication number Priority date Publication date Assignee Title
WO1996010581A1 (fr) * 1994-09-30 1996-04-11 Galilaeus Oy Procedes de production des anthracyclines et de leurs intermediaires

Non-Patent Citations (1)

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Title
TORKKELL S. ET AL: "Characterization of Streptomyces Nogalater Genes Encoding Enzymes Involved in Glycosylation Steps in Nogalamycin Biosynthesis", MOL. GEN GENET., vol. 256, 1997, pages 203 - 209, XP002922379 *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002074800A1 (fr) * 2001-03-19 2002-09-26 Galilaeus Oy Batterie de genes pour la biosynthese de la rabelomycine, et leur utilisation pour obtenir des composes en vue du criblage de medicaments
WO2013053857A3 (fr) * 2011-10-12 2013-08-01 Sanofi Grappe de gènes pour la biosynthèse de la grisélimycine et de la méthylgrisélimycine
US9422566B2 (en) 2011-10-12 2016-08-23 Sanofi Gene cluster for biosynthesis of griselimycin and methylgriselimycin
EA029209B1 (ru) * 2011-10-12 2018-02-28 Санофи Кластер генов биосинтеза гризелимицина и метилгризелимицина
EP2586791A1 (fr) * 2011-10-28 2013-05-01 Sanofi Groupes de gène pour la biosynthèse de griselimycine et de méthylgriselimycine
CN110713992A (zh) * 2019-11-07 2020-01-21 凯莱英医药化学（阜新）技术有限公司 酮还原酶突变体及生产手性醇的方法
CN110713992B (zh) * 2019-11-07 2021-08-03 凯莱英医药化学（阜新）技术有限公司 酮还原酶突变体及生产手性醇的方法

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FI982295L (fi) 2000-04-24
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JP2002528068A (ja) 2002-09-03
FI107053B (fi) 2001-05-31
WO2000024775A9 (fr) 2000-08-24

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