EP1137796A2 - Gene aus -i(micromonospora echinospora), welche für enzyme der calicheamicin-biosynthese kodieren und eigenresistenz dagegen - Google Patents

Gene aus -i(micromonospora echinospora), welche für enzyme der calicheamicin-biosynthese kodieren und eigenresistenz dagegen

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Publication number
EP1137796A2
EP1137796A2 EP99972435A EP99972435A EP1137796A2 EP 1137796 A2 EP1137796 A2 EP 1137796A2 EP 99972435 A EP99972435 A EP 99972435A EP 99972435 A EP99972435 A EP 99972435A EP 1137796 A2 EP1137796 A2 EP 1137796A2
Authority
EP
European Patent Office
Prior art keywords
nucleic acid
calicheamicin
acid molecule
gene
isolated nucleic
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP99972435A
Other languages
English (en)
French (fr)
Other versions
EP1137796A4 (de
Inventor
Jon Thorson
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Memorial Sloan Kettering Cancer Center
Original Assignee
Sloan Kettering Institute for Cancer Research
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Sloan Kettering Institute for Cancer Research filed Critical Sloan Kettering Institute for Cancer Research
Publication of EP1137796A2 publication Critical patent/EP1137796A2/de
Publication of EP1137796A4 publication Critical patent/EP1137796A4/de
Withdrawn legal-status Critical Current

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Classifications

    • 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
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/44—Preparation of O-glycosides, e.g. glucosides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/44—Preparation of O-glycosides, e.g. glucosides
    • C12P19/60—Preparation of O-glycosides, e.g. glucosides having an oxygen of the saccharide radical directly bound to a non-saccharide heterocyclic ring or a condensed ring system containing a non-saccharide heterocyclic ring, e.g. coumermycin, novobiocin
    • C12P19/62—Preparation of O-glycosides, e.g. glucosides having an oxygen of the saccharide radical directly bound to a non-saccharide heterocyclic ring or a condensed ring system containing a non-saccharide heterocyclic ring, e.g. coumermycin, novobiocin the hetero ring having eight or more ring members and only oxygen as ring hetero atoms, e.g. erythromycin, spiramycin, nystatin

Definitions

  • the present invention relates to a biosynthetic gene cluster of Micromonospora
  • calicheamicin s aryltetrasaccharide and aglycone, and the gene conferring
  • the present invention also relates to isolated genes of the
  • the invention relates to
  • the invention also relates to expression vectors containing the biosynthetic gene
  • Enediyne antibiotics were originally derived by
  • microorganisms including Micromonospora, Actinomedura, and
  • chromophore core structure which also requires a specific associated protein for
  • the second category of enediyne is classified as non-
  • chromoprotein enediynes contain a 10-membered ring, which requires
  • This enediyne ring structure is often referred to as the
  • warhead induces DNA damage, which is frequently a double-stranded
  • the 9-membered ring chromoprotein enediyne subfamily is comprised of:
  • pluricolorescens (Yamaguchi. T.. et al., J Antibiot.. XXIII 369-372 (1970));
  • the non-chromophore enediyne subfamily is comprised of calicheamicin from
  • lithostrotum lithostrotum
  • esperamicin from Actinomadura verrucosospora
  • dynemicin from Actinomadura verrucosospora
  • Micromonospora chersina Micromonospora chersina.
  • Enediyne antibiotics have potential as anticancer agents because of their ability to
  • Calicheamicin has two distinct structural regions: the aryltetrasaccharide and the
  • aglycone also known as the warhead.
  • the aryltetrasaccharide displays a highly unusual
  • calicheamicin consists of a highly functionalized
  • CMA-676 calicheamicin-antibody conjugates
  • calicheamicin analogs random mutagenesis of M. echinospora and screening for mutant
  • Nacelle's procedure only provides approximately a 0.007% yield and requires
  • calicheamicin DNA opens the door for genetic analysis of calicheamicin
  • DNA For example, one can study calicheamicin biosynthesis by mutagenesis of M.
  • calicheamicin biosynthesis and the subsequent analysis of their defective or partial calicheamicin products. Additionally, particular enzyme could be overexpressed or
  • biosynthetic genes can ultimately result in increased yields of the gene product by cloning
  • calicheamicin i.e. aromatization of the bicyclo[7.3.1]tridecadiynene core
  • this invention relates to the first identification, isolation, and cloning of a
  • calicheamicin self-resistance gene and protein have been isolated as have the genes and resulting enzymes for steps within the calicheamicin
  • the invention also provides for construction of enediyne overproducing strains
  • the present invention thus, also relates to a biosynthetic modification of bioactive
  • ligands which serve as molecular recognition elements critical for biological activity.
  • the present invention utilizes the fact that glycosyltransferases,
  • invention discloses a method using the recruitment and collaborative action of sugar
  • the present invention provides an isolated nucleic acid molecule from
  • biosynthetic gene cluster the protein coding region of the gene or a biologically active
  • the present invention provides an isolated nucleic acid
  • invention also relates to nucleic acids capable of hybridizing with a nucleic acid molecule
  • nonchromoprotein enediyne biosynthetic gene cluster In a further embodiment the
  • invention provides an expression vector comprising an isolated nucleic acid molecule
  • the invention provides a cosmid comprising an
  • nucleic acid molecule from Micromonospora echinospora comprising a nucleic
  • the invention provides the isolated nucleic acid sequence encoding for a nonchromoprotein enediyne biosynthetic gene cluster.
  • the invention provides the isolated nucleic acid
  • the present invention provides a host cell
  • Host cells can optionally
  • the host cell is the bacterium
  • the invention is directed to a
  • the invention provides a transformed host cell with an
  • the invention further provides a method of expressing a protein by culturing a
  • nonchromoprotein enediyne biosynthetic gene cluster incubating the host cell for a
  • invention provides a method of purifying calicheamicin using affinity chromatography.
  • sample containing calicheamicin is contacted with an affinity matrix having the protein CalC bound thereto, for a time and under conditions allowing calicheamicin to bind to the
  • the invention further provides a method of conferring calicheamicin resistance to
  • a subject comprising obtaining cells from the subject, transforming the cells with the
  • calicheamicin self-resistance gene and returning the cells to the subject.
  • the calicheamicin self-resistance gene can be targeted and delivered to the desired host
  • Figure 1 depicts the summary of the cosmid clones isolated from M. echinospora
  • genomic library This figure illustrates the results of the screening of the genomic library
  • Figure 2 shows a restriction map of a portion of cosmid clones 4b, 13a, and 56 and
  • Figure 3 is a table of the open reading frames ("orfs " ) in the calicheamicin
  • biosynthetic cluster This table lists the polypeptides that the genes encode for as well as
  • Figure 4 is a graph of the UV-visible absorption spectra of purified mbp-CalC.
  • the purified mpb-CalC was analyzed in the following solution: 52 ⁇ M mpb-CalC; 10
  • Figure 4(b) provides the results of the mbp-CalC in vitro assay.
  • FIG. 5 depicts the postulated routes for the biosynthesis of required nucleotide
  • E ep epimerase
  • E met methyltransferase
  • E od 4,6-dehydratase
  • E ox
  • E p nucleotidyltransferase
  • E red reductase
  • E sh sulfhydrytransferase
  • Figure 6 illustrates a schematic representation of the in vivo production of
  • Figure 7 depicts the Streptomyces Venezuela methymycin/pikromycin gene cluster.
  • Figure 8 illustrates calicheamicin's (6) four unique sugars which are crucial to
  • Sugar (9) is derived from 4-amino-4,6-dideoxyglucose (8) and is part
  • Compound 8 is derived from
  • calicheamicin biosynthetic cluster This cluster encodes the genes that encode the
  • the calicheamicin biosynthetic gene cluster comprises the following genes: calA.
  • calB calC
  • calD calE
  • calF calG
  • calH call, call, calK, calL, ca M, calN, calO, cal?
  • Orf3 (209 amino acids).
  • Orf4 (521 amino acids), Orf5 (175 amino acids),
  • Orf6 (139 amino acids), Orf7 (187 amino acids), and IS-element (402 amino acids).
  • the cosmid library was generated by isolating chromosomal DNA of
  • calicheamicin aglycone would be polyketide derived.
  • Polyketide metabolites encompass
  • PKS polyketide synthase
  • sequence homology (from pathway to pathway and organism to organism) and are often
  • the second screening was based on the assumption that calicheamicin's
  • biosynthetic cluster would also contain genes encoding for deoxysugar ligand synthesis.
  • dehydratase gene encoding the putative enzymes E p and E od . respectively. See figure 5.
  • nucleotide transferase from Salmonella has been characterized as an
  • calicheamicin synthesis would begin from a similar precursor found in E. coli,
  • DNA probe (designated ⁇ od ') was designed from the conserved NAD -binding site of
  • E od ' probe revealed cross-hybridization with clones 4b, 10a, 13a. 56. and 60.
  • hybridization established similarity between clones 3a, 4a. 4b, 10a, 13a. 16a and 56.
  • the positive cosmid clones corresponded to a continuous region of the M. echinospora
  • the present invention thus provides for cosmids having
  • nucleic acid molecule from Micromonospora echinospora encoding for a
  • genes participating in the construction of the aryltetrasaccharide include: a)
  • genes encoding nucleotide sugar biosynthesis (calG H, K, O, Q, and S); b) genes encoding for aryltetrasaccharide assembly (calE and N); and c) genes encoding for
  • One aspect of the invention relates to transformation of a host cell with M.
  • invention further provides that the host cell can be but is not limited to bacteria, yeast,
  • plant or mammalian cells are performed by methods known in the art.
  • One aspect of the invention relates to an isolated
  • present invention also relates to an isolated protein CalC, having the amino acid sequence,
  • the invention further provides for calC gene fragments coding for a
  • CalC bioactive CalC.
  • the polypeptide, CalC confers calicheamicin resistance and has 181
  • the invention also provides for CalC fragments conferring calicheamicin
  • the calC locus was isolated by identifying calicheamicin genomic cosmid clones
  • LB luria bertani
  • iron metalloprotein that functions via inhibition of calicheamicin-induced DNA cleavage
  • Another aspect of the invention is an expression vector containing calC or a
  • host cell preferably bacteria, more preferably, E. coli containing calC or a fragment of
  • the present invention provides for the transformation of human cells with the
  • calC gene This allows bone marrow cells, for example, to be removed from a patient
  • calicheamicin or allows the patient to receive higher doses of calicheamicin as the
  • Another aspect of the invention relates to an isolated DNA strand containing the
  • caM. gene having the DNA sequence S ⁇ Q ID. No: 3.
  • the invention also relates to the
  • polypeptide CalH having amino acid sequence S ⁇ Q ID. No. 4.
  • the invention furthermore, having amino acid sequence S ⁇ Q ID. No. 4.
  • CalH is involved in the
  • CalH were overexpressed as a (histidine), 0 -fusion protein and subsequently
  • TDP-perosamine TDP-4.6-dideoxy-4-amino-D-mannose
  • one aspect of the present invention further relates to the construction of a
  • invention further provides an expression vector having a calicheamicin gene operably
  • the regulatory sequence is a Streptomyces promoter.
  • the present invention also provides a Streptomyces promoter.
  • Compound 11 has the formula:
  • Compound 12 has the formula:
  • One aspect of the invention relates to an isolated DNA strand containing the calG
  • Another aspect of the invention is a gene and having the DNA sequence SEQ ID. NO.: 5. Another aspect of the invention is a gene and having the DNA sequence SEQ ID. NO.: 5. Another aspect of the invention is a gene and having the DNA sequence SEQ ID. NO.: 5. Another aspect of the invention is a gene and having the DNA sequence SEQ ID. NO.: 5. Another aspect of the invention is a gene and having the DNA sequence SEQ ID. NO.: 5. Another aspect of the invention is
  • CalG appears to be a TDP-D-glucose 4,6-dehydratase which catalyzes the conversion of
  • a transformed host cell preferably bacteria, more
  • E. coli. containing calG or a fragment of calG encoding for a bioactive
  • CalS appears to be a P450-oxidase
  • the oxidation may occur at the nucleotide sugar level or hydroxylamine formation after
  • a transformed host cell preferably bacteria, more preferably, E. coli,
  • the present invention allows genetic manipulation of the biosynthetic gene cluster
  • the present invention provides for producing
  • calicheamicin analogs by constructing deletions or substitutions of the genes involved in
  • the invention further provides for in vitro
  • glycosylation by altering the glycosylation pattern of calicheamicin (via a
  • glycosyltransferase to produce additional analogs.
  • the invention also provides for
  • the invention provides for a method of purifying calicheamicin through affinity
  • the invention relates to the expression of the genes located in the biosynthetic gene
  • the present invention also provides a gene to produce the protein encoded by the inserted gene.
  • biosynthetic gene cluster which encode for biologically active proteins
  • thermocycle sequencing was performed by:
  • sequence data was acquired using two Applied Biosystems automated 310 genetic
  • Brujene. MacVector is a commercially available software package which provides the
  • calicheamicin (0.25 ⁇ g ml "1 ).
  • six clones (3a, 4a, 4b, 10a. 13a and 16a)
  • the proximal 1 kb of this fragment carried a single orf (calD).
  • CalD its respective protein
  • IPTG Isopropyl Beta-D-thiogalactoside
  • the protein mbp-CalC was overexpressed and purified for further analysis.
  • mbp-CalC was purified from pRE7/E. coli to homogeneity as judged by SDS-PAG ⁇ .
  • buffer A 50mM Tris-Cl, pH 7.5, 200 mM NaCl, ImM ⁇ DTA
  • ICP-MS inductively coupled plasma atomic mass spectrometry
  • nucleotide sequence of the mbp-calC gene fusion which is consistent with the determined
  • hydrolysate was subsequently determined by ICP-MS on four distinct mbp-CalC
  • calicheamicin-induced DNA cleavage assay would inhibit DNA cleavage.
  • pBS pBS
  • DTT dithiothreitol
  • DNA fragmentation was assessed by electrophoresis on a 1% agarose gel
  • FeSO 4 Fe ⁇ 2
  • FeCl 3 Fe *3
  • the 1.2 kb calH gene was amplified by polymerase chain reaction (PCR) from
  • pJSTl 192 pn7 which is a subclone containing a 7.0 kb Kpnl fragment of cosmid 13a.
  • amplified gene was cloned into the EcoR /Xbal site of the expression vector pDHS617.
  • This expression vector contains an apramycin resistance marker.
  • pLZ-C242 (containing the cal ⁇ gene insert and the promoter sequence) was introduced by
  • the culture was centrifuged to remove cellular debris and mycella.
  • the supernatant was adjusted to pH 9.5 with concentrated KOH. followed by chloroform extraction.
  • the crude product was extracted from the crude cells.

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  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Health & Medical Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Biomedical Technology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Molecular Biology (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Plant Pathology (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Saccharide Compounds (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Enzymes And Modification Thereof (AREA)
EP99972435A 1998-12-07 1999-12-07 Gene aus -i(micromonospora echinospora), welche für enzyme der calicheamicin-biosynthese kodieren und eigenresistenz dagegen Withdrawn EP1137796A4 (de)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US11132598P 1998-12-07 1998-12-07
US111325P 1998-12-07
PCT/US1999/029110 WO2000037608A2 (en) 1998-12-07 1999-12-07 Micromonospora echinospora genes encoding for biosynthesis of calicheamicin and self-resistance thereto

Publications (2)

Publication Number Publication Date
EP1137796A2 true EP1137796A2 (de) 2001-10-04
EP1137796A4 EP1137796A4 (de) 2005-05-25

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EP99972435A Withdrawn EP1137796A4 (de) 1998-12-07 1999-12-07 Gene aus -i(micromonospora echinospora), welche für enzyme der calicheamicin-biosynthese kodieren und eigenresistenz dagegen

Country Status (4)

Country Link
EP (1) EP1137796A4 (de)
JP (1) JP2002533067A (de)
CA (1) CA2354030A1 (de)
WO (1) WO2000037608A2 (de)

Families Citing this family (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6733998B1 (en) 1998-12-07 2004-05-11 Sloan-Kettering Institute For Cancer Research Micromonospora echinospora genes coding for biosynthesis of calicheamicin and self-resistance thereto
US7257562B2 (en) 2000-10-13 2007-08-14 Thallion Pharmaceuticals Inc. High throughput method for discovery of gene clusters
JP2005506050A (ja) * 2000-11-28 2005-03-03 スローン−ケッタリング インスティテュート フォー キャンサー リサーチ カリケマイシンの生合成およびそれに対する自己耐性をコードするミクロモノスポラ・エチノスポラ（Ｍｉｃｒｏｍｏｎｏｓｐｏｒａｅｃｈｉｎｏｓｐｏｒａ）遺伝子
EP1390507A2 (de) * 2001-05-21 2004-02-25 Ecopia Biosciences Inc. Gene und proteine mit bezug zur enediyne ring-struktur-biosynthese
SI2029750T1 (sl) * 2006-06-22 2011-11-30 Dsm Ip Assets Bv Proizvajanje pravastatina

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5276159A (en) * 1990-08-01 1994-01-04 The Scripps Research Institute Dynemicin analogs: syntheses, methods of preparation and use
US5712146A (en) * 1993-09-20 1998-01-27 The Leland Stanford Junior University Recombinant combinatorial genetic library for the production of novel polyketides

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
BORDERS D. B.; ROTHSTEIN D. M.: "Enediyne antibiotics as Antitumor Agents" 1995, MARCEL DEKKER , NEW YORK , XP002309022 * pages 107-126; see particularly page 119-122 * *
HOPWOOD D A: "Genetic contributions to understanding polyketid synthases" CHEMICAL REVIEWS, AMERICAN CHEMICAL SOCIETY. EASTON, US, vol. 97, no. 7, November 1997 (1997-11), pages 2465-2497, XP002130647 ISSN: 0009-2665 *
LIN L-S ET AL: "MUTATIONS IN THE P1 PROMOTER REGION OF MICROMONOSPORA-ECHINOSPORA" JOURNAL OF BACTERIOLOGY, vol. 174, no. 10, 1992, pages 3111-3117, XP002309020 ISSN: 0021-9193 *
LOVE S F ET AL: "CONDITIONS FOR PROTOPLASTING REGENERATING AND TRANSFORMING THE CALICHEAMICIN PRODUCER MICROMONOSPORA-ECHINOSPORA" APPLIED AND ENVIRONMENTAL MICROBIOLOGY, vol. 58, no. 4, 1992, pages 1376-1378, XP002309021 ISSN: 0099-2240 *
See also references of WO0037608A2 *
THORSON J S ET AL: "Enedyine biosynthesis and self-resistance: A progress report" BIOORGANIC CHEMISTRY, ACADEMIC PRESS INC., NEW YORK, NY, US, vol. 27, no. 2, 1999, pages 172-188, XP002222216 ISSN: 0045-2068 *

Also Published As

Publication number Publication date
EP1137796A4 (de) 2005-05-25
CA2354030A1 (en) 2000-06-29
WO2000037608A3 (en) 2000-11-23
JP2002533067A (ja) 2002-10-08
WO2000037608A2 (en) 2000-06-29

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