WO2000070056A9 - Chloramphenicol biosynthetic pathway and gene cluster characterization - Google Patents
Chloramphenicol biosynthetic pathway and gene cluster characterizationInfo
- Publication number
- WO2000070056A9 WO2000070056A9 PCT/US2000/013394 US0013394W WO0070056A9 WO 2000070056 A9 WO2000070056 A9 WO 2000070056A9 US 0013394 W US0013394 W US 0013394W WO 0070056 A9 WO0070056 A9 WO 0070056A9
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- chloramphenicol
- gene cluster
- microbe
- nucleotide sequence
- venezuelae
- Prior art date
Links
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
- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/02—Amides, e.g. chloramphenicol or polyamides; Imides or polyimides; Urethanes, i.e. compounds comprising N-C=O structural element or polyurethanes
-
- 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
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1034—Isolating an individual clone by screening libraries
-
- 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
- Chloramphenicol is an N-dichloroacyl phenylpropanoid antibiotic produced by Streptomyces venezuelae.
- Other strains which produce chloramphenicol include Streptomyces pheochromogenes and Streptomyces venezuelae 13S.
- Corynebacterium hydrocarboclastus makes a related metabolite called corynecin.
- Chloramphenicol is a broad spectrum antibiotic, and although it demonstrates some side effects in humans, it is a clinically important drug that is especially effective against typhoid, meningitis, and other microbially related diseases.
- Chloramphenicol is synthesized by S. venezuelae as follows:
- Chloramphenicol biosynthesis genes are located on the chromosome of S. venezuelae ATCC 10712. Mutants blocked in the production of chloramphenicol have been generated in the labs of L. C. Nining and C. Stuttard at Dalhousie University (Doull et al., 1985). The cml mutations present in these mutants have been used to define the organization of cml genes in this organism. Conjugation and transductional analysis has indicated that all cml genes form a tight cluster on the chromosome ( Figure 1, Nats et al. 1987).
- the present invention provides a chloramphenicol synthesis gene cluster.
- the gene cluster is produced by the process comprising: (a) forming a genomic clone library of a chloramphenicol producing microbe; (b) transfecting clones from said library into donor host cells; (c) mating the transfected donor host cells with a chloramphenicol producing microbe, said microbe comprising a mutation in the chloramphenicol gene cluster; (d) screening the resulting recombinant clones for production of chloramphenicol; and (e) isolating said gene cluster from the clones positive for chloramphenicol production.
- the donor host cell is E. coli.
- the chloramphenicol producing microbe is S. venezuelae.
- the mutation in the gene cluster results in the inactivation of a gene encoding a protein involved in the chloramphenicol synthesis pathway, and/or the production of a non-functional protein involved in such pathway.
- the present invention further provides a method of producing a clone containing a chloramphenicol gene cluster, the method comprising: (a) forming a genomic clone library of a chloramphenicol producing microbe; (b) transfecting clones from said library into donor host cells; (c) mating the transfected donor host cells with a chloramphenicol producing microbe, said microbe comprising a mutation in the chloramphenicol gene cluster; (d) screening the resulting recombinant clones for production of chloramphenicol; and (e) isolating clones positive for chloramphenicol production.
- the present invention also provides a method of producing chloramphenicol comprising: (a) culturing the clone produced according to the method described herein; and (b) isolating the resulting chloramphenicol.
- the production is increased relative to the wild-type strain of S. venezuelae.
- Figure 1 is a schematic representation of the relative positions of the cml genes within the cml gene cluster.
- Figure 2 shows results of exconjugants grown on a bioassay plate containing MYM agar and apramycin.
- Figure 3 shows h.p.l.c. analysis comparing chloramphenicol production.
- Figure 4 shows results of exconjugants grown on a bioassay plate containing MYM agar and apramycin, and bioassayed with Micrococcus luteus.
- the strategy used to clone cml genes was to complement cml-5 or cml-12 mutations and to examine for chloramphenicol production.
- NS153 (trpC, cml-5) is a chloramphenicol non-producing strain derived from NS35 by mutagenesis.
- NS35 is derived from the wild type strain (Stuttard, C, FEMS Microbiol. Lett. 20:467-470 (1983)). It is blocked in a step before /. -aminophenylserine and after /.-aminophenylalanine intermediates in the pathway.
- NS503 (cml-12, pdx-4, hsp-11) is a chloramphenicol non-producing strain that is derived from the wild type strain (Sushma Nats, 1987 Ph.D. Thesis, Dalhousie University, Suite, ⁇ .S., Canada). It is blocked in a step before /.-aminophenylalanine formation.
- DS154 (trpC, is derived from NS153 by introduction of the jad- l: hyg mutation.
- the knockout was constructed by inserting hygromycin resistance gene (hyg) (Zalacain et al., 1986) within orfl (jad-1).
- hyg hygromycin resistance gene
- the jad l::hyg mutation was introduced in strain NS153 to stop jadomycin production, which is another antibiotic produced by S. venezuelae (See also, Han et al, 1994).
- Genomic D ⁇ A of S. venezuelae ATCC10712 was isolated using standard methods (Hopwood et al., 1985, Genetic Manipulations of Streptomyces, a laboratory manual, Norwich, UK, John Innes Foundation), and partially digested with S ⁇ w3Al.
- the cut genomic DNA was size-fractionated on a sucrose gradient and fragments more than 24 kb in size were ligated to the cosmid pOJ446 arms (Bierman et al., 1992).
- the cosmid arms were prepared by first cutting pOJ446 with Hpa ⁇ , then treating with shrimp alkaline phosphatase followed by digestion with Bam ⁇ l.
- STR611 is a Diversa strain which is used as a donor strain for mating DNA libraries into S. venezuelae. It is derived from strain GM2163 (dam) by incorporating the mutations mcrC-mrr, and introducing the mobilizing plasmid pUZ8002. The DNA in strain STR611 is non-methylated and is thus introduced into Streptomyces venezuelae at high efficiency. b. Introduction ofS. venezuelae genomic library into DS154
- the STR611 E. coli containing S. venezuelae library was mated into S. venezuelae DS154 (cml-5) strain by a mating protocol as follows: 100 ⁇ l of spores of S. venezuelae were suspended in 0.4 ml of MYM liquid medium (maltose-yeast extract-malt extract) and heat shocked at 50 C for 10 min. The spores were spun down in a centrifuge and then washed once with 0.4 ml of MYM liquid medium. 0.2 ml of the E.
- MYM liquid medium maltose-yeast extract-malt extract
- coli library cells were resuspended in 0.5 ml of LB+kan+apr+cml (Luria broth + kanamycin + apramycin + chloramphenicol) and incubated in a shaker for 15 min at 37°C, after which the cells were mixed with the heat treated S. venezuelae spores. The S. venezuelae-E. coli cell mixture was then centrifuged and the pellet washed with 0.5 ml MYM once and the cell mixture was resuspended in 1.1 ml MYM medium.
- the strain DS154 (clone 10) was grown in MYM liquid medium (plus apramycin) for 48 hours and the mycelia was used to prepare plasmid D ⁇ A using methods described in the Streptomyces manual (Hopwood et al., 1985). The resulting plasmid D ⁇ A preparation was used to transform electrocompetent DH10B cells. Of several apramycin- resistant colonies obtained, twenty colonies were examined for plasmid content. Four colonies contained plasmid carrying large insert D ⁇ A. Out of these one plasmid, pOJ446:10-4 was first introduced into the E. coli donor strain STR611 and from there mated into S. venezuelae DS154 (cml-5) and S.
- venezuelae NS503 (cml-12).
- the cosmid pOJ446 (without insert) was also mated from STR611 into S. venezuelae strains DS154 and DS503.
- Fourteen exconjugants each of DS154 (pOJ446), DS154 (pOJ446:clone 10- 4), NS503 (pOJ446) and NS503 (pOJ446:clone 10-4) were patched on MYM agar + apramycin, grown for 4-5 days and bioassayed with M. luteus for detecting chloramphenicol production.
- cosmid 10 contains D ⁇ A fragment that complements cml-12 and cml-5 mutations, and therefore other chloramphenicol biosynthesis genes are very likely to be present on cosmid 10.
- the cosmid 10-4 is currently being analyzed for D ⁇ A sequence.
- the resulting 2.8 kb BamUl-Sacl fragment was sequenced. Codon preference analysis showed one complete ORF encoding a polypeptide of 670 amino acids. Comparison of the deduced amino acid sequence with database proteins indicated that the ⁇ - and C-terminal regions resembled PabA and PabB, respectively, of numerous bacteria. The gene product showed overall sequence similarity to the product of a fused pabAB gene associated with secondary metabolism in Streptomyces griseus. Insertion of an apramycin resistance gene into pabAB cloned in a segregationally unstable vector and replacement of the S.
- Genomic D ⁇ A libraries of Streptomyces venezuelae ISP5230 and of a mutant blocked at the chlorination step of chloramphenicol biosynthesis were probed by hybridization with a synthetic oligonucleotide corresponding to the ⁇ -terminal amino acid sequence of a bromoperoxidase-catalase purified from the wild-type strain. Hybridizing fragments obtained from the two strains were cloned and sequenced. Analysis of the nucleotide sequences demonstrated that the fragments contained the same 1449 bp open reading frame with no differences in nucleotide sequence.
- Replacement of the bca gene in the wild-type strain of S. venezuelae with a copy disrupted by insertion of a D ⁇ A fragment encoding apramycin resistance did not prevent chloramphenicol production.
- Plasmid pJN4 containing a 2.4-kilobase pair insert of genomic D ⁇ A from the chloramphenicol (Cm) producer Streptomyces venezuelae ISP5230, confers resistance when introduced by transformation into the Cm-sensitive host Streptomyces lividans M252 (Mosher, R. H. Ranade, N. P., Schrempf, H., and Nining, L. C. (1990) J. Gen. Microbiol. 136, 293-301). Transformants rapidly metabolized Cm to one major product, which was isolated and purified by reversed phase chromatography.
- the metabolite was identified by nuclear magnetic resonance spectroscopy and mass spectrometry as 3'-O- phospho-Cm, and was shown to have negligible inhibitory activity against Cm-sensitive Micrococcus luteus.
- the nucleotide sequence of the S. venezuelae D ⁇ A insert in pJN4 contains an open reading frame (ORF) that encodes a polypeptide (19 kDa) with a consensus motif at its ⁇ H2 terminus corresponding to a nucleotide-binding amino acid sequence (motif A or P-loop; Walker, J. E., Saraste, M., Runswick, M. J., and Gay, N. J. (1982) EMBO J. 1, 945-951).
- Chloramphenicol resistance in Streptomyces cloning and characterization of a chloramphenicol hydrolase gene from Streptomyces venezuelae.
- a 6.5 kb DNA fragment containing a chloramphenicol-resistance gene of Streptomyces venezuelae ISP5230 was cloned in Streptomyces lividans M252 using the high-copy-number plasmid vector pIJ702.
- the gene was located within a 2.4 kb Kpnl- Sstl fragment of the cloned DNA and encoded an enzyme (chloramphenicol hydrolase) that catalysed removal of the dichloroacetyl moiety from the antibiotic.
- the deacylated product, p-nitrophenylserinol was metabolized to p-nitrobenzyl alcohol and other compounds by enzymes present in S.
- Auxotrophs isolated from two chloramphenicol-nonproducing mutants of Streptomyces venezuelae included three requiring pyridoxal (Pxl-), NS248 (cml-11 pdx- 2), NS253 (cml-11 pdx-3), and NS258 (cml-12 pdx-4), and one requiring thiosulfate, NS263 (cml-12 cys-28).
- Streptomyces venezuelae fertility defined as chromosomal gene recombination, was enhanced over 1000-fold when one parent in a biparental conjugational cross lacked the physically-undetected plasmid SNPl, as compared with crosses in which both parents carried SNPl.
- SNPl and at least two other fertility plasmids, SNP2 and SNP3 was detected in S. venezuelae by 'lethal zygosis' elicited by a plasmid-plus mycelium in contact with a plasmid-minus mycelium. Conjugational crosses were used to construct a linkage map of S.
- Streptomyces venezuelae strain 13s contained extrachromosomal D ⁇ A detectable by agarose gel electrophoresis and cesium chloride-ethidium bromide density gradient centrifugation.
- the single 17-megadalton plasmid present in this strain was indistinguishable from plasmid pUC3 previously isolated from mutagenized cultures.
- Strains selected for their inability to produce chloramphenicol after treatment with acriflavine or ethidium bromide still contained a plasmid that had the same electrophoretic mobility as plasmid pUC3 and yielded similar fragments when digested with restriction endonucleases.
- strain PC51-5 By regenerating protoplasts of strain 13s and screening for isolates lacking extrachromosomal DNA, strain PC51-5 was obtained. The absence of plasmid pUC3 sequences in this strain was confirmed by Southern hybridization using 32P-labeled plasmid as a probe. Since the plasmidless strain produced as much chloramphenicol as did the parent strain, pUC3 contains neither structural nor regulatory genes for antibiotic production. Evidence from electrophoretic analysis of Bam ⁇ l digests of total cellular DNA from wild-type and dye- treated nonproducing progeny indicated that acriflavin caused structural changes in the chromosome.
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Abstract
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Priority Applications (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CA002376722A CA2376722A1 (en) | 1999-05-14 | 2000-05-15 | Chloramphenicol biosynthetic pathway and gene cluster characterization |
EP00935986A EP1203083A1 (en) | 1999-05-14 | 2000-05-15 | Chloramphenicol biosynthetic pathway and gene cluster characterization |
AU51362/00A AU5136200A (en) | 1999-05-14 | 2000-05-15 | Chloramphenicol biosynthetic pathway and gene cluster characterization |
JP2000618462A JP2003521883A (en) | 1999-05-14 | 2000-05-15 | Characterization of chloramphenicol biosynthetic pathway and gene cluster |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US13432399P | 1999-05-14 | 1999-05-14 | |
US60/134,323 | 1999-05-14 |
Publications (2)
Publication Number | Publication Date |
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WO2000070056A1 WO2000070056A1 (en) | 2000-11-23 |
WO2000070056A9 true WO2000070056A9 (en) | 2001-11-29 |
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Application Number | Title | Priority Date | Filing Date |
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PCT/US2000/013394 WO2000070056A1 (en) | 1999-05-14 | 2000-05-15 | Chloramphenicol biosynthetic pathway and gene cluster characterization |
Country Status (3)
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EP (1) | EP1203083A1 (en) |
AU (1) | AU5136200A (en) |
WO (1) | WO2000070056A1 (en) |
Families Citing this family (1)
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CN100465277C (en) * | 2005-07-01 | 2009-03-04 | 中国科学院上海有机化学研究所 | Chlorothricin biological synthesis gene cluster and its uses |
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2000
- 2000-05-15 AU AU51362/00A patent/AU5136200A/en not_active Abandoned
- 2000-05-15 WO PCT/US2000/013394 patent/WO2000070056A1/en not_active Application Discontinuation
- 2000-05-15 EP EP00935986A patent/EP1203083A1/en not_active Withdrawn
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WO2000070056A1 (en) | 2000-11-23 |
EP1203083A1 (en) | 2002-05-08 |
AU5136200A (en) | 2000-12-05 |
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