EP1228218A2 - Nouveaux composes - Google Patents

Nouveaux composes

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Publication number
EP1228218A2
EP1228218A2 EP00975883A EP00975883A EP1228218A2 EP 1228218 A2 EP1228218 A2 EP 1228218A2 EP 00975883 A EP00975883 A EP 00975883A EP 00975883 A EP00975883 A EP 00975883A EP 1228218 A2 EP1228218 A2 EP 1228218A2
Authority
EP
European Patent Office
Prior art keywords
clavam
gene
lat
deleted
genes
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Withdrawn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
EP00975883A
Other languages
German (de)
English (en)
Inventor
Barry GlaxoSmithKline BARTON
Philip Andrew GlaxoSmithKline GREAVES
Alison Michelle GlaxoSmithKline GRIFFIN
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.)
SmithKline Beecham Ltd
Original Assignee
SmithKline Beecham Ltd
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
Priority claimed from GBGB9925137.3A external-priority patent/GB9925137D0/en
Priority claimed from GBGB9925135.7A external-priority patent/GB9925135D0/en
Application filed by SmithKline Beecham Ltd filed Critical SmithKline Beecham Ltd
Publication of EP1228218A2 publication Critical patent/EP1228218A2/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • 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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C12N15/76—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Actinomyces; for Streptomyces
    • 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
    • C12N1/00—Microorganisms; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/18—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing at least two hetero rings condensed among themselves or condensed with a common carbocyclic ring system, e.g. rifamycin
    • C12P17/188—Heterocyclic compound containing in the condensed system at least one hetero ring having nitrogen atoms and oxygen atoms as the only ring heteroatoms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/465—Streptomyces

Definitions

  • the present invention relates to new organisms for producing increased levels of clavulanic acid or other 5R clavams, including those with strong ⁇ -lactamase inhibitory activity, in combination with low or undetectable levels of 5S clavams.
  • the invention also relates to processes for using said microorganisms for the production of 5R clavams, in particular clavulanic acid.
  • Microorganisms in particular Streptomyces species, produce a number of antibiotics including clavulanic acid and other clavams, cephalosporins, cephamycins, tunicamycin, holomycin, polyketides and penicillins.
  • antibiotics including clavulanic acid and other clavams, cephalosporins, cephamycins, tunicamycin, holomycin, polyketides and penicillins.
  • antibiotics In the cephalosporin metabolic pathway in, for example, Streptomyces clavuligerus three important antibiotics can be produced namely penicillin N, O- carbarn oyldeacetylcephalosporin C and cephamycin C. These antibiotics are synthesised from the tripeptide precursor d-(L-a-aminoadipyl)-L-cysteinyl-D-valine; ACV (J.F. Martin et al (1990), "Clusters of genes involved in Penicillin and cephalosporin biosynthesis" in "50 years of Penicillin applications, history and trends").
  • the recognised first dedicated step in the biosynthesis of both penicillins and cephalosporins in S. clavuligerus is the conversion of L-lysine to L- ⁇ -aminoadipic acid catalysed by the enzyme lysine- ⁇ -amino transferase (LAT) which is encoded by the lat gene.
  • LAT lysine- ⁇ -amino transferase
  • the nucleotide sequence of the S. clavuligerus lat gene, and consequently the derived amino acid sequence of the encoded protein, are known (Tobin, M.B et al., (1991) J. Bacteriol, 173, 6223-6229).
  • S clavams include clavam-2-carboxylate (C2C), 2- hydroxymethylclavam (2HMC), 2-(3-alanyl)clavam, valclavam and clavaminic acid (GB 1585661 , Rohl, F. et al. Arch. Microbiol.
  • clavaminic acid a 5S clavam which is also a clavulanic acid precursor
  • clavaminic acid synthase is produced by the action of clavaminic acid synthase in the clavulanic acid biosynthetic pathway in S. clavuligerus .
  • Gene cloning experiments have identified that S. clavuligerus contains two clavaminic acid synthase isoenzymes, casl and cas2 (Marsh, E.N. et al Biochemistry 31, 12648-657, (1992)) both of which can contribute to clavulanic acid production under certain nutritional conditions (Paradkar, A.S. et al., J. Bact. 177(5), 1307-14 (1995)).
  • Clavaminic acid synthase activity has also been detected in other clavulanic acid producing micro-organsims, ie. S. umonjinensis (Nidal, CM., ES 550549, (1987)) and S. katsurahamanus (Kitano, K. et al, JP 53-104796, (1978)) as well as S. antibioticos, a producer of the 5S clavam, valclavam (Baldwin, J.E. et al, Tetrahedron Letts. 35(17), 2783-86, (1994)).
  • S. antibioticos to have proclavaminic acid amidino hydrolase activity, another enzyme known to be involved in clavulanic acid biosynthesis.
  • 5S clavams for example clavulanic acid
  • C2C W098/33896, SmithKline Beecham pic and the Governors of the University of Alberta
  • the invention relates to a 5R clavam producing organism comprising: a) a cephalosporin biosynthetic pathway, or a part thereof, wherein the lat gene has been deleted, disrupted or otherwise made defective; and b) a clavam biosynthetic pathway, wherein one or more genes that are specific for 5S clavam biosynthesis have been deleted, disrupted or otherwise made defective.
  • the organism is a Streptomycete, preferably S. clavuligerus.
  • the gene specific for 5S clavam biosynthesis which is disrupted or otherwise made defective is selected from the group consisting of cvml, cvm2, cvm3, cvm4, cvm5 and cvm6, preferably cvml, cvm4 or cvm5, most preferably cvml.
  • the 5R clavam has ⁇ -lactamase inhibitory activity and most preferably the 5R clavam is clavulanic acid.
  • the term “deleted” means a total or partial deletion of the gene.
  • a partial deletion can involve the removal of any amount of DNA from the target gene, from 1 base pair (bp) up to almost the entire polypeptide coding region of the gene.
  • a total deletion involves the removal of the entire coding region of the gene with or without flanking sequences, which may or may not include regulatory elements that are required for gene function, for example transcriptional promoters. Furthermore the deletion may result in the removal of just a regulatory region, such as a promoter, leaving the coding region intact. The result is that no mRNA can be produced and so the gene is rendered defective.
  • the removal of just one or two base pairs from the coding region of the gene results in a frameshift mutation and consequently a truncated gene product.
  • the truncated gene product will have no residual activity that may be possessed by the complete polypeptide. In this respect, deletions closer to the 5' end of the coding region are preferred.
  • the term "otherwise made defective” is understood to include, but is not limited to, such methods as inserting a strong transcriptional terminator downstream from the promoter to prevent transcription of the gene or part of the gene; the use of antisense (eg. O'Connor, J Neurochem (1991) 56:560) or ribozymes (eg Usman, N, et al., Curr. Opin. Struct. Biol (1996) 6(4), 527-33) to block expression at the post- transcriptional level by binding to or cleaving the mRNA respectively; or inhibiting transcription by using triplex-forming oligonucleotides to bind to the specific DNA sequence of the gene to be transcriptional ly silenced (eg. Dervan et al, Science (1991) 251 : 1360).
  • the skilled man would be able perfom these aspects of the invention using methods that are well known in the art. Other methods for rendering the target gene defective are understood to fall within this definition.
  • the invention relates to a process for producing a 5R clavam, comprising growing the organism of the invention under fermentation conditions that facilitates the biosynthesis of the 5R clavam.
  • the invention relates to the 5R clavam, preferably clavulanic acid, produced using the process of the invention.
  • the starting point for preparing the organisms of the invention is a "parent" organism.
  • the parent organism may be a Streptomycete, for example a strain of S. clavuligerus (available for example from the American Type Culture Collection - ATCC 27064) or any modified strain derived therefrom.
  • Such parent strains include those which have been engineered, or selected by established strain improvement techniques, to produce higher levels of clavulanic acid.
  • Such parent strains comprise a cephalosporin biosynthetic pathway, or a part thereof.
  • a part of a biosynthetic pathway is where one or more genes encoding one or more enzymes in that biosynthetic pathway are absent, for example by deletion, or have been otherwise made defective.
  • the absence of a biosynthetic pathway gene could be a consequence of a natural event, or could be engineered using methods well known in the art (Sambrook, J., Fritsch, E.F. and Maniatis,T. (1989) Molecular Cloning A Laboratory Manual (2nd Edition)).
  • the parent strain also comprises a pathway for the biosynthesis of a 5R clavam, most preferably clavulanic acid.
  • the lat gene and 5S clavam specific genes of the parent organism may be deleted, disrupted or otherwise made defective using well known methods (for example the methods disclosed in Sambrook, et al (1989) supra).
  • the genes may be disrupted using random mutagenesis methods, for example exposure to UN radiation or mutagenic chemicals (ethidium bromide or ⁇ TG), followed by selection of mutants disrupted in the lat or 5S clavam specific genes.
  • mutagenic chemicals ethidium bromide or ⁇ TG
  • the genes, or parts of the genes may be deleted or otherwise mutated using standard in-vitro mutagenesis techniques.
  • the lat or 5S clavam specific gene or genes can be cloned, by PCR or library screening techniques, and mutated in-vitro (for example by single or multiple nucleotide deletion, substitution or insertion), and then reintroduced into a parent organism by replacing the existing wild-type gene.
  • Methods of gene disruption are preferred (for example by inactivation by insertion of an apramycin resistance gene (Paradkar and Jensen (1995) J.Bacteriol. 177(5) 1307-1314) and methods of gene deletion are particularly preferred.
  • Gene replacement in vivo may be facilitated by, for example, double cross-over events using methods well known in the art (see for example Aidoo, KA et al (1994) Gene 147 p41-46).
  • the cvml gene may be disrupted by insertional inactivation using an apramycin resistance gene, as described in W098/33896, or more preferably by deletion of the entire, or part of, the cvml gene.
  • a 654bp portion of the cvml gene, between two Aatll restriction sites, is deleted.
  • the lat gene may be disrupted by insertional mutagenesis, as described in W096/41886, wherein recombinant DNA techniques were used to introduce an apramycin resistance gene within the coding region of the lat gene.
  • the lat gene, or a part thereof, is deleted.
  • the organism of the invention can be constructed by deleting, disrupting or otherwise making defective the lat gene in a first step, followed by a second step wherby the cvml gene is deleted, disrupted or otherwise made defective.
  • the cvml gene may be deleted or disrupted in the first step followed by the deletion or disruption of the lat gene in a second step.
  • the cvm 1 gene is deleted or disrupted in the first step.
  • the organism of the invention is used to produce a 5R clavam with reduced amounts, or undetectable levels of 5S clavam.
  • the 5R clavam is clavulanic acid, preferably increased levels of 5R clavam are produced compared with the parent strain.
  • the processes of the invention may be carried out in flasks at the laboratory scale, in fermentors at pilot plant scale, or in fermentors at production scale.
  • Example 1 Construction of a strain of S. clavuligerus containing gene disruptions of the lat and cvml genes
  • the plasmid pCECO ⁇ l was prepared from a wild type strain of S.clavuligerus and used to transform S. clavuligerus strain SB 203 (a high titre variant of the S.clavuligerus strain ATCC 27064) using methods described in WO 94/18326 (except that in addition to thiostrepton the overpour used to select the transformants also contained apramycin (20ug/ml final concentration).
  • the transformants which were found to be resistant to both thiostrepton and apramycin were then subclutured through two successive rounds of sporulation on solid agar media which had not been supplemented with either thiostrepton or apramycin (as described in W098/33896). Individual colonies were then replica plated onto antibiotic containing media to identify apramycin-resistant and thiostrepton-sensitive transformants. From this process 84 putative mutants were chosen for further analysis.
  • the plasmid p4861atap was prepared from a wild type strain of S.clavuligerus and used to transform S. clavuligerus strain GEM 1801 as described in WO 94/18326. Since GEM 1801 already contained the apramycin gene inserted into its chromosome, transformants were selected for thiostrepton resistance only. The transformants which were found to be resistant to thiostrepton and were then subclutured through four successive rounds of sporulation on solid agar media which had not been supplemented with either thiostrepton or apramycin (as described in the previous example). Individual colonies were then replica plated to antibiotic containing media to identify strains which were apramycin-resistant and thiostrepton-sensitive. From this process 9 putative mutants were chosen for further analysis.
  • Example 2 Clavulanic acid productivity of the cvml/lat double disrupted strains
  • Seven strains identified from example 1 as being inactivated in both the lat and cvm l genes (strains GEM 1894, 1895, 1896, 1897,1898 18100, and 18101) were analysed in shake flask fermentations for clavulanic acid productivity.
  • the shake flask fermentations and assays for clavulanic acid productivity were carried out as in WO 94/18326. From the results shown in table 1 it is clear that the strains containing both of these mutations have increased clavulanic acid productivity compared to parental control. The improvements range from 6% to 16% above the productivity of the parental strain.
  • Example 3 Construction of a strain of 5. clavuligerus containing gene deletions of the cvml and lat genes
  • PCR product 1 was generated using primers 1 and 2. This product is approximately 1Kb and contains the 3' region of cvml from the second AatlX site and downstream regions.
  • PCR product 2 was generated using primers 3 and 4. This product is approximately 1.1Kb and contains the 5' region of cvml from the first AatXX site and upstream regions.
  • PCR product 2 was ligated to Srfi digested pCR- Script Amp SK(+) as per instruction manual (Strategene Ltd, Cambridge Science Park, Milton Road, Cambridge CB4 4GF).
  • the ligation mixture was used to transform Epicurian E.coli XL1- Blue MRF' Kan supercompetent cells (available from Strategene)- to ampicillin resistance (as per manufacturers instructions). Plasmid DNA was isolated from the resulting transformants and DNA restriction analysis using SalXI HindXll and
  • PCR product 1 was digested with .Pstl and AatXl and the resultant DNA fractionated by agarose gel electrophoresis.
  • the 1Kb fragment was excised and eluted from the gel using the Sephaglas band prep kit. (Pharmacia, St. Albans, Herts, ALI 3 AW).
  • the isolated fragment was then ligated to Aatll and Pstl digested pSB 1.
  • the ligation mixture was used to transform competent cells of E.coli XLl-Blue (available from Strategene) to ampicillin resistance (as per manufacturers instructions).
  • Plasmid DNA was isolated from the resulting transformants and restriction analysis using Notl/EcoRI, Sr ⁇ , Pstl and Sail revealed that 1 clone contained plasmid into which PCR product 1 had been ligated. This plasmid which now contained a cloned copy of the cvml gene with a 654 nucleotide deletion was designated pSB2.
  • plasmid D ⁇ A of pSB2 was digested with EcoRX and HindXXX and ligated into the
  • Streptomyces vector pIJ486 which encodes a thiostrepton resistance gene (6.2Kb: Ward et al.,(1986) Mol. Gen. Genet. 203:468-478) and was also digested with EcoXU and HindXll. The ligation mixture was used to transform E.coli (JM109) competent cells (Strategene Ltd) to ampicillin resistance. Plasmid DNA was isolated from the resulting transformants and restriction analysis using EcoTU., Sail and BglXX revealed 6 clones possessed pSB2 containing pIJ486. One of these plasmids was designated pSB3. The plasmid pSB3 was used to transform a derivative of the S.clavuligerus strain
  • the DNA sequence of the lat gene and its surrounding DNA is known (Tobin et al J.Bact 173, 6223-9 (1991) and Perez-llarena et al J.Bact 2053-9 1997).
  • a PCR strategy was used to create an approximately lkb deletion of the lat gene as discribed below.
  • PCR was used to amplify the 3' region of the open reading frame (orfl2) adjacent to the lat gene using the primers:
  • FZl [5'- dGTTAAAGCTTCCGATACAGACAAGGGGTTCCTTCAC-3'] - SEQ ID N0.5; and RZ1 [5'-dATTAGGATCCCGAGCTGTTGCTCAGCTTCGTGTTCA-3'] - SEQ ID NO:6
  • PCR was further used to amplify the 3' region of the lat gene from S.clavuligerus chromosomal DNA using the following oligonucleotide primers FL1 [5'-dATATGAATTCTGCAGCGGCTCTGCCACGAGAACG-3'] - SEQ ID NO:7 and;
  • Plasmids were prepared from each transformation, pCR-lat for the lat fragment and pCR-Z for the orfl2 fragment were digested with EcoRI. This linearised the plasmid pCR-Z but in the case of pCR-lat two DNA fragments were produced one of which contained the 3' portion of the lat gene. The digests were electrophoresed in low melting point agarose and DNA fragments recovered as Sambrook et al. (1989) supra.
  • the gel purified pCR-Z EcoRI cut vector was ligated to the gel purified EcoRI DNA fragment from pCR-lat containing the 3'portion of the lat gene.
  • the ligation mix was then used to transform competent JM109 cells using the CaCl 2 method as Sambrook et al. 1989. Transformants were selected on Luria agar containing 100 ug/ml ampicillin.
  • the resultant plasmid, pSB13 was then further modified to remove a small amount of sequence (111 nucleotides) upstream of the deleted copy of the lat gene. This was achieved by digesting pSB13 with the DNA restriction enzyme Notl and religating the plasmid together.
  • the ligation mix was used to transform competent JM109 cells using the CaCl 2 method as Sambrook et al. (1989) supra. Transformants were selected on Luria agar plus 100 ug/ml ampicillin.
  • This plasmid was named pSB486.
  • This plasmid pSB486 was then introduced into SB82 by transformation. From the transformation, 88 thiostrepton resistant transformants were selected and tested for cephalosporoate production. Several of these primary transformants were found to have reduced cephalosporoate productivity compared to SB 82. These primary transformants were then put through a further round of sporulation on non-selective media and screened for the loss of thiostrepton resistance. This was carried out to identify those transformants where the plasmid had been lost.
  • strains identified from example 3 as being inactivated in both the lat and cvml genes as described above were analysed in shake flask fermentations for clavulanic acid productivity.
  • the shake flask fermentations and assays for clavulanic acid productivity were carried out as in WO 94/18326. From the results shown in table 1 it is clear that the strains containing both of these mutations have increased clavulanic acid productivity compared to parental control. The improvements range from 9% to 19% above the productivity of the parental strain.

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  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
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  • Microbiology (AREA)
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  • Tropical Medicine & Parasitology (AREA)
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Abstract

L'invention concerne des organismes qui produisent des taux accrus d'acide clavulanique et d'autres clavames 5R, notamment ceux présentant une forte activité inhibitrice de la β-lactamase, en combinaison avec des taux faibles ou indétectables de clavames 5S.
EP00975883A 1999-10-22 2000-10-18 Nouveaux composes Withdrawn EP1228218A2 (fr)

Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
GBGB9925137.3A GB9925137D0 (en) 1999-10-22 1999-10-22 Novel compounds
GBGB9925135.7A GB9925135D0 (en) 1999-10-22 1999-10-22 Novel compounds
GB9925135 1999-10-22
GB9925137 1999-10-22
PCT/EP2000/010283 WO2001030977A2 (fr) 1999-10-22 2000-10-18 Nouveaux composes

Publications (1)

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EP1228218A2 true EP1228218A2 (fr) 2002-08-07

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EP00975883A Withdrawn EP1228218A2 (fr) 1999-10-22 2000-10-18 Nouveaux composes

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EP (1) EP1228218A2 (fr)
JP (1) JP2003512832A (fr)
AU (1) AU1385701A (fr)
WO (1) WO2001030977A2 (fr)

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GB0308696D0 (en) * 2003-04-15 2003-05-21 Glaxo Group Ltd New process
GB0402799D0 (en) * 2004-02-09 2004-03-10 Sandoz Ag Pharmaceutical composition

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GB9511679D0 (en) * 1995-06-09 1995-08-02 Smithkline Beecham Plc Novel process
GB9702218D0 (en) * 1997-02-04 1997-03-26 Smithkline Beecham Plc Novel product

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* Cited by examiner, † Cited by third party
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See references of WO0130977A2 *

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JP2003512832A (ja) 2003-04-08
AU1385701A (en) 2001-05-08
WO2001030977A3 (fr) 2001-12-13

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