WO2000077222A1 - Genes codant pour des enzymes dans la biosynthese de pimaricine et application associee - Google Patents

Genes codant pour des enzymes dans la biosynthese de pimaricine et application associee Download PDF

Info

Publication number
WO2000077222A1
WO2000077222A1 PCT/EP2000/006227 EP0006227W WO0077222A1 WO 2000077222 A1 WO2000077222 A1 WO 2000077222A1 EP 0006227 W EP0006227 W EP 0006227W WO 0077222 A1 WO0077222 A1 WO 0077222A1
Authority
WO
WIPO (PCT)
Prior art keywords
cell
polynucleotide
pimaricin
polypeptide
streptomyces
Prior art date
Application number
PCT/EP2000/006227
Other languages
English (en)
Inventor
Juan Fransisco Martin
Jesus F Aparicio
Angel Jose Colina
Rudolf Gijsbertus Marie Luiten
Original Assignee
Dsm N.V.
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 Dsm N.V. filed Critical Dsm N.V.
Priority to AU54074/00A priority Critical patent/AU5407400A/en
Priority to EP00938825A priority patent/EP1185668A1/fr
Publication of WO2000077222A1 publication Critical patent/WO2000077222A1/fr

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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
    • C12N9/0077—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14) with a reduced iron-sulfur protein as one donor (1.14.15)
    • 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
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
    • 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
    • C12P19/626—Natamycin; Pimaricin; Tennecetin

Definitions

  • the invention relates to novel genes encoding enzymes which are fundamental in the biosynthesis of pimaricin.
  • the invention further relates the application of said gene for modifying the biosynthesis of pimaricin. It also relates to the biosynthesis of new compounds.
  • Polyketides such as pimaricin (in the literature also referred to as natamycin, see for its structure Fig. 3A) , form a large and highly diverse group of natural products.
  • Members of the said group include compounds having antibacterial, antifungal, anticancer, antiparasitic and immunosuppressant activities.
  • PKS polyketide synthase
  • the process resembles fatty acid biosynthesis, except that the ⁇ -keto function introduced at each elongation step may undergo all, part or none of a reductive cycle comprising ⁇ -ketoreduction, dehydration and enoylreduction .
  • Structural variety of polyketides arises from the choice of monomers, the extent of ⁇ -ketoreduction and dehydration, and the stereochemistry of each chiral center.
  • Yet further diversity is produced by functionalization of the polyketide chain by the action of glycosylases , methyltransferases and oxidative enzymes.
  • Bioconversion of simple organic compounds i.e. compounds with no or single reactive centers, has been known for some time and has been widely applied. Examples are the oxidation of long chain alkanes using alkane hydroxylation systems of Pseudomonas , and epoxidation of alkenes using enzyme systems from various micro-organisms.
  • ⁇ -lactam antibiotics for example, ⁇ -lactam antibiotics, polyketide antibiotics, anticancer agents, or peptide antibiotics
  • the large amounts of reactive groups present in those molecules are problematic for even the simplest treatments, such as hydrolysis of specific bonds. More complicated treatments frequently completely destroy the molecule.
  • the present invention is based on the identification and isolation of three genes which encode enzymes which facilitate specific oxidative conversions in the biosynthesis of pimaricin.
  • the present invention thus provides the means to perform specific conversions in complex biomolecules, in particular in polyketides, without applying the harsh conditions often related to chemical modifications.
  • the said conversions can be carried as part of a biosynthesis of said biomolecules, for instance in micro-organisms .
  • polynucleotides of the invention in different micro- organisms, can lead to the biosynthesis of different biomolecules . It has further been found that expression of the said polynucleotides may be switched off (or knocked out) in Streptomyces which is usually used for the biosynthesis of pimaricin. In this embodiment, no pimaricin is produced by said Streptomyces, but instead a modified biomolecule is produced. In addition, it has been found that the polynucleotides may be overexpressed in Streptomyces , leading to an increase in the biosynthesis of pimaricin in the said Streptomyces .
  • a polynucleotide comprising: i) a nucleic acid sequence set out in SEQ ID NO: 5, 7 or 9 or a sequence complementary thereto; or ii) a homologue or fragment of a sequence defined in i) .
  • the invention also provides: a polypeptide encoded by a polynucleotide of the invention which is preferably isolated and/or purified; a polypeptide obtainable by a polynucleotide of the invention in a cell which is a Streptomyces (including e.g. S .
  • na tal ens is) cell or a cell of a heterologous species a polypeptide comprising the amino acid sequence set out in SEQ ID NO: 6, 8 or 9 or a homologue or fragment thereof ; - a recombinant cell comprising at least one additional copy of a polynucleotide of the invention, wherein the cell naturally possesses at least one said polynucleotide ; a recombinant cell, wherein a polynucleotide of the invention which naturally occurs in the cell has been inactivated; a recombinant cell comprising a polynucleotide according to the invention which polynucleotide does not naturally occur in that cell or where the polynucleotide is heterologous to that cell; - a method for overexpressing a polynucleotide encoding a polypeptide according to the invention in Streptomyces cell which method comprises: i) attaching a promoter sequence to the said polynucle
  • Figure 1 Physical map of part of the Pimaricin biosynthetic cluster. Genes: locations of the genes encoding polyketide synthases and oxidative genes involved in Pimaricin production (not drawn to scale) ;
  • Probes 0.7 indicates the location of the 0.7 kb fragment used to identify the extent of polyketide synthase encoding regions; 3.3 indicates the location of the 3.3 kb fragment used in polyketide synthase gene disruption; Cosmids : sizes and numbers of available cosmids covering the chromosomal region encompassing the oxidative genes.
  • Figure 2 Detailed physical map of the chromosomal regions including the oxidative genes.
  • Figure 3A Molecular structure of Pimaricin.
  • Figure 3B Molecular structures of Pimaricin derivatives with a reduced oxidation state of C4 and C5 and/or the carboxyl group at C12.
  • Figure 5 illustrates the conversion of the triketide lactone to it oxidized form by the action of pORFl and pORF2
  • SEQ ID 1 shows the nucleotide sequence and derived amino acid sequence of a first Pimaricin biosynthesis associated polyketide synthase gene
  • SEQ ID 2 shows the amino acid sequence of a first Pimaricin biosynthesis associated polyketide synthase
  • SEQ ID 3 shows the nucleotide sequence and derived amino acid sequence of a second Pimaricin biosynthesis associated polyketide synthase gene
  • SEQ ID 4 shows the amino acid sequence of a second Pimaricin biosynthesis associated polyketide synthase
  • SEQ ID 5 shows the nucleotide sequence and derived amino acid sequence of ORF1, an oxidative gene involved in
  • SEQ ID 6 shows the amino acid sequence of an oxidation enzyme pORFl involved in Pimaricin biosynthesis
  • SEQ ID 7 shows the nucleotide sequence and derived amino acid sequence of ORF2 , an oxidative gene involved in Pimaricin biosynthesis
  • SEQ ID 8 shows the amino acid sequence of an oxidation enzyme pORF2 involved in Pimaricin biosynthesis
  • SEQ ID 9 shows the nucleotide sequence and derived amino acid sequence of ORF3 , an oxidative gene involved in Pimaricin biosynthesis
  • SEQ ID 10 shows the amino acid sequence of an oxidation enzyme pORF3 involved in Pimaricin biosynthesis
  • SEQ ID 11 shows a synthetic oligonucleotide (forward primer) for isolation by PCR of the ermE promoter of Saccharopolyspora erythraea
  • SEQ ID 12 shows a synthetic oligonucleotide (reverse primer) for isolation by PCR of the ermE promoter of Saccharopolyspora erythraea
  • SEQ ID 13 shows a synthetic oligonucleotide (forward primer) for isolation by PCR of the N-terminal region of ORF1
  • SEQ ID 14 shows a synthetic oligonucleotide (reverse primer) for isolation by PCR of the N-terminal region of 0RF1
  • ORFs open-reading frames
  • Pimaricin PKS associated genes were initially pursued by comparing their derived amino acid sequences with those present in public databases like EMBL, Genbank, NBRF/PIR, or Swissprot.
  • ORF1 appeared to resemble cholesterol oxidases from several Streptomyces species .
  • the close association of ORF1 with the Pimaricin PKS suggests an oxidative step in Pimaricin tailoring.
  • a methyloxidase encoding gene has not been observed previously in a polyketide biosynthesis gene cluster.
  • ORF2 and ORF3 resemble cytochrome P450 dependent monooxygenases from various sources.
  • P450 dependent monooxygenases have been identified before in association with polyketide gene clusters, e.g. in the Erythromycin and Rapamycin biosynthesis gene clusters.
  • the invention provides a polynucleotide which comprises : i) a nucleic acid sequence set out in SEQ ID NO: 5, 7, or 9 or a sequence complementary thereto; or ii) a homologue or fragment of a sequence defined in i) .
  • Polynucleotides of the invention may comprise DNA or RNA.
  • the invention also provides double stranded polynucleotides comprising a polynucleotide of the invention and its complement.
  • Homologues of a nucleic acid sequence set out in SEQ ID NO: 5, 7 or 9 are polynucelotideds which do not share 100% sequence identity with a sequence set out in SEQ ID NO: 5, 7, or 9 , but which do encode polypeptides having a similar enzyme activity to a polypeptide encoded by a nucleic acid sequence set out in SEQ ID NO: 5, 7 or 9.
  • a homolog of a polypeptide encoded by SEQ ID NO: 5 will typically encode a polypeptide which has methyl oxidase or methyloxidase-like activity.
  • a homologue of a polypeptide encoded by SEQ ID NO: 7 or 9 will typically encode a polynucleotide which has cytochrome P-450 monocxygenase activity or cytochrome P-450 monooxygenase-like activity.
  • a homologue of the invention will generally at least 90%, at least 95%, at least 98% or at least 99% sequence identity to the sequence of SEQ ID NO : 5, 7 or 9 over a region of at least 60, more preferably at least 100 contiguous nucleotides or most preferably over the full length of SEQ ID NO : 5, 7 or 9 (for determination of sequence identity see D.J. Lipman, W.R. Pearson. 1985. Science 227, pl435) .
  • polynucleotides of the invention Any combination of the above mentioned degrees of sequence identity and minimum sizes may be used to define polynucleotides of the invention, with the more stringent combinations (i.e. higher sequence identity over longer lengths) being preferred.
  • a polynucleotide which has at least 90% sequence identity over 60 forms one aspect of the invention, as does a polynucleotide which has at least 95% sequence identity over 100 nucleotides.
  • the sequence of SEQ ID NO: 5, 7 or 9 may be modified by nucleotide substitutions, for example from 1, 2 or 3 to 10 or 25 substitutions.
  • the polynucleotide of SEQ ID NO : 5, 7 or 9 may alternatively or additionally be modified by one or more insertions and/or deletions and/or by an extension at either or both ends.
  • the modified polynucleotide generally encodes a polypeptide which has methyl oxidase or cytochrome P-450 monooxygenase activity.
  • Polynucleotides of the invention include fragments of a sequence set out in SEQ ID NO: 5, 7 or 9.
  • polynucleotides of the invention may be used as a primer, e.g. a PCR primer, a primer for an alternative amplification reaction, a probe e.g. labeled with a revealing label by conventional means using radioactive or non-radioactive labels, or the polynucleotides may be cloned into vectors (M.A. Innis et al..l990. PCR Protocols, Academic Press Inc) .
  • Such primers, probes and other fragments will preferably be at least 10, preferably at least 15 or at least 20, for example at least 25, at least 30 or at least 40 nucleotides in length. They will typically be up to 40, 50, 60, 70, 100, or 150 nucleotides in length. Probes and fragments can be longer than 150 nucleotides in length, for example up to 200, 300, 400, 500, 600, 700 nucleotides in length, or even up to a few nucleotides, such as five or ten nucleotides, short of the full length of the sequence of SEQ ID NO: 5, 7 or 9.
  • Polynucleotides such as DNA polynucleotide and primers according to the invention may be produced recombinantly, synthetically, or by any means available to those of skill in the art. They may also be cloned by standard techniques. The polynucleotides are typically provided in isolated and/or purified form.
  • primers will be produced by synthetic means, involving a stepwise manufacture of the desired nucleic acid sequence one nucleotide at a time. Techniques for accomplishing this using automated techniques are readily available in the art.
  • Longer polynucleotides will generally be produced using recombinant means, for example using PCR (polymerase chain reaction) cloning techniques.
  • a polypeptide of the invention comprises the amino acid sequence set out in SEQ ID NO: 6, 8 or 10 or a substantially homologous sequence, or a fragment of the said sequences and typically has methyl oxidase or cytochrome P- 450 monooxygenase activity.
  • the naturally occurring amino acid sequence shown in SEQ ID NO: 6, 8 or 10 is preferred.
  • a polypeptide of the invention may comprise:
  • a homologue may occur naturally, for example, in a bacterium and will function in a substantially similar manner to the protein of SEQ ID NO: 6, 8 or 10, for example it acts as a methyl oxidase in the case of a homologue of
  • SEQ ID NO: 6 or a cytochrome P-450 monooxygenase in the case of a homologue of SEQ ID NO : 8 or 10.
  • Homologues can be obtained by following the procedures described herein for the production of the polypeptides of SEQ ID NO: 6, 8 or 10 and performing such procedures on a suitable cell source e.g. a bacterial cell. It will also be possible to use a probe as defined above to probe libraries made from bacterial cells in order to obtain clones encoding homologues. The clones can be manipulated by conventional techniques to generate a polypeptide of the invention which can then be produced by recombinant or synthetic techniques known per se .
  • a homologue of a polypeptide of the invention preferably has at least 80% sequence identity to the protein of SEQ ID NO: 6, 8 or 10, or more preferably at least 90%, at least 95%, at least 97% or at least 99% sequence identity thereto over a region of at least at least 40, preferably at least 60, for instance at least 100 contiguous amino acids or over the full length of SEQ ID NO : 6, 8 or 10.
  • the sequence of the polypeptide of SEQ ID NO: 6, 8 or 10 and of homologues can thus be modified to provide polypeptides of the invention.
  • Amino acid substitutions may be made, for example from 1, 2 or 3 to 10 or 20. substitutions.
  • the modified polypeptide generally retains activity as a methyl oxidase or cytochrome P-450 monooxygenase. Conservative substitutions may be made, for example according to the following Table. Amino acids in the same block in the second column and preferably in the same line in the third column may be substituted for each other.
  • Polypeptides of the invention also include fragments of the above-mentioned full length polypeptides. Such fragments typically retain activity as a methyl oxidase or cytochrome P-450 monooxygenase.
  • Polynucleotides of the invention can be incorporated into a recombinant replicable vector.
  • the vector may be used to replicate the nucleic acid in a compatible host cell .
  • the invention provides a method of making polypeptides of the invention by introducing a polynucleotide of the invention into a replicable vector, introducing the vector into a compatible host cell, and growing the host cell under conditions which bring about replication of the vector.
  • the vector may be recovered from the host cell .
  • a polynucleotide of the invention in a vector is operably linked to a control sequence which is capable of providing for the expression of the coding sequence by the host cell, i.e. the vector is an expression vector.
  • the term "operably linked” refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner.
  • a regulatory sequence, such as a promoter, "operably linked" to a coding sequence is positioned in such a way that expression of the coding sequence is achieved under conditions compatible with the regulatory sequence.
  • Vectors of the invention may be transformed into a suitable host cell to provide for expression of a polypeptide of the invention.
  • the invention provides a process for preparing a polypeptide according to the invention which comprises cultivating a host cell transformed or transfected with an expression vector encoding the polypeptide and recovering the polypeptide.
  • Each of the genes ORF1, ORF2 and ORF3 can be used for various purposes separately or in combination. This will be discussed in detail below.
  • Targeted inactivation of one or more of the present genes interferes with at least one (oxidation) step in the Pimaricin biosynthetic route.
  • molecules can be created lacking the epoxide function at carbons C4 and C5 , or molecules with a modified oxidation state of the carboxyl group at C12 resulting in an aldehyde, alcohol, or methyl group at this position.
  • Disruption of chromosomally encoded genes can be accomplished by gene replacement strategies. Gene replacement is preferably carried out using suicide plasmid vectors or defective phage vectors carrying modified target genes and detection or selection marker genes. The various elements useful for such strategies, and how to employ them, are described below.
  • Target gene modification can be accomplished by disruption of a coding sequence by insertion or deletion of nucleotides or nucleotide stretches. Such insertions or deletions may be of any suitable size. Preferably, they are of a size of at least 2 nucleotides, for example up to 5, up to 10, up to 25 or up to 50 nucleotides in length, excepting deletions which are multiples of 3.
  • the coding region of the target gene may be replaced by that of a (marker) gene.
  • a (marker) gene This confers an easily detectable phenotype on cells transformed with such a construct .
  • replacement genes are lacZ, xylE, Green Fluorescent Protein, and genes for the biosynthesis of antibiotics, such as erythromycin, apramycin, hygromycin, and thiostrepton, and metabolite analogues, such as fluoroacetamide .
  • Transfer of a disrupted target gene to a Pimaricin production host, resulting in in vivo gene inactivation can be accomplished by using e.g. suicide vector systems, a defective phage containing a fragment internal to the coding region of the target gene, or a variant of the gene inactivated through deletion or insertion of DNA stretches as described above, and optionally a detection or selection marker.
  • Suicide vectors and defective phages are characterized by their inability to propagate autonomously in the strain to be transformed and thus cannot be stably maintained by themselves.
  • Strep omyce es in general several suicide systems are available and suicide vectors can be chosen from the group of extrachromosomal element based cloning vectors available for E.
  • Streptomyces plasmids characterized by a limited host range can be selected that are incapable of stable maintenance in the desired host strain. Examples of such narrow host range plasmids are SLP1.2 and SCP2 , and cloning vectors derived from these plamids . Still another possibility is to use temperature sensitive variants of -Stre tomyces wide host range plasmids. These plamids are characterized by their inability to replicate above a certain (restrictive) temperature.
  • suicide constructs can be introduced in a desired host cell using transformation procedures with isolated DNA, by conjugation from a donor microorganism, e.g. an E. coli or Streptomyces strain harboring the construct, or via transfection by phage particles. All of these methods are well within the knowledge of the person skilled in the art .
  • a donor microorganism e.g. an E. coli or Streptomyces strain harboring the construct, or via transfection by phage particles. All of these methods are well within the knowledge of the person skilled in the art .
  • stable maintenance of the introduced genetic information is only possible by integration of the construct in the host chromosome, preferably by homologous recombination with the chromosomal copy of the target gene.
  • Strains having integrated the construct in the chromosome can be detected by the expression of a co-introduced marker.
  • a detection marker transformed colonies can be screened for acquired properties such as conversion of a colorless substrate into a colored compound (applicable with e.g. the genes lacZ, or xylE) or fluorescence (by expression of e.g. Green Fluorescent Protein) .
  • a marker can be used which allows selection of transformed strains by acquired resistance to e.g. antibiotics or toxic metabolite analogues. The latter method usually is employed more frequently because only cells with the acquired resistance will be able to grow in media containing the antibiotic or toxic metabolite analogue.
  • an internal fragment of the target gene is used for the construction of the suicide vector or defective phage, integration of the construct into the chromosomal copy of the target gene will result in inactivation immediately.
  • the suicide construct or defective phage contains the complete target gene or a fragment including the N-terminal or C-terminal coding region, though inactivated through smaller insertions or deletions, only integration of the construct will result in the presence of an active and ' inactive copy of the gene, separated by vector DNA.
  • a second homologous recombination has to take place removing the vector sequences and the active copy of the target gene. Strains having undergone this second homologous recombination can be detected by the loss of the acquired property encoded by the co- introduced marker gene.
  • Another application of the present genes from the Pimaricin gene cluster lies in overexpression of one or more of these genes in the natural host, Streptomyces natalensis .
  • the expression of the individual genes within the cluster is tightly regulated by the cell physiology and/or cluster specific regulatory genes . This internal control may be appropriate for production of the antibiotic in the natural environment, but is undesirable for industrial production.
  • Pimaricin production This increase may be in the form of increased Pimaricin titre in the culture broth or a higher product yield on substrate consumed.
  • enhanced expression of certain genes can also be combined with inactivation of other genes, thus effecting improved production of variants of Pimaricin as described above.
  • Strains containing additional copies of target genes can be obtained through introduction of complete genes including expression signals (promoters and optionally enhancers) into the host chromosome. Suitable techniques include suicide vectors and defective phage, as described above. Alternatively, autonomously replicating DNA molecules derived from phage genomes or extrachromosomal elements, for example plasmids, can be used to carry the additional genes. Suitable cloning vectors include those derived from plasmids pIJlOl and SCP2.
  • vectors can be constructed based on the plasmid naturally occurring in Streptomyces natal ensis , as disclosed in GB patent application nr 2210619, using selection and/or detection markers similar to those employed for the pIJlOl derived vectors, such as pIJ702, pIJ486, with or without added markers as described above.
  • promoters For gene expression, a large variety of promoters efficiently directing transcription of genes in Streptomyces is available.
  • An example of a constitutive promoter is the ermE promoter, which directs expression of the erythromycin resistance gene from Saccharopolyspora erythraea .
  • the agarase gene promoter from S . coeli color, the promoter of the glycerol utilization operon, or the tipA promoter are examples of promoters inducible by specific substrates .
  • additional promoters can be obtained, e.g. promoters endogenous to
  • the degree of overexpression can be manipulated by the choice of the promoter, by the amount of inducing compound, or by the choice of the autonomously replicating vector systems.
  • predetermined plasmid copy numbers can range from 1 or 2 to about 500. It is well within the expertise of the normal person skilled in the art to adjust the vector system to the desired degree of overexpression.
  • inactivation to obtain new variants of Pimaricin and overexpression to increase Pimaricin productivity can also be applied to strains producing structurally similar bioactive compounds for instance polymer antibiotics such as Amphotericin B ⁇ Streptomyces nodo ⁇ u ⁇ ) , Nystatin ⁇ Streptomyces noursei ) (see Figure 4) to obtain variants of these compounds and/or to improve productivity
  • polymer antibiotics such as Amphotericin B ⁇ Streptomyces nodo ⁇ u ⁇ ) , Nystatin ⁇ Streptomyces noursei ) (see Figure 4) to obtain variants of these compounds and/or to improve productivity
  • Using the present genes to inactivate the corresponding genes in Streptomyces species other than Streptomyces na tal ensis will result in new derivatives of, inter alia , nystatin and amphotericin B which are altered in their oxidative state.
  • a further application of the polynucleotides of the invention is the heterologous expression and exploitation of the enzymatic activity encoded by one or more of the said polynucleotides .
  • other microorganisms preferably Strep tomycetes species for instance the strain Streptomyces lividans or Streptomyces coeli color, can be genetically transformed and thus acquire new oxidative enzymatic activity.
  • This route is particularly useful for application of the enzymatic activities of polypeptides of the invention to the oxidative modification of other, preferable bioactive, compounds.
  • Examples include secondary metabolites, antibiotics and anticancer agents etc., which often are highly functionalized chemical entities.
  • a strain having acquired a gene or genes encoding oxidative enzymatic activity from the Pimarcin biosynthetic gene cluster will then be able to introduce, for example, epoxide functions or alcohol, aldehyde, or carboxyl groups into metabolites previously not modified in such a way. In this way it is possible to oxidize a methyl group which is not part of an linear alkane.
  • a methyl group forming part of an aliphatic ring of an organic compound or biocompound can be oxidized by one or more of the polypeptides of the invention.
  • the polypeptides of the invention can be isolated or purified from rDNA transformed hosts in which one or more of the polynucleotides of the invention are introduced.
  • the polynucleotide are heterologous to the host.
  • the transformed host as such may be used for the oxidative conversion.
  • Example 1 Isolation and identification of Pimaricin biosynthetic enes.
  • Streptomyces na tal ensis strain ATCC27448 was grown in YEME medium (D.A. Hopwood, M.J. Bibb, K.F. Chater, T. Kieser, C.J. Bruton, H.M. Kieser, D.J. Lydiate, C.P. Smith, J.M. Ward, H. Schrempf, Genetic Manipulation of
  • a cosmid library of S . na talensis DNA in E. coli was obtained.
  • the cosmid library was screened for the presence of polyketide synthase (PKS) related sequences by hybridization with radioactively labeled fragments from known PKS genes from the Rapamycin biosynthesis cluster from Streptomyces hygroscopi cus (T.Schwecke, J. F.
  • Example 3 Detailed sequence analysis of non-PKS genes; preliminary identification.
  • PKS genes of Example 1 revealed the presence of additional open reading frames (ORF) potentially encoding proteins functional in Pimaricin biosynthesis.
  • ORFl showed a clear homology with previously identified cholesterol oxidases and ORF2 and ORF3 were similar to cytochrome P-450 monooxygenase proteins. Also, genes encoding accessory proteins for the P- 450 enzymes seem to be present i.e. ferredoxin type.
  • SEQ ID numbers 5- 10 Detailed information on the chromosomal regions enompassing the three open reading frames (ORF's) is presented in Figure 2.
  • ORFl a 7kb Sp l fragment containing the complete ORFl was cloned into pUC19, the resulting plasmid was digested with Bglll, the cohesive ends were filled in by treatment with Klenow polymerase and religated. This new plasmid was used as a source for DNA for the gene replacement .
  • the 2.9 kb BamHI-Pstl fragment from the plasmid was cloned into the BamHI-Pstl sites of KC515.
  • the recombinant phage was propagated in S . lividans , and used to infect the wildtype S . na talensis strain. Lysogens were obtained by selection for thiostrepton .
  • the second recombination event was searched for by the loss of thiostrepton resistance.
  • the insertion and subsequent loss of the phage as well as the final structure of the disruptred gene was confirmed by Southern hybridization.
  • ORF3 disruption was accomplished by insertion of a 667 bp PvuII-S-mal fragment internal to ORF3 in HinCII cut pUC19; The fragment was excised using Ba-mHI and Pstl and ligated into similarly digested phage vector KC515. Transformation of the ligation mixture to S . lividans yielded recombinant phage 06D4-lparticles . After transfection of S . na tal ensis , lysogens were isolated as described above. Disruption of ORF3 in S . na talensis mutant D4 was confirmed by Southern hybridization
  • Example 5 Analysis of ORFl and ORF3 gene disruptants of S. natalensis .
  • ORFl ORF2 and ORF3 was achieved by placing each gene under the direction of the ermE promoter from Saccharopolyspora erythraea (M.J. Bibb, G.R. Janssen, J.M. Ward. 1985. Gene 38: 215-226).
  • a useful derivative of this promoter, having a number of cloning sites attached was obtained by PCR using the following oligonucleotides : SEQ ID 11:
  • SEQ ID 12 AAACTGCAGCTCTAGATGCCCGGGTATCGATCGTCGACGGCATGCGGATCCTACCAACCG GCACGATTG
  • the 225 bp PCR fragment obtained was digested with Pstl, purified by agarose gel electrophoresis and inserted into Pstl digested pUC19, yielding pUCermE
  • ORFl was inserted in pUCermE as a 2.2 kb Sphl - Clal fragment encompassing the complete coding sequence.
  • ORF2 a 3.5 kb Clal -Nrul fragment was used, and for ORF3 a 2.8 kb Sall -Kpnl fragment was used.
  • Each ermE promoter-ORF combination was subsequently excised as a Pstl fragment, inserted in Pstl digested phage vector KC515 and introduced in S. natalensis essentially as described in Example 4.
  • Example 7 Expression of S . natalensis ORFl. ORF2 , and ORF3 in S . coelicolor and S . lividans
  • ORFl and ORF2 A 223 bp Ndel -EcoRI fragment, corresponding to the 5 'end of ORFl from the ATG to the first EcoRI site was obtained using the Polymerase Chain Reaction such that an Ndel site was created coinciding with the ATG initiation codon of ORFl.
  • the oligonuleotide ⁇ used for this PCR were 5 ' -AGGATTACCCATATGTTCGAGAACCAGCAT-3 ' (forward; SEQ ID NO 13) and 5 ' -GCATGAGCGTGGGAATTCCG -3" (reverse; SEQ ID NO 14) .
  • the PCR product was digested with Ndel and -BcoRI cloned into similarly digested vector pT7-7 (S. Tabor, C.C. Richardson. 1985. P ⁇ AS 82, 1074) to yield plasmid pJA56.
  • pJA56 was digested with EcoRI and S-mal , and ligated to an EcoRI -NruI fragment encompassing ORFl and ORF2 , yielding plasmid pJA57.
  • pJA57 was digested with Ndel and ligated to Ndel - digested pIJ6021 (E . Takano et al . 1995. Gene 166, 133).
  • the resulting plasmid was named pJA58. Both ORFl and ORF2 are now under the direction of the thiostrepton inducible tipA promoter. Plasmid pJA58 was transformed into strain S. coelicolor A(3) 2 and S . lividans
  • ORF3 The ORF3 expression vector has been construc- ted by cloning a 3.7 kb Kpnl fragment containing the complete ORF3 into the unique Kpnl site of pHZ1351 (Bao et al .. 1997. ISBA Meeting abstract 4P15). The resulting plasmid (pJA50) was transformed to strain S . coel i color A (3) 2 and S. lividans 1326. Expression of ORF3 is directed by its own promoter.
  • Example 8 Activity of cell-free extracts of S . coelicolor expressing ORFl. ORF2 , and ORF3.
  • Mycelium was harvested by centrifugation at 5000xg/4 c C for 10 minutes and washed with 1 volume of 50mM Tris-HCl pH 7.5, ImM DTT, 10% glycerol .
  • the mycelium was resuspended in 0.2 volume of 50mM Tris-HCl pH 7.5, ImM DTT, 10% glycerol; 1 tablet of protease inhibitor cocktail (Boehringer Mannheim) was added per 25 ml of extract.
  • Cell extracts were prepared by sonication. After sonication cell debris were removed by centrifugation at lOOOOxg / 4°C for 10 minutes.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Plant Pathology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

La présente invention concerne un polynucléotide comprenant la séquence d'acides nucléiques figurant dans les SEQ ID NO: 5, 7 or 9, ou une séquence homologue ou un fragment de ces séquences ou une séquence complémentaire. Les polynucléotides de l'invention peuvent être utilisés afin de modifier la biosynthèse de la pimaricine ainsi que dans la biosynthèse de nouveaux composés.
PCT/EP2000/006227 1999-06-14 2000-06-14 Genes codant pour des enzymes dans la biosynthese de pimaricine et application associee WO2000077222A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
AU54074/00A AU5407400A (en) 1999-06-14 2000-06-14 Genes encoding enzymes in the biosynthesis of pimaricin and the application thereof
EP00938825A EP1185668A1 (fr) 1999-06-14 2000-06-14 Genes codant pour des enzymes dans la biosynthese de pimaricine et application associee

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
EP99201893.7 1999-06-14
EP99201893 1999-06-14

Publications (1)

Publication Number Publication Date
WO2000077222A1 true WO2000077222A1 (fr) 2000-12-21

Family

ID=8240310

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2000/006227 WO2000077222A1 (fr) 1999-06-14 2000-06-14 Genes codant pour des enzymes dans la biosynthese de pimaricine et application associee

Country Status (3)

Country Link
EP (1) EP1185668A1 (fr)
AU (1) AU5407400A (fr)
WO (1) WO2000077222A1 (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001059126A2 (fr) 2000-02-08 2001-08-16 Norges Teknisk Naturvitenskapelige Universitet Nouveaux genes codant une polyketide de nystatine synthase, manipulation et utilisation
WO2006080648A1 (fr) * 2004-10-04 2006-08-03 Hanson Biotech Co., Ltd. Amorce pour la detection d'hydroxylase du cytochrome p450 specifique au polyene
US20110124088A1 (en) * 2009-11-20 2011-05-26 Pei-Fen Yang Expression vector for expressing recombinant protein in Cyanobacterium

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995001098A2 (fr) * 1993-06-28 1995-01-12 Monsanto Co Methode de controle de la proliferation des insectes
US5672497A (en) * 1986-03-21 1997-09-30 Eli Lilly And Company Method for increasing the antibiotic-producing ability of antibiotic-producing microorganisms
WO1998011230A1 (fr) * 1996-09-13 1998-03-19 Bristol-Myers Squibb Company Synthases polyketides dans la biosynthese de la pradimicine et sequences d'adn les codant

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5672497A (en) * 1986-03-21 1997-09-30 Eli Lilly And Company Method for increasing the antibiotic-producing ability of antibiotic-producing microorganisms
WO1995001098A2 (fr) * 1993-06-28 1995-01-12 Monsanto Co Methode de controle de la proliferation des insectes
WO1998011230A1 (fr) * 1996-09-13 1998-03-19 Bristol-Myers Squibb Company Synthases polyketides dans la biosynthese de la pradimicine et sequences d'adn les codant

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
APARICIO ET AL.: "The biosynthetic gene cluster for the 26-membered ring polyene macrolide pimaricin. A new polyketide synthase organization encoded by two subclusters separated by functionalization genes", JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 274, no. 15, 9 April 1999 (1999-04-09), pages 10133 - 10139, XP002120719 *
BRAUTASET ET AL: "Biosynthesis of the polyene antifungal antibiotic nystatin in Streptomyces noursei ATCC 11455: analysis of the gene cluster and deduction of the biosynthetic pathway", CHEMISTRY AND BIOLOGY, vol. 7, no. 6, 23 May 2000 (2000-05-23), pages 395 - 403, XP000953274 *
DATABASE EMBL EBI; 25 May 2000 (2000-05-25), BRAUTASET ET AL.: "Streptomyces noursei ATCC 11455 nystatin biosynthetic gene cluster, complete sequence", XP002151299 *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001059126A2 (fr) 2000-02-08 2001-08-16 Norges Teknisk Naturvitenskapelige Universitet Nouveaux genes codant une polyketide de nystatine synthase, manipulation et utilisation
WO2001059126A3 (fr) * 2000-02-08 2002-03-14 Norges Teknisk Naturvitenskape Nouveaux genes codant une polyketide de nystatine synthase, manipulation et utilisation
US7141660B2 (en) 2000-02-08 2006-11-28 Sinvent As Genes encoding a nystatin polyketide synthase and their manipulation and utility
US7709231B2 (en) 2000-02-08 2010-05-04 Sinvent As Genes encoding a nystatin polyketide synthase and their manipulation and utility
US7851183B2 (en) 2000-02-08 2010-12-14 Sinvent As Genes encoding a nystatin polyketide synthase and their manipulation and utility
WO2006080648A1 (fr) * 2004-10-04 2006-08-03 Hanson Biotech Co., Ltd. Amorce pour la detection d'hydroxylase du cytochrome p450 specifique au polyene
US20110124088A1 (en) * 2009-11-20 2011-05-26 Pei-Fen Yang Expression vector for expressing recombinant protein in Cyanobacterium

Also Published As

Publication number Publication date
AU5407400A (en) 2001-01-02
EP1185668A1 (fr) 2002-03-13

Similar Documents

Publication Publication Date Title
Han et al. Cloning and characterization of polyketide synthase genes for jadomycin B biosynthesis in Streptomyces venezuelae ISP5230
Decker et al. Cloning and characterization of a polyketide synthase gene from Streptomyces fradiae Tü2717, which carries the genes for biosynthesis of the angucycline antibiotic urdamycin A and a gene probably involved in its oxygenation
Faust et al. Two new tailoring enzymes, a glycosyltransferase and an oxygenase, involved in biosynthesis of the angucycline antibiotic urdamycin A in Streptomyces fradiae Tu2717
Stassi et al. Identification of a Saccharopolyspora erythraea gene required for the final hydroxylation step in erythromycin biosynthesis
Perlova et al. Identification and analysis of the chivosazol biosynthetic gene cluster from the myxobacterial model strain Sorangium cellulosum So ce56
Smanski et al. Engineered Streptomyces platensis strains that overproduce antibiotics platensimycin and platencin
Chen et al. Identification and utility of FdmR1 as a Streptomyces antibiotic regulatory protein activator for fredericamycin production in Streptomyces griseus ATCC 49344 and heterologous hosts
CA2731760C (fr) Gene de biosynthese de pyripyropene a
Schupp et al. A Sorangium cellulosum (myxobacterium) gene cluster for the biosynthesis of the macrolide antibiotic soraphen A: cloning, characterization, and homology to polyketide synthase genes from actinomycetes
Hidaka et al. Cloning and nucleotide sequence of fosfomycin biosynthetic genes of Streptomyces wedmorensis
Shah et al. Cloning, characterization and heterologous expression of a polyketide synthase and P-450 oxidase involved in the biosynthesis of the antibiotic oleandomycin
JP2000515390A (ja) 新規ポリケチド誘導体およびそれを製造するための組換え方法
Yang et al. Accumulation of the angucycline antibiotic rabelomycin after disruption of an oxygenase gene in the jadomycin B biosynthetic gene cluster of Streptomyces venezuelae
JPS6371183A (ja) ホスフィノトリシン耐性遺伝子及びその使用
Kong et al. Elucidation of the biosynthetic gene cluster and the post-PKS modification mechanism for fostriecin in Streptomyces pulveraceus
Oja et al. Characterization of the alnumycin gene cluster reveals unusual gene products for pyran ring formation and dioxan biosynthesis
EP3164485A1 (fr) Microorganismes et procédés de production de vanilline
Novakova et al. Cloning and characterization of a polyketide synthase gene cluster involved in biosynthesis of a proposed angucycline-like polyketide auricin in Streptomyces aureofaciens CCM 3239
Suwa et al. Identification of two polyketide synthase gene clusters on the linear plasmid pSLA2-L in Streptomyces rochei
JPWO2006126723A1 (ja) 遺伝子組換え微生物およびそれらの微生物を用いるマクロライド系化合物の製造方法
Cooper et al. Shift to Pseudomonic acid B production in P. fluorescens NCIMB10586 by mutation of mupirocin tailoring genes mupO, mupU, mupV, and macpE
Akhgari et al. Single cell mutant selection for metabolic engineering of actinomycetes
US7153667B2 (en) Discrete acyltransferases associated with type I polyketide synthases and methods of use
US20030068788A1 (en) Methods and compositions for making emamectin
EP1185668A1 (fr) Genes codant pour des enzymes dans la biosynthese de pimaricine et application associee

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY CA CH CN CR CU CZ DE DK DM DZ EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT TZ UA UG US UZ VN YU ZA ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
WWE Wipo information: entry into national phase

Ref document number: 2000938825

Country of ref document: EP

WWP Wipo information: published in national office

Ref document number: 2000938825

Country of ref document: EP

REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

NENP Non-entry into the national phase

Ref country code: JP

WWW Wipo information: withdrawn in national office

Ref document number: 2000938825

Country of ref document: EP