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WO1999067374A1 - Procedes de transfert de la capacite de production d'un produit naturel dans un hote de production approprie - Google Patents

Procedes de transfert de la capacite de production d'un produit naturel dans un hote de production approprie Download PDF

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WO1999067374A1
WO1999067374A1 PCT/EP1999/004079 EP9904079W WO9967374A1 WO 1999067374 A1 WO1999067374 A1 WO 1999067374A1 EP 9904079 W EP9904079 W EP 9904079W WO 9967374 A1 WO9967374 A1 WO 9967374A1
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coli
actinomycete
plasmid
streptomyces
host
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PCT/EP1999/004079
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English (en)
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Stefano Donadio
Margherita Sosio
Francesco Giusino
Carmela Cappellano
Anna Maria Puglia
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Biosearch Italia S.P.A.
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Priority to AU46096/99A priority Critical patent/AU743003B2/en
Priority to IL14002199A priority patent/IL140021A0/xx
Priority to EP99929199A priority patent/EP1090113A1/fr
Priority to JP2000556019A priority patent/JP2002518045A/ja
Priority to KR1020007014621A priority patent/KR20010083061A/ko
Priority to CA002330543A priority patent/CA2330543A1/fr
Publication of WO1999067374A1 publication Critical patent/WO1999067374A1/fr

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    • 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
    • C12P17/189—Heterocyclic compound containing in the condensed system at least one hetero ring having nitrogen atoms and oxygen atoms as the only ring heteroatoms containing the rifamycin nucleus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
    • 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
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • 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
    • 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
    • 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 novel approach for drug discovery. More particularly, the invention relates to a system for improving the process of lead optimization and development of compounds, when these compounds are natural products produced by microorganisms belonging to the order Actinomycetales or chemical derivatives of these compounds.
  • the invention relates to a system for transferring the capability to produce a natural product from a microorganism belonging to the order Actinomycetales into a defined host, where said natural product can be optimally produced and its biosynthetic pathway suitably modified.
  • Natural products are complex molecules with important uses in medicine. Examples include: antibacterial agents, such as erythromycin, teicoplanin, tetacycline; antitumor compounds, such as dauxorubicin; antihelmintic compounds, such as avermectin; immunosuppressive agents, such as cyclosporin and FK506; antifungal compounds, such as amphotericin and nystatin; etc. Natural products are produced as secondary metabolites by a wide range of living organisms. Although many secondary metabolites have been identified, there remains the need to obtain novel structures with new activities or enhanced properties. Current methods of obtaining such molecules include screening of natural isolates and chemical modification of existing ones.
  • HTS high throughput screening
  • a natural product may have one or more potential therapeutic properties, including but not limited to antibacterial, antifungal, antiviral, antitumor, immunomodulating or other pharmacological properties.
  • Natural products have long constituted a source of interesting, structurally original and "imaginative" molecules endowed with potent biological activities.
  • recent observations indicate that only a small fraction of the microbial flora present in environmental samples, ranging from 0.01 to 1% according to the estimates, is related to known species. Microorganisms belonging to the order Actinomycetales represent thus far the group of producers unsurpassed for chemical and biological diversity.
  • Microorganisms employ intricate biosynthetic machineries to make natural products: for example, synthesis of the macrolide antibiotic erythromycin, a secondary metabolite in the medium-range structural complexity, requires the participation of over 40 different enzymatic activities (Katz and Donadio, 1995, Macrolides, in Genetics and Biochemistry of Antibiotic Production, Vining and Stuttard eds . , Butterworth-Heinemann, Boston CT, p. 385-420) . Biosynthetic pathways can often be redirected through manipulation of the fermentation conditions or of the biosynthesis genes, in order to produce desired analogs of the original structure.
  • the present invention describes a general method for transferring the capability to produce any secondary metabolite from the original actinomycete producer to an established and genetically anipulatable production host.
  • the general concept of the invention is illustrated in Fig. 1. Conditions for optimal growth, metabolite production and maintenance need therefore to be developed for one host.
  • the availability of the cloned genes in a genetically manipulatable and well characterized host allows the utilization of all the genetic tools developed for these strains for the creation of novel derivatives of the natural product after genetic intervention.
  • the present invention provides a system for producing and manipulating natural products produced by a large group of bacteria for the purpose of drug discovery, development and production.
  • the method of the invention transfers the ability to produce a secondary metabolite from an actinomycete that is the original producer of the natural product, to another production host that has desirable characteristics .
  • the invention involves the construction of a library from a donor organism, the producer of a natural product, in an Artificial Chromosome that can be shuttled between a convenient, neutral cloning host, such as the bacterium Escherichia coli, and a production host, such as the actinomycetes Streptomyces lividans or Streptomyces coelicolor .
  • a convenient, neutral cloning host such as the bacterium Escherichia coli
  • a production host such as the actinomycetes Streptomyces lividans or Streptomyces coelicolor .
  • the invention involves the reconstruction of a large segment that directs the synthesis of a natural product, starting from smaller DNA fragments cloned from the genome of a donor organism.
  • This reconstruction occurs in an Artificial Chromosome that can be maintained in a convenient neutral host, such as the bacterium Escherichia coli, and subsequently transferred into an actinomycete production host.
  • the reconstructed genomic segment in the Artificial Chromosome is transferred into the production host where said natural product is synthesized.
  • the present invention also relates to Escherichia coli- Streptomyces Artificial Chromosomes, recombinant DNA vectors useful for shuttling the genetic information necessary to synthesize a given natural product between a donor actinomycete producer and a production host.
  • Chromosome is a recombinant DNA construct that can maintain very large DNA inserts in an Escherichia coli host and that can be introduced and maintained in an actinomycete production host.
  • Chromosome is a library of different recombinant constructs carrying very large DNA inserts that can be maintained in an Escherichia coli host and introduced and maintained in an actinomycete production host .
  • a pESAC is a vector used to construct an "Escherichia coli- Streptomyces Artificial Chromosome” or an ESAC library.
  • a "natural product” is a secondary metabolite made by a microorganism through a series of biosynthetic steps. This natural product may or may not have any useful biological activity .
  • a “complex” is the mixture of related natural products with similar properties and biological activity that are often produced by the same biosynthetic pathway.
  • a “donor organism” is the original producer of a natural product, where the synthesis of said compound is governed by a defined number of genetic elements .
  • a “gene cluster”, a “cluster”, a “biosynthesis cluster” all designate a contiguous segment of the donor organism's genome that contains all the genes required for the synthesis of a natural product.
  • a "production host” is a microorganism where the formation of a natural product is directed by a gene cluster derived from a donor organism.
  • Ab antibiotic
  • Ap ampicillin
  • a ttB chromosomal attachment site
  • attP phage or plasmid attachment site
  • bp base pair
  • ca . circa (i.e. "about”
  • Cm chloramphenicol
  • £. Escheri chia
  • ESAC E . coli- Streptomyces Artificial
  • Figure 1 Scheme of the invention.
  • the general concept of the invention whereby the gene cluster required for the synthesis of a natural product in a donor organism is established as an ESAC in an Escherichia coli host, and then transferred into a desired production host, where it integrates into the chromosome and directs production of the secondary metabolite.
  • the hexagon represents the natural product, the twisted thin line the bacterial chromosomes, and the thick line the desired gene cluster.
  • the pESAC episome is represented by a circle.
  • FIG. 1 E . coli- Streptomyces Artificial Chromosome vectors.
  • Vectors pPAC-Sl and pPAC-S2 differ solely for the orientation of the int- tsr cassette. Relevant features of the vectors are illustrated.
  • Kmr indicates resistance to kanamycin; sacB indicates sensitivity to sucrose.
  • Suitable cloning sites are shown as: B, BamHI; S, Seal; X, Xbal .
  • the replicating function of bacteriophage PI are indicated by the thick bars.
  • FIG. 3 General scheme of the invention, top-down approach. High molecular weight DNA from the donor organism is cloned into a pESAC.The resulting library in E . coli is screened with the required probes, and the relevant ESACs are identified. These are introduced into the desired production host strain, where they integrate site- specifically into the host chromosome. Symbols and abbreviations are as in Fig. 1. Figure 4. General scheme of the invention, bottom-up approach. A cosmid library is prepared with DNA from the donor organism and screened with the required probes. The overlapping inserts from the positive cosmids, which consitute the correct contig,are assembled into a pESAC via homologous recombination in E. coli . The reconstructed ESAC is introduced into the desired production host, where it integrates site-specifically into the host chromosome. Symbols and abbreviations are as in Fig. 1.
  • FIG. 5 Scheme of assemblage.
  • the figure illustrates a hypothetical genomic segment from a donor organism that is covered by the inserts from three overlapping clones.
  • the relevant fragments A and D, which denote the ends of the segment, and B and C, which represent regions of overlap, are indicated with their relative orientation (thick side on the fragment rectangle) .
  • the bottom part illustrates the reconstructed ESAC.
  • Figure 6 Constructs required for cluster assemblage.
  • the plasmids indicated are generated by routine in vitro DNA manipulations. Fragments A, B, C and D are as in Fig. 5. Fragment pairs are in this example separated by a marker, indicated as Ab R for antibiotic resistance. Selective markers present on the two compatible replicons are, as an example: Cm R and Km R .
  • FIG. 7 Interplasmid insert exchange.
  • Each of the Cm R derivatives, as of Fig. 6, is introduced in the same E . coli cell as the cognate clone of Fig. 5 (for example a cosmid that carries a Km R marker) . Formation and then resolution of the cointegrate leads to the transfer of the cosmid' s insert, indicated here by a looping line, in the Cm R replicon.
  • FIG. 8 Sequel of assembling steps. A series of interplasmid cointegration and resolution events is conducted. Only the growing ESAC is indicated. The starting construct (Fig. 6) is recombined with plasmid pAB2 (Fig. 7), leading to the insertion of the segment flanked by fragments A and B. Next, the Ab R marker from pBCl (Fig. 6) is introduced between fragments B and C, and subsequently replaced by the insert from pBC2 (Fig. 7) . Finally, the Ab R marker from pCDl (Fig. 6) is introduced between fragments C and D, and subsequently replaced by the insert from pCD2
  • FIG. 7 Figure 9. A gene cluster from Planobispora rosea .
  • the extension of a gene cluster from P. rosea ATCC 53733 is reported, together with the cosmids pRPl ⁇ , pRP31 and pRP58.
  • the fragments A, B, C and D used for assemblage are highlighted. Restriction sites are abbreviated as: M, Smal ; P, Pstl ; S, Sstl .
  • FIG. 10 Site-specific integration of an ESAC. PFGE analysis of S . lividans ZX7 transformed with ESAC-70.
  • Lanes 1 and 2 S . coelicolor M145; lane 3: S . lividans ZX7 DNA; lane 4: ZX7 a ttB : : ESAC-70 DNA, colony 1; lane 5: ZX7 a t tB : :ESAC-70 DNA, colony 2; lane 6: 50-kb ladder, size marker. All DNAs in lanes 1-5 are digested with Dral . Conditions for PFGE are: 200 Volts, 70 s switching for 7 15 h, 120 s switching for 11 h.
  • FIG. 11 Characterization of S . lividans transformants . Southern hybridization of S . lividans a t tB : ;PAD6, grown with (lane 1) or without (lane 2) thiostrepton. P. rosea DNA is shown as control (lane 3) .
  • Lane 4 contains 1-kb ladder. All DNAs are digested with Bamtil and probed with labeled PAD6.
  • the present invention entails a general procedure for constructing a Streptomyces host producing any natural product after selective transfer of the relevant genes from the original actinomycete producer, the donor strain. This general procedure is outlined in Fig. 1.
  • the present invention can be applied with only limited information on the structure of the natural product and very little knowledge of the original producer's genetics.
  • the present invention has a substantial impact on the process of drug discovery involving natural products or their structural derivatives.
  • the transfer of the producing capability to a well characterized host can substantially improve several portions of the process of lead optimization and development: the titer of the natural product in the producing strain can be more effectively increased; the purification of the natural product can be carried out in a known background of possible interfering activities; the composition of the complex can be more effectively controlled; altered derivatives of the natural product can be more effectively produced through manipulation of the fermentation conditions or by pathway engineering.
  • the titer of the natural product in the producing strain can be more effectively increased; the purification of the natural product can be carried out in a known background of possible interfering activities; the composition of the complex can be more effectively controlled; altered derivatives of the natural product can be more effectively produced through manipulation of the fermentation conditions or by pathway engineering.
  • the level of production of a natural product depends on the composition of the growth medium; on the presence of appropriate precursors or on the absence of specific inhibitors; on the expression level and timing of genes controlling the biosynthetic pathway and competing routes; and on the level and specific activity of key enzymes in the pathway.
  • the productivity of the original strain is usually increased by an empirical process, which may include, among other things, one or more of the following steps: strain purification, selection of phenotypic variants arising spontaneously or after mutagenic treatment of the strain, variation in the fermentation medium or in the fermentation parameters; genetic engineering of the producing strain. Fundamental knowledge about the physiology of the producing strain and the variables affecting titer must be achieved for an effective improvement of productivity. This knowledge is very scant in a newly identified producer strain.
  • a natural product may be produced by a microorganism as a complex of a few or tens of molecules with minor structural differences, designated congeners.
  • congeners are usually biologically active, only one or a few may represent the desired product: for example, one congener may be substantially more active than the others; it may possess better physico-chemical properties; or it may be a better substrate for chemical modification.
  • the composition of a complex can be somehow controlled by intervening on the fermentation parameters.
  • the most effective way is usually the altered expression of selected genes by genetic engineering (e.g. Sezonov et al . , 1997, Nature Biotechnol . 15:349-353).
  • Examples include the introduction of additional genetic information (Epp et al., 1989, Gene 85:293-301), the targeted inactivation of selected genes or portion thereof (Donadio et al . , 1993, Proc. Natl. Acad. Sci. USA 90:7119-7123), the "mixing and matching" of genes or portions thereof from different pathways (McDaniel et al . , 1994, Nature 375:549-554).
  • pathway engineering The process by which a given organism is genetically manipulated in order to alter the type, quality or quantity of a natural product is referred to as pathway engineering.
  • pathway engineering The ability to perform pathway engineering in a newly isolated microorganism producing a bioactive molecule with promising characteristics can therefore considerably expedite the optimization of a lead structure and the development process.
  • Pathway engineering can be schematized as a sequel of three steps: a) isolation of the genes of interest; b) performing on selected gene(s) the manipulations required by the specific objective; and c) introduction of the modified gene(s) in suitable form in an appropriate host.
  • biosynthesis of the natural products erythromycin (an antibiotic) , avermectin (an antihelmintic agent) and rapamycin (an immunosuppressant ) requires 55, 90 and 95 kb, respectively, of genetic information (Katz and Donadio, 1993, Annu. Rev. Microbiol. 47:875-912; MacNeil, 1995, Avermectins, in Genetics and Biochemistry of Antibiotic Production, Vining and Stuttard eds . , Butterworth- Heinemann, Boston CT, p.421-442; Schwecke et al . , 1995, Proc. Natl. Acad. Sci. USA 92:7839-7843).
  • Other natural products may require even larger extent of genetic information. Therefore, in order to isolate an entire cluster in a single piece, cloning vectors capable of accepting and maintaining large DNA segments are necessary.
  • Manipulations relevant to pathway engineering can include some or all of the following: site directed mutagenesis, gene inactivation, gene fusions, modification of regulatory sequences, etc.
  • Techniques for the in vitro manipulation of DNA and for the propagation of the mutated alleles in E . coli are well developed and can be applied to DNA from virtually any source (Sambrook et al., 1989, In Molecular Cloning: A labora tory Manual , 2nd edn, Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press) .
  • the final step in pathway engineering requires the introduction of modified or heterologous gene(s), in suitable form, in a strain where these genes can be appropriately expressed.
  • This strain is often the strain producing the natural product whose quantity, quality or type one wants to alter.
  • the genes of interest must be carried on appropriate vectors : according to the particular objective of pathway engineering, one may need, among others, vectors that can be stably maintained as single or multicopy episomes; that can insert into the host chromosome at a fixed location; that allow replacement of an endogenous gene with an in vi tro modified allele; that allow deletion of selected genes from the host chromosome.
  • vectors that can be stably maintained as single or multicopy episomes; that can insert into the host chromosome at a fixed location; that allow replacement of an endogenous gene with an in vi tro modified allele; that allow deletion of selected genes from the host chromosome.
  • one must have means for introducing heterologous DNA and selecting for its presence.
  • Naive hosts have been shown to produce the appropriate natural product or its intermediate (s) when the relevant DNA was introduced into them (Malpartida and Hopwood, 1984, Nature 309:462-464; Hong et al . , 1997, J. Bacteriol. 179:470-476; Kao et al . , 1994, Science 265:509-512; McGowan et al., 1996, Mol . Microbiol. 22:415-426; Kealey et al . , 1998; Proc. Natl. Acad. Sci. USA 95:505-509). However, the examples reported thus far represent special cases.
  • Streptomyces vectors currently available have an upper limit of ca. 40 kb (Hopwood et al . , 1987, Methods Enzymol . 153:116-167) .
  • the present invention rests on the fact that the genes required for the formation of a natural product are found as gene clusters of a defined size; that these gene clusters can be conveniently isolated, manipulated and transferred among different actinomycete strains; that they are expressed in a heterologous host; and on the fact that all the primary metabolite precursors required for the formation of a particular natural product are either produced by selected enzymes encoded by cluster-specific genes, or are present and available in the heterologous host at the time of formation of the natural product.
  • the present invention addresses also the crucial aspect of natural product formation in actinomycetes: i.e. synthesis of many natural products may require over 100 kb of genetic information.
  • transferring all the genes necessary for the production of any natural product requires cloning vectors capable of accommodating fragments as large as 150 kb, and possibly more.
  • An object of the present invention is therefore represented by vectors capable of accommodating such large fragments which are also capable of being stably maintained in a suitable microbial host, such as a Streptomyces host.
  • vectors are designated with the generic name pESAC. They are derived from bacterial artificial chromosomes (Shizuya et al., 1992, Proc. Natl. Acad. Sci. USA 89:8794-8797; Sicilnou et al . , 1994, Nature Genet. 6:84-89)and can carry inserts up to 300 kb, or more.
  • the exemplary organism chosen as the donor organism is the actinomycete P. rosea , belonging to one of the lesser characterized genera of actinomycetes (Goodfellow, 1992, In The Prokaryotes, 2nd edn., Balows, Trueper, Dworkin, Harder and Schleifer eds, Springer-Verlag, New York, NY, USA) .
  • This organism produces the natural product GE2270 (Selva et al., 1991, J.
  • Antibiotics 44:693-701 an antibacterial agent. This particular case therefore describes the general applicability of the present invention, since very little information is available on the donor organism, on its genetics and physiology, and on the gene clusters present in its genome. Further examples described herein illustrate the application of the principles and methodologies of the present invention to other gene clusters described in the literature.
  • the present invention relating to a general method for transferring the capability to produce any natural product from the original actinomycete to an established and genetically manipulatable Streptomyces host, can be schematized in a series of passages summarized as: 1) design of suitable vectors; 2) construction of a large- insert library in said vectors; 3) selection of the desired clones with appropriate probes; 4) insertion of the selected clones into a convenient Streptomyces host; and 5) growth of the recombinant strain under appropriate conditions to produce the natural product.
  • Actinomycetes produce a large number of natural products with important applications.
  • Important classes of microbial producers include, among others, filamentous fungi, bacilli, mixobacteria, pseudomonas and cyanobacteria .
  • the series of passages described above can therefore be applied to other important classes of microbial producers, provided that two requisites are met: the synthesis of the desired natural product is governed by a gene cluster; suitable production host(s) exist; and appropriate selective marker (s) and maintenance function (s) are introduced into the Artificial Chromosome .
  • a neutral cloning host This host, as described in the present invention, is the bacterium Escherichia coli . In a preferred example of such a host, a high cloning efficiency can be obtained, and many of the analyses of the ESACs can be quickly performed. However, it is evident to one of ordinary skill in this art that any other host that allows high cloning efficiency can be used as neutral cloning host. Additionally, the use of such a host is not a conditio sine qua non for the applicability of the present invention. In fact, when it is possible to establish directly a library in a production host, there is no need for an intermediate neutral cloning host.
  • the present invention consists of a method for transferring the production of a natural product from an actinomycete donor organism that is the original producer of said natural product to a different actinomycete host, where this transfer is achieved by means of an E . coli-Streptomyces Artificial Chromosome that carries a gene cluster governing the biosynthesis of said natural product derived from said donor organism.
  • This method comprises the steps of :
  • step (c) constructing an E . coli-Streptomyces Artificial Chromosome by inserting said large fragments of chromosomal DNA of step (a) into the above said vector of step (b) and selecting the E . coli-Streptomyces Artificial Chromosome comprising the entire gene cluster construct that directs the biosynthesis of the above said natural product;
  • step (d) transforming an actinomycete host different from the donor actinomycete host with the E . col i -Streptomyces Artificial Chromosome of step (c) that carries the gene cluster governing the biosynthesis of said natural product wherein the actinomycete host carries a region which is specific for the integration of the E. col i -Streptomyces Artificial Chromosome.
  • Plasmids, Bacterial Strains and Growth Conditions Plasmids pUCBM20, pUCBM21, pBR322 and pUC18 are obtained from Boheringer Mannheim; plasmid pIJ39 and ⁇ C31 DNA have been described (Hopwood et al . , 1985, Genetic Manipula tion of Streptomyces : A Labora tory Manual , The John Innes Foundation, Norwich, UK) and are available from prof. David Hopwood, The John Innes Centre, Norwich, UK; plasmid pCYPAC2 has been described (Ioannou et al . , 1994, Nature Genetics 6:84-89) and is available from prof.
  • plasmid pMAK705 has been described (Hamilton, et al . , 1989, J. Bacteriol, 171:4617) and is available from prof. Sidney Kushner, University of Georgia, Athens, USA; cosmid Lorist6 has been described (Gibson et al . , 1987, Gene, 53:283-286) and is from prof. Stewart Cole, Pasteur Institute, Paris, France.
  • E coli strains are obtained from commercial sources: DH5 ⁇ (Life Technologies), DH10B (Life Technologies) , C600 ⁇ E .
  • Planobispora rosea ATCC 53733, Streptomyces hygroscopicus ATCC 29253, Amycola topsis mediterranei ATCC 13685 and Saccharopolyspora erythraea NRRL2338 are from the ATCC culture collection. All other materials are from commercial sources. Media for cultivation of E . coli (Sambrook et al . , 1989, In Molecular Cloning: A labora tory Manual , 2nd edn, Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press) and Streptomyces (Hopwood et al .
  • DNA Manipulations are performed following described procedures, using the appropriate E . coli strains as cloning hosts (Sambrook et al . , 1989, In Molecular Cloning: A labora tory Manual , 2nd edn, Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press) . Genomic DNA from actinomycetes is prepared as described (Hopwood et al . , 1985, Genetic Manipula tion of Streptomyces : A Labora tory Manual , The John Innes Foundation, Norwich, UK) . A cosmid library of P. rosea DNA is constructed in the cosmid vector Lorist6 following published procedures (Sambrook et al .
  • Probes Pep6 and Pep8 are derived from conserved motifs in peptide synthetase gene sequences (Turgay and Marahiel, 1994, Pept . Res. 7:238-241). Oligonucleotide probe Pep6 consists of an equimolar mixture of 5 ' -GCSTACATCATCTACACSTCSGGSACSACS-GGSAAGCCSAAGGG-
  • Oligonucleotide probe Pep8 consists of an equimolar mixture of 5 ' -AKGCTGTCSCCSCCSAGSNNGAAG-AAGTYGTCGTCGATSCC-3 ' ( SEQID N°3) and 5 ' -AKGGAGTCSCCSCCSAGSNNGAAGAAGTYGTCGTCGATSCC- 3' (SEQID N°4) .
  • S indicates G or C
  • K indicates G or T
  • N any base
  • Hybridizations are performed with a hybridization stringency set at 2xSSC, 55 °C, and a final wash set at the same stringency.
  • Preparation of high molecular weight DNA Procedures for the preparation of high molecular weight DNA from actinomycetes for PFGE have been described (Dyson, 1993, Trends Genet. 9:72; Kieser et al . , 1992, J. Bacteriol . 174:5496-5507). They are modified for constructing libraries as described in the Examples.
  • the present invention consists in a series of passages, involving the design of suitable vectors; the introduction of large DNA inserts in said vectors employing genomic DNA from the donor organism; the selection of clones carrying the cluster specifying the synthesis of the desired natural product; the introduction of selected clone (s) into the appropriate production host; and the growth of the recombinant strain under appropriate conditions for metabolite production.
  • These passages are described in detail in the Examples reported herein. These Examples outline the steps necessary to accomplish each passage, for the overall purpose of the present invention: the production of a natural product in a different host. They serve to illustrate the principles and methodologies of the present invention, and are not meant to restrict its scope to the Examples specified herein.
  • Bacterial Artificial Chromosomes are circular plasmids that can be easily propagated in and prepared from E . coli cells by standard miniprep methods (Shizuya et al . , 1992, Proc.
  • Site-specific integration therefore allows the introduction of foreign DNA in single copy at a defined genetic locus.
  • Several systems capable of directing site-specific integration of incoming circular DNA into the chromosome of a Streptomyces host have been described.
  • a convenient system that can be used in the present invention is for istance the int-a ttP system derived from the temperate bacteriophage ⁇ C31 (Kuhstoss and Rao, 1991, J. Mol . Biol . 222:897-908), which directs, during lysogen formation, integration of the 41-kb phage genome at the a t tB site, located in a stable segment of the S . coelicolor chromosome (Redenbach et al .
  • Vectors pPAC-Sl and pPAC-S2 are 22 kb in size and differ solely for the orientation of the int- tsr cassette. After release of the stuffer pUC19 segment, the vector size is reduced to 19.7 kb .
  • the vector When cloning in the BamHI site, the vector can be released by digestion with Dral , resulting in vector fragments of 7.4, 4.2 and 0.6 kb . The additional 7.5 kb of vector DNA will be associated with the insert. Dral rarely cuts in the high-GC genome of actinomycetes, so that the insert size can be easily calculated.
  • Two pairs of PCR primers 5 ' -TTTTTGGTACCTGACGTCCCGAAGG- CGTG-3' (SEQID N°5) and 5 ' -CAGCTTGTCCATGGCGGA-3 ' (SEQID N°6); and 5 ' -TCTGTCCGCCATGGACAAGC-3 ' (SEQID N°7) and 5'- TTTTTGGATCCGGCTAACTAACTAAACCGAGA-3' (SEQID N°8), are used to amplify the in -containing fragments of 1.3 and 0.9 kb, respectively.
  • the template is ⁇ C31 DNA.
  • the amplified fragments are digested with Kpnl + Ncol and Ncol + BamRI , respectively, and recovered from an agarose gel.
  • Plasmid pI ⁇ T The 1.3 and 0.9 kb fragment, prepared as described in Example 1, are ligated to pUCMB21, digested with Kpnl + BamRI . The resulting mixture contains the desired plasmid pI ⁇ T.
  • plasmid pINT prepared as described in Example 2, are used to transform E . coli DH5 ⁇ and a few of the resulting Ap R colonies that appear on the LB-agar plates are analyzed for their plasmid content.
  • One colony is found to carry pINT, as verified by the observation, upon agarose gel-electrophoresis, of fragments of 4.0 and 0.9 kb after digestion of the plasmid with Ncol + BamRI .
  • Plasmids pUITl The 1.8 kb BamRI fragment containing the tsr gene is isolated from pIJ39 and ligated to pI ⁇ T, prepared as described in Example 3 and previously digested with BainHI . The resulting mixture contains the desired plasmids pUITl.
  • plasmid pUITl prepared as described in Example 4, are used to transform E . coli DH5 ⁇ and a few of the resulting Ap R colonies that appear on the LB-agar plates are analyzed for their plasmid content.
  • One colony is found to carry pUITl, as verified by the observation, upon agarose gel-electrophoresis, of fragments of 4.9 and 1.8 kb after BamRI digestion of the plasmid.
  • the 3.7 kb Apal fragment, containing the int- tsr cassette, is isolated from plasmid pUITl, prepared as described in Example 5, and ligated to pUCBM21 digested with Apal. The resulting mixture contains the desired plasmid pUIT3.
  • plasmid pUIT3 prepared as described in Example 6, are used to transform E . coli DH5 ⁇ and a few of the resulting Ap R colonies that appear on the LB-agar plates are analyzed for their plasmid content.
  • One colony is found to carry pUIT3, as verified by the observation, upon agarose gel-electrophoresis, of fragments of of 4.2 and 2.2 kb after BamRI digestion of the plasmid.
  • Plasmid pUIT3 prepared as described in Example 7, is partially digested with BamRI , followed by filling-in of the resulting ends, and treated with DNA ligase. The resulting mixture contains the desired plasmid pUIT4.
  • plasmid pUIT4 prepared as described in Example 8, are used to transform E . coli DH5 ⁇ and a few of the resulting Ap R colonies that appear on the LB-agar plates are analyzed for their plasmid content.
  • One colony is found to carry pUIT4, as verified by the observation, upon agarose gel-electrophoresis, of a 6.4 kb fragment after BamRI digestion of the plasmid.
  • the 3.7 kb Apal fragment from pUIT4, prepared as described in Example 9, is mixed with pCYPAC2, previously digested with Nhel . After filling-in of the ends, DNA ligase is added. The resulting mixture contains the desired plasmids pPAC-Sl and pPAC-S2.
  • E . coli K12 DHlOB/pPAC-Sl and DH10B/pPAC-S2 Approximately 10 ng of plasmids pPAC-Sl and pPAC-S2, prepared as described in Example 10, are used to transform E . coli DH10B and a few of the resulting Km R colonies that appear on the LB-agar plates are analyzed for their plasmid content. One colony is found to carry pPAC-Sl, as verified by the observation, upon agarose gel-electrophoresis, of fragments of 8.1, 4.8, 4.6, 2.2, 2.2, 0.5 and 0.1 kb after EcoRI digestion of the plasmid.
  • the first methodology can be referred to as the top-down approach and is depicted in Fig. 3. It consists of directly cloning the desired gene cluster into a pESAC through the construction of a genomic library of DNA fragments of average size of 100 kb, or more. The library is then screened with suitable probes (Section 7.3) in order to identify the desired cluster.
  • the second methodology can be considered a bottom- up approach and is illustrated in Fig. 4. It consists of assembling the desired gene cluster from pre-existing smaller segments of cloned, overlapping DNA, through the iterative use of homologous recombination in E . coli .
  • the desired overlapping clones encompass the desired gene cluster and are identified as described in Section 7.3. Both methodologies fall within the scope of this invention. Depending on factors such as previous knowledge about the biosynthesis cluster, the extent of characterization of the producing strain, the existence of other natural products of interest produced by the original microorganism, one methodology may be preferred over the other. However, the two methodologies are not mutually exclusive.
  • the growth time should allow formation of a sufficient quantity of biomass; however, long incubation times should be avoided, since mycelia are generally more resistant to lysis as they age.
  • the mycelium is pelleted, washed and embedded in agarose for the subsequent lytic steps. Lysis of the cells is achieved by a combination of enzymatic (e.g., incubation with lysozyme and/or achromopeptidase) and mild physical treatments (e.g., SDS) .
  • the concentrations of reagents and the incubation times need to be optimized for each strain.
  • a good starting point is represented by the conditions described in Example 12.
  • the quality of the DNA preparation is checked by PFGE under appropriate conditions.
  • the DNA can be digested as described in Example 13.
  • the exact incubation time and the units of restriction endonuclease are adjusted to the particular DNA preparation for optimizing the size and yield of the bulk of digested DNA, which should exceed 150 kb .
  • the partially digested DNA is size-fractionated on a PFGE gel, without exposure to ethidium bromide or UV light, in order to avoid damage to the DNA.
  • the gel slice containing the desired DNA fraction is localized by staining the marker-containing portion of the gel and cut. All subsequent manipulations are performed with great care (Birren and Lai, 1993, Pulsed Field Gel Electrophoresis : A Practical Guide, Academic Press, New York, NY) .
  • the size-selected DNA is ligated to an appropriately prepared pESAC (see Example 14) employing a high molar excess of vector to insert (ca. 10:1) in order to minimize the formation of chimeric clones (i.e. those constituted by the religation of two uncontiguous inserts) . Subsequent steps are performed using published procedures for the cloning in Bacterial Artificial Chromosomes, as described in Examples 16 and 17.
  • ESAC DNA is prepared from a representative number of clones obtained after electroporation of a ligation mixture and analyzed for determining the frequency of insert-carrying clones and their average size.
  • all insert containing clones can be analyzed by miniprep procedure (Birren and Lai, 1993, Pulsed Field Gel Electrophoresis : A Practical Guide, Academic Press, New York, NY, USA) and clones carrying inserts below a certain threshold can be discarded.
  • the number of clones carrying insert of the appropriate size can be estimated after analysis of a representative number of ESACs .
  • the quality of the library can be evaluated by probing with cloned genes from the strain (if available) , or from highly conserved "housekeeping" genes from a strain with a similar GC content, such as S. coelicolor.
  • S. coelicolor strain M145 is grown in YEME medium containing 0.5% (wt/vol) glycine for 40 h at 30°C on an orbital shaker (ca. 200 rpm) .
  • the mycelium is pelleted by centrifugation, washed with 10.3% sucrose and the chromosomal DNA is extracted from the mycelium embedded in 0.75% LMP agarose by treatment with 1 mg/ml lysozyme and with 1 mg/ml proteinase K in 0.1% SDS for 40 h at 50°C.
  • coelicolor M145 chromosomal DNA prepared as described in Example 12 and embedded in LMP agarose plugs, is partially digested by limiting the magnesium concentration for 20 min with 4 U of Sau3AI .
  • the resulting DNA fragments are resolved by PFGE and the size-selected genomic DNA fraction (larger than 100 kb) is recovered and released from the agarose gel by digestion with gelase.
  • the vector pPAC-Sl prepared as described in Example 11, is cut with Seal and then treated with calf intestinal phosphatase.
  • the recovered DNA is then digested with BamRI and treated with an excess of calf intestinal phosphatase.
  • the short Scal-BamHI linker fragments are removed by spin dialysis .
  • the resulting ligation mixture contains the desired ESAC library, consisting of fragments S. coelicolor DNA inserted into the pPAC-Sl vector.
  • the ligation mixture prepared as described in Example 15, is drop-dialyzed against 0.5 X TE for 2 h using 0.025 mm type VS membranes (Millipore) and a few ⁇ l are used to electroporate 40 ⁇ l of electrocompetent E . coli DH10B cells.
  • the electroporation conditions are: 2.5 kV, 100 ⁇ and 25 mFa employing the Biorad Gene Pulser II.
  • the cells are plated on LB-agar plates containing 25 ⁇ g/ml Km and 5% sucrose to select for recombinant cells harboring insert- carrying pPAC-Sl.
  • ESAC DNA is isolated using the alkaline extraction procedure (Sambrook et al . , 1989, In Molecular Cloning: A labora tory Manual , 2nd edn, Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press)) without the phenol extraction step.
  • the DNA is analyzed, after digestion with Dral, by PFGE. Three bands of 7.4, 4.2 and 0.6 kb are common to all clones and represent vector DNA.
  • an ESAC library of the rapamycin producer Streptomyces hygroscopicus ATCC 29253 can be constructed, employing the procedures reported for PFGE analysis (Ruan et al., 1997, Gene 203:1-9) and applying the principles and methodologies described in Examples 12 through 17.
  • strains from actinomycete genera other than Streptomyces can be used as a source of DNA for constructing an ESAC library. These strains can belong to any genus of the order Actinomycetales , which include but are not limited to the genera reported in Table 1.
  • an ESAC library of the erythromycin producer Saccharopolyspora erythraea can be constructed, employing the procedures reported for PFGE analysis (Reeves et al . , 1998, Microbiology 144:2151-2159) and applying the principles and methodologies described in Examples 12 through 17.
  • Amy cola tops is Ex iguoba cterium Micro raspora
  • the bottom-up strategy of assembling large fragments from a set of pre-existing smaller segments of partially overlapping DNA cloned from the genome of the actinomycete donor organism is described in this section.
  • This methodology makes use of the same pESAC described in the present invention under Section 7.1.
  • the desired cluster is assembled from existing partially overlapping clones by the iterative use of homologous recombination in E . coli .
  • clones include leftward fragment "A" unique to clone 1; fragment “B” common to clones 1 and 2; fragment “C” common to clones 2 and 3; and rightward fragment “D” unique to clone 3.
  • These fragments can range from a few hundred bp to a few kb, and are thus amenable to routine in vitro DNA manipulations.
  • the number of overlapping clones encompassing the cluster may vary. However, if n is the number of overlapping clones that cover the desired genomic segment, the number of fragments to consider will be equal to n + 1. In the example illustrated in Fig. 5, four fragments are required.
  • Fragments A and B are cloned in a ts vector, as shown in Fig. 6, which carries a selectable marker, Cm R as exemplified in Fig. 6. The same is done with fragment pairs B-C and C-D (Fig. 6) .
  • the relative orientation of each fragment pair in the ts vector must be the same as in the gene cluster.
  • the fragments in each pair may be separated by a selectable marker, designated Ab R in Fig. 6, to monitor interplasmid insert exchange. Therefore, three constructs in the ts vector, designated pABl, pAB2 and pAB3, are required.
  • the A-B-C-D four-fragment cassette is cloned in a pESAC (Fig. 6) .
  • the relative orientation of the four fragments in the pESAC must be the same as in the gene cluster.
  • a selectable marker may separate any of two fragments to monitor interplasmid insert exchange.
  • the original clone for example, a cosmid, which carries a selectable marker, Km R as exemplified in Fig. 7
  • the original clone for example, a cosmid, which carries a selectable marker, Km R as exemplified in Fig. 7
  • the original clone containing part of the cluster and the cognate ts construct (Fig. 7) are introduced into the same E . coli cell.
  • the interplasmid cointegrate between the original clone and the ts construct is selected at the non-permissive temperature for the ts replicon.
  • a selectable marker is introduced in the growing ESAC between the next fragment pair, again through the use of two rounds of single, reciprocal homologous recombination mediated by plasmid pBCl, leading to the appearance of Km R Ab R colonies.
  • the interplasmid exchange with pBC2 leads to the introduction of the genomic segment comprised between fragments B and C.
  • the use of pCDl first and subsequently of pCD2 leads to the reconstruction of the genomic segment into the pESAC. Therefore, through the use of alternating steps, the Ab R marker first and the genomic segment later are introduced between any fragment pair, as schematized in Fig. 8.
  • a cosmid library of P. rosea DNA prepared in the vector Lorist6 is screened with oligonucleotide probes Pep ⁇ and Pep ⁇ , according to the conditions described under Section 6. Among the positive colonies identified, several cosmids were found to span the ca . 90 kb segment of the P. rosea chromosome reported in Fig. 9. Signature sequences close to the left and right ends of this segment are reported as SEQID N°9 and SEQID N°10, respectively. Three cosmids are chosen for further studies.
  • Cosmids pRP16, pRP31 and pRP58 exhibits, after digestion with BamRI and resolution by agarose gel-electrophoresis, fragments of 7.5, 7.2, 5.6, 5.2, 2.7, 2.0, 1.9, 1.9, 1.8, 1.6, 1.4, 0.9 and 0.7 kb; of 10.5, 6.2, 3.1, 2.8, 2.6, 2.5, 2.1, 1.9, 1.9, 1.5, 1.4, 1.2, 1.0, 1.0, 0.9, 0.9, 0.7, 0.6, 0.5, 0.5, 0.1 and 0.1 kb; and of 10.0, 7.6, 6.7, 6.2, 3.4, 3.0, 2.8, 2.1, 1.0, 1.0, 0.9, 0.9, 0.7, 0.6, 0.5, 0.5, 0.3 and 0.1 kb; respectively.
  • the 0.9 kb Smal-Sstl fragment comprised between map coordinates 2.0-2.9 kb of Fig. 9, is obtained from cosmid pRP16, prepared as described in Example 18, and ligated to pUC18 previously digested with Sstl and Yale .
  • the resulting mixture contains the desired plasmid pUAl .
  • plasmid pUAl prepared as described in Example 19, are used to transform E . coli XLlblue and a few of the resulting Ap R colonies that appear on the LB-agar plates are analyzed for their plasmid content.
  • One colony is found to carry pUAl, as verified by the observation, upon agarose gel-electrophoresis, of fragments of 2.7 and 0.9 kb after digestion of the plasmid with BamHI + Sstl.
  • the resulting mixture contains the desired plasmid pUA2.
  • E. coli K12 XLlblue/pUA2 Approximately 10 ng of plasmid pUA2 , prepared as described in Example 21, are used to transform E . coli XLlblue and a few of the resulting Ap R colonies that appear on the LB-agar plates are analyzed for their plasmid content. One colony is found to carry pUA2, as verified by the observation, upon agarose gel-electrophoresis, of fragments of 2.7 and 0.9 kb after digestion of the plasmid with BcoRI + Sstl.
  • Plasmid pUBl The 1.8 kb Sstl-BamHI fragment, comprised between map coordinates 33.4-35.2 of Fig. 9, is obtained from cosmid pRP16, prepared as described in Example 18, and ligated to pUCl ⁇ previously digested with Sstl + BamHI .
  • the ligation mixture contains the desired plasmid pUBl .
  • plasmid pUBl prepared as described in Example 23, are used to transform E. coli XLlblue and a few of the resulting Ap R colonies that appear on the LB-agar plates are analyzed for their plasmid content.
  • One colony is found to contain plasmid pUBl as verified by the observation, upon agarose gel electrophoresis, of fragments 2.7 and 1.8 kb after digestion with Sstl + Xbal .
  • the 6.2 kb BamHI fragment comprised between map coordinates 54.2-60.4 kb of Fig. 9, is obtained from cosmid pRP58, prepared as described in Example 18, and ligated to pUC18 previously digested with BamHI.
  • the ligation mixture contains the desired plasmid pUCl.
  • plasmid pUCl prepared as described in Example 25, are used to transform E . coli XLlblue and a few of the resulting Ap R colonies that appear on the LB-agar plates are analyzed for their plasmid content.
  • One colony is found to carry pUCl, as verified by the observation, upon agarose gel-electrophoresis, of fragments of 4.9 and 4.0 kb after digestion of the plasmid with Pstl.
  • N°ll and 5 ' -CCCCCAAGCTTA-3 ' are annealed and ligated to the 1.5 kb Pstl-BamHI fragment, comprised between map coordinates 89.5-91.0 kb of Fig. 9 and obtained from cosmid pRP58, prepared as described in Example 18.
  • the ligation mixture is digested with Bindlll and ligated to pUC18 previously digested with Pstl + Bindlll.
  • the resulting mixture contains the desired plasmid pUDl .
  • E . coli K12 XLlblue/pUDl Approximately 10 ng of plasmid pUDl, prepared as described in Example 27, are used to transform E. coli XLlblue and a few of the resulting Ap R colonies that appear on the LB-agar plates are analyzed for their plasmid content. One colony is found to contain plasmid pUDl as verified by the observation, upon agarose gel-electrophoresis, of fragments of 2.7 and 1.5 kb after digestion with Pstl + Hindlll.
  • the 0.9 kb EcoRI-Sstl fragment from plasmid pUA2 prepared as described in Example 22, and the 1.8 kb Sstl-BamHI fragment from pUBl, prepared as described in Example 24, are ligated to pUC18 previously digested with BcoRI + BamRI .
  • the ligation mixture contains the desired plasmid pUABl.
  • E . coli K12 XLlblue/pUABl Approximately 10 ng of plasmid pUABl, prepared as described in Example 29, are used to transform E . coli XLlblue and a few of the resulting Ap R colonies that appear on the LB-agar plates are analyzed for their plasmid content. One colony is found to carry pUABl, as verified by the observation, upon agarose gel-electrophoresis, of two fragments of 2.7 kb after digestion of the plasmid with BcoRI + Xbal .
  • the 1.6 kb fragment containing the tet gene is isolated after PCR amplification of pBR322 DNA using oligonucleotides 5 ' -GAGCTCTCATGTTTGACAGCT-3 ' (SEQID N°13) and 5'-GAGCTCTGACTTCCGCGTTTCCAG-3' (SEQID N°14) as primers, followed by digestion with Sstl.
  • Plasmid pUABl prepared as described in Example 30, is digested with Sstl and ligated to the tet fragment prepared as described in Example 31.
  • the ligation mixture contains the desired plasmid pUAB2.
  • plasmid pUAB2 prepared as described in Example 32, are used to transform E . coli DH5 ⁇ and a few of the resulting Tc R Ap R colonies that appear on the LB-agar plates are analyzed for their plasmid content.
  • One colony is found to carry pUAB2, as verified by the observation, upon agarose gel-electrophoresis, of fragments of 4.3 and 2.7 kb after digestion of the plasmid with EcoRI + Xbal .
  • the 1.8 kb Sstl-Xfoal fragment obtained from plasmid pUBl, prepared as described in Example 24, and the 4.0 kb Xbal- Pstl fragment obtained from plasmid pUCl, prepared as described in Example 26, are ligated to pUCl ⁇ previously digested with Sstl + Pstl.
  • the ligation mixture contains the desired plasmid pUBCl.
  • plasmid pUBCl prepared as described in Example 34, are used to transform E. coli XLlblue and a few of the resulting Ap R colonies that appear on the LB-agar plates are analyzed for their plasmid content.
  • One colony is found to carry pUBCl, as verified by the observation, upon agarose gel-electrophoresis, of fragments of 5.8 and 2.7 kb after digestion of the plasmid with BcoRI + Hindlll .
  • Plasmid pUBCl prepared as described in Example 35 and previously digested with Xbal, and the tet fragment, prepared as described in Example 31, are treated with T4 DNA polymerase and T4 DNA ligase.
  • the ligation mixture contains the desired plasmid pUBC2.
  • plasmid pUBC2 prepared as described in Example 36, are used to transform E . coli DH5 ⁇ and a few of the resulting Tc R Ap R colonies that appear on the LB-agar plates are analyzed for their plasmid content.
  • One colony is found to carry pUBC2, as verified by the observation, upon agarose gel-electrophoresis, of fragments of 5.6 and 4.5 kb after digestion of the plasmid with Hindlll.
  • Example 39 The 4.0 kb Xbal-Pstl fragment obtained from plasmid pUCl, prepared as described in Example 26, and the 1.5 kb Pstl- Hindlll fragment isolated from plasmid pUDl, prepared as described in Example 28, are ligated to pUCl ⁇ previously digested with Xbal and Hindlll. The mixture contains the desired plasmid pUCDl .
  • Example 39 The 4.0 kb Xbal-Pstl fragment obtained from plasmid pUCl, prepared as described in Example 26, and the 1.5 kb Pstl- Hindlll fragment isolated from plasmid pUDl, prepared as described in Example 28, are ligated to pUCl ⁇ previously digested with Xbal and Hindlll. The mixture contains the desired plasmid pUCDl .
  • plasmid pUCDl prepared as described in Example 36, are used to transform E . coli XLlblue and a few of the resulting Ap R colonies that appear on the LB-agar plates are analyzed for their plasmid content.
  • One colony is found to carry pUCDl, as verified by the observation, upon agarose gel-electrophoresis, of fragments of 5.5 and 2.7 kb after digestion of the plasmid with Xbal + Hindlll.
  • Plasmid pUCDl prepared as described in Example 39 and previously digested with Pstl, and the tet fragment prepared as described in Example 31, are treated with T4 DNA polymerase and T4 DNA ligase.
  • the ligation mixture contains the desired plasmid pUCD2.
  • plasmid pUCD2 prepared as described in Example 40, are used to transform E . coli DH5 ⁇ and a few of the resulting Tc R Ap R colonies that appear on the LB-agar plates are analyzed for their plasmid content.
  • One colony is found to carry pUCD2, as verified by the observation, upon agarose gel-electrophoresis, of fragments of 6.7 and 3.1 kb after digestion of the plasmid with Hindlll.
  • the 4.3 kb £coRI-XfoaI fragment obtained from plasmid pUAB2, prepared as described in Example 33, and the 5.5 Xbal- Hindl ⁇ l fragment from plasmid pUCDl, prepared as described in Example 39, are ligated to pUCl ⁇ , previously digested with BcoRI + Hindlll.
  • the ligation mixture contains the desired plasmid pUADl .
  • plasmid pUADl prepared as described in Example 42, are used to transform E . coli DH5 ⁇ and a few of the resulting Tc R Ap R colonies that appear on the LB-agar plates are analyzed for their plasmid content.
  • One colony is found to carry pUADl, as verified by the observation, upon agarose gel-electrophoresis, of fragments of 8.9 and 3.6 kb after digestion of the plasmid with Hindlll.
  • the 4.3 kb EcoRI-Xbal fragment obtained from plasmid pUAB2 , prepared as described in Example 33, is treated with T4 DNA Polymerase and ligated to pMAK705 previously digested with Hindi .
  • the ligation mixture contains the desired plasmid pMABl .
  • plasmid pMABl prepared as described in Example 44, are used to transform E . coli C600 and a few of the resulting Cm R Tc R colonies that appear on the LB-agar plates are analyzed for their plasmid content.
  • One colony is found to carry pMABl, as verified by the observation, upon agarose gel-electrophoresis, of fragments of 4.1, 3.4, 1.4 and 0.9 kb after digestion of the plasmid with Hindlll + BcoRI .
  • the 7.1 kb fragment from plasmid pUBC2, prepared as described in Example 37, is obtained after partial digestion with Pstl, treated with T4 DNA polymerase and ligated to pMAK705 previously digested with Hindi .
  • the ligation mixture contains the desired plasmid pMBCl .
  • plasmid pMBCl prepared as described in Example 46, are used to transform E . coli C600 and a few of the resulting Cm R Tc R colonies that appear on the LB-agar plates are analyzed for their plasmid content.
  • One colony is found to carry pMBCl, as verified by the observation, upon agarose gel-electrophoresis, of fragments of 9.5, 1.5, 1.3 and 0.3 kb after digestion of the plasmid with BamRI .
  • Plasmid pMCDl The 7.1 kb fragment from plasmid pUCD2, prepared as described in Example 41, is obtained by complete digestion with Xbal and partial digestion with Hindlll, treated with T4 DNA polymerase and ligated to pMAK705, previously digested with Hindi .
  • the ligation mixture contains the desired plasmid pMCDl .
  • plasmid pMCDl prepared as described in Example 46, are used to transform E. coli C600 and a few of the resulting Cm R Tc R colonies that appear on the LB-agar plates are analyzed for their plasmid content.
  • One colony is found to carry pMCDl, as verified by the observation, upon agarose gel-electrophoresis, of fragments of ⁇ .6 and 4.3 kb after digestion of the plasmid with BamHI .
  • Example 50 Construction of plasmid pPADl
  • the ligation mixture contains the desired plasmid pPADl .
  • plasmid pPADl prepared as described in Example 50, are used to transform E . coli C600 and a few of the resulting Km R Tc R colonies that appear on the LB-agar plates are analyzed for their plasmid content.
  • One colony is found to carry pPADl, as verified by the observation, upon agarose gel-electrophoresis, of fragments of 19.7, 5.6, 3.1 and 1.2 kb after digestion of the plasmid with BamHI . After digestion with Dral and resolution by PFGE, pPADl yields fragments of 17.4, 7.4, 4.2 and 0.6 kb .
  • E . coli C600/pMABl prepared as described in Example 45, is transformed with ca . 50 ng of pRP16, prepared as described in Example 18.
  • the Cm R Km R colonies that appear at 30 °C on the LB-agar plates are grown at 30°C in LB broth containing Km and Cm, aliquots are withdrawn at various times and appropriate dilutions plated. Few of the Cm R Km R colonies that appear on the LB-agar plates after overnight incubation at 44°C are grown in LB broth containing Km and Cm for 16 h at 44°C and analyzed for their plasmid content.
  • E . coli C600/pMABl : :pRP16 prepared as described in Example 52, are grown individually in LB broth containing Cm for 24 h at 30°C, diluted 1:100 and incubated for further 8 h. Appropriate dilutions are plated. Few of the resulting Cm R Km s Tc s colonies that appear at 30°C are analyzed for their plasmid content. One colony is found to carry pMAB2 , as verified by the observation, upon agarose gel-electrophoresis, of fragments of 37 and 1.5 kb after digestion of the plasmid with BcoRI .
  • E. coli DHl/pMBCl : :pRP31 prepared as described in Example 54, are grown individually in LB broth containing Cm for 24 h at 30°C, diluted 1:100 and incubated for further 8 h. Appropriate dilutions are plated. Few of the resulting Cm R Km s Tc s colonies that appear at 30°C are analyzed for their plasmid content. One colony is found to carry pMBC2, as verified by the observation, upon agarose gel-electrophoresis, of fragments of 14.4, 14.1 and 1.5 kb after digestion of the plasmid with EcoRI .
  • E . coli K12 DHl/pMCD2 Construction of E . coli K12 DHl/pMCD2 Several colonies of E. coli DHl/pMCDl : :pRP58 , prepared as described in Example 56, are grown individually in LB broth containing Cm for 24 h at 30°C, diluted 1:100 and incubated for further 8 h. Appropriate dilutions are plated. Few of the resulting Cm R Km s Tc s colonies that appear at 30°C are analyzed for their plasmid content. One colony is found to carry pMCD2, as verified by the observation, upon agarose gel-electrophoresis, of fragments of 42 and 1.5 kb after digestion of the plasmid with EcoRI .
  • E . coli C600/pMAB2 prepared as described in Example 53, is transformed with ca . 50 ng of plasmid pPADl, prepared as described in Example 51.
  • the Cm R Km R colonies that appear at 30°C on the LB-agar plates are grown at 30°C in LB broth containing Km and Cm, aliquots are withdrawn at various times and appropriate dilutions plated. Few of the Cm R Km R colonies that appear on the LB-agar plates after overnight incubation at 44°C are grown in LB broth containing Km and Cm for 16 h at 44°C and analyzed for their plasmid content.
  • E . coli C600/pMAB2 : :pPADl prepared as described in Example 58, are grown individually in LB containing Km for 24 h at 30°C, diluted 1:100 and incubated for further 8 h at 44°C. Appropriate dilutions are plated. Few of the resulting Km R Cm s Tc s colonies that appear at 37°C are analyzed for their plasmid content.
  • PAD2 One colony is found to carry PAD2, as verified by the observation, upon agarose gel-electrophoresis, of fragments of 19.7, 7.2, 5.6, 5.5, 5.2, 2.7, 1.9, 1.9, 1.8, 1.8, 1.6, 1.4, 0.9 and 0.7 kb after digestion of the plasmid with BamHI . After Dral digestion and resolution by PFGE, PAD2 yields fragments of 45, 7.4, 4.2 and 0.6 kb .
  • Plasmid pMCD3 The 1.4 kb Kpnl-XhoII fragment obtained from plasmid pCYPAC2 after digestion with XhoII, treatment with T4 DNA polymerase and digestion with Kpnl , and the 7.1 kb Xbal- Hindlll fragment from pUCD2, prepared as described in Example 40 and obtained after partial digestion with Hindlll, complete digestion with Xbal and treatment with T4 DNA polymerase, are ligated to pMAK705, previously digested with Kpnl + Hindi .
  • the ligation mixture contains the desired plasmid pMCD3.
  • plasmid pMCD3 prepared as described in Example 60, are used to transform E. coli C600 and a few of the resulting Cm R Tc R colonies that appear on the LB-agar plates are analyzed for their plasmid content.
  • One colony is found to carry pMCD3, as verified by the observation, upon agarose gel-electrophoresis, of fragments of 9.8 and 4.3 kb after digestion of the plasmid with BamHI .
  • E . coli C600/PAD2, prepared as described in Example 59, is transformed with ca . 10 ng of plasmid pMCD3, prepared as described in Example 61.
  • the Cm R Km R colonies that appear at 30°C on the LB-agar plates are grown at 30°C in LB broth containing Km and Cm, aliquots are withdrawn at various times and appropriate dilutions plated. Few of the Cm R Km R colonies that appear on the LB-agar plates after overnight incubation at 44°C are grown in LB broth containing Km and Cm for 16 h at 44°C and analyzed for their plasmid content.
  • E. coli C600/PAD3 prepared as described in Example 63, is transformed with ca . 50 ng of plasmid pMCD2, prepared as described in Example 57.
  • the Cm R Km R colonies that appear at 30°C on the LB-agar plates are grown at 30°C in LB broth containing Km and Cm, aliquots are withdrawn at various times and appropriate dilutions plated. Few of the Cm R Km R colonies that appear on the LB-agar plates after overnight incubation at 44°C are grown in LB broth containing Km and Cm for 16 h at 44°C and analyzed for their plasmid content.
  • E. coli C600/PAD3 : :pMCD2 prepared as described in Example 64, are grown individually in LB broth containing Km for 24 h at 30°C, diluted 1:100 and incubated for further 8 h at 44°C. Appropriate dilutions are plated. Few of the resulting Km R Cm ⁇ Tc s colonies are analyzed for their plasmid content.
  • E . coli C600/PAD4 prepared as described in Example 65, is transformed with ca . 10 ng of plasmid pMBCl, prepared as described in Example 47.
  • the Cm R Km R colonies that appear at 30°C on the LB-agar plates are grown at 30°C in LB broth containing Km and Cm, aliquots are withdrawn at various times and appropriate dilutions plated. Few of the Cm R Km R colonies that appear on the LB-agar plates after overnight incubation at 44°C are grown in LB broth containing Km and Cm for 16 h at 44°C and analyzed for their plasmid content.
  • E . coli C600/PAD4 : :pMBCl prepared as described in Example 66, are grown individually in LB broth containing Km for 24 h at 30°C, diluted 1:100 and incubated for further 8 h at 44°C. Appropriate dilutions are plated. Few of the resulting Km R Cm s Tc R colonies are analyzed for their plasmid content.
  • E . coli C600/PAD5 prepared as described in Example 67, is transformed with ca . 50 ng of plasmid pMBC2, prepared as described in Example 55.
  • the Cm R Km R colonies that appear at 30°C on the LB-agar plates are grown at 30°C in LB broth containing Km and Cm, aliquots are withdrawn at various times and appropriate dilutions plated. Few of the Cm R Km R colonies that appear on the LB-agar plates after overnight incubation at 44°C are grown in LB broth containing Km and Cm for 16 h at 44°C and analyzed for their plasmid content.
  • E . coli C600/PAD5 : :pMBC2 prepared as described in Example 68, are grown individually in LB broth containing Km for 24 h at 30°C, diluted 1:100 and incubated for further 8 h at 44°C. Appropriate dilutions are plated. Few of the resulting Km R Cm s Tc s colonies are analyzed for their plasmid content.
  • One colony is found to carry the correct ESAC, designated PAD6, as verified by the observation, upon agarose gel-electrophoresis, of fragments of 47, 46, 8.1, 4.6, 2.2, 0.5 and 0.1 kb after digestion of the plasmid with EcoRI . After digestion with Dral and resolution by PFGE, PAD6 yields fragments of 102, 4.2 and 0.6 kb.
  • the cluster of Fig. 9 can be assembled using A-B-C-D fragments other than those specified in the Examples. Any A fragment, such that no useful genes are present to its left (using the orientation of Fig. 9) can be used for assembling the cluster. Similarly, any D fragment, such that no useful genes are present to its right (using the orientation of Fig. 9) can also be used. Furthermore, any fragment common to pRPl ⁇ and pRP31, or to pRP31 and pRP58, can be used in place of the fragments B and C, respectively, described above. It will also occur to those skilled in the art that other methods for obtaining these fragments, such as use of different segments from the cluster of Fig.
  • intermediate vectors other than the pUC- series used in the above Examples, can be used for subcloning fragments A through D, and the use of these intermediate vectors is merely instrumental to the transfer of the fragment pairs into the ts vector. Some or all of the fragment pairs could therefore be cloned directly into a ts vector.
  • cosmids other than pRP16, pRP31 and pRP58 can be used to achieve equivalent results, provided that they encompass the entire gene cluster and they have overlapping segments.
  • pMAK705, Lorist ⁇ and pPAC-Sl are merely examples of ts, cosmid and pESAC, respectively. Any of the several cosmid vectors described in the literature, other ts replicons derived from pMAK705 or other sources, and any of the pESAC other than pPAC-Sl, which include the possible vectors described in Section 7.1, can be used for obtaining equivalent results.
  • ts replicon the purpose of a ts replicon is to select for interplasmid cointegrates at the non-permissive temperature.
  • cointegrate formation can occur between any two replicons, and cointegrate can be isolated after transformation of suitable hosts with a plasmid preparation made from an E . coii cell harboring both replicons. Selection for the antibiotic resistance markers carried by both replicons can lead to the isolation of cointegrates from the resulting transformants .
  • tet marker between the A-B, B-C and C-D fragment pairs serves solely the scope of recognizing insert exchange after resolution of the interplasmid cointegrate.
  • Selectable markers other than tet can be equally effective, as long as they are not present in the vectors.
  • the presence of a selectable marker within the fragment cassettes is not absolutely necessary, as insert exchange can be observed by other methods, such as selective hybridization or PCR.
  • different E. coli hosts other than those used in the above Examples can be also employed.
  • Section 7.2.2 following the principles and methodologies described in Section 7.2.2. Furthermore, it will occur to those skilled in the art that the principles and methodologies of Section 7.2.1 and 7.2.2 can complement each other. For example, after constructing an ESAC library of P. rosea DNA, inserts from individual ESACs may be enlarged by applying the principles and methodologies of Section 7.2.2, using, for example, cosmids overlapping the cognate ESACs.
  • the rapamycin gene cluster from S. hygroscopicus is contained within three overlapping cosmids designated cos58, cos25 and cos2 (Schwecke et al . , 1995, Proc. Natl. Acad. Sci. USA 92:7839-7843).
  • the Examples described herein report the preparation of the appropriate fragments A, B, C and D; the construction of the plasmids containing the A-B, B-C and C-D cassettes; and the cloning approach to obtain constrcuts equivalent to those reported in Fig. 6.
  • Fragments A and B prepared as described in Example 70, are ligated to pUCl ⁇ digested with EcoRI + Xbal .
  • the resulting mixture contains the desired plasmid pURl .
  • E. coli K12 DHl/pURl Approximately 10 ng of plasmid pURl, prepared as described in Example 71, are used to transform E . coli DH1 and a few of the resulting Ap R colonies that appear on the LB-agar plates are analyzed for their plasmid content. One colony is found to carry pURl, as verified by the observation, upon agarose gel-electrophoresis, of fragments of 2.7 and 2.4 kb after digestion of the plasmid with BcoRI + Xbal .
  • Fragments B and C prepared as described in Example 70, are ligated to pUC18 digested with BamHI + Pstl. The resulting mixture contains the desired plasmid pUR2.
  • plasmid pUR2 prepared as described in Example 73, are used to transform E . coli DHl and a few of the resulting Ap R colonies that appear on the LB-agar plates are analyzed for their plasmid content.
  • One colony is found to carry pUR2, as verified by the observation, upon agarose gel-electrophoresis, of fragments of 2.7 and 2.4 kb after digestion of the plasmid with BamHI + Pstl.
  • Fragments C and D prepared as described in Example 70, are ligated to pUCl ⁇ digested with Xjbal + Hindlll. The resulting mixture contains the desired plasmid pUR3.
  • plasmid pUR3 prepared as described in Example 75, are used to transform E . coli DHl and a few of the resulting Ap R colonies that appear on the LB-agar plates are analyzed for their plasmid content.
  • One colony is found to carry pUR3, as verified by the observation, upon agarose gel-electrophoresis, of fragments of 2.7 and 2.1 kb after digestion of the plasmid with BcoRI + Hindlll.
  • Plasmid pURl prepared as described in Example 72 and previously digested with BamHI, and the tet fragment, prepared as described in Example 31, are treated with T4 DNA Polymerase and DNA ligase.
  • the ligation mixture contains the desired plasmid pURll.
  • plasmid pURll prepared as described in Example 77, are used to transform E. coli DHl and a few of the resulting Tc R Ap R colonies that appear on the LB-agar plates are analyzed for their plasmid content.
  • One colony is found to carry pURll, as verified by the observation, upon agarose gel-electrophoresis, of fragments of 3.9 and 2. ⁇ kb after digestion of the plasmid with Hindlll.
  • Plasmid pUR2 prepared as described in Example 74 and previously digested with Xbal, and the tet fragment, prepared as described in Example 31, are treated with T4 DNA Polymerase and DNA ligase.
  • the ligation mixture contains the desired plasmid pUR21.
  • plasmid pUR21 prepared as described in Example 79, are used to transform E . coli DHl and a few of the resulting Tc R Ap R colonies that appear on the LB-agar plates are analyzed for their plasmid content.
  • One colony is found to carry pUR21, as verified by the observation, upon agarose gel-electrophoresis, of fragments of 3.9 and 2.8 kb after digestion of the plasmid with Hindlll.
  • Example 81 is found to carry pUR21, as verified by the observation, upon agarose gel-electrophoresis, of fragments of 3.9 and 2.8 kb after digestion of the plasmid with Hindlll.
  • Plasmid pUR3, prepared as described in Example 76 and digested with Pstl, and the tet fragment, prepared as described in Example 31, are treated with T4 DNA Polymerase and DNA ligase.
  • the ligation mixture contains the desired plasmid pUR31.
  • plasmid pUR31 prepared as described in Example 81, are used to transform E . coli DHl and a few of the resulting Tc R Ap R colonies that appear on the LB-agar plates are analyzed for their plasmid content.
  • One colony is found to carry pUR31, as verified by the observation, upon agarose gel-electrophoresis, of fragments of 3.9 and 2.5 kb after digestion of the plasmid with Hindlll.
  • the 4.0 kb EcoRI-Xfoal fragment obtained from plasmid pURll, prepared as described in Example 78, and the 2.1 kb Xbal- Hindlll fragment obtained from plasmid pUR3, prepared as described in Example 76, are ligated to pUCl ⁇ digested with BcoRI + Hindlll.
  • the ligation mixture contains the desired plasmid pUR13.
  • plasmid pUR13 prepared as described in Example 63, are used to transform E . coli DHl and a few of the resulting Tc R Ap R colonies that appear on the LB-agar plates are analyzed for their plasmid content.
  • One colony is found to carry pUR13, as verified by the observation, upon agarose gel-electrophoresis, of fragments of 4.9 and 3.9 kb after digestion of the plasmid with Hindlll.
  • the plasmids constructed above can be used for transfering the two-fragment cassettes present in pURll, pUR21 and pUR31 into a ts vector. This can be achieved by recovering the 4.0 kb insert from pURll, the 4.0 kb insert from pUR21, and the 3.7 kb insert from pUR31, after digestion with BcoRI + Xbal, EcoRI + Pstl, and BcoRI + Ndel , respectively.
  • the 6.1 kb four-fragment cassette present in plasmid pUR13 can be easily transfered into pPAC-Sl after digestion with BcoRI + Ndel .
  • Synthetic oligonucleotides 5 ' -CATGGGAATTCGGGGG-3 ' (SEQID N°23) and 5 ' -CCCCCGAATTCC-3 ' (SEQID N°24) are annealed and ligated to the 1.2 kb NcoI-BamHI fragment isolated from cosmid p3B2. The resulting mixture is digested with EcoRI + BamHI .
  • Fragment A prepared as described in Example 85, and fragment B, prepared as described in Example 86, are ligated to pUCl ⁇ digested with EcoRI + XJbal .
  • the resulting mixture contains the desired plasmid pUEl.
  • plasmid pUEl prepared as described in Example 87, are used to transform £. coli DHl and a few of the resulting Ap R colonies that appear on the LB-agar plates are analyzed for their plasmid content.
  • One colony is found to carry pURl, as verified by the observation, upon agarose gel-electrophoresis, of fragments of 2.7 and 2.2 kb after digestion of the plasmid with EcoRI + Xbal .
  • Fragments B and C prepared as described in Example 86, are ligated to pUCl ⁇ digested with BamHI + Pstl. The resulting mixture contains the desired plasmid pUE2.
  • plasmid pUE2 prepared as described in Example 89, are used to transform E. coli DHl and a few of the resulting Ap R colonies that appear on the LB-agar plates are analyzed for their plasmid content.
  • One colony is found to carry pUE2, as verified by the observation, upon agarose gel-electrophoresis, of fragments of 2.7 and 2.1 kb after digestion of the plasmid with BamHI + Pstl.
  • Fragments C and D prepared as described in Example 86, are ligated to pUCl ⁇ digested with Xbal + Hindlll. The resulting mixture contains the desired plasmid pUE3.
  • E. coli K12 DHl/pUE3 Approximately 10 ng of plasmid pUE3, prepared as described in Example 91, are used to transform E. coli DHl and a few of the resulting Ap R colonies that appear on the LB-agar plates are analyzed for their plasmid content. One colony is found to carry pUE3, as verified by the observation, upon agarose gel-electrophoresis, of fragments of 2.7 and 2.2 kb after digestion of the plasmid with EcoRI + Hindlll.
  • Plasmid pUEl prepared as described in Example ⁇ and previously digested with BamHI, and the tet fragment, prepared as described in Example 31, are treated with T4 DNA Polymerase and DNA ligase.
  • the ligation mixture contains the desired plasmid pUEll.
  • E . coli K12 DHl/pUEll Approximately 10 ng of plasmid pUEll, prepared as described in Example 93, are used to transform E. coli DHl and a few of the resulting Tc R Ap R colonies that appear on the LB-agar plates are analyzed for their plasmid content. One colony is found to carry pUEll, as verified by the observation, upon agarose gel-electrophoresis, of fragments of 3.9 and 2.6 kb after digestion of the plasmid with Hindlll.
  • Plasmid pUE2 prepared as described in Example 90 and previously digested with Xbal , and the tet fragment, prepared as described in Example 31, are treated with T4 DNA Polymerase and DNA ligase.
  • the ligation mixture contains the desired plasmid pUE21.
  • plasmid pUE21 prepared as described in Example 95, are used to transform £. coli DHl and a few of the resulting Tc R Ap R colonies that appear on the LB-agar plates are analyzed for their plasmid content.
  • One colony is found to carry pUE21, as verified by the observation, upon agarose gel-electrophoresis, of fragments of 3.7 and 2.7 kb after digestion of the plasmid with Hindlll.
  • Plasmid pUE3, prepared as described in Example 92 and digested with Pstl, and the tet fragment, prepared as described in Example 31, are treated with T4 DNA Polymerase and DNA ligase.
  • the ligation mixture contains the desired plasmid pUE31.
  • plasmid pUE31 prepared as described in Example 97, are used to transform E. coli DHl and a few of the resulting Tc R Ap R colonies that appear on the LB-agar plates are analyzed for their plasmid content.
  • One colony is found to carry pUE31, as verified by the observation, upon agarose gel-electrophoresis, of fragments of 3. ⁇ and 2.7 kb after digestion of the plasmid with Hindlll.
  • plasmid pUE13 prepared as described in Example 99, are used to transform E. coli DHl and a few of the resulting Tc R Ap R colonies that appear on the LB-agar plates are analyzed for their plasmid content.
  • One colony is found to carry pUE13, as verified by the observation, upon agarose gel-electrophoresis, of fragments of 4.8 and 3.9 kb after digestion of the plasmid with Hindlll.
  • the plasmids constructed above can be used for transfering the two-fragment cassettes present in pUEll, pUE21 and pUE31 into a ts vector. This can be achieved by recovering the 3.8 kb insert from pUEll, the 3.7 kb insert from pUE21, and the 3. ⁇ kb insert from pUE31, after digestion with EcoRI + Xbal, EcoRI + Pstl, and EcoRI + Ndel , respectively.
  • the 6.0 kb four-fragment cassette present in plasmid pUE13 can be easily transfered into pPAC-Sl after digestion with EcoRI + Ndel .
  • TTTTTAAGCTTCAACAAGCCATCCGGGTC-3' (SEQID N°38), are used to amplify fragments of 1.2, 1.2, 1.2 and 1.0 kb, respectively, from A. medi terranei genomic DNA. Fragments A, B, C and D are then digested with EcoRI + BamRI , BamHI + Xbal, Xbal + Pstl, and Pstl + Hindlll, respectively.
  • fragments generated from the rifamycin gene cluster contain the same restriction sites as those generated from the rapamycin and erythromycin gene clusters, so that the same cloning strategies for generating the pUCl ⁇ derivatives containing the A-B, B-C and C-D cassettes, described above in Examples 72-77 for the rapamycin cluster and 88-93 for the erythromycin cluster, can also be applied to the rifamycin cluster.
  • the rifamycin fragments A, B, C and D have been selected so that the same cloning methodologies described above for inserting tet within the A-B, B-C and C-D cassettes from the rapamycin and erythromycin clusters, described in Examples 78-83 and 94-99, respectively, can be applied in this instance as well.
  • the construction of the four-fragment cassette can also make use of the same cloning strategy. Therefore, following the same principles and methodologies described in detail for the rapamycin and erythromycin clusters in Examples 72-85 and 88-101, respectively, plasmids equivalent to those reported in Fig. 6 can be constructed for assembling the rifamycin cluster into the pPAC-Sl.
  • interplasmid insert exchange can be conducted between any plasmid containing the desired region and the cognate ts construct.
  • .Plasmids corresponding to pAB2, pBC2 and pCD2 can therefore be derived from any cluster.
  • the principles and methodologies illustrated in Fig. 8 can be applied employing the appropriate A-B-C-D cassette and the cognate pMAK705 derivatives prepared according to the scheme of Fig. 7.
  • the principles and methodologies illustrated in Fig. 7 and Fig. 8 and described in Examples 52-69 can therefore be extended to other clusters .
  • n is the number of overlapping clones that encompass the desired genomic segment
  • n will also be the number of homologous recombination rounds that introduce cluster DNA into the pESAC. If an Ab R marker is used to facilitate monitoring insert exchange, the total number of rounds of homologous recombination will be equal to 2n - 1 .
  • Interplasmid homologous recombination has been described to introduce large DNA segments into a desired vector (O'Connor et al., 1989, Science 244:1307-1312; Kao et al., 1994, Science 265:509-512) or to target a smaller segment into a large episome (Yang et a., 1997, Nature Biotechnol . 15:859-665).
  • O'Connor et al., 1989, Science 244:1307-1312; Kao et al., 1994, Science 265:509-512 or to target a smaller segment into a large episome (Yang et a., 1997, Nature Biotechnol . 15:859-665).
  • these procedures could be applied iteratively for the precise reconstruction of very large DNA segments .
  • the principles and methodologies described in Section 7.2 for obtaining an entire gene cluster in a pESAC rely on the identification of the desired genomic segment.
  • the desired clones are identified by screening an ESAC library with one of the possible strategies described below.
  • the desired clones are identified in a genomic library, such as a cosmid library, with one of the possible strategies described below, and then assembled into pESAC.
  • the principles and methodologies for identifying the genes responsible for the biosynthesis of natural products are well described in the literature and are reported here solely to illustrate the fact that they represent a necessary step in the overall scope of the present invention.
  • genes involved in the biosynthesis of natural products in actinomycetes are invariably found as gene clusters in the chromosome of the producing organism, often associated with one or more resistance determinants. Consequently, identifying one gene allows ready access to all the others.
  • One or more genes responsible for the biosynthesis of a natural product could have been described, or the entire cluster could be known.
  • biosynthesis clusters from actinomycetes have been reported and other clusters are likely to be described in the future. Suitable probes from the cluster extremities can be derived from published clusters, when available.
  • fragments A and D, described in Example 70 can be used as probes to screen an ESAC library prepared from S. hygroscopicus DNA.
  • ESACs positive to both probes will contain the rapamycin cluster. Similar strategies can be applied to ESAC libraries prepared from Sac . erythraea and A . mediterranei DNA, screened with fragments A and D, prepared as described in Examples 85-86 and 101, respectively . If no biosynthesis genes are known, different strategies for identifying them can be applied. These strategies are well described in the literature and are summarized below.
  • One possible strategy involves the isolation of the resistance gene(s) after cloning in a heterologous host that is sensitive to that natural product (for example, Stanzak et al . , 1986, Bio/Technol. 4:229- 232).
  • Another possible strategy is based on reverse genetics: a particular biosynthetic enzyme is purified, and from its partial protein sequence (s) the corresponding gene is isolated via PCR or hybridization (for example, Fishman et al., 1987, Proc. Natl. Acad. Sci. USA 84:6246-8252).
  • Another approach relies on the complementation of mutants blocked in one or more biosynthesis steps, after introduction of a DNA library constructed in a suitable vector into the wild type strain (for example, Malpartida and Hopwood, 1984, Nature 309:462-464).
  • Another approach involves the construction of an expression library in a suitable vector in an appropriate host, where the gene product is sought after using specific antibodies or looking for a particular enzymatic activity (for example, Jones and Hopwood, 1984, J. Biol . Chem. 259:14151-14157).
  • Another possible approach makes use of heterologous probes derived from biosynthesis, resistance or regulatory genes. Natural products can be broadly grouped into classes according to their biosynthetic origin, and for many of them suitable probes are available. For example, genes encoding aromatic or modular polyketide synthases can be effectively identified through the use of heterologous hybridization probes (Malpartida et al., 1987, Nature 325:818-821; Schwecke et al . , 1995, Proc.
  • the desired gene cluster can be identified in any library. If an ESAC library is used, the identified cluster is ready for transfer into the production host. If a smaller fragment library is employed, the cluster can be assembled into a pESAC.
  • any ESAC can be selected from said library and transferred into a production host. Therefore, a single donor organism can be utilized as the source of several biosynthesis clusters that can be mobilized into a production host. Similarly, an ESAC library needs not be constructed from a single donor organism.
  • Streptomyces transformants are selected for Th R , specified by the tsr marker present in the pESAC. Since the incoming DNA is incapable of self- replication in Streptomyces, site-specific integration occurs at the chromosomal a ttB site, mediated by the int- a ttP function specified by the pESAC. That integration has occurred at the proper site can be verified by Southern hybridization or by PFGE analysis of the transformants .
  • Fig. 10 illustrates a PFGE separation of a S. lividans derivative carrying an ESAC with a 70 kb insert integrated into its chromosome.
  • ESAC-120, and ESAC-140, respectively are used to transform protoplasts of S. lividans ZX7.
  • the colonies that appear on the R2YE plates, after overlaying with Th, are analyzed for their Th R by streaking them on fresh R2YE plates .
  • S. lividans ZX7 a t tB : :ESAC-70 Individual colonies of S. lividans ZX7 a ttB : :ESAC-70, prepared as described in Example 102, are grown in YEME and total genomic DNA is prepared. The DNA is digested with BamHI, resolved by agarose gel-electrophoresis, and transferred onto a membrane. Hybridization to labeled pPAC- Sl DNA, prepared as described in Example 11, reveals the appearance of three bands of approximately 16, 8 and 2.7 kb . PFGE analysis of genomic DNA reveals the disappearance of a 2.5 Mb Dral fragment present in ZX7 and the appearance of two fragments of 1.4 and 1.1 Mb (Fig. 10).
  • a few hundred ng of PAD6, prepared as described in Example 69, are used to transform protoplasts of S. lividans ZX7.
  • the colonies that appear on the R2YE plates, after overlaying with Th, are analyzed for their Th R by streaking them on fresh R2YE plates.
  • S. lividans ZX7 a ttB : :PAD6 contains the expected number and size of bands expected from transfer of the cluster of Fig. 9 via PAD6.
  • the rapamycin, erythromycin and rifamycin clusters assembled in pESAC can be used to transform S . lividans .
  • other S. lividans strains can be equally used as hosts for transformation with ESACs.
  • ⁇ C31 can lysogenize other Streptomyces species, in addition to S. lividans .
  • ⁇ C31 attB site may be engineered into Streptomyces species or other actinomycetes that are not naturally lysogenized by phage ⁇ C31. Therefore, any ESAC, prepared according to the principles and methodologies of Section 7.2, and any natural or engineered actinomycete host, fall within the scope of the present invention.
  • the recombinant production hosts are cultivated in a suitable medium and the presence of the relevant metabolites is determined following appropriate procedures, which may include biological assays, chromatographic properties, MS, NMR, etc. It will occur to those skilled in the art that ESACs, containing the relevant biosynthesis cluster derived from any donor actynomycete, can be used to transform S. lividans . The resulting transformants will produce the corresponding natural product. For example, an ESAC carrying the rapamycin, erythromycin or rifamycin cluster, prepared according to the principles of Section 7.2, can be used to transform S.
  • the present invention describes principles and methodologies for optimizing and speeding up the process of lead optimization and development in drug discovery. These can be applied since the early stages of drug discovery as briefly summarized herein.
  • a natural product produced by a donor organism has an interesting property, such as antibacterial, antifungal, antitumor, antihelmintic, herbicidal, immunosuppressive, or other pharmacological activity.
  • the potential is seen for increasing the productivity of the producing organism, and/or for improving the biological or physico-chemical properties of said natural product after manipulating its structure.
  • the biosynthetic pathway for the natural product is inferred from its chemical structure. This leads to a hypothesis on the genes involved, including the approximate size of the corresponding cluster.
  • a large insert library is constructed in the pESAC vectors described herein using genomic DNA prepared from the donor organism. Through a judicious choice of hybridization probes and PCR primers, the desired cluster is identified in the library. Alternatively, the cluster is assembled into the pESAC vectors described herein from overlapping cosmid clones identified by hybridization as above. The selected clone (s) are transferred into S. lividans, S . coelicolor or other suitable strain, and the resulting transformants are evaluated for production of the natural product.
  • the desired genetic, physiological and technological manipulations can be performed on the production host, employing well-developed methodologies.
  • the bioactive molecule is purified from a known host, amid a background of known metabolites. If necessary, ad hoc mutations can be conveniently introduced in the production host to eliminate unwanted, interfering products. Because of the deeper knowledge on the physiological processes and regulatory networks for secondary metabolism in the production host compared to the donor organism, targeted approaches to strain improvement, using classical and molecular techniques, can be applied.
  • well-characterized mutant strains are available for the production host, and the desired ESAC could be easily introduced into different genetic backgrounds.
  • the biosynthetic pathway can be easily manipulated, because of the availability of the cloned genes and the efficient genetic tools for the production host.
  • additional specialized genes or even entire clusters can be introduced into the production host, further expanding the possible applications of the present invention.
  • a further object of this invention is to provide a process for the procduction of a natural product by cultivating an actinomycete strain capablre of producing said natural product in the presence of a nutrient medium, isolating and purifying said natural product, characterized in that the actinomycete strain capable of producing said natural product ( production host) is an actinomycete strain modified by means of an E. coli-Streptomyces Artificial Chromosome that carries a gene cluster governing the biosynthesis of said natural product derived from an actinomycete donor organism which is the original producer of said natural product, according to the method described herein.
  • said modified actinomycete strain shall be a Streptomyces lividans or Streptomyces coelicolor strai .
  • the present invention provides significant advantages over the existing process of drug discovery and development, including production. It exploits the fact that the host where the natural product will be produced is an organism commonly used for process development and genetic manipulation, with substantial information available, including safety for handling it.

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Abstract

L'invention concerne un système de production et de modification de produits naturels obtenus à partir d'un groupe important de bactéries, dans un but de découverte, développement et production de médicaments. Le procédé de l'invention consiste à transférer la capacité de production d'un métabolite secondaire, à partir d'un actinomycète constituant le producteur original du produit naturel, à un hôte de production différent et possédant les caractéristiques voulues. Ce système implique la construction d'un segment du chromosome du producteur original, dans un chromosome artificiel qui peut être maintenu de manière stable dans un hôte de production approprié. L'invention concerne encore des vecteurs d'ADN de recombinaison, utiles en tant que navettes des informations génétiques nécessaires à la synthèse d'un produit naturel donné, entre un organisme donneur et un hôte de production. Ces procédés sont utiles pour améliorer le rendement, le procédé de purification et la modification structurelle d'un produit naturel.
PCT/EP1999/004079 1998-06-23 1999-06-14 Procedes de transfert de la capacite de production d'un produit naturel dans un hote de production approprie WO1999067374A1 (fr)

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AU46096/99A AU743003B2 (en) 1998-06-23 1999-06-14 Methods for transferring the capability to produce a natural product into a suitable production host
IL14002199A IL140021A0 (en) 1998-06-23 1999-06-14 Methods for transferring the capability to produce a natural product into a suitable production host
EP99929199A EP1090113A1 (fr) 1998-06-23 1999-06-14 Procedes de transfert de la capacite de production d'un produit naturel dans un hote de production approprie
JP2000556019A JP2002518045A (ja) 1998-06-23 1999-06-14 天然物を生産する能力を適当な生産宿主中に移す方法
KR1020007014621A KR20010083061A (ko) 1998-06-23 1999-06-14 천연 생산물 생산 능력을 적합한 생산 숙주로 전달하는 방법
CA002330543A CA2330543A1 (fr) 1998-06-23 1999-06-14 Procedes de transfert de la capacite de production d'un produit naturel dans un hote de production approprie

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EP98111506 1998-06-23
EP98111506.6 1998-06-23
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WO2001040497A3 (fr) * 1999-11-29 2002-10-17 Aventis Pharma Sa Procede d'obtention d'acides nucleiques a partir d'un echantillon de l'environnement
US6591954B2 (en) 2001-09-28 2003-07-15 Eaton Corporation Clutch brake
US7119250B2 (en) 1997-06-03 2006-10-10 The University Of Chicago Plant centromere compositions
US7193128B2 (en) 1997-06-03 2007-03-20 Chromatin, Inc. Methods for generating or increasing revenues from crops
US7227057B2 (en) 1997-06-03 2007-06-05 Chromatin, Inc. Plant centromere compositions
US7235716B2 (en) 1997-06-03 2007-06-26 Chromatin, Inc. Plant centromere compositions
US7255989B1 (en) 1999-11-29 2007-08-14 Aventis Pharma S.A. Method for obtaining nucleic acids from an environment sample, resulting nucleic acids and use in synthesis of novel compounds
JP2009017884A (ja) * 2001-05-30 2009-01-29 Chromos Molecular Systems Inc 染色体に基くプラットホーム
US20110262971A1 (en) * 2010-04-21 2011-10-27 Tufts University Genetically Modified E. coli Strains for Producing Erythromycin
US9096909B2 (en) 2009-07-23 2015-08-04 Chromatin, Inc. Sorghum centromere sequences and minichromosomes
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EP1413626A1 (fr) 2002-10-23 2004-04-28 Vicuron Pharmaceuticals, Inc. Gènes et protéines pour la biosynthèse de l'antibiotique glycopeptidique A40926
JP6130224B2 (ja) * 2013-05-27 2017-05-17 公益財団法人微生物化学研究会 新規化合物レンツトレハロース、その製造方法、及びその用途、並びに、新規微生物

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US8759086B2 (en) 1997-06-03 2014-06-24 University Of Chicago Methods for generating or increasing revenues from crops
US7456013B2 (en) 1997-06-03 2008-11-25 Chromatin, Inc. Plant centromere compositions
US7235716B2 (en) 1997-06-03 2007-06-26 Chromatin, Inc. Plant centromere compositions
US7119250B2 (en) 1997-06-03 2006-10-10 The University Of Chicago Plant centromere compositions
US7193128B2 (en) 1997-06-03 2007-03-20 Chromatin, Inc. Methods for generating or increasing revenues from crops
US7226782B2 (en) 1997-06-03 2007-06-05 Chromatin, Inc. Plant centromere compositions
US7227057B2 (en) 1997-06-03 2007-06-05 Chromatin, Inc. Plant centromere compositions
WO2001040497A3 (fr) * 1999-11-29 2002-10-17 Aventis Pharma Sa Procede d'obtention d'acides nucleiques a partir d'un echantillon de l'environnement
US7255989B1 (en) 1999-11-29 2007-08-14 Aventis Pharma S.A. Method for obtaining nucleic acids from an environment sample, resulting nucleic acids and use in synthesis of novel compounds
WO2001081567A3 (fr) * 2000-04-26 2002-03-21 Wisconsin Alumni Res Found Methode de production de bibliotheques genomiques et bibliotheques genomiques obtenues
JP2009017884A (ja) * 2001-05-30 2009-01-29 Chromos Molecular Systems Inc 染色体に基くプラットホーム
US6591954B2 (en) 2001-09-28 2003-07-15 Eaton Corporation Clutch brake
US9096909B2 (en) 2009-07-23 2015-08-04 Chromatin, Inc. Sorghum centromere sequences and minichromosomes
US20110262971A1 (en) * 2010-04-21 2011-10-27 Tufts University Genetically Modified E. coli Strains for Producing Erythromycin
WO2016097957A3 (fr) * 2014-12-16 2016-08-18 Centro De Investigación Y De Estudios Avanzados Del Instituto Politécnico Nacional Système génétique pour produire un inhibiteur de protéases de type petit aldéhyde peptidique
US10414796B2 (en) 2014-12-16 2019-09-17 Centro De Investigación Y De Estudios Avanzados Del Instituto Politécnico Nacional Genetic system for producing a proteases inhibitor of a small peptide aldehyde type

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IL140021A0 (en) 2002-02-10
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KR20010083061A (ko) 2001-08-31
EP1090113A1 (fr) 2001-04-11
CA2330543A1 (fr) 1999-12-29

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