WO2002029035A2 - Procede destine a modifier des fragments de sucre - Google Patents

Procede destine a modifier des fragments de sucre Download PDF

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Publication number
WO2002029035A2
WO2002029035A2 PCT/US2001/031255 US0131255W WO0229035A2 WO 2002029035 A2 WO2002029035 A2 WO 2002029035A2 US 0131255 W US0131255 W US 0131255W WO 0229035 A2 WO0229035 A2 WO 0229035A2
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host cell
recombinant host
nucleic acid
sugar
modified recombinant
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PCT/US2001/031255
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English (en)
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WO2002029035A3 (fr
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Hung-Wen Liu
David H. Sherman
Lishan Zhao
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Regents Of The University Of Minnesota
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Priority to AU2001296652A priority Critical patent/AU2001296652A1/en
Priority to US10/398,605 priority patent/US20040161839A1/en
Priority to EP01977540A priority patent/EP1325134A2/fr
Priority to JP2002532605A priority patent/JP2004534502A/ja
Priority to CA002424567A priority patent/CA2424567A1/fr
Publication of WO2002029035A2 publication Critical patent/WO2002029035A2/fr
Publication of WO2002029035A3 publication Critical patent/WO2002029035A3/fr

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    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C12P19/28—N-glycosides
    • C12P19/30—Nucleotides
    • C12P19/305—Pyrimidine nucleotides
    • 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

  • Glycosyltransferases responsible for the final glycosylation of certain secondary metabolites show a high degree of promiscuity towards the nucleotide sugar donor (Zhao et al., 1998a; Zhao et al., 1998b; Borisova et al., 1999; Weber et al., 1991; Decker et al., 1995, Sasaki et al., 1996; Solenberg et al., 1997; Madduri et al., 1998; Salah-Bey et al., 1998; Gaisser et al., 1998; Wohlert et al., 1998).
  • the invention provides a method to alter the sugar structure diversity for a particular metabolite via the recruitment and collaborative action of sugar genes from a variety of sugar biosynthetic pathways to yield a metabolite comprising a non-natural sugar, e.g., a novel glycosylated polyketide.
  • This alteration can be accomplished in vivo through genetic engineering.
  • the method of the invention provides a modified recombinant bacterial host cell that is genetically engineered to produce novel polyketides having non- natural sugar structures.
  • a sugar biosynthetic gene(s) from a heterologous (e.g., non-native or different) sugar biosynthetic pathway, or one that is modified in vitro and encodes an enzyme having an activity or specificity that is different than the native (wild type) enzyme is introduced into a recombinant host cell that produces a substrate for the enzyme(s) encoded by that gene(s) to yield a modified recombinant host cell that produces a novel product, i.e., one not produced by the corresponding recombinant host cell.
  • the product from the modified recombinant host cell comprises a sugar(s) that is significantly different than the sugar on the naturally occurring product from the corresponding wild type cell, e.g., the sugar on the modified product is not a stereoisomer of the sugar on the naturally occurring product.
  • the recombinant host cell and the modified recombinant host cell are genetically modified so that at least one gene for sugar biosynthesis, for example, in a sugar biosynthetic gene cluster, in that cell is disrupted, e.g., via an insertion or deletion, resulting in the accumulation of an intermediate in the biosynthetic pathway which is disrupted.
  • the disruption may be in a nucleic acid sequence present in the genome of the cell or present in an extrachromosomal element in the cell.
  • the invention is useful to generate libraries of polyketides and other sugar-containing molecules that are biologically active or can be activated.
  • a deacetylase may be employed to render the product biologically active.
  • the availability of such libraries can greatly decrease the time for drug discovery.
  • calH. 4-ketohexose aminotransferase gene from the calicheamicin pathway of Micromonospora echinospora spp. calichensis was introduced into a mutant strain of Streptomyces venezuelae in which the 4-dehydrase gene (des ⁇ ) in the methymycin/pikromycin pathway was deleted. Deletion of des ⁇ gene led to the accumulation of 4-keto-6-deoxyglucose intermediate which is the substrate of CalH. Consequently, heterologous expression of calH in this mutant resulted in the production of two methymycin/pikromycin-calicheamicin hybrids.
  • heterologous expression of selected genes from the L- dihydrostreptose pathway for example, thestr and strL genes o ⁇ Streptomyces griseus that encode a 6-deoxy-4-hexulose 3,5-epimerase and a dihydrostreptose synthase, respectively, was accomplished in aS. venezuelae mutant. Growth of the engineered S. venezuelae strain resulted in the accumulation of a set of methymycin/pikromycin analogs, each carrying a L-rhamnose.
  • the invention provides a modified recombinant bacterial host cell comprising at least one nucleic acid segment which encodes at least one sugar biosynthetic enzyme.
  • a nucleic acid segment of the invention does not encode a glycosyltransferase or any other non-sugar biosynthetic sequences such as polyketide synthase sequences.
  • the modified recombinant host cell may include more than one nucleic acid segment, each encoding a different enzyme, or one nucleic acid segment encoding one or more enzymes.
  • the modified recombinant host cell also preferably comprises a disrupted nucleic acid sequence, which corresponds to a nucleic acid sequence in a wild type host cell that encodes at least one sugar biosynthetic enzyme from a pathway that is different than the pathway of the enzyme(s) encoded by the nucleic acid segment.
  • the nondisrupted wild type nucleic acid sequence may encode a dehydrase, a reductase, a TDP-sugar synthase, a TDP-sugar dehydratase, an amino transferase, a N-methyltransferase, and/or a tautomerase.
  • the disruption results in the accumulation of a substrate(s) for the enzyme(s) encoded by the nucleic acid segment thus yielding a novel sugar.
  • the modified recombinant host cell also preferably produces a product having the novel sugar linked thereto, e.g., the native (endogenous) glycosyltransferase(s) transfers the novel sugar to another molecule, e.g., a polyketide such as an aglycone, to yield a novel product such as a macrolide.
  • nucleic acid molecule encoding a glycosyltransferase having relaxed substrate specificity may also be introduced to the recombinant host cell so as to provide an enzyme which attaches the novel sugar to another molecule in the modified recombinant host cell.
  • Preferred cells for use in the invention include any cell which produces a metabolite such as a polyketide, anticancer agent or antibiotic that has or can be modified to accommodate a sugar.
  • Antibiotic-producing cells include but are not limited to Actinoplanes, Actinomadura, Bacillus, Cephalosporium, Micromonospora, Penicillium, Nocardia, and Streptomyces, which either produce an antibiotic or contains genes which, if expressed, would produce an antibiotic or other biologically active compound, e.g., any cell which contains the genes sno, str, tyl, car, srm, tet, act, gra, tcm, mit/mmc, elm, sal, rifi grs, srfi bac, dau, sty, dnr, sna, fren, avr, ole, urd, ery, or any combination thereof.
  • actinomycetes that naturally produce polyketides include but are not limited to Micromonospora rosaria, Micromonospora megalomicea, Saccharopolyspora eiythraea, Streptomyces antibioticus, Streptomyces albereticuli, Streptomyces ambofaciens, Streptomyces avermitilis, Streptomyces fi'adiae, Streptomyces griseus, Streptomyces hydroscopicus, Streptomyces tsukulubaensis, Streptomyces my carofasciens, Streptomyces platenesis, Streptomyces violaceoniger, Streptomyces violaceoniger, Streptomyces thermotolerans, Streptomyces rimosus, Streptomyces peucetius, Streptomyces coelicolor, Streptomyces glaucescens, Streptomyces roseofulvus, Streptept
  • Streptomyces spp. include but are not limited to Streptomyces venezuelae (e.g., ATCC 15439, ATCC 15068, MCRL 0306, SC 2366 or 3629), Streptomyces narbonensis (e.g., ATCC 19790), Streptomyces eurocidicus, Streptomyces zaomyceticus (MCRL 0405), Streptomyces flavochromogens, Streptomyces sp. AM400, Streptomyces felleus, Streptomyces fradiae, Streptomyces argillaceus, Streptomyces olivaceus, Streptomyces peucetius, and Streptomyces griseus.
  • Streptomyces spp. include but are not limited to Streptomyces venezuelae (e.g., ATCC 15439, ATCC 15068, MCRL 0306, SC 2366 or 3629), Streptomyces
  • any cell which encodes a sugar biosynthetic gene is a source for the nucleic acid segments of the invention.
  • a source for nucleic acid segments are cells which produce a compound having a sugar including but not limited to cells that produce streptomycin, carbomycin, tylosin, spiramycin, streptothricin, erythromycin, vancomycin, teicoplanin, chloroerpmycin, methymycin, pikromycin, uramycin, granaticin, oleandomicin, landomycin, tetracenomycin, doxorubicin, mithramycin, epirubicin, and daunoribicin, or other sugar-containing compounds such as calicheamicin or nystatin, are included within the scope of the nucleic acid segments for use in the practice of the invention.
  • a recombinant host cell in which a nucleic acid sequence encoding at least one of the enzymes in desosamine biosynthesis is disrupted so as to alter desosamine synthesis, and is augmented with a nucleic acid segment which encodes a homolog of the enzyme encoded by the nondisrupted form of the nucleic acid sequence, yielding a modified recombinant host cell.
  • the modified recombinant host cell does not have a disruption is desl and does not consist of a calH nucleic acid segment.
  • a "homolog" of a reference sugar biosynthetic enzyme is an enzyme which can recognize the substrate of the reference biosynthetic enzyme and catalyze a reaction.
  • TylB is a homolog of Desl
  • CalH is a homolog of Desl
  • StrL and StrM together are a homolog of Desl
  • TylM2 is a homolog of DesNI.
  • Preferred homologs catalyze a reaction that produces a product, such an intermediate in sugar biosynthesis, that is different than the product of the reference enzyme.
  • Homologs can be identified functionally using methods such as those described herein. Generally, a homolog has at least about 28% amino acid sequence identity to the reference enzyme.
  • Other methods to identify a nucleic acid segment for use in the invention is by hybridization or computer assisted sequence alignments, e.g., using default settings.
  • the nucleic acid sequence of the invention hybridizes under low, moderate or stringent hybridization conditions to the nucleic acid segment of the invention.
  • Low, moderate and stringent hybridization conditions are well known to the art, see, for example sections 9.47-9.51 of Sambrook et al. (Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, Y (1989).
  • stringent conditions are those that (1) employ low ionic strength and high temperature for washing, for example, 0.015 M ⁇ aCl/0.0015 M sodium citrate (SSC); 0.1% sodium lauryl sulfate (SDS) at 50°C, or (2) employ a denaturing agent such as formamide during hybridization, e.g., 50% formamide with 0.1 % bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mM NaCl, 75 mM sodium citrate at 42°C.
  • SSC sodium lauryl sulfate
  • a denaturing agent such as formamide during hybridization, e.g., 50% formamide with 0.1 % bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mM NaCl, 75 mM sodium citrate at 42°C.
  • Exemplary moderate stringency conditions include hybridization in 40 to 45% formamide, 1.0 M NaCl, 1% SDS at 37°C, and a wash in 0.5X to IX SSC at 50 to 60°C.
  • Exemplary high stringency conditions include hybridization in 50%> formamide, 1 M NaCl, 1% SDS at 37°C, and a wash in O.lX SSC at 60 to 65°C.
  • the invention also provides an isolated and purified nucleic acid segment comprising a nucleic acid sequence comprising a sugar (desosamine) biosynthetic gene cluster, a biologically active variant or fragment thereof, wherein the nucleic acid sequence is not derived from the eryC gene cluster of Saccharopolyspora erythraea.
  • the isolated nucleic acid segment comprising the gene cluster preferably includes a nucleic acid sequence comprising SEQ ID NO:3 (see PCT/US 99/14398, which is incorporated by reference herein), or a fragment or variant thereof.
  • the cluster was found to encode nine polypeptides including Desl (e.g., SEQ ID NO:8 encoded by SEQ ID NO:7), DesII (e.g., SEQ ID NO: 10 encoded by SEQ ID NO:9), DesIII (e.g., SEQ ID NO: 12 encoded by SEQ ID NO:l 1), DesIN (e.g., SEQ ID NO:14 encoded by SEQ ID NO:13), DesN (e.g., SEQ ID NO: 16 encoded by SEQ ID NO: 15), DesNI (e.g., SEQ ID NO: 18 encoded by SEQ ID NO: 17), DesVII (e.g., SEQ ID NO:20 encoded by SEQ ID NO:19), DesNIII (e.g., SEQ
  • nucleic acid segment of the invention encoding DesR is not derived from the et ⁇ B gene cluster of Saccharopolyspora erythraea or the oleD gene from Streptomyces antibioticus.
  • nucleic acid segment comprising the desosamine biosynthetic gene cluster hybridizes under moderate, or more preferably stringent, hybridization conditions to SEQ ID NO:3, or a fragment thereof.
  • the invention also provides a variant polypeptide having at least about
  • a preferred variant polypeptide, or a subunit or fragment of a polypeptide, of the invention includes a variant or subunit polypeptide having at least about 1%, more preferably at least about 10%, and even more preferably at least about 50%>, the activity of the polypeptide having the amino acid sequence comprising SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:22, or SEQ ID NO:24.
  • glycosyltransferase activity of a polypeptide of SEQ ID NO:20 can be compared to a variant of SEQ ID NO:20 having at least one amino acid substitution, insertion, or deletion relative to SEQ ID NO:20.
  • a variant nucleic acid sequence of the invention has at least about 80%, more preferably at least about 90%>, and even more preferably at least about 95%, but less than 100%>, contiguous nucleic acid sequence identity to a nucleic acid sequence comprising SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO: 11 , SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:21 , SEQ ID NO:23, or a fragment thereof.
  • an expression cassette comprising a nucleic acid sequence comprising a desosamine biosynthetic gene cluster, a biologically active variant or fragment thereof operably linked to a promoter functional in a host cell, as well as host cells comprising an expression cassette of the invention.
  • the expression cassettes of the invention are useful to express individual genes within the cluster, e.g., the desR gene which encodes a glycosidase or the desVII gene which encodes a glycosyltransferase having relaxed substrate specificity for polyketides and deoxysugars, i.e., the glycosyltransferase processes sugar substrates other than TDP-desosamine.
  • the desVII gene can be employed in combinatorial biology approaches to synthesize a library of macrolide compounds having various polyketide and deoxysugar structures.
  • the expression of a glycosylase in a host cell which synthesizes a macrolide antibiotic may be useful in a method to reduce toxicity of, e.g., inactivate, the antibiotic.
  • a host cell which produces the antibiotic is transformed with an expression cassette encoding the glycosyltransferase.
  • the recombinant glycosyltransferase is expressed in an amount that reversibly inactivates the antibiotic.
  • the antibiotic preferably the isolated antibiotic which is recovered from the host cell, is contacted with an appropriate native or recombinant glycosidase.
  • the nucleic acid segment encoding desosamine in the expression cassette of the invention is not derived form the eryC gene cluster of Saccharopolyspora erythraea.
  • Preferred host cells are prokaryotic cells, although eukaryotic host cells are also envisioned. These host cells are useful to express desosamine, analogs or derivatives thereof as well as individual polypeptides which can then be isolated from the host cell. Also provided is an expression cassette or host cell comprising antisense sequences from at least a portion of the desosamine biosynthetic gene cluster.
  • Another embodiment of the invention is a recombinant host cell, e.g., a bacterial cell, in which at least a portion of a nucleic acid sequence encoding desosamine in the host chromosome is disrupted, e.g., deleted or interrupted (e.g., by an insertion) with heterologous sequences, or substituted with a variant nucleic acid sequence of the invention, so as to alter, preferably so as to result in a decrease or lack of, desosamine synthesis and/or so as to result in the synthesis of an analog or derivative of desosamine.
  • a recombinant host cell e.g., a bacterial cell, in which at least a portion of a nucleic acid sequence encoding desosamine in the host chromosome is disrupted, e.g., deleted or interrupted (e.g., by an insertion) with heterologous sequences, or substituted with a variant nucleic acid sequence of the invention, so as
  • the nucleic acid sequence which is disrupted is not derived from the eryC gene cluster of Saccharopolyspora erythraea.
  • the recombinant host cell of the invention has at least one gene, i.e., desl, desll, desIII, desIV, desV, desVI, desVII, desVIII or desR, which is disrupted.
  • One embodiment of the invention includes a recombinant host cell in which the des VI gene, which encodes an N- methyltransferase, is disrupted, for example, by replacement with an antibiotic resistance gene.
  • such a host cell produces an aglycone having anN- acetylated aminodeoxy sugar, 10-deoxy-methylonide, a compound of formula (7), a compound of formula (8), or a combination thereof.
  • the deletion or disruption of the des VI gene may be useful in a method for preparing novel sugars.
  • Another preferred embodiment of the invention is a recombinant bacterial host cell in which the desR gene, which encodes a glycosidase such as ⁇ -glucosidase, is disrupted.
  • the host cell synthesizes C-2' ⁇ - glucosylated macrolide antibiotics, for example, a compound of formula (13), a compound of formula (14), or a combination thereof. Therefore, the invention further provides a compound of formula (8), (9), (13) or (14).
  • each atom of the compounds of the invention having a chiral center may exist in and be isolated in optically active and racemic forms. Some compounds may exhibit polymo ⁇ hism.
  • the present invention encompasses any racemic, optically active, polymorphic or stereoisomeric form, or mixtures thereof, of a compound of the invention, which possess the useful properties described herein, it being well known in the art how to prepare optically active forms (for example, by resolution of the racemic form by recrystallization techniques, by synthesis from optically active starting materials, by chiral synthesis, or by chromatographic separation using a chiral stationary phase) and how to determine activity using the standard tests described herein, or using other similar tests which are well known in the art.
  • the method comprises introducing into a polyketide- producing microorganism a D ⁇ A sequence encoding enzymes for sugar biosynthesis, e.g., desosamine biosynthesis such as a D ⁇ A sequence comprising SEQ ID NO:3, a variant or fragment thereof, so as to yield a microorganism that produces specific glycosylation-modified polyketides.
  • a D ⁇ A sequence encoding enzymes for sugar biosynthesis e.g., desosamine biosynthesis such as a D ⁇ A sequence comprising SEQ ID NO:3, a variant or fragment thereof.
  • an anti- sense DNA sequence of the invention may be employed.
  • the glycosylation-modified polyketides are isolated from the microorganism. It is preferred that the DNA sequence is modified so as to result in the inactivation of at least one enzymatic activity in sugar biosynthesis or in the attachment of the sugar to a polyketide.
  • the compounds (products) produced by the recombinant host cells and modified recombinant host cells of the invention may be particularly useful as biologically active agents, such as those useful to prepare a medicament for the treatment of a pathological condition or a symptom in a mammal, e.g., a human.
  • the products include pharmaceuticals such as chemotherapeutic agents, immunosuppressants, agents to treat asthma, chronic obstructive pulmonary disease as well as other diseases involving respiratory inflammation, cholesterol- lowering agents, or macrolide-based antibiotics which are active against a variety of organisms, e.g., bacteria, including multi-drug-resistant pneumococci and other respiratory pathogens, as well as viral and parasitic pathogens; or as crop protection agents (e.g., fungicides or insecticides).
  • pharmaceuticals such as chemotherapeutic agents, immunosuppressants, agents to treat asthma, chronic obstructive pulmonary disease as well as other diseases involving respiratory inflammation, cholesterol- lowering agents, or macrolide-based antibiotics which are active against a variety of organisms, e.g., bacteria, including multi-drug-resistant pneumococci and other respiratory pathogens, as well as viral and parasitic pathogens; or as crop protection agents (e.g., fungicides or insecticides).
  • Methods employing these compounds e.g., to treat a mammal, bird or fish in need of such therapy, such as a patient having a bacterial, viral or parasitic infection, cancer, respiratory disease, or in need of immunosuppression, e.g., during cell, tissue or organ transplantation, are also envisioned.
  • Figure 1 Schematic diagram of the desosamine biosynthetic pathway and the enzymatic activity associated with each of the desosamine biosynthetic polypeptides.
  • Figure 2 Schematic of the conversion of the inactive (diglycosylated) form of methymycin and pikromycin to the active form of methymycin and pikromycin.
  • FIG. 1 Schematic diagram of the desosamine biosynthetic pathway.
  • Figure 4 Pathway for the synthesis of a compound of formula 7 and 8 in desNI " mutants of Streptomyces.
  • FIG. 1 Structure and biosynthesis of methymycin, pikromycin, and related compounds in Streptomyces venezuelae ATCC 15439.
  • Polyketide synthase components PikAI, PikAII, PikAIII, PikAIN, and PikAN are represented by solid bars. Each circle represents an enzymatic domain in the Pik PKS system.
  • KS ⁇ -ketoacyl-ACP synthase
  • AT acyltransferase
  • ACP acyl carrier protein
  • KR ⁇ -ketoacyl-ACP reductase
  • DH ⁇ -hydroxyl-thioester dehydratase
  • ER enoyl reductase
  • KS Q a KS-like domain
  • KR with a cross nonfunctional KR
  • TE thioesterase domain
  • TEII type II thioesterase.
  • PikC is the cytochrome P450 monooxygenase responsible for hydroxylation at R, , R 2 , and R 3 positions (Xue et al., 1998).
  • Figure 6. Organization of the pik cluster in S. venezuelae. Each arrow represents an open reading frame (ORF). The direction of transcription and relative sizes of the ORFs deduced from nucleotide sequence are indicated. The cluster is composed of four genetic loci: pikA, pikB (des), pikC, and pikR. Cosmid clones are denoted as overlapping lines.
  • Figure 7. Conversion of YC-17 and narbomycin by PikC P450 hydroxylase.
  • FIG. 1 Nucleotide sequence (SEQ ID NO:3) and inferred amino acid sequence (SEQ ID NO:4) of the desosamine gene cluster.
  • Figure 9. Exemplary and preferred amino acid substitutions.
  • Figure 10. Pathway for desosamine biosynthesis.
  • FIG. 1 Schematic of pathway leading to methymycin/neomethymycin analogs 18 and 19.
  • Figure 13 Products produced by desl mutant.
  • Figure 14 Macrolides produced in a desl mutant which expresses CalH.
  • Figure 15. Natural substrate for and product of CalH, and structure of calicheamicin.
  • a "Type I polyketide synthase” is a single polypeptide with a single set of iteratively used active sites. This is in contrast to a Type II polyketide synthase which employs active sites on a series of polypeptides.
  • a “module” is one of a series of repeated units in a multifunctional protein, such as a Type I polyketide synthase or a fatty acid synthase.
  • a "premature termination product” is a product which is produced by a recombinant multifunctional protein which is different than the product produced by the non-recombinant multifunctional protein.
  • the product produced by the recombinant multifunctional protein has fewer acyl groups.
  • a "recombinant" nucleic acid or protein (polypeptide) molecule is a molecule where the nucleic acid molecule which encodes the protein has been modified in vitro, so that its sequence is not naturally occurring, or corresponds to naturally occurring sequences that are not positioned as they would be positioned in a genome which has not been modified.
  • a "recombinant" host cell of the invention has been genetically manipulated so as to alter, e.g., decrease or disrupt, or, alternatively, increase, the function or activity of at least one gene in a sugar biosynthetic pathway.
  • the manipulation may occur in an extrachromosomal genetic element which comprises the at least one gene or in the genome of the cell.
  • a "wild type” or “nonrecombinant” cell has not been genetically manipulated.
  • the genetic manipulation in the recombinant cell preferably results in the absence of a product (compound) that is produced by the corresponding wild type cell or the production of a product that is not produced by the corresponding wild type cell.
  • a "modified" recombinant host cell of the invention is a recombinant host cell that has been genetically manipulated so as to express at least one isolated nucleic acid segment, preferably in the form of an expression cassette which includes a promoter, that is introduced to the recombinant cell to form the modified recombinant host cell.
  • the genetic manipulation in the modified recombinant host cell preferably results in the production of a product (compound) that is not produced by the corresponding recombinant host cell or the corresponding wild type cell.
  • a DNA that is "derived from" a gene or gene cluster is a
  • DNA that has been isolated and purified in vitro from genomic DNA or synthetically prepared on the basis of the sequence of genomic DNA.
  • the "pUF or "pik/met" gene cluster includes sequences encoding a polyketide synthase (pikA), desosamine biosynthetic enzymes (pikB, also referred to as des), a cytochrome P450 (pikC), regulatory factors (pikD) and enzymes for cellular self-resistance (pikR).
  • pikA polyketide synthase
  • pikB desosamine biosynthetic enzymes
  • pikC cytochrome P450
  • pikD regulatory factors
  • enzymes for cellular self-resistance pikR
  • isolated and/or purified refer to in vitro isolation of a DNA or polypeptide molecule from its natural cellular environment, and from association with other components of the cell, such as nucleic acid or polypeptide, so that is can be sequenced, replicated and/or expressed.
  • an isolated DNA molecule encoding an enzyme for desosamine biosynthesis or a fragment thereof is RNA or DNA containing greater than 7, preferably 15, and more preferably 20 or more sequential nucleotide bases that encode a biologically active polypeptide, fragment, or variant thereof, that is complementary to the non-coding, or complementary to the coding strand, of a RNA encoding at least one enzyme for desosamine biosynthesis, or hybridizes to the RNA or DNA comprising the desosamine biosynthetic gene cluster and remains stably bound under low, moderate or preferably stringent conditions, as defined by methods well known to the art, e.g., in Sambrook et al., 1989.
  • An "antibiotic” as used herein is a substance produced by a microorganism which, either naturally or with limited chemical modification, will inhibit the growth of or kill another microorganism or eukaryotic cell.
  • an "antibiotic biosynthetic gene” is a nucleic acid, e.g., DNA, segment or sequence that encodes an enzymatic activity which is necessary for an enzymatic reaction in the process of converting primary metabolites into antibiotics.
  • An "antibiotic biosynthetic pathway” includes the entire set of antibiotic biosynthetic genes necessary for the process of converting primary metabolites into antibiotics. These genes can be isolated by methods well known to the art, e.g., see U.S. Patent No. 4,935,340.
  • Antibiotic-producing organisms include any organism, including, but not limited to, Actinoplanes, Actinomadura, Bacillus, Cephalosporium, Micromonospora, Penicillium, Nocardia, and Streptomyces, which either produces an antibiotic or contains genes which, if expressed, would produce an antibiotic.
  • An antibiotic resistance-conferring gene is a DNA segment that encodes an enzymatic or other activity which confers resistance to an antibiotic.
  • polyketide refers to a large and diverse class of natural products, including but not limited to antibiotic, antifungal, anticancer, and anti-helminthic compounds.
  • Polyketides include but are not limited to macrolides, anthracyclines, angucyclins, avermectins, milbemycins, tetracyclines, polyenes, polyethers, ansamycins and isochromanequinones and the like.
  • Polyketide antibiotics include, but are not limited to anthracyclines and macrolides of different types (polyenes and avermectins as well as classical macrolides such as erythromycins). Macrolides are produced by, for example, S.
  • glycosylated in the context of another molecule refers to a molecule that contains one or more sugar residues.
  • sugar refers to a polyhydroxylated aldehyde or ketone.
  • the polyhydroxylated aldehyde or ketone can optionally be linked to lipids, peptides and/or proteins.
  • Sugars may have additional substituents such as amino, sulfate or phosphate groups, in addition to the carbon-hydrogen-oxygen core.
  • a polymer consisting of two to ten saccharide units is termed an oligosaccharide (OS), e.g., monosaccharides, disaccharides, e.g., sucrose, and trisaccharides, and those consisting of more than ten saccharide units is termed a polysaccharide (PS).
  • OS oligosaccharide
  • PS polysaccharide
  • Sugars include, e.g., trioses, pentoses and hexoses, ribose, glucose, as well as deoxy sugars such as fructose, rhamnose, and deoxyribose, and 6-, 2,6-, 3,6-, 4,6-, 2,3,6-deoxysugars, such as olivose, oliose, mycarose, rhodinose, mycinose, and other modified sugars (e.g., amino sugars including mycaminose, desosamine, vancosamine and daunosamine).
  • modified sugars e.g., amino sugars including mycaminose, desosamine, vancosamine and daunosamine.
  • Saccharide derivatives can conveniently be prepared as described in International Patent Applications Publication Numbers WO 96/34005 and 97/03995.
  • the term "glycosylation-modified" as it relates to a particular molecule refers to a molecule having a changed glycosylation pattern or configuration relative to that particular molecule's unmodified or native state.
  • polyketide-producing microorganism includes any microorganism that can produce a polyketide naturally or after being suitably engineered (i.e., genetically).
  • actinomycetes that naturally produce polyketides include but are not limited to Micromonospora rosaria, Micromonospora megalomicea, Saccharopolyspora erythraea, Streptomyces antibioticus, Streptomyces albereticuli, Streptomyces ambofaciens, Streptomyces avermitilis, Streptomyces fradiae, Streptomyces griseus, Streptomyces hydroscopicus, Streptomyces tsukulubaensis, Streptomyces mycarofasciens, Streptomyces platenesis, Streptomyces violaceoniger, Streptomyces violaceoniger, Streptomyces thermotolerans, Streptomyces rimosus, Streptomyces rimos
  • sugar biosynthesis genes refers to nucleic acid sequences or segments from organisms such as Micromonospora, Streptomyces venezuelae, Str'eptomyces fradiae, Streptomyces griseus, Streptomyces peucetius, Streptomyces argillaceous, and Streptomyces olivaceus that encode sugar biosynthesis enzymes, and is intended to include sugar biosynthetic DNA from other polyketide-producing microorganisms.
  • sugar biosynthesis enzymes refers to polypeptides which are involved in the biosynthesis and/or attachment of polyketide-associated sugars and their derivatives and intermediates.
  • polyketide-associated sugar refers to a sugar that is known to attach to polyketides or that can be attached to polyketides.
  • sugar derivative refers to a sugar which is naturally associated with a polyketide but which is altered relative to the unmodified or native state, including but not limited to N-3- ⁇ -desdimethyl D-desosamine.
  • sugar intermediate refers to an intermediate compound produced in a sugar biosynthesis pathway.
  • the term "derivative" means that a particular compound (product) produced by a host cell of the invention or prepared in vitro using polypeptides encoded by the nucleic acid molecules of the invention, is modified so that it comprises other moieties, e.g., peptide or polypeptide molecules, such as antibodies or fragments thereof, nucleic acid molecules, sugars, lipids, fats, a detectable signal molecule such as a radioisotope, e.g., gamma emitters, small chemicals, metals, salts, synthetic polymers, e.g., polylactide and polyglycolide, surfactants and glycosaminoglycans, which are covalently or non-covalently attached or linked to the compound.
  • moieties e.g., peptide or polypeptide molecules, such as antibodies or fragments thereof, nucleic acid molecules, sugars, lipids, fats, a detectable signal molecule such as a radioisotope, e.
  • each atom of the compounds of the invention having a chiral center may exist in and be isolated in optically active and racemic forms. Some compounds may exhibit polymo ⁇ hism.
  • the present invention encompasses any racemic, optically active, polymo ⁇ hic or stereoisomeric form, or mixtures thereof, of a compound of the invention, which possess the useful properties described herein, it being well known in the art how to prepare optically active forms (for example, by resolution of the racemic form by recrystallization techniques, by synthesis from optically active starting materials, by chiral synthesis, or by chromatographic separation using a chiral stationary phase) and how to determine activity using the standard tests described herein, or using other similar tests which are well known in the art.
  • sequence homology or “sequence identity” means the proportion of base matches between two nucleic acid sequences or the proportion amino acid matches between two amino acid sequences.
  • sequence homology is expressed as a percentage, e.g., 50%, the percentage denotes the proportion of matches over the length of sequence that is compared to some other sequence. Gaps (in either of the two sequences) are permitted to maximize matching; gap lengths of 15 bases or less are usually used, 6 bases or less are preferred with 2 bases or less more preferred.
  • the sequence homology between the target nucleic acid and the oligonucleotide sequence is generally not less than 17 target base matches out of 20 possible oligonucleotide base pair matches (85%); preferably not less than 9 matches out of 10 possible base pair matches (90%), and more preferably not less than 19 matches out of 20 possible base pair matches (95%).
  • Two amino acid sequences are homologous if there is a partial or complete identity between their sequences and/or have the same or similar activity. For example, 85% homology means that 85% of the amino acids are identical when the two sequences are aligned for maximum matching.
  • Gaps in either of the two sequences being matched are allowed in maximizing matching; gap lengths of 5 or less are preferred with 2 or less being more preferred.
  • two protein sequences or polypeptide sequences derived from them of at least 30 amino acids in length are homologous, as this term is used herein, if they have an alignment score of at more than 5 (in standard deviation units) using the program ALIGN with the mutation data matrix and a gap penalty of 6 or greater (Dayhoff, 1972).
  • the two sequences or parts thereof are more preferably homologous as used herein if their amino acids are greater than or equal to 29% identical.
  • reference sequence is a defined sequence used as a basis for a sequence comparison; a reference sequence may be a subset of a larger sequence, for example, as a segment of a full-length cDNA or gene sequence given in a sequence listing, or may comprise a complete cDNA or gene sequence. Generally, a reference sequence is at least 20 nucleotides in length, frequently at least 25 nucleotides in length, and often at least 50 nucleotides in length.
  • two polynucleotides may each (1) comprise a sequence (i.e., a portion of the complete polynucleotide sequence) that is similar between the two polynucleotides, and (2) may further comprise a sequence that is divergent between the two polynucleotides
  • sequence comparisons between two (or more) polynucleotides are typically performed by comparing sequences of the two polynucleotides over a "comparison window" to identify and compare local regions of sequence similarity.
  • a “comparison window”, as used herein, refers to a conceptual segment of at least 20 contiguous nucleotides and wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) of 20 percent or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences.
  • Optimal alignment of sequences for aligning a comparison window may be conducted by the local homology algorithm of Smith and Waterman (1981) by the homology alignment algorithm of Needleman and Wunsch (1970), by the search for similarity method of Pearson and Lipman (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by inspection, and the best alignment (i.e., resulting in the highest percentage of homology over the comparison window) generated by the various methods is selected.
  • default settings are employed to identify homologs using computerized algorithms.
  • sequence identity means that two polynucleotide sequences are identical (i.e., on a nucleotide-by-nucleotide basis) over the window of comparison.
  • percentage of sequence identity means that two polynucleotide sequences are identical (i.e., on a nucleotide-by-nucleotide basis) over the window of comparison.
  • percentage of sequence identity is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, U, or I) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity.
  • the identical nucleic acid base e.g., A, T, C, G, U, or I
  • the term "substantial identity” or “homology” means that two peptide sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, share at least about 29 percent sequence identity, preferably at least about 35 percent sequence identity and/or have the same or similar activity, i.e., recognize one or more common substrate(s) and thereby produce a product.
  • a modified recombinant host cell derived from a recombinant host cell, the genome of which is altered, optionally to disrupt sugar biosynthesis that occurs in the corresponding wild type cell.
  • the modified recombinant host cell is augmented with a nucleic acid segment that encodes at least one sugar biosynthetic enzyme that is a homolog of an enzyme encoded by the wild type cell which is absent or present in a reduced amount in the recombinant host cell as a result of the disruption.
  • the modified recombinant host cell includes a least one expression cassette comprising at least one isolated and purified nucleic acid segment which encodes a sugar biosynthetic enzyme(s) that recognizes the substrate of an enzyme(s) encoded by the wild type cell and which is not expressed, or expressed in a reduced amount, in the recombinant cell.
  • the enzyme(s) encoded by the nucleic acid segment produces a substrate for another sugar biosynthetic enzyme or for a glycosyltransferase.
  • the invention described herein can be used for the production of a diverse range of novel compounds including glycosylated polyketides, e.g., antibiotics, through genetic redesign of sugar biosynthetic DNA such as that found in Streptomyces spp. as well as other polyketide producing organisms. This gene allows for the selective production of particular compounds, including the production of novel compounds.
  • combinational biosynthetic- based modification of compounds may be accomplished by selective activation or disruption of specific genes within the sugar gene cluster and expressing other sugar biosynthetic genes into biosynthetic libraries which are assayed for a wide range of biological activities, to derive greater chemical diversity.
  • a further example includes the introduction of biosynthetic gene(s) into a particular host cell so as to result in the production of a novel compound due to the activity of the biosynthetic gene(s) on other metabolites, intermediates or components of the host cells.
  • nucleic acid sequences and segments employed in the invention include those that hybridize under low, moderate or stringent hybridization conditions to the genes encoding sugar biosynthetic enzymes, such as those set forth herein, and/or encode enzymes that have the same or similar activity.
  • a nucleic acid molecule, segment or sequence of the present invention can also be an RNA molecule, segment or sequence which corresponds to, is complementary to or hybridizes under low, moderate, or stringent conditions to any of the DNA segments or sequences described herein.
  • the invention includes nucleic acid sequences and segments that encode a homolog of a particular sugar biosynthetic enzyme, including a polypeptide that has at least one amino acid substitution (Figure 9; Alberts et al., 1989), relative to a wild type polypeptide, e.g., the homolog may have at least 29% identity to the wild type polypeptide, as long as the homolog can recognize and catalyze a reaction with a substrate for the wild type enzyme.
  • the homolog may be a naturally occuring enzyme or one that is prepared recombinantly.
  • mutations can be made to a native (wild type) nucleic acid segment or sequence of the invention to yield a variant nucleic acid segment or sequence, and such variants may be used in place of the native segment or sequence, so long as the variant encodes an enzyme(s) that functions with other molecules to collectively catalyze the synthesis of an identifiable glycosylatedmolecule such as a glycosylated polyketide or macrolide.
  • Such mutations can be made to the native sequences using conventional techniques such as by preparing synthetic oligonucleotides including the mutations and inserting the mutated sequence into the gene using restriction endonuclease digestion (see, e.g., Kunkel, 1985; Geisselsoder et al., 1987).
  • the mutations can be effected using a mismatched primer (generally 10-20 nucleotides in length) which hybridizes to the native nucleotide segment or sequence, at a temperature below the melting temperature of the mismatched duplex.
  • the primer can be made specific by keeping primer length and base composition within relatively narrow limits and by keeping the mutant base centrally located (Zoller and Smith, 1983).
  • Primer extension is effected using DNA polymerase, the product cloned and clones containing the mutated DNA, derived by segregation of the primer extended strand, selected. Selection can be accomplished using the mutant primer as a hybridization probe.
  • the technique is also applicable for generating multiple point mutations. See, e.g., Dalbie-McFarland et al. (1982). PCR mutagenesis will also find use for effecting the desired mutations.
  • Random mutagenesis of the nucleotide sequence can be accomplished by several different techniques known in the art, such as by altering sequences within restriction endonuclease sites, inserting an oligonucleotide linker randomly into a plasmid, by irradiation with X-rays or ultraviolet light, by inco ⁇ orating incorrect nucleotides during in vitro DNA synthesis, by error- prone PCR mutagenesis, by preparing synthetic mutants or by damaging plasmid DNA in vitro with chemicals.
  • Chemical mutagens include, for example, sodium bisulfite, nitrous acid, hydroxylamine, agents which damage or remove bases thereby preventing normal base-pairing such as hydrazine or formic acid, analogues of nucleotide precursors such as nitrosoguanidine, 5-bromouracil, 2- aminopurine, or acridine intercalating agents such as proflavine, acriflavine, quinacrine, and the like.
  • plasmid DNA or DNA fragments are treated with chemicals, transformed into E. coli and propagated as a pool or library of mutant plasmids.
  • the gene sequences can be inserted into one or more expression vectors, using methods known to those of skill in the art.
  • Expression vectors may include control sequences operably linked to the desired genes.
  • Suitable expression systems for use with the present invention include systems which function in eukaryotic and prokaryotic host cells. Prokaryotic systems are preferred, and in particular, systems compatible with Streptomyces spp. are of particular interest.
  • Control elements for use in such systems include promoters, optionally containing operator sequences, and ribosome binding sites. Particularly useful promoters include control sequences derived from the gene clusters of the invention.
  • promoters such as those derived from sugar metabolizing enzymes, such as galactose, lactose (lac) and maltose, will also find use in the expression cassettes encoding desosamine.
  • Preferred promoters are Streptomyces promoters, including but not limited to the ermE*,pikA and tip A promoters. Additional examples include promoter sequences derived from biosynthetic enzymes such as tryptophan (frp), the ⁇ -lactamase (bla) promoter system, bacteriophage lambda PL, and T5.
  • synthetic promoters such as the tac promoter (U.S. Pat. No. 4,551,433), which do not occur in nature, also function in bacterial host cells.
  • regulatory sequences may also be desirable which allow for regulation of expression of the genes relative to the growth of the host cell. Regulatory sequences are known to those of skill in the art, and examples include those which cause the expression of a gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound. Other types of regulatory elements may also be present in the vector, for example, enhancer sequences.
  • Selectable markers can also be included in the recombinant expression vectors.
  • a variety of markers are known which are useful in selecting for transformed cell lines and generally comprise a gene whose expression confers a selectable phenotype on transformed cells when the cells are grown in an appropriate selective medium.
  • markers include, for example, genes which confer antibiotic resistance or sensitivity to the plasmid.
  • sequences or segments of interest can be cloned into one or more recombinant vectors as individual cassettes, with separate control elements, or under the control of, e.g., a single promoter.
  • the sequences or segments can include flanking restriction sites to allow for the easy deletion and insertion of other sequences or segments.
  • the design of such unique restriction sites is known to those of skill in the art and can be accomplished using the techniques described above, such as site-directed mutagenesis and PCR.
  • the choice of vector depends on the pool of mutant sequences, i.e., donor or recipient, with which they are to be employed. Furthermore, the choice of vector determines the host cell to be employed in subsequent steps of the claimed method. Any transducible cloning vector can be used as a cloning vector for the donor pool of mutants. It is preferred, however, that phagemids, cosmids, or similar cloning vectors be used for cloning the donor pool of mutant encoding nucleotide sequences into the host cell.
  • Phagemids and cosmids are advantageous vectors due to the ability to insert and stably propagate therein larger fragments of DNA than in Ml 3 phage and ⁇ phage, respectively.
  • Phagemids which will find use in this method generally include hybrids between plasmids and filamentous phage cloning vehicles.
  • Cosmids which will find use in this method generally include ⁇ phage-based vectors into which cos sites have been inserted.
  • Recipient pool cloning vectors can be any suitable plasmid.
  • the cloning vectors into which pools of mutants are inserted may be identical or may be constructed to harbor and express different genetic markers (see, e.g., Sambrook et al., supra). The utility of employing such vectors having different marker genes may be exploited to facilitate a determination of successful transduction.
  • the cloning vector employed may be anE. coli/Streptomyces shuttle vector (see, for example, U.S. Patent Nos. 4,416,994, 4,343,906, 4,477,571, 4,362,816, and 4,340,674), a cosmid, a plasmid, an artificial bacterial chromosome (see, e.g., Zhang and Wing, 1997; Schalkwyk et al., 1995; and Monaco and Lavin, 1994), or a phagemid
  • the host cell may be a bacterial cell such as E. coli, Penicillium patulum, and Streptomyces spp. such as S. lividans, S. venezuelae, or S. lavendulae, or a eukaryotic cell such as fungi, yeast or a plant cell, e.g., monocot and dicot cells, preferably cells that are regenerable.
  • recombinant polypeptides having a particular activity may be prepared via "gene-shuffling". See, for example, Crameri et al., 1998; Patten et al., 1997, U.S. Patent Nos. 5,837,458, 5,834,252, 5,830,727, 5,811,238, 5,605,793.
  • phagemids upon infection of the host cell which contains a phagemid, single-stranded phagemid DNA is produced, packaged and extruded from the cell in the form of a transducing phage in a manner similar to other phage vectors.
  • clonal amplification of mutant encoding nucleotide sequences carried by phagemids is accomplished by propagating the phagemids in a suitable host cell. Following clonal amplification, the cloned donor pool of mutants is infected with a helper phage to obtain a mixture of phage particles containing either the helper phage genome or phagemids mutant alleles of the wild-type encoding nucleotide sequence.
  • Infection, or transfection, of host cells with helper phage is generally accomplished by methods well known in the art (see., e.g., Sambrook et al., supra; and Russell et al., 1986).
  • the helper phage may be any phage which can be used in combination with the cloning phage to produce an infective transducing phage.
  • the helper phage will necessarily be a ⁇ phage.
  • the cloning vector is a phagemid and the helper phage is a filamentous phage, and preferably phage Ml 3.
  • the transducing phage can be separated from helper phage based on size difference (Barnes et al., 1983), or other similarly effective technique.
  • Recipient cells which may be employed in the method disclosed and claimed herein may be, for example, E. coli, or other bacterial expression systems which are not recombination deficient.
  • a recombination deficient cell is a cell in which recombinatorial events is greatly reduced, such as rec ⁇ mutants of E. coli (see, Clark et al, 1965).
  • transductants can now be selected for the desired expressed protein property or characteristic and, if necessary or desirable, amplified.
  • transductants may be selected by way of their expression of both donor and recipient plasmid markers.
  • the recombinants generated by the above-described methods can then be subjected to selection or screening by any appropriate method, for example, enzymatic or other biological activity.
  • the above cycle of amplification, infection, transduction, and recombination may be repeated any number of times using additional donor pools cloned on phagemids.
  • the phagemids into which each pool of mutants is cloned may be constructed to express a different marker gene.
  • Each cycle could increase the number of distinct mutants by up to a factor of 10 6 .
  • the probability of occurrence of an inter-allelic recombination event in any individual cell is f (a parameter that is actually a function of the distance between the recombining mutations)
  • the transduced culture from two pools of IO 6 allelic mutants will express up to IO 12 distinct mutants in a population of 10 12 /f cells.
  • Str-eptomyces venezuelae contain desosamine as their sole sugar component, the organization of the sugar biosynthetic genes in the methymycin/neomethymycin gene cluster may be less complicated.
  • this system was chosen for the study of the biosynthesis of desosamine, aN,N-dimethylamino-3,4,6- trideoxyhexose, which also exists in the erytliromycin structure (Flinn et al., 1954).
  • a DNA library was constructed by partially digesting the genomic DNA of S.
  • ORFs open reading frames downstream of the PKS genes
  • Figure 1 Based on sequence similarities to other sugar biosynthetic genes, especially those derived form the erythromycin cluster (Gaisser et al., 1997; Summers et al., 1997), eight of these nine ORFs are believed to be involved in the biosynthesis of TDP-D- desosamine.
  • the ery cluster lacks homologs of the tylAl and tylA2 genes that are responsible for the first two steps in desosamine pathway.
  • erythromycin biosynthetic machinery may rely on a general cellular pool of TDP-4-keto-6-deoxy-D-glucose for mycarose and desosamine formation.
  • Depicted in Figure 1 is a biosynthetic pathway for TDP-D- desosamine.
  • a disruption plasmid (pBL1005) derived from pKCl 139 (containing an apramycin resistance marker) (Bierman et al., 1992) was constructed in which a 1.0 kb Ncol/Xho ⁇ fragment of the desR gene was deleted and replaced by the thiostrepton resistance (tsr) gene (1.1 kb) (Bibb et al., 1985) via blunt-end ligation.
  • This plasmid was used to transform E. coli SI 7-1,- which serves as the donor strain to introduce the pBLl 005 construct through conjugal transfer into the wild-type S.
  • the double crossover mutants in which chromosomal desR had been replaced with the disrupted gene were selected according to their thiostrepton-resistant and apramycin-sensitive characteristics. Southern blot hybridization analysis was used to confirm the gene replacement.
  • the desired mutant was first grown at 29°C in seed medium for 48 hours, and then inoculated and grown in vegetative medium for another 48 hours (Cane et al., 1993). After the fermentation broth was centrifuged at 10,000 g to remove cellular debris and mycelia, the supernatant was adjusted to pH 9.5 with concentrated KOH, and extracted with an equivolume of chloroform (four times).
  • the organic layer was dried over sodium sulfate and evaporated to dryness.
  • the amber oil-like crude products were first subjected to flash chromatography on silica gel using a gradient of 0-40% methanol in chloroform, followed by HPLC purification on a C 18 column eluted isocratically with 45% acetonitrile in 57 mM ammonium acetate (pH 6.7).
  • methymycin a compound of formula (1)
  • neomethymycin a compound of formula (2)
  • two new products were isolated.
  • the yield of a compound of formula (13) and a compound of formula (14) was each in the range of 5-10 mg/L of fermentation broth.
  • the antibiotic activity of a compound of fomiula (13) and (14) against Streptococcus pyogenes was examined by separately applying 20 ⁇ L of each sample (1.6 mM in MeOH) to sterilized filter paper discs which were placed onto the surface of S. pyogenes grown on Mueller-Hinton agar plates (Mangahas, 1996). After being grown overnight at 37°C, the plates of the controls (a compound of formula (1) and (2)) showed clearly visible inhibition zones. In contrast, no such clearings were discernible around the discs of a compound of formula (13) and (14). Evidently, ⁇ -glucosylation at C-2' of desosamine in methymycin/neomethymycin renders these antibiotics inactive.
  • glycosyltransferases Although the genes of the aforementioned glycosyltransferases have been cloned in a few cases, such as mgtA of S. lividans and oleD of S. antibioticus, the whereabouts of macrolide ⁇ -glycosidase genes remain obscure. Interestingly, the recently released eryBI sequence, which is part of the erythromycin biosynthetic cluster, is highly homologous to desR (55% identity) (Gaisser et al., 1997).
  • desR a macrolide ⁇ -glucosidase gene
  • the accumulation of deactivated compounds of formula (13) and (14) after desR disruption provides direct molecular evidence indicating that a similar self-defense mechanism via glycosylation/deglycosylation may also be operative inS. venezuelae.
  • glucosylation of desosamine may not be the primary self-resistance mechanism in S. venezuelae.
  • the translated desR gene has a leader sequence characteristic of secretory proteins (von Heijne, 1986; von Heijne, 1989).
  • DesR may be transported through the cell membrane and hydrolyze the modified antibiotics extracellularly to activate them (Figure 2). Summary
  • the entire desosamine biosynthetic gene cluster from the methymycin and neomethymycin producing strain Streptomyces venezuelae was cloned, sequenced, and mapped. Eight of the nine mapped genes were assigned to the biosynthesis of TDP-D-desosamine based on sequence similarities to those derived from the erythromycin cluster. The remaining gene, designated desR, showed strong sequence homology to ⁇ -glucosidases.
  • a disruption mutant was constructed in which aNcol/Xltol fragment of the desR gene was deleted and replaced by the thiostrepton resistance (tsr) gene.
  • tsr thiostrepton resistance
  • two new products were isolated from the fermentation of the mutant strain. These two new compounds, which are biologically inactive, were found to be C-2' ⁇ -glucosylated methymycin and neomethymycin.
  • the DesR protein may be an extracellular ⁇ - glucosidase capable of removing the added glucose from the modified antibiotics to activate them.
  • the desR gene can be used as a prqbe to identify homologs in other antibiotic biosynthetic pathways.
  • Deletion of the corresponding macrolide glycosidase gene in other antibiotic biosynthetic pathways may lead to the accumulation of the glycosylated products which may be used as prodrugs with reduced cytotoxicity.
  • Glycosylation also holds promise as a tool to regulate and/or minimize the potential toxicity associated with new macrolide antibiotics produced by genetically engineered microorganisms.
  • macrolide glycosidases which can be used for the activation of newly formed antibiotics that have been deliberately deactivated by engineered glycosyltransferases, may be useful in the development of novel antibiotics using the combinatorial biosynthetic approach (Hopwood et al., 1990; Katz et al., 1993; Hutchinson et al., 1995; Carreras et al., 1997; Kramer et al., 1996; Khosla et al., 1996; Jacobsen et al., 1997; Marsden et al, 1998).
  • Example 2 Deletion of the desVI Gene of the Desosamine Biosynthetic Gene Cluster
  • the emergence of pathogenic bacteria resistant to many commonly used antibiotics poses a serious threat to human health and has been the impetus of the present resurgent search for new antimicrobial agents (Box et al., 1997; Davies, 1996; Service, 1995). Since the first report on using genetic engineering techniques to create "hybrid" polyketides (Hopwood et al., 1995), the potential of manipulating the genes governing the biosynthesis of secondary metabolites to create new bioactive compounds, especially macrolide antibiotics, has received much attention (Kramer et al., 1996; Khosla et al., 1996).
  • This class of clinically important drugs consists of two essential structural components: a polyketide aglycone and the appended deoxy sugars (Omura, 1984).
  • the aglycone is synthesized via sequential condensations of acyl thioesters catalyzed by a highly organized multi-enzyme complex, polyketide synthase (PKS) (Hopwood et al., 1990; Katz, 1993; Hutchinson et al., 1995; Carreras et al., 1997).
  • PKS polyketide synthase
  • D-desosamine a compound of formula (3)
  • desosamine is the only sugar attached to the macrolactone of formula (1) and (2), identification of the sugar biosynthetic genes within the methymycin/neomethymycin gene cluster should be possible with much more certainty.
  • the desVl gene which has been predicted to encode the N- methyltransferase, was chosen as a target (Gaisser et al., 1997; Summers et al., 1997).
  • the deduced desVI product is most closely related to that of eryCVI from the erythromycin producing strain Saccharopolyspora erythraea (70%) identity), and also strongly resembles the predicted products of rdrnD from the rhodomycin cluster of Str'eptomyces purpurascens ( ⁇ iemi et al., 1995), smiX from the spiromycin cluster of Streptomyces ambofaciens (Geistlich et al., 1992), and tylMl from the tylosin cluster o ⁇ Streptomyces fradiae (Gandecha et al., 1997).
  • All of these enzymes contain the consensus sequence LLDV(I)ACGTG (SEQ ID ⁇ O:25) (Gaisser et al., 1997; Summers et al., 1997), near their N- terminus, which is part of the S-adenosylmethionine binding site (Ingrosso et al., 1989; Haydock et al., 1991).
  • deletion o ⁇ desVI should have little polar effect (Lin et al., 1984) on the expression of other desosamine biosynthetic genes because the ORF (desR) lying immediately downstream from desVI is not directly involved in desosamine formation, and those lying further downstream are transcribed in the opposite direction.
  • KdesVI-21 and KdesVI-22 with phenotypes of thiostrepton resistance (Thio R ) and apamycin sensitivity (Apm s ) were obtained.
  • Southern blot hybridization using tsr or a 1.1 kb Hindi fragment from the des VII region further confirmed that the desVI gene was indeed replaced by tsr on the chromosome of these mutants.
  • the KdesVI-21 mutant was first grown at 29°C in seed medium (100 mL) for 48 hours, and then inoculated and grown in vegetative medium (3 L) for another 48 hours (Cane et al., 1993).
  • E. coli DH5 was used as a cloning host.
  • E. coli LE392 was the host for a cosmid library derived from S. venezuelae genomic DNA.
  • LB medium was used in E. coli propagation.
  • Streptomyces venezuelae ATCC 15439 was obtained as a freeze-dried pellet from ATCC.
  • Media for vegetative growth and antibiotic production were used as described (Lambalot et al., 1992). Briefly, SGGP liquid medium was for propagation of S. venezuelae mycelia.
  • Sporulation agar (SPA) was used for production of S. venezuelae spores.
  • Methymycin production was conducted in either SCM or vegetative medium and pikromycin production was performed in Suzuki glucose-peptone medium.
  • Plasmid vectors for gene disruption were either pGM160 (Muth et al., 1989) or pKCl 139 (Bierman et al., 1992). Plasmid, cosmid, and genomic DNA preparation, restriction digestion, fragment isolation, and cloning were performed using standard procedures (Sambrook et al., 1989; Hopwood et al., 1985). The cosmid library was made according to instructions from the Packagene ⁇ -packaging system (Promega).
  • Plasmids for insertional inactivation were constructed by cloning a kanamycin resistance marker into target genes, and plasmid for gene deletion/replacement was constructed by replacing the target gene with a kanamycin or thiostrepton resistance gene in the plasmid.
  • Disruption plasmids were introduced into S. venezuelae by either PEG-mediated protoplast transformation (Hopwood et al., 1985) or RK2-mediated conjugation (Bierman et al., 1992). Then, spores from individual transformants or transconjugants were cultured on non-selective plates to induce recombination. The cycle was repeated three times to enhance the opportunity for recombination.
  • Double crossovers yielding targeted gene disruption mutants were selected and screened using the appropriate combination of antibiotics and finally confirmed by Southern hybridization.
  • Antibiotic Extraction and Analysis Methymycin, pikromycin, and related compounds were extracted following published procedures (Cane et al., 1993). Thin layer chromatography (TLC) was routinely used to detect methymycin, neomethymycin, narbomycin and pikromycin. Further purification was conducted using flash column chromatography and HPLC, and the purified compounds were analyzed by ⁇ , 13 C NMR spectroscopy and MS spectrometry. Results
  • Nucleotide Sequence of the pik Cluster The nucleotide sequence of the pik cluster was completely determined and shown to contain 18 open reading frames (ORFs) that span approximately 60 kb. Central to the cluster are four large ORFs,pikAI, pikAII, pikAIII, and p ⁇ kAIV, encoding a multifunctional PKS ( Figure 5). Analysis of the six modules comprising the pik PKS indicated that it would specify production of narbonolide, the 14-membered ring aglycone precursor of narbomycin and pikromycin ( Figure 5). Initial analysis unveiled two significant architectural differences in the pikA-encoded PKS.
  • PikA may produce the 12-membered ring macrolactone 10-deoxymethynolide as well.
  • the domain organization of PikAI - AIII (module L-5) is consistent with the predicted biosynthesis of 10- deoxymethynolide except for the absence of a TE function at the C-terminus of Pik module 5 (PikAIII).
  • the lack of a TE domain in PikAIII may be compensated by the type II TE (encoded by p ⁇ kAV) immediately downstream of pikAIV.
  • the genetic locus for desosamine biosynthesis and glycosyl transfer are immediately downstream o ⁇ pikA. Seven genes, desl, desll, desIII, desIV, desV, desVI, and desVIII, are responsible for the biosynthesis of the deoxysugar, and the eighth gene, desVII, encodes a glycosyltransferase that apparently catalyzes transfer of desosamine onto the alternate (12- and 14-membered ring) polyketide agly cones. The existence of only one set of desosamine genes indicates that DesVIII can accept both 10-deoxymethynolide and narbonolide as substrates (Jacobsen et al., 1997). The largest ORF in the des locus, desR, encodes a ⁇ - glycosidase that is involved in a drug inactivation-reactivation cycle for bacterial self-protection.
  • PikC Just downstream of the des locus is a gene (pikC) encoding a cytochrome P450 hydroxylase similar to eryF (Andersen et al., 1992), and eryK (Stassi et al., 1993), PikC, and a gene (pikD) encoding a putative regulator protein, PikD (Figure 5).
  • PikC is the only P450 hydroxylase identified in the entire pik cluster, suggesting that the enzyme can accept both 12- and 14- membered ring macrolide substrates and, more remarkably, it is active on both C-10 and C-12 of the YC-17 (12-membered ring intermediate) to produce methymycin and neomethymycin ( Figure 7).
  • PikD is a putative regulatory protein similar to ORFH in the rapamycin gene cluster (Schwecke et al., 1995).
  • the combined functionality coded by the eighteen genes in thepik cluster predicts biosynthesis of methymycin, neomethymycin, narbomycin and pikromycin (Table 1). Flanking thepik cluster locus are genes presumably involved in primary metabolism and genes that may be involved in both primary and secondary metabolism. An S-adenosyl-methionine synthase gene is located downstream o ⁇ pikD that may help to provide the methyl group in desosamine synthesis. A threonine dehydratase gene was identified upstream o ⁇ pikRl that may provide precursors for polyketide biosynthesis. It is not apparent that any of these genes are dedicated to antibiotic biosynthesis and they are not directly linked to thepik cluster. Table 1. Deduced function of ORFs in the pik cluster
  • PikAI the first putative enzyme involved in the biosynthesis of 10-deoxymethynolide and narbonolide was inactivated by insertional mutagenesis.
  • the resulting mutant, AX903 produced neither methymycin or neomethymycin, nor narbomycin or pikromycin, indicating that p ⁇ kA encodes a PKS required for both 12- and 14-membered ring macrolactone formation.
  • mutant LZ3001 in which mutation in an enzyme downstream o ⁇ p ⁇ kA V accumulated 10-deoxymethynolide and narbonolide.
  • mutant AX905 failed to accumulate these intermediates suggested that the polyketide chains were not efficiently released from this PKS protein in the absence of Pik TEII. Therefore, Pik TEII plays a crucial role in polyketide chain release and cyclization, and it presumably provides the mechanism for alternative termination in pik polyketide biosynthesis.
  • PikC the P450 hydroxylase
  • p ⁇ kA evolved in a line analogous to ery A and oleA since each of these PKSs specify the synthesis of 14-membered ring macrolactones. Therefore, pik may have acquired the capacity to generate methymycin when a mutation in the primordial pikAIII-pikAIV linker region caused splitting of Pik module 5 and 6 into two separate gene products. This notion is raised by two features of the nucleotide sequence.
  • the intergenic region between pikAIII and pikAIV which is 105 bp, may be the remanent of an intramodular linker peptide of 35 amino acids.
  • the potential for independently regulated expression of pikAIV ' is implied by the presence of a 100 nucleotide region at the 5' end of the gene that is relatively AT-rich (62% as comparing 74% G+C content in coding region).
  • the mutation in an original ORF encoding the bimodular multifunctional protein (PikAIII-PikAIV) occurred, so too may have evolved a mechanism for regulated synthesis of the new gene product (PikAIV).
  • Pik TEII in alternative termination of polyketide chain elongation intermediates provides a unique aspect of diversity generation in natural product biosynthesis.
  • Engineered polyketides of different chain length are typically generated by moving the TE catalytic domain to alternate positions in a modular PKS (Cortes et al., 1995). Repositioning of the TE domain necessarily abolishes production of the original full-length polyketide so only one macrolide is produced each time.
  • the independent Pik TEII polypeptide presumably has the flexibility to catalyze termination at different stages of polyketide assembly, therefore enabling the system to produce multiple products of variant chain length.
  • Combinatorial biology technologies can now exploit this system for generating molecular diversity through construction of novel PKS systems with TEIIs for simultaneous production of several new molecules as opposed to the TE domains alone that limit catalysis to a single termination step.
  • Pik TEII sequences similar to Pik TEII are found in almost all known polyketide and non-ribosomal polypeptide biosynthetic systems (Marahiel et al., 1997).
  • thepik TEII is the first to be characterized in a modular PKS.
  • recent work on a' TEII gene in the lipopeptide surfactin biosynthetic cluster demonstrated that sr ⁇ TEII plays an important role in polypeptide chain release, and may suggest that srf- TEII reacts at multiple stages in peptide assembly as well (Marahiel et al., 1997).
  • the enzymes involved in post-polyketide assembly of 10- deoxymethynolide and narbonolide are particularly interesting, especially the glycosyltransferase, DesVII, and P450 hydroxylase, PikC. Both have the remarkable ability to accept substrates with significant structural variability. Moreover, disruption of des VI demonstrated that DesVII also tolerates variations in deoxysugar structure. Likewise, PikC has recently been shown to convert YC-17 to methymycin/neomethymycin and narbomycin to pikromycin in vitro. Targeted gene disruption of ORF 1 abolished both pikromycin and methymycin production, indicating that the single cluster is responsible for biosynthesis of both antibiotics.
  • TE2 in contrast to the position-fixed TE1 domain, has the capacity to release polyketide chain at different points during the assembly process, thereby producing polyketides of different chain length.
  • the pik cluster represents the least complex yet most versatile modular PKS system so far investigated. This simplicity provides the basis for a compelling expression system in which novel active ketoside products are engineered and produced with considerable facility for discovery of a diverse range of new biologically active compounds. Summary
  • PKSs represent one of the most amenable systems for combinatorial technologies because of their inherent genetic organization and ability to produce polyketide metabolites, a large group of natural products generated by bacteria (primarily actinomycetes and myxobacteria) and fungi with diverse structures and biological activities.
  • Complex polyketides are produced by multifunctional PKSs involving a mechanism similar to long-chain fatty acid synthesis in animals (Hopwood et al., 1990).
  • Pioneering studies (Cortes et al., 1990; Donadio et al., 1991) on the erythromycin PKS in Saccharopolyspora erythraea revealed a modular organization.
  • Streptomyces venezuelae ATCC 15439 is notable in its ability to produce two distinct groups of macrolide antibiotics.
  • Methymycin and neomethymycin are derived from the 12-membered ring macrolactone 10-deoxymethynolide, while narbomycin and pikromycin are derived from the 14-membered ring macrolactone, narbonolide.
  • the cloning and characterization of the biosynthetic gene cluster for these antibiotics reveals the key role of a type II thioesterase in forming a metabolic branch through which polyketides of different chain length are generated by the pikromycin multifunctional polyketide synthase (PKS).
  • PKS pikromycin multifunctional polyketide synthase
  • pikA a set of genes for desosamine (des) biosynthesis and macrolide ring hydroxylation.
  • the glycosyl transferase encoded by desVIII
  • the/>t£C-encoded P450 hydroxylase provides yet another layer of structural variability by introducing regiochemical diversity into the macrolide ring systems.
  • the glycosyltransferase (DesVII) of this pathway is capable of recognizing and processing the keto sugar intermediate 17
  • the macrolide product(s) produced by the KdesV-41 mutant should have an attached 3 -keto sugar.
  • the two products isolated were the methymycin/neomethymycin analogues 18 and 19, each carrying a 4,6-dideoxyhexose ( Figure 12). While this result demonstrated a relaxed specificity for the glycosyltransferase toward its sugar substrate, it also indicated the existence of a pathway-independent reductase in S. venezuelae that can stereospecifically reduce the C-3 keto group of the sugar metabolite.
  • Kdesl- 80 was selected and grown at 29°C in seed medium (100 mL) for 48 hours and then inoculated and grown in vegetative medium (5 L) for another 48 hours (Cane et al., 1993).
  • the fermentation broth was centrifuged to remove cellular debris and mycelia, and the supernatant was adjusted to pH 9.5 with concentrated potassium hydroxide solution.
  • the resulting solution was extracted with chloroform, and the pooled organic extracts were dried over sodium sulfate and evaporated to dryness.
  • the yellow oil was subjected to flash chromatography on silica gel using a gradient of 0-12% methanol in chloroform, and the isolated products were further purified by HPLC using a C 18 column eluted isocratically with 50% acetonitrile in water.
  • 10-deoxymethynolide 23 was found as the major product (approximately 600 mg).
  • Significant quantities of methynolide 24 (approximately 40 mg) and neomethynolide 25 (approximately 2 mg) were also isolated (Figure 13).
  • a new macrolide 15 containing D-quinovose (3.2 mg) was produced by this mutant.
  • cytochrome P450 hydroxylase which catalyzes the hydroxylation of 10-deoxy-methynolide at either its C-10 or C-12 position (Xue et al, 1998), is sensitive to structural variations in the appended sugar. It could be argued that the presence of the 4-OH group in the sugar moiety is somehow responsible for decreasing or preventing hydroxylation of the macrolide.
  • the results demonstrate the feasibility of combining pathway-dependent genetic manipulations and pathway-independent enzymatic reactions to engineer a sugar of designed structure. It is conceivable that the pathway- independent enzymes could also be used in concert with the natural biosynthetic machinery to generate further structural diversity, which can provide an array of random compounds.
  • Example 5 Engineering a Hybrid Macrolide
  • Streptomyces venezuelae met/pik gene cluster was selected as the parent system and a gene from the calicheamicin biosynthetic gene cluster (from Micromonospora echinospora spp. Calichensis) as the foreign gene.
  • the parent cluster encodes the biosynthetic enzymes for methymycin, neomethymycin, pikromycin, and narbomycin, of which all are macrolides containing desosamine as the sole sugar component for antibiotic activity (Xue et al., 1998; Zhao et al, 1998) Eight open reading frames (desI-desVIII) in this cluster have been assigned as genes involved in desosamine biosynthesis (Figure 15).
  • the antitumor agent calicheamicin (26) contains four unique sugars crucial to tight DNA binding (K a about 10 6 -10 8 ), one of which (29) is derived from 4-amino-4,6-dideoxyglucose (28) and is part of the unusually restricted N-O connection between sugars A and B ( Figure 16) (Ding et al., 1991; Drak et al., 1991; Walker et al., 1991; EUestad et al.; Borders et al., 1995).
  • Compound 28 is anticipated to be derived from the corresponding 4-ketosugar 27 via a transamination reaction, and recent work has led to the assignment of a gene (calH) as encoding a C-4 aminotransferase ( Figure 16) (Alhert et al).
  • the proposed substrate for CalH, 27, is also an intermediate in the desosamine pathway and is expected to exist in a tautomerase (DesVIII)-mediated equilibrium with the substrate for Desl (Chen et al., 1999).
  • DesVIII tautomerase
  • Heterologous expression of calH in this mutant may reconstitute a hybrid pathway towards new methymycin/pikromycin derivatives which carry the 4-amino-4,6-dideoxy glucose derived from 26.
  • the 1.2 kb calH gene was amplified by polymerase chain reaction (PCR) from pJSTl 192 Kpn70Kb , a subclone containing a 7.0 kb Kpnl fragment of cosmid 13a (Thorson et al., 1999).
  • the amplified gene was cloned into the EcoRI/Xbal sites of the expression vector pDHS617, which contains an apramycin resistance marker.
  • pDHS617 is derived from pOJ446 (Bierman et al., 1992), and a promoter sequence from met/pik (Xue et al., 1998).
  • pLZ-C242 The resulting plasmid, pLZ-C242, was introduced by conjugal transfer using Escherichia coli S 17-1 (Bierman et al., 1992) into a previously constructed S. venezuelae mutant (Kdesl) (Borisova et al., 1999) in which des/ was replaced by the neomycin resistance gene that also confers resistance to kanamycin.
  • the pLZ-C242 containing S. venezuelae-Kdesl colonies were identified on the basis of their resistance to apramycin antibiotic (Apr 11 ).
  • KdesI/calH-1 One of the engineered strains, KdesI/calH-1, was first grown in 100 mL of seed medium at 29°C for 48 hours and then inoculated and grown in vegetative medium (5 L) for another 48 hours (Cane et al., 1993). The fermentation broth was centrifuged to remove the cellular debris and mycelia, and the supernatant was adjusted to pH 9.5 with concentrated KOH followed by chloroform extraction. The crude products (700 mg) were subjected to flash chromatography on silica gel using a gradient of 0- 20%) methanol in chloroform. A major product, 10-deoxymethynolide, and a mixture oftwo minor macrolide compounds were obtained.
  • the two macrolides were further purified by HPLC on a C 18 column using an isocratic mobile phase of acetonitrile/H 2 O (1:1). They were later identified as 31 (11.0 mg) and 32 (1.5 mg) by spectral analyses.
  • aglycone of the isolated macrolide 31 was 10-deoxymethynolide instead of methymycin and neomethymycin analogues that are hydroxylated.
  • the aglycone of 32 was the 14-membered narbonolide that is also devoid of hydroxylation.
  • the cytochrome P450 hydroxylase (PikC) which catalyzes the hydroxylation of 10-deoxymethynolide and narbonolide (Xue et al., 1998) is sensitive to structural variations on the appended sugar.
  • the strM gene may encode a 3,5-epimerase responsible for the conversion of 33 to 34, while the product o ⁇ strL gene is speculated to catalyze the ring contraction of 34 to give 35 (Pisowotzki et al., 1991; Distler et al. 1992). Since the proposed substrate for StrM, 33, is also an intermediate in the desosamine pathway, heterologous expression of StrM, StrL, or StrM/StrL in the S. venezuelae desl-mutant in which 33 accumulates, may reconstitute hybrid pathways toward new methymycin/pikromycin derivatives carrying an L- pyranose or an L-furanose.
  • str (0.8 kb) and strL (1.0 kb) genes were separately amplified by polymerase chain reaction (PCR) from the genomic DNA of S. griseus.
  • the amplified strM gene was cloned into the EcoRI/Nsil sites of the expression vector pDHS702 (Xue et al, 2000), which contains a thiostrepton resistance marker.
  • the strL gene was cloned into the EcoRl/Xbal ⁇ sites of the vector pDHS617, which has an apramycin resistance marker.
  • Each plasmid was transformed into Escherichia coli S 17-1 (Bierman et al., 1992) and then introduced separately by conjugal transfer into the previously constructed mutant S.
  • KdesI/strM and KdesI/strL were identified on the basis of their resistance to the corresponding antibiotics.
  • the strJ-containing plasmid was further engineered into the KdesI/strM mutant to produce the recombinant strain Kdes strM/strL, which confers resistance to both apramycin and thiostrepton.
  • KdesI/strM/strL-8 was chosen to grow in 150 mL of seed medium at 29°C for 48 hours, and then inoculated and grown in vegetative medium (6 L) for another 48 hours (Cane et al., 1993).
  • the fermentation broth was centrifuged, and the supernatant was extracted with chloroform. After concentration, the residual yellow oil (1.5 g) was subjected to flash chromatography on silica gel using 10%) methanol in chloroform as eluent.
  • the crude products were further purified by HPLC on a C 18 column eluted with a linear gradient of 0-50% acetonitrile in water over 20 minutes to yield four new macrolide derivatives, 38 (31.1 mg), 39 (6.3 mg), 40 (3.0 mg), and 41 (3.9 mg).
  • venezuelae Kdesl contains a pathway- independent D-hexulose reductase that can reduce 33 to TDP-D-quinovose (46), but lacks an L-hexulose reductase of its own to reduce 34.
  • the StrM catalyzed epimerization is expected to be reversible.
  • the equilibrium between 33 and 34 in the KdesI/strM strain will be shifted toward 33, which after reduction gives quinovose as observed in the product.
  • StrL could also serve as a sugar reductase capable of reducing an L-6-deoxy-4- hexulose such as 34 to TDP-L-rhamnose (48).
  • the translated sequence of desl shows high homology to B 6 -dependent enzymes and is 24% identical to that of E l5 and the translated desll sequence contains a conserved motif of CXXXCXXC (SEQ ID NO:50) characteristic for a [4Fe-4S] center (Ruzicka et al., 2000), the C-4 deoxygenation has been postulated to follow a path similar to that catalyzed by E x and E 3 ( Zhao et al., 1998; and see Gaisser et al., 1997; Summers et al, 1997 .
  • the reaction may be initiated by a tautomerization step presumably catalyzed by DesVIII to convert 6, a common precursor for 6-deoxyhexoses, to 3-keto-6-deoxyhexose 7.
  • Desl and Desll may then effect the removal of 4-OH from 7 to give the 3-keto-4,6-dideoxyhexose product (8) which has earlier been confirmed as the substrate of the next enzyme in the pathway, DesV (Zhao et al., 1998).
  • This proposal is supported by the fact that 4-OH is retained in the appended sugar (D-quinovose, 9) of the modified methymycin and pikromycin derivatives produced by the desl deleted mutant (Borisova et al., 1999).
  • Desl is a 4-aminotransferase
  • 4- amination is the initial step of 4-deoxygenation.
  • the desl gene was amplified by PCR and cloned into the pET-28b(+) expression vector ( ⁇ ovagen) with a His 6 -tag at the N-terminus.
  • the produced Desl protein was purified to near homogeneity by a ⁇ i- ⁇ TA column (Qiagen) followed by FPLC on a MonoQ column.
  • the subunit M r of Desl was estimated to be 45 kDa, which agrees well with the calculated molecular mass of 45 765 Da (plus the His 6 tag). Further analysis by size exclusion chromatography revealed aM r of 95.6 kDa for Desl. Therefore, Desl exists as a homodimer in solution.
  • the UV - vis spectrum of purified Desl is transparent above 300 nm; however, that of the more concentrated sample shows the presence of trace amount of PLP.
  • TDP-3-keto-deoxy-D-glucose (7) was incubated with the purified Desl in the presence of L-glutamate, no consumption of 7 and no new product were discemable by HPLC analysis.
  • This new compound was purified by FPLC on a MonoQ column and characterized as the TDP-4-amino-4,6-dideoxy-D-glucose (10).
  • ethanolamine ammonia lyase and adenosylcobalamin (AdoCbl)-dependent enzyme that catalyzes the degradation of ethanolamine to ammonia and acetaldehyde
  • AdoCbl adenosylcobalamin
  • lysine 2,3-aminomutase which catalyzes the interconversion of L-lysine and L- ⁇ -lysine via 1,2-migration of the amino group
  • the latter enzyme from Clostridium subterminale SB4 contains an iron-sulfur center and is PLP-as well as S-adenosylmethionine (SAM)-dependent. Both reactions are believed to involve a putative 5'-deoxyadenosyl radical which is generated by a reductive cleavage of SAM in lysine 2,3-aminomutase, or a homolytic cleavage of the Co-C bond of adenosylcob(III)alamin in ethanolamine ammonia lyase. This adenosyl radical then abstracts a hydrogen atom from the substrate to initiate the isomerization.
  • SAM S-adenosylmethionine
  • Desl is a PLP enzyme and Desll has recently been identified as a member of radical SAM superfamily by sequence analyses (Sofia et al., 2001), the Desl and Desll enzymes may work together to catalyze a 1,2-amino migration analogous to that of lysine 2,3- aminomutase (see Scheme 2, Figure 20) to achieve C-4 deoxygenation.
  • Desll may act alone by abstracting a 3-H » directly from 10 to generate a radical intermediate which, after deprotonation of OH, is converted to a ketyl equivalent. Subsequence ⁇ elimination of 4-amino group followerd by a H " return and tautomerization can also afford 8).

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Abstract

L'invention concerne un procédé destiné à modifier la structure de sucres.
PCT/US2001/031255 2000-10-05 2001-10-05 Procede destine a modifier des fragments de sucre WO2002029035A2 (fr)

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JP2002532605A JP2004534502A (ja) 2000-10-05 2001-10-05 糖部分を変更するための方法
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109593740A (zh) * 2018-12-07 2019-04-09 广东省微生物研究所（广东省微生物分析检测中心） 一种糖基转移酶及其应用

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997023630A2 (fr) * 1995-12-21 1997-07-03 Abbott Laboratories Genes de biosynthese de sucres associes a des polyketides
WO1999005283A2 (fr) * 1997-07-25 1999-02-04 Hoechst Marion Roussel Genes de biosynthese et de transfert des 6-desoxyhexoses chez saccharopolyspora erythraea et chez streptomyces antibioticus et leur utilisation
WO2000000620A2 (fr) * 1998-06-26 2000-01-06 Regents Of The University Of Minnesota Adn codant pour la methymycine et la pikromycine

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997023630A2 (fr) * 1995-12-21 1997-07-03 Abbott Laboratories Genes de biosynthese de sucres associes a des polyketides
WO1999005283A2 (fr) * 1997-07-25 1999-02-04 Hoechst Marion Roussel Genes de biosynthese et de transfert des 6-desoxyhexoses chez saccharopolyspora erythraea et chez streptomyces antibioticus et leur utilisation
WO2000000620A2 (fr) * 1998-06-26 2000-01-06 Regents Of The University Of Minnesota Adn codant pour la methymycine et la pikromycine

Non-Patent Citations (11)

* Cited by examiner, † Cited by third party
Title
DOUMITH M ET AL.: "Interspecies complementation in Saccharopolyspora erythraea: Elucidation of the function of oleP1, oleG1 and oleG2 from the oleandomycin biosynthetic gene cluster of Streptomyces antibioticus and generation of new erythromycin derivatives." MOLECULAR MICROBIOLOGY, vol. 34, no. 5, December 1999 (1999-12), pages 1039-1048, XP002210931 ISSN: 0950-382X *
GAISSER S ET AL.: "A defined system for hybrid macrolide biosynthesis in Saccharopolyspora erythraea." MOLECULAR MICROBIOLOGY, vol. 36, no. 2, April 2000 (2000-04), pages 391-401, XP002210930 ISSN: 0950-382X *
GAISSER S ET AL.: "New erythromycin derivatives from Saccharopolyspora erythraea using sugar O-methyltransferases from the spinosyn biosynthetic gene cluster." MOLECULAR MICROBIOLOGY, vol. 41, no. 5, September 2001 (2001-09), pages 1223-1231, XP002210934 ISSN: 0950-382X *
GAISSER S ET AL.: "Sugaring the pill by design." NATURE BIOTECHNOLOGY, vol. 16, no. 1, January 1998 (1998-01), pages 19-20, XP002210928 ISSN: 1087-0156 *
HUTCHINSON C R: "Combinatorial biosynthesis for new drug discovery" CURRENT OPINION IN MICROBIOLOGY, vol. 1, no. 3, 1998, pages 319-329, XP000993550 ISSN: 1369-5274 *
LIU H-W ET AL: "Pathways and mechanisms in the biogenesis of novel deoxysugars by bacteria" ANNUAL REVIEW OF MICROBIOLOGY, vol. 48, 1994, pages 223-256, XP002061259 ISSN: 0066-4227 *
MADDURI K ET AL.: "Production of the antitumor drug epirubicin (4'-epidoxorubicin) and its precursor by a genetically engineered strain of Streptomyces peucetius." NATURE BIOTECHNOLOGY, vol. 16, no. 1, January 1998 (1998-01), pages 69-74, XP002210929 ISSN: 1087-0156 *
See also references of EP1325134A2 *
XUE Y ET AL.: "A gene cluster for macrolide antibiotic biosynthesis in Streptomyces venezuelae: Architecture of metabolic diversity." PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES, vol. 95, no. 21, 13 October 1998 (1998-10-13), pages 12111-12116, XP002210932 ISSN: 0027-8424 *
YAMASE H ET AL.: "Engineering a Hybrid Sugar Biosynthetic Pathway: Production of L-Rhamnose and Its Implication on Dihydrostreptose Biosynthesis" JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, vol. 122, no. 49, 13 December 2000 (2000-12-13), pages 12397-12398, XP002210933 *
ZHAO L ET AL.: "Engineering a methymycin/pikromycin-calicheamicin hybrid: Construction of two new macrolides carrying a designed sugar moiety" JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, vol. 121, no. 42, 27 October 1999 (1999-10-27), pages 9881-9882, XP002210927 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109593740A (zh) * 2018-12-07 2019-04-09 广东省微生物研究所（广东省微生物分析检测中心） 一种糖基转移酶及其应用

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EP1325134A2 (fr) 2003-07-09
JP2004534502A (ja) 2004-11-18
WO2002029035A3 (fr) 2003-01-30
CA2424567A1 (fr) 2002-04-11

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