WO2001009155A1 - Cloning of the streptomyces avermitilis genes for glycosylation of avermectin aglycones - Google Patents

Cloning of the streptomyces avermitilis genes for glycosylation of avermectin aglycones Download PDF

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Publication number
WO2001009155A1
WO2001009155A1 PCT/US2000/020331 US0020331W WO0109155A1 WO 2001009155 A1 WO2001009155 A1 WO 2001009155A1 US 0020331 W US0020331 W US 0020331W WO 0109155 A1 WO0109155 A1 WO 0109155A1
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Prior art keywords
polynucleotide
avermectin
protein
genes
sequence
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PCT/US2000/020331
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French (fr)
Inventor
Douglas J. Macneil
James Occi
Keith M. Gewain
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Merck & Co., Inc.
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Application filed by Merck & Co., Inc. filed Critical Merck & Co., Inc.
Priority to CA002380639A priority Critical patent/CA2380639A1/en
Priority to EP00950713A priority patent/EP1206477A4/en
Priority to JP2001513962A priority patent/JP2003506022A/en
Publication of WO2001009155A1 publication Critical patent/WO2001009155A1/en

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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
    • A61P33/00—Antiparasitic agents
    • A61P33/10—Anthelmintics

Definitions

  • the invention is in the field of the genetics of biocatalysis and biosynthesis of secondary metabolites.
  • Streptomyces are gram positive bacteria which undergo temporal differentiation from substrate mycelia to aerial mycelia and, later to, spores. Streptomyces produce a wide variety of secondary metabolites, including most of the known antibiotics. In order to better understand the biology of secondary metabolism, many genetic techniques have been developed for Streptomyces (reviewed by Hopwood, 1967; Chater and Hopwood, 1984). In addition, in order to isolate and study the function of Streptomyces genes involved in antibiotic production, recombinant DNA procedures have been developed (Hopwood et al., 1985).
  • Avermectins are potent anthelmintic compounds which are active against many endoparasites of animals and humans, including
  • the avermectins are also active against arthropod ectoparasites (Fisher et al, 1984) and are effective in controlling numerous agricultural pests (Putter et al., 1981).
  • the semi -synthetic avermectin, ivermectin is a major compound in use world wide for control of animal parasites. Therefore, it is commercially important to know how many genes are involved in the biosynthesis of the avermectins, how the genes are regulated, and what the genes' functions are. Efficient procedures for transformation of S.
  • avermitilis have been developed (Klapko & MacNeil, 1987) and a variety of plasmid vectors have been identified which replicate in 5. avermitilis (Klapko & MacNeil, 1987; MacNeil, 1988; MacNeil & Gibbons, 1986).
  • avermitilis that have altered pathways of avermectin biosynthesis have been described. These includes a mutant which fails to close the furan ring of avermectin (Gogelman et al., 1983), a mutant which produces avermectin aglycones (Schulman et al., 1985), and mutants which are deficient in O- methylation of avermectin (Ruby et al., 1986; Schulman et al., 1987). Ikeda et al. (1987) reported the isolation of two classes of S. avermitilis mutants.
  • NPA mutants nonproducers
  • AGL mutants aglycone producers
  • OMT mutants which lack the ability to methylate the O at C-5
  • GMT mutants which lack the ability to methylate the O at C-3' and C-3" of the oleandrose moiety. Ikeda et al. used a natural fertility system to show linkage between the mutations in these classes, indicating that at least some of the genes for avermectin biosynthesis are clustered.
  • Complementation analysis using various restriction fragments from one end of the avermectin gene cluster, has identified 3 complementation classes involved in the synthesis and/or attachment of oleandrose to the avermectin aglycone (MacNeil et al., (1992)).
  • the present invention extends the genetic analysis of the avermectin genes involved in glycosylation.
  • the invention provides polynucleotides of eight ORFs that correlate to seven glycosylation deficiency complementation classes.
  • the invention further provides eight polypeptides encoded by the ORFs.
  • Aspects of this invention are isolated nucleic acid fragments of the 11 kb fragment of the S. avermitilis genome disclosed herein.
  • the fragments preferable encode at least one of the proteins encoded on the genomic fragment.
  • Any such polynucleotide includes but is not necessarily limited to nucleotide substitutions, deletions, additions, amino-terminal truncations and carboxy-terminal truncations such that these mutations encode an ORF that can be expressed as a protein or protein fragment of enzymatic, biochemical, biosynthetic or diagnostic use.
  • the isolated nucleic acid molecule of the present invention can be a deoxyribonucleic acid molecule (DNA), such as genomic DNA and complementary DNA (cDNA), which can be single (coding or noncoding strand) or double stranded, as well as synthetic DNA, such as a synthesized, single stranded polynucleotide.
  • DNA deoxyribonucleic acid molecule
  • cDNA complementary DNA
  • synthetic DNA such as a synthesized, single stranded polynucleotide.
  • the isolated nucleic acid molecule of the present invention can also be a ribonucleic acid molecule (RNA).
  • the nucleic acid can include the entire sequence of the gene cluster, the sequence of any one of the ORFs, a sequence encoding an ORF and an associated promoter, or smaller sequences useful for expressing peptides, polypeptides or full length proteins encoded in the fragment of the S. avermitilis genome disclosed herein.
  • the nucleic acid can have natural, non-natural or modified nucleotides or internucleotide linkages or mixtures of these. Aspects of the present invention include nucleotide probes and primers derived from the nucleotide disclosed herein.
  • probes and primers are used to identify or isolate polynucleotides encoding the avermectin pathway proteins disclosed herein or mutant or polymorphic forms of the proteins.
  • Probe and primers can be highly specific for the nucleotide sequences disclosed herein.
  • An aspect of this invention is a substantially purified form of a protein described herein.
  • the proteins have the amino acid sequence disclosed herein and set forth in SEQ ID NOs.
  • aspects of the present invention include fragments, polymorphs and/or mutants of the polypeptides disclosed herein, including but not necessarily limited to amino acid substitutions, deletions, additions, amino terminal truncations and carboxy-terminal truncations such that these mutations provide for active proteins or active protein fragments or protein fragments of diagnostic use.
  • aspects of the present invention include recombinant vectors and recombinant hosts which contain the nucleic acid molecules disclosed throughout this specification
  • the vectors and hosts can be prokaryotic or eukaryotic.
  • the hosts express peptides, polypeptides, protems or fusion proteins of the avermectin pathway polypeptides disclosed herein.
  • the host cells are used as a source of expression products
  • aspects of the invention are polyclonal and monoclonal antibodies raised in response to either the entirety of a polypeptide disclosed herein, or only a fragment, or a single epitope thereof.
  • nucleic acids or proteins disclosed herein include the use of the nucleic acids or proteins disclosed herein, and their active polypeptide fragments, together, individually, or in combination with other enzymatically active polypeptides to perform combinatorial biocatalysis in vitro and in vivo in an approp ⁇ ate host cell.
  • the nucleic acids or polypeptides disclosed herein are used to perform biotransformations of macrolide compounds, including the glycosylation of avermectin or other macrolide aglycones.
  • the nucleic acid and proteins can be used in vivo in a bacte ⁇ al host, in vitro in combination with an actinomycete fermentation, or in vitro in combination with enzymatically active polypeptides that are not from the avermectin biosynthetic pathway to effect the synthesis of a pharmaceutically active compound, including but not limited to an antibiotic compound.
  • a pharmaceutically active compound including but not limited to an antibiotic compound.
  • FIG. 1 A map showing the location of the 8 avermectin genes on the 11 kb Pstl fragment and indicating the subclones of the region used m the complementation analysis.
  • the present invention provides nucleotide sequences of eight genes of the Streptomyces avermitilis avermectin biosynthesis pathway.
  • the genes are a cluster of genes involved in the synthesis and addition of oleandrose to avermectin aglycone.
  • the invention also provides the polypeptides encoded by these genes.
  • the genes and polypeptides can be used to glycosylate avermectin aglycones, other macro des or other hydroxy compounds.
  • the genes and polypeptides can be used in combination with other biosynthetic genes to produce known or novel compounds.
  • a preferred aspect of the present invention is a nucleic acid that encodes at least one polypeptide encoded by the sequence disclosed below.
  • a preferred embodiment is a nucleic acid that encodes at least one polypeptide encoded by the sequence disclosed below and has the same sequence from that segment of the sequence disclosed below follows:
  • the sequence SEQ ID NO: 1 is characterized by the following open reading frames (ORFs) noted below.
  • ORFs open reading frames
  • Each ORF encodes a protein in the avermectin biosynthetic pathway.
  • Avermectin glycosylation genes AvrB, C, and D were identified by complementation analysis previously (MacNeil et al Gene (1992) 111:61-68 and map to ORF2, ORF3b and ORF3a respectively.
  • Newly identified genes for avermectin glycosylation are designated AvrE, F, G, H, and I.
  • An isolated nucleic acid molecule of the present invention can include a deoxyribonucleic acid molecule (DNA), such as genomic DNA and complementary DNA (cDNA), which can be single (coding or noncoding strand) or double stranded, as well as synthetic DNA, such as a synthesized, single stranded polynucleotide.
  • the isolated nucleic acid molecule of the present invention can also include a ribonucleic acid molecule (RNA).
  • polynucleotide is a nucleic acid of more than one nucleotide.
  • a polynucleotide can be made up of multiple polynucleotide units that are referred to by description of the unit.
  • a polynucleotide can comprise within its bounds a polynucleotide(s) having a coding sequence(s), a polynucleotide(s) that is a regulatory region(s) and/or other polynucleotide units commonly used in the art.
  • the present invention also relates to recombinant vectors and recombinant hosts, both prokaryotic and eukaryotic, which contain the substantially purified nucleic acid molecules disclosed throughout this specification.
  • the DNA sequences of the present invention encoding a polypeptide disclosed herein, in whole or in part, can be linked with other DNA sequences, i.e., a sequences to which the nucleic acid is not naturally linked, to form "recombinant DNA molecules" a nucleic acid disclosed herein.
  • the novel DNA sequences of the present invention can be inserted into vectors in order to direct recombinant expression of polypeptides disclosed herein.
  • Such vectors may be comprised of DNA or RNA; for most purposes DNA vectors are preferred.
  • Typical vectors include plasmids, modified viruses, bacteriophage, cosmids, yeast artificial chromosomes, and other forms of episomal or integrated DNA that can encode a polypeptide disclosed herein.
  • plasmids modified viruses
  • bacteriophage modified viruses
  • cosmids modified viruses
  • yeast artificial chromosomes yeast artificial chromosomes
  • Other forms of episomal or integrated DNA that can encode a polypeptide disclosed herein.
  • One skilled in the art can readily determine an appropriate vector for a particular use.
  • an "expression vector” is a polynucleotide having regulatory regions operably linked to a coding region such that, when in a host cell, the regulatory regions can direct the expression of the coding sequence.
  • the use of expression vectors is well known in the art. Expression vectors can be used in a variety of host cells and, therefore, the regulatory regions are preferably chosen as appropriate for the particular host cell. Preferred expression vectors can be those particularly designed for use in actinomycetes or the particular host chosen for a particular application of a gene or protein disclosed herein.
  • a “regulatory region” is a polynucleotide that can promote or enhance the initiation or termination of transcription or translation of a coding sequence.
  • a regulatory region includes a sequence that is recognized by the RNA polymerase, ribosome, or associated transcription or translation initiation or termination factors of a host cell. Regulatory regions that direct the initiation of transcription or translation can direct constitutive or inducible expression of a coding sequence. Preferred regulatory regions can be those particularly designed for use in actinomycetes or the particular host chosen for a particular application of a gene or protein disclosed herein.
  • Polynucleotides of this invention contain full length or partial length sequences of ORFs disclosed herein. Polynucleotides of this invention can be single or double stranded.
  • the polynucleotides can be a coding, "sense,” strand or a complementary, “antisense,” strand.
  • Antisense strands can be useful as modulators of the receptor by interacting with RNA encoding the receptor.
  • Antisense strands are preferably less than full length strands having sequences unique or highly specific for RNA encoding the receptor.
  • the polynucleotides can include deoxyribonucleotides, ribonucleotides or mixtures of both.
  • the polynucleotides can be produced by cells, in cell-free biochemical reactions or through chemical synthesis.
  • Non-natural or modified nucleotides including inosine, methyl-cytosine, deaza-guanosine, and others known to those of skill in the art, can be present.
  • Natural phosphodiester internucleotide linkages can be appropriate.
  • polynucleotides can have non-natural linkages between the nucleotides.
  • Non-natural linkages are well known in the art and include, without limitation, methylphosphonates, phosphorothioates, phosphorodithionates, phosphoroamidites and phosphate ester linkages.
  • Dephospho-linkages are also known, as bridges between nucleotides. Examples of these include siloxane, carbonate, carboxymethyl ester, acetamidate, carbamate, and thioether bridges.
  • Plastic DNA having, for example, N-vinyl, methacryloxytethyl, methacrylamide or ethyleneimine internucleotide linkages, can be used.
  • PNA Peptide Nucleic Acid
  • PNA is also useful and resists degradation by nucleases. These linkages can be mixed in a polynucleotide.
  • nucleic acids claimed herein can be present in whole cells or in cell lysates or in a partially, substantially or wholly purified form.
  • a polynucleotide is considered purified when it is purified away from environmental contaminants.
  • a polynucleotide isolated from cells is considered to be substantially purified when purified from cellular components by standard methods while a chemically synthesized nucleic acid sequence is considered to be substantially purified when purified from its chemical precursors.
  • nucleotide sequences that hybridize to the sequences disclosed herein under stringent conditions.
  • a procedure using conditions of high stringency is as follows: Prehybridization of filters containing DNA is carried out for 2 hr. to overnight at 65°C in buffer composed of 6X SSC, 5X Denhardt's solution, and 100 ⁇ g/ml denatured salmon sperm DNA. Filters are hybridized for 12 to 48 hrs at 65 °C in prehybridization mixture containing 100 ⁇ g/ml denatured salmon sperm DNA and 5-20 X l ⁇ 6 cpm of 32p_ ⁇ a beled probe. Washing of filters is done at 37°C for 1 hr in a solution containing 2X SSC, 0.1% SDS. This is followed by a wash in 0.1X SSC, 0.1% SDS at 50°C for 45 min. before autoradiography.
  • Preferred aspects of the present invention are substantially purified forms of the polypeptides encoded by the fragment of the S. avermitilis genome disclosed herein.
  • Preferred embodiments of these aspects of the invention proteins that have an amino acid sequence which is set forth in SEQ ID NOs:2-10 and disclosed as follows in single letter code:
  • the present invention also relates to fragments and mutant or polymo ⁇ hic forms of the protems set forth in SEQ ID NOs:2-10, including but not necessa ⁇ ly limited to amino acid substitutions, deletions, additions, ammo terminal truncations and carboxy-terminal truncations such that these provide for protems or protein fragments of enzymatic, biocatalytic, biosynthetic or diagnostic use.
  • polynucleotide and polypeptide sequences provided herein to isolate polynucleotides encoding naturally occurring forms of the proteins disclosed herein, one of skill in the art can determine whether such naturally occurring forms are mutant or polymo ⁇ hic forms by sequence comparison.
  • mutant or polymo ⁇ hic forms, or fragments of any protein disclosed herein are biologically active by routine testing of the protein or fragment in a in vitro or in vivo assay for the biological activity of the full length version of the protein as encoded by the nucleotide sequence disclosed herein. For example, one can express N-terminal or C-terminal truncations, or internal additions or deletions of a protein in a host cell and test whether the altered form can perform the same enzymatic step as performed by the full-length polypeptide disclosed herein.
  • this invention is also directed to those DNA sequences encode RNA comprising alternative codons which code for the eventual translation of the identical amino acid sequence of any of the avermectin pathway proteins disclosed herein. Therefore, the present invention includes nucleic acid sequences that vary because of codon redundancy which can result in differing DNA molecules expressing an identical protein.
  • this invention includes modified polypeptides which have amino acid deletions, additions, or substitutions but that still retain substantially the same biological activity as proteins disclosed herein. Also included within the scope of this invention are polypeptides having changes which do not substantially alter the ultimate physical or functional properties of the expressed protein.
  • a "conservative amino acid substitution” refers to the replacement of one amino acid residue by another, chemically similar, amino acid residue.
  • DNA sequences coding for a peptide can be altered so as to code for a peptide having properties that are different than those of the naturally occurring peptide. Methods of altering the DNA sequences include but are not limited to site directed mutagenesis. Examples of altered properties include but are not limited to changes in the affinity of an enzyme for a substrate or a receptor for a ligand.
  • naturally occurring, or wild-type protein has an amino acid sequence shown as SEQ ID NOs:2-10 and is encoded by the particular nucleic acid sequences disclosed herein.
  • a "functional equivalent" of a wild-type protein possesses a biological activity that is substantially the same biological activity of the wild type protein.
  • a polypeptide has "substantially the same biological activity" as a wild-type if that polypeptide has a Krj for a ligand that is no more than 5-fold greater than the Kd of the wild-type for the same ligand.
  • the term “functional derivative” is intended to include those "fragments,” “mutants,” “variants,” “degenerate variants,” “analogs,” “homologues” or “chemical derivatives” of the wild type protein that exhibit substantially the same biological activity.
  • fragment is meant to refer to any polypeptide subset of wild-type protein disclosed herein.
  • mutant is meant to refer to a molecule that may be substantially similar to the wild-type form but possesses distinguishing biological characteristics. Such altered characteristics include but are in no way limited to altered substrate binding, altered substrate affinity and altered sensitivity to chemical compounds affecting biological activity of the wild-type.
  • variant is meant to refer to a molecule substantially similar in structure and function to either the entire wild-type protein or to a fragment thereof. As used herein in reference to a gene or encoded protein, a
  • polymo ⁇ hic form that is naturally found as an allele in the population at large.
  • a polymo ⁇ hic form can have a different nucleotide sequence from the particular nucleic acid or protein disclosed herein. However, because of silent mutations, a polymo ⁇ hic gene can encode the same or different amino acid sequence as that disclosed herein. Further, some polymo ⁇ hic forms will exhibit biological characteristics that distinguish the form from wild-type protein activity, in which case the polymo ⁇ hic form is also a mutant. Polymo ⁇ hic forms encompass allelic variants.
  • a protein or fragment thereof is considered purified or isolated when it is obtained at a concentration at least about five-fold to ten-fold higher than that found in nature.
  • a protein or fragment thereof is considered substantially pure if it is obtained at a concentration of at least about 100-fold higher than that found in nature.
  • a protein or fragment thereof is considered essentially pure if it is obtained at a concentration of at least about 1000-fold higher than that found in nature.
  • the present invention also relates to recombinant vectors and recombinant hosts, both prokaryotic and eukaryotic, which contain the substantially purified nucleic acid molecules disclosed throughout this specification. Therefore, the present invention also relates to methods of expressing the proteins and their biological equivalents described herein and reactions employing these recombinantly expressed gene products, including in vivo or in vitro biosynthetic, biocatalytic or biotransformation reactions employing the genes, proteins, vectors and host cells disclosed herein.
  • a variety of expression vectors can be used to express recombinant proteins in host cells. Expression vectors are defined herein as DNA sequences that are arranged for the transcription of cloned DNA and the translation of their mRNAs in an appropriate host. Such vectors can be used to express the nucleotide sequences of this invention in a variety of hosts such as bacteria, blue-green algae, plant cells, insect cells and animal cells. Specifically designed vectors allow the shuttling of
  • An appropriately constructed expression vector should contain: an origin of replication for autonomous replication in host cells, selectable markers, a limited number of useful restriction enzyme sites, optionally a potential for high copy number, and promoters.
  • a promoter is defined as a DNA sequence operably linked to a coding region so that it interacts with cellular proteins to direct RNA polymerase to bind to DNA and initiate mRNA synthesis.
  • a strong promoter is one which causes mRNAs to be initiated at high frequency.
  • a promoter can be inducible.
  • Expression vectors can include, but are not limited to, cloning vectors, modified cloning vectors, specifically designed plasmids or viruses.
  • mammalian expression vectors which can be suitable for recombinant protein expression, include but are not limited to, pcDNA3.1 (Invitrogen), pLLTMUS28, pLITMUS29, pLLTMUS38 and pLLTMUS39 (New England Biolabs), pcDNAI, pcDNAIamp (Invitrogen), pcDNA3 (Invitrogen), pMClneo (Stratagene), pXTl (Stratagene), pSG5 (Stratagene), EBO-pSV2-neo (ATCC 37593) pBPV-l(8-2) (ATCC 37110), pdBPV-MMTneo(342-12) (ATCC 37224), pRSVgpt (ATCC 37199), pRSVneo (ATCC 37198), pSV2-dhfr (ATCC 37146), pUCTag (ATCC 37460), and 1ZD35 (ATCC 37565).
  • bacterial expression vectors can be used to express recombinant protein in bacterial cells.
  • Commercially available bacterial expression vectors which are suitable for recombinant expression include, but are not limited to pQE (Qiagen), pETl la (Novagen), lambda gtl 1 (Invitrogen), and pKK223-3 (Pharmacia).
  • Preferrred vectors include vectors designed for expression of proteins in actinomycetes including but not limited to the pIJ series developed at the John Innes Institute and described in Hopwood, D.A. et al., 1985. Genetic Manipulation of Streptomyces, A Laboratory Manual. F.
  • fungal cell expression vectors can be used to express recombinant protein in fungal cells.
  • Commercially available fungal cell expression vectors which are suitable for recombinant expression include but are not limited to pYES2 (Invitrogen) and Pichia expression vector (Invitrogen).
  • insect cell expression vectors can be used to express recombinant receptor in insect cells.
  • Commercially available insect cell expression vectors which are suitable for recombinant expression include but are not limited to pBlueBacHI and pBlueBacHis2 (Invitrogen), and pAcG2T (Pharmingen).
  • An expression vector containing DNA encoding a protein can be used for expression of the protein in a recombinant host cell.
  • Recombinant host cells can be prokaryotic or eukaryotic, including but not limited to bacteria such as E.
  • coli or Streptomycetes fungal cells such as yeast, mammalian cells including but not limited to cell lines of human, bovine, porcine, monkey and rodent origin, and insect cells including but not limited to Drosophila- and silkworm-derived cell lines.
  • L cells L-M(TK") ATCC CCL 1.3
  • L cells L-M ATCC CCL 1.2
  • Saos-2 ATCC HTB-85
  • 293 ATCC CRL 1573
  • Raji ATCC CCL 86
  • CV-1 ATCC CCL 70
  • COS-1 ATCC CRL 1650
  • COS-7 ATCC CRL 1651
  • CHO-Kl ATCC CCL 61
  • 3T3 ATCC CCL 92
  • NIH/3T3 ATCC CRL 1658
  • HeLa ATCC CCL 2
  • C127I ATCC CRL 1616
  • BS-C-1 ATCC CCL 26
  • MRC-5 ATCC CCL 171
  • CPAE ATCC CCL 209
  • the appropriateness of any cell line for any particular pu ⁇ ose can be assessed by simply testing the expression of a protein of this invention in the cell line.
  • the expression vector can be introduced into host cells via any one of a number of techniques including but not limited to transformation, transfection, protoplast fusion, and electroporation.
  • the expression vector-containing cells are analyzed to determine whether they produce protein Identification of expressing cells can be done by several means, including but not limited to immunological reactivity with antibodies, labeled ligand binding and the presence of host cell-associated recombinant protein activity
  • the cloned DNA obtained through the methods desc ⁇ bed herein can be recombinantly expressed by molecular cloning into an expression vector containing a suitable promoter and other approp ⁇ ate transc ⁇ ption regulatory elements, and transferred into prokaryotic or eukaryotic host cells to produce recombinant protein.
  • Techniques for such manipulations can be found desc ⁇ bed in Sambrook, et al , supra , and are well known and easily available to the one of ordinary skill in the art
  • Synthetic mRNA can be efficiently translated in va ⁇ ous cell-free systems, including but not limited to wheat germ extracts and reticulocyte extracts
  • molecules including but not limited to the following can be constructed- a DNA fragment containing the full-length open reading frame for a protein as well as va ⁇ ous constructs containing portions of the DNA encoding only specific domains of the protein or rearranged domains of the protein.
  • the expression levels and activity of the protein can be determined following the introduction, both singly and in combination, of these constructs into approp ⁇ ate host cells Following determination of the DNA cassette yielding optimal expression in transient assays, this construct is transferred to a va ⁇ ety of expression vectors, including but not limited to those for mammalian cells, plant cells, insect cells, oocytes, bacte ⁇ a, and yeast cells where expression is assessed.
  • Protein and polypeptides can be pu ⁇ fied from cell lysates and extracts, or from conditioned culture medium, by va ⁇ ous combinations of, or individual application of methods including ultrafiltration, acid extraction, alcohol precipitation, salt fractionation, ionic exchange chromatography, phosphocellulose chromatography, lecithin chromatography, affinity (e.g., antibody or His-Ni) chromatography, size exclusion chromatography, hydroxylapatite adso ⁇ tion chromatography and chromatography based on hydrophobic or hydrophilic interactions.
  • protein denaturation and refolding steps can be employed.
  • High performance liquid chromatography (HPLC) and reversed phase HPLC can also be useful. Dialysis can be used to adjust the final buffer composition.
  • the present invention also relates to polyclonal and monoclonal antibodies raised in response to a protein disclosed herein, or a fragment thereof. It is preferable to raise antibodies to epitopes which show the least homology to other known proteins.
  • An antibody is specific for an epitope if one of skill in the art can use standard techniques to determine conditions under which one can detect a polypeptide of this invention in a Western Blot of a sample from a host cell that expresses a protein of this invention.
  • the blot can be of a native or denaturing gel as appropriate for the epitope.
  • An antibody is highly specific for an epitope if no nonspecific background binding is visually detectable.
  • An antibody can also be considered highly specific if the binding of the antibody to the protein can not be competed by non- homologous peptides, polypeptides or proteins, but can be competed by homologous peptides or polypeptides or the full length form of the relevant protein as disclosed herein.
  • Recombinant protein can be separated from other cellular proteins by use of an immunoaffinity column made with monoclonal or polyclonal antibodies specific for full-length protein, or polypeptide fragments of protein. Additionally, polyclonal or monoclonal antibodies can be raised against a synthetic peptide (usually from about 9 to about 25 amino acids in length) from a portion of a protein disclosed in SEQ ID NOs:2-10. Monospecific antibodies are purified from mammalian antisera containing antibodies reactive against a protein or are prepared as monoclonal antibodies reactive with a protein using the technique of Kohler and Milstein (1975, Nature 256: 495-497). Monospecific antibody as used herein is defined as a single antibody species or multiple antibody species with homogenous binding characteristics for a particular protein.
  • Homogenous binding refers to the ability of the antibody species to bind to a specific antigen or epitope, such as those associated with a protein described herein.
  • Specific antibodies are raised by immunizing animals such as mice, rats, guinea pigs, rabbits, goats, horses and the like, with an appropriate concentration of a protein described herein or a synthetic peptide generated from a portion of the desc ⁇ bed proteins with or without an immune adjuvant
  • Preimmune serum is collected p ⁇ or to the first immunization.
  • Each animal receives between about 0.1 mg and about 1000 mg of protein associated with an acceptable immune adjuvant
  • acceptable adjuvants include, but are not limited to, Freund's complete, Freund's incomplete, alum-precipitate, water in oil emulsion containing Corynebacterium parvum and tRNA
  • the initial immunization consists of injecting protein or peptide fragment thereof, preferably in Freund's complete adjuvant, at multiple sites either subcutaneously (SC), intrape ⁇ toneally (IP) or both
  • SC subcutaneously
  • IP intrape ⁇ toneally
  • Each animal is bled at regular intervals, preferably weekly, to determine antibody titer
  • the animals may or may not receive booster injections following the initial immunization Those animals receiving booster injections are generally given an equal amount of protein m Freund's incomplete adjuvant by the same route.
  • Booster injections are given at about three week intervals until maximal titers are obtained At about 7 days after each booster immunization or about weekly after a single immunization, the animals are bled, the serum collected, and ahquots are stored at about -20°C.
  • Monoclonal antibodies (mAb) reactive with a protein are prepared by immunizing mbred mice, preferably Balb/c, with the protein.
  • the mice are immunized by the IP or SC route with about 1 mg to about 100 mg, preferably about 10 mg, of protein m about 0.5 ml buffer or saline inco ⁇ orated an equal volume of an acceptable adjuvant, as discussed herein. Freund's complete adjuvant is preferred.
  • the mice receive an initial immunization on day 0 and are rested for about 3 to about 30 weeks. Immunized mice are given one or more booster immunizations of about 1 to about 100 mg of protein m a buffer solution such as phosphate buffered saline by the intravenous (IV) route.
  • IV intravenous
  • Lymphocytes from antibody positive mice, preferably splenic lymphocytes, are obtained by removing spleens from immunized mice by standard procedures known in the art.
  • Hyb ⁇ doma cells are produced by mixing the splenic lymphocytes with an approp ⁇ ate fusion partner, preferably myeloma cells, under conditions which will allow the formation of stable hyb ⁇ domas.
  • Fusion partners can include, but are not limited to: mouse myelomas P3/NSl/Ag 4-1; MPC-11; S-194 and Sp 2/0, with Sp 2/0 being preferred.
  • the antibody producing cells and myeloma cells are fused in polyethylene glycol, about 1000 mol. wt., at concentrations from about 30% to about 50%.
  • Fused hyb ⁇ doma cells are selected by growth in hypoxanthme, thymidme and ammopte ⁇ n supplemented Dulbecco's Modified Eagles Medium (DMEM) by procedures known in the art.
  • DMEM Dulbecco's Modified Eagles Medium
  • Supernatant fluids are collected form growth positive wells on about days 14, 18, and 21 and are screened for antibody production by an immunoassay such as solid phase immunoradioassay (SPIRA) using the protein as the antigen.
  • SPIRA solid phase immunoradioassay
  • the culture fluids are also tested in the Ouchterlony precipitation assay to determine the isotype of the mAb.
  • Hybridoma cells from antibody positive wells are cloned by a technique such as the soft agar technique of MacPherson, 1973, Soft Agar Techniques, in Tissue Culture Methods and Applications, Kruse and Paterson, Eds., Academic Press.
  • Monoclonal antibodies are produced in vivo by injection of pristine primed Balb/c mice, approximately 0.5 ml per mouse, with about 2 x 106 to about 6 x l ⁇ 6 hybridoma cells about 4 days after priming. Ascites fluid is collected at approximately 8-12 days after cell transfer and the monoclonal antibodies are purified by techniques known in the art.
  • mAb In vitro production of mAb is carried out by growing the hybridoma in DMEM containing about 2% fetal calf serum to obtain sufficient quantities of the specific mAb.
  • the mAb are purified by techniques known in the art.
  • Antibody titers of ascites or hybridoma culture fluids are determined by various serological or immunological assays which include, but are not limited to, precipitation, passive agglutination, enzyme-linked immunosorbent antibody (ELISA) technique and radioimmunoassay (RIA) techniques. Similar assays are used to detect the presence of the protein in a biological sample or in an in vitro biocatalysis reaction.
  • serological or immunological assays include, but are not limited to, precipitation, passive agglutination, enzyme-linked immunosorbent antibody (ELISA) technique and radioimmunoassay (RIA) techniques. Similar assays are used to detect the presence of the protein in a biological sample or in an in vitro biocatalysis reaction.
  • Antibody affinity columns are made, for example, by adding the antibodies to Affigel-10 (Biorad), a gel support which is pre-activated with N- hydroxysuccinimide esters such that the antibodies form covalent linkages with the agarose gel bead support. The antibodies are then coupled to the gel via amide bonds with the spacer arm. The remaining activated esters are then quenched with 1M ethanolamine HC1 (pH 8). The column is washed with water followed by 0.23 M glycine HC1 (pH 2.6) to remove any non-conjugated antibody or extraneous protein.
  • the column is then equilibrated in phosphate buffered saline (pH 7.3) and the cell culture supernatants or cell extracts containing full-length protein or protein fragments are slowly passed through the column.
  • the column is then washed with phosphate buffered saline until the optical density (A28O) falls to background, then the protein is eluted with 0.23 M glyc e-HCl (pH 2.6).
  • the pu ⁇ fied protein is then dialyzed against phosphate buffered saline.
  • Levels of recombinant protein in host cells is quantified by a va ⁇ ety of techniques including, but not limited to, immunoaffinity and/or ligand affinity techniques.
  • Specific-antibody affinity beads or specific antibodies are used to isolate 35S-meth ⁇ onme labeled or unlabelled recombinant protein.
  • Labeled recombinant protein is analyzed by SDS-PAGE.
  • Unlabelled protein is detected by Western blotting, ELISA or RIA assays employing either protein specific antibodies and/or antiphosphotyrosme antibodies.
  • pVE650 a 47.8 kb plasmid was isolated from a library of 5. avermitilis by its ability to complement a mutant producing non-glycosylated avermectins. Five overlapping cosmid clones of S. avermitilis genomic DNA were isolated using a fragment of pVE650 as a probe. Subclones from pVE650 and an overlapping cosmid were used in complementation studies with 23 mutants defective in the glycosylation of avermectin aglycone. Seven complementation classes were identified.
  • a 11-kb Pstl fragment of S. avermitilis genomic DNA complemented all 23 mutants, indicating the genes for avermectin glycosylation were clustered.
  • the 11 kb Pstl fragment can be cloned from a deposited strain, ATCC 67890, which contains plasmid ⁇ VE859
  • the 11 kb Pstl fragment of the avermectin gene cluster from S. avermitilis was subcloned into an integration vector, pVE1053.
  • the resulting plasmid, pVE1190 could complement all the mutants known to us that are defective in glycosylation.
  • Upon sequencing 10 kb region of the fragment it was discovered that the fragment contained nine open reading frames.
  • the 11-kb subclone was mutagenized with Tn5 and Tn5seql. Fourteen insertions were transferred to S. avermitilis and used in complementation analysis. An eighth complementation class was identified. Sequencing of an 10-kb region identified nine ORFs and an additional partial ORF. Eight of the nine ORFs were correlated to seven glycosylation complementation classes confirming that these eight genes are involved in the biosynthesis and attachment of oleandrose to avermectin aglycones.
  • genes from this cluster or the encoded polypeptides could be used to glycosylate avermectin aglycones or other macrolide aglycones.
  • US patent US 5,312,753 describes the glucosylation of the C13 and C14a positions of avermectin derivatives by a S. avermitilis strain.
  • Another use of the polynucleotides and polypeptides would be to use them separately and in combination with other cloned genes or expressed proteins to make and attach known and novel sugars to known and novel macrolides or to other hydroxyl containing compounds.
  • lividans strain 1326 and its SLP2-SLP3 " derivative TK21 (Hopwood et al, 1983) were obtained from D. Hopwood (John Innes Institute, Norwich, UK).
  • pBR322 was obtained from BRL (Bethesda, MD) and pIJ922 was obtained from D. Hopwood (Hopwood et al, 1985).
  • pVE616 is a 4.4 kb Amp R derivative of pBR322 which contains a 1.8 kb Bam ⁇ fragment which expresses thiostrepton-resistance in Streptomyces (Gene). Cultures were preserved by adding 0.1 ml of dimethyl sulfoxide (Aldrich Chemical Co., Milwaukee, WI) to 0.9 ml of culture and quick freezing the mixture at -70°C.
  • E. coli was grown in LB (Miller, 1972). Solid media containing 1.5% agar included LB for E. coli (Miller, 1972), R2YE for S. lividans (Thompson, et al, 1982), RM14 for S. avermitilis (MacNeil & Klapko, 1987), and YME-TE for S. avermitilis .
  • YME-TE contained per liter: yeast extract 3.0 g, malt extract 10.0 g, dextrose 4.0 g and 4 ml of a trace element solution (per liter: HC1 (37.3%) 49.7 ml, MgSO4-7H2O 61. lg, CaCO3 2.0g, FeCl3-6H2O 5.4 g, ZnSO4-7H2O 1.44 g, MnS ⁇ 4-H2 ⁇ 1.11 g, CuSO4-5H2O 0.25 g, H3BO3 0.062g, Na2MoO4-2H2O 0.49 g). YME-TE was adjusted to pH 7.0 with NaOH before autoclaving.
  • Fermentation medium A contained, per liter: glucose 20.0 g, yeast extract 20.0g, Hy-Case SF 20.0 g/ml, MgSO4-7H2O (12.5%), NaCl (12.5%), MnS ⁇ 4-H2 ⁇ (0.5%), ZnSO4-7H2O (1.0%), CaCl2"2H2 ⁇ (2.0%), FeSO4-7H2O 0.025 g, and KNO3 2.0 g.
  • Fermentation medium B which was adjusted to pH 7.2 with NaOH before autoclaving contained, per liter, peptonized milk 20.0 g, Ardamine pH 4.0 g, glucose 90.0 g, MgSO4-7H2O 0.5 g, CUSO4 5H2O (0.06 mg/ml) 1 ml, ZnSO 4 -6H O (1 mg/ml) 1 ml, CoCl 2 -6H 2 O (0.1 mg/ml) 1ml, and FeCl -6H 2 O (3 mg/ml) 1 ml.
  • TE buffer (10 mM Tris, pH 7.9, 1 mM EDTA) was used to store and dilute DNA.
  • Polyethylene glycol 1000 PEG
  • agarose polyethylene glycol 1000
  • ampicillin obtained from Sigma Chemical Co., St Louis, MO.
  • Formamide was obtained from EBI (New Haven, CT).
  • Thiostrepton gift from E. R. Squibb & Sons, Princeton, NJ was added to a final concentration of 5 ⁇ g/ml in liquid medium, 10 ⁇ g/ml in solid medium, and 15 ⁇ g/ml when added as an overlay to select transformants.
  • Ampicillin was added to a final concentration of 100 ⁇ g/ml.
  • 0.05% diethyl pyrocarbonate was added, mixed, and centrifuged at 12,000 x g for 15 minutes at 4°C.
  • the DNA pellet was dried and the DNA was dissolved in 0.5 ml of 0.3 M ammonium acetate.
  • the solution was transferred to a 1.5 ml Eppendorf tube, mixed with 400 ⁇ l of phenol, previously equilibrated with 1 M Tris pH 7.9, and the aqueous phase separated by centrifugation in a microfuge for 3 minutes. The aqueous phase was removed to another Eppendorf tube and extracted with 400 ⁇ l of chloroform.
  • aqueous DNA solution was precipitated with 2 volumes of ethanol, washed with 70% ethanol, and the plasmid DNA resuspended in 100 ⁇ l of TE.
  • Large scale plasmid preparations were isolated from 1 1 YEME cultures of Streptomyces by a scaled up alkaline lysis procedure except that the DNA precipitated by isopropanol was resuspended in a CsCl solution and subjected to two bandings. Chromosomal DNA from Streptomyces was prepared as described by Hopwood et al, 1985.
  • Transformations with plasmid DNA Transformations with plasmid DNA.
  • Restriction enzymes were obtained from New England Biolabs (Beverly, MA), Bethesda Research Labs (Bethesda, MD), or IBI (New Haven, CT) and were used according to the manufactures directions. Agarose gels were prepared and electrophoresis performed as described (Maniatis et al, 1982).
  • OMT- cultures produce predominantly the B avermectins. Mutants unable to glycosylate avermectin aglycones also produce 4 bands, however, since aglycones are a better substrate for the C5-Omethyltransferae, mostly the A- aglycones are produced. In contrast in the OMT- strains, residual C5-O-methyltransferase only methylates about 1/2 the aglycones resulting in 4 bands. The aglycones run faster in the TLC system than the corresponding glycosylated avermectins.
  • the order, from fastest to slowest band is, avermectin aglycone Aia+b, avermectin aglycone A2a+b, avermectin Ai a+b and avermectin aglycone Bi a+b, avermectin A2a+b and avermectin aglycone B2a+b, avermectin Bia+b, and avermectin B2a+b.
  • the cosmid library of S. avermitilis was constructed in the 6.7 kb, double lambda cos vector, pVE328, and consists of 2016 cosmid clones stored as individual cultures in 21 microtiter dishes. Replicates of the library were made on LB plates containing ampicillin, colonies were transferred to Biotrans nylon membranes (1.5 ⁇ M pore size), and colonies processed to release and fix DNA to the filters (Maniatis et al., ). The resulting 21 filters were individually hybridized with 3 p labeled probes. Preparation of probes, hybridizations and autoradiography were as described above for Southern analysis.
  • Putative hybridizing clones were retested by patching duplicates to LB plates with ampicillin, lifting the colonies to nitrocellulose (Schleicher & Schuell, Keene, NH), fixing the DNA to the filters and hybridizing with the probe. Plasmid DNA was isolated from the cosmid clones which retested positive, restricted with BamKL, and confirmed by a Southern analysis.
  • avermitilis DNA was constructed and screened for complementation of two mutants defective in avermectin biosynthesis (Avr).
  • One mutant was a C-5 O-methyltransferase mutant (OMT), which produces predominantly avermectin Bi a+b and B2a+b.
  • OMT O-methyltransferase mutant
  • MA6278 an avermectin aglycone producer.
  • Several overlapping plasmids were isolated which complemented OMT mutants (Streicher et al). When the plasmids which complemented OMT mutants were introduced into several mutants altered in, or defective in, avermectin biosynthesis, no other mutants were complemented (Streicher et al).
  • Approximately 3000 transformants of MA6278 were screened for avermectin production by small scale fermentation and TLC analysis of methanol extracts of each transformant. One transformant complemented the defect in MA6278.
  • a plasmid was isolated from this transformant and designated pVE650.
  • avermectin glycosylation genes on pVE650 was confirmed by retransforming MA6278 by pVE650 and detecting glycosylated avermectins by TLC. Most aglycone producing mutants (21/26) were complemented by pVE650.
  • a restriction map was determined for pVE650 see MacNeil et al, 1992.
  • the insert in pVE650 is delimited by Bam ⁇ . sites, no sites were found in the 24 kb insert for the following enzymes: Asel, Dral, EcoRV, HindUl, Hpal, Ndel, Nhel, Spel, Sspl, and Xbal. No common restriction bands were found between pVE650 and pATl, a plasmid which complements OMT mutants (Streicher et al).
  • the insert in pVE650 was found to be colinear with the chromosome of S. avermitilis by Southern analysis.
  • the 9 BamHl fragments greater than 400 bp were used as probes against BamHl and Sstl digestions of genomic DNA from avermectin producing and nonproducing strains. Seven of the nine Z? ⁇ mHI fragments hybridized to a band identical in size to the BamHl fragment used as probes. Therefore, the seven BamHl fragments do not appear to have undergone rearrangement to form pVE650. This was confirmed by the Sstl digestions in which adjacent BamHl fragments hybridize to an overlapping Sstl fragment.
  • FIG. 1 indicates the subclones which were used to successfully complement AGL" mutants. Table 1 identifies the mutants in each complementation class and presents the complementation results with key subclones. Twenty-one aglycone producing mutants, representing complementation Classes I, ⁇ , and HI, were complemented after introduction of pVE650, but 5 Agl " mutants and two GMT- mutants were not.
  • Class I mutants were complemented when they contained pVE650, or subclone pVE908 (2.4 EcoRl-BglU fragment).
  • Class II mutants were complemented by pVE650 or subclone pVE807 (2.6 kb Bgl l fragment), but not by pVE908.
  • Class HI mutants were not complemented by pVE807 or pVE908. Although we can not exclude the occurrence of intragenic complementation, it is likely that each complementation class represents at least one gene for avermectin glycosylation.
  • cosmid contained all the DNA represented by pVE650 and additional DNA from each end. Collectively the cosmids represent 60 kb of S. avermitilis DNA. None of the cosmids overlapped sequences on pATl. From one cosmid, pVE859, we identified a 15 kb BglR fragment which contained the 470 bp EcoRl to BamHl fragment near the end of pVE650.
  • this 15 kb fragment represents the chromosomal BglR fragment that is adjacent to the 140 bp BglR fragment of pVE650 and extends 13 kb beyond the DNA contained on pVE650.
  • This fragment was cloned into pIJ922 to yield pVE941.
  • pVE941 contains all the S. avermitilis DNA on pVE807 and, as expected, complements Class II aglycone producers.
  • pVE941 also complemented all 5 AGL " mutants not complemented by pVE650 and two GMT" strains.
  • the genes for glycosylation of avermectin are clustered since all the mutants defective in synthesis or addition of oleandrose to avermectin aglycone are complemented by pVE650 and/or pVE941.
  • pVEllll (4.1 EcoRI fragment of pVE650 plus the 1.8 kb Ec ⁇ RI fragment of pVE941) complemented Class I, II and Class HI mutants.
  • the mutants in Class HI are be defective in a gene, designated ⁇ vrC, located between avrB and avrD.
  • MA6057 and MA6622 were complemented by only pVE941 and pVEl 115 and are designated class IV.
  • pVE1019 which contained the 3.5 kb BamHl fragment from pVE941, complemented the defects in the two GMT mutants and AGL " strain MA6590. This later mutant was designated Class V.
  • pVE650 Only AGL" mutants are complemented by pVE650
  • pVE650 was introduced into mutants representing each phenotypic class of S. avermitilis defective or altered in avermectin biosynthesis. No complementation was observed in MA6238 (C-22, C-23 dehydrase [DH”]), MA5218 (C-6, C-8' furan ring formation [FUR”]), MA6316 (C-3', C-3" O-methyltransferase or glycosyl O- methyltransferase [GMT “ ]), MA6262, (nonproducer of avermectin [NPA ]), or MA6233 (OMT " ).
  • pVEl 115 which contains a 12 kb Pstl fragment from S. avermitilis , complemented all 26 mutants which fail to glycosylate avermectin and 2 mutants which fail to methylate hydroxyls at the C-3', C- 3" positions.
  • pVE1115 may contain all the genes for synthesis and for attachment of oleandrose to avermectin aglycone.
  • it is possible our collection of mutants does not include defects in all the genes involved m avermectin glycosylation. If this is so, then pVEl 115 may not contain all the glycosylation genes.
  • the AvrB and AvrC genes are convergently transc ⁇ bed and their coding regions overlap for 95 nt.
  • the AvrD and AvrC genes are co-transc ⁇ bed but encode proteins in different reading frames and overlap for 16 nts.
  • a compa ⁇ son of the open reading frames in the sequence to the clones used in complementation analysis results in the identification of 8 genes essential for avermectin glycosylation.
  • ORF1 showed simila ⁇ ty to keto-reductases.
  • ORF2 showed greater than 30% identity to glycosyl-transferases.
  • ORF3a was greater than 60% identical to TDP-glucose-4,6-dehydratases, ORF3b was greater than 60% identical to several TDP-glucose synthases, and ORF4 showed weak homology to keto reductases.
  • ORF5 had greater than 50% identity to hexose 3,5 epimerases.
  • ORF7 was identified as a glycosyl methyltransferase since that ORF could complement the GMT- mutants.
  • Macrohdes contain many unusual sugars (Omura, S Macrolide Antibiotics, Academic Press, 1984).
  • a biochemical study of the mutants and cloned genes will help elucidate the biochemical pathway for synthesis of oleandrose.
  • the cloned genes for synthesis and addition of oleandrose to avermectin aglycone can be useful in mtergenic complementation studies to identify genes involved in glycosylation of other macrohdes
  • the cloned DNA can be useful as a probe to identify genes involved in the synthesis and/or addition of other sugar moieties to other macrohdes.
  • the acil gene of S. coehcolor which is required for synthesis of actmorhodin, has been useful as a probe to identify putative polyketide synthetases from other species (Bergh and Uhlen, 1992)
  • avermectins are much more potent antiparasitic agents than avermectin aglycones (Campbell, W. Ivermectm and Abamect , Sp ⁇ nger-Verlag, 1989) or the non-glycosylated, but similar milbemycms (Omura, S Macrolide Antibiotics, Academic Press, 1984), it is evident that the oleandrose disaccha ⁇ de moiety enhances the potency of avermectin.
  • genes desc ⁇ bed herein for synthesis and attachment of oleandrose to avermectin aglycone can be useful for the construction of hyb ⁇ d antibiotics.
  • the introduction of a plasmid containing at least one gene of the present invention into strains that produce antibiotics with a hydroxyl group may result in hyb ⁇ d glycosylated antibiotics.
  • Potentially useful substrates for glycosylation are other macrohdes (Omura, 1984).
  • LL-F28249 antibiotic complex A new family of antiparasitic macrocyclic lactones isolation, characterization, and structures of LL- F28249. J. Antibiotics 41:519-529.
  • Avermectins novel insecticides, acaricides, and nematicides from a soil microorganism. Experientia 37:963-964.
  • MA6 5 79 MA6581 , MA6589, MA6 5 91.
  • Plasmids used are shown in FIG. 1.
  • PVE650 has been described (MacNeil et al., 1992)
  • pVEl 115 contains the 11 kb Pstl fragment which complements all avermectin aglycone producing mutants. In most cases, at least 6 transformants of each plasmid into each mutant were tested for avermectin production by Microferm and TLC analysis.

Abstract

A cluster of genes involved in the synthesis and/or addition of oleandrose to avermectin aglycones has been cloned. A 11-kb PstI clone complemented 28 avermectin glycosylation mutants in seven complementation classes. Sequencing of an 10-kb region identified 9 ORFs and an additional partial ORF. Eight of the ORFs were correlated to the seven glycosylation complementation classes. Sequence comparison to Genbank databases identified 6 genes: dTDP-glucose synthase; dTDP-glucose 4,6 dehydrase; dTDP-4-keto-hexose reductase; dTDP-hexose 3,5 epimerase; dTDP-hexose 3' O-methylase; and an avermectin aglycone-dTDP-oleandrose glycosyltransferase. The ninth ORF was essential for biosynthesis of the avermectin aglycones. The partial ORF encoded part of an avermectin polyketide synthase module 7.

Description

TΓΓLE OF THE INVENTION
CLONING OF THE STREPTOMYCES AVERMITILIS GENES FOR
GLYCOSYLATION OF AVERMECTIN AGLYCONES
CROSS-REFERENCE TO RELATED APPLICATIONS
Not applicable.
STATEMENT REGARDING FEDERALLY-SPONSORED R&D Not applicable.
REFERENCE TO MICROFICHE APPENDD
Not applicable.
FIELD OF THE INVENTION The invention is in the field of the genetics of biocatalysis and biosynthesis of secondary metabolites.
BACKGROUND OF THE INVENTION
Streptomyces are gram positive bacteria which undergo temporal differentiation from substrate mycelia to aerial mycelia and, later to, spores. Streptomyces produce a wide variety of secondary metabolites, including most of the known antibiotics. In order to better understand the biology of secondary metabolism, many genetic techniques have been developed for Streptomyces (reviewed by Hopwood, 1967; Chater and Hopwood, 1984). In addition, in order to isolate and study the function of Streptomyces genes involved in antibiotic production, recombinant DNA procedures have been developed (Hopwood et al., 1985).
The commercially important Streptomycete, S. avermitilis , produces a series of eight related oleandrose containing, polyketide macrolides, termed the avermectins (Burg et al, 1979). Avermectins are potent anthelmintic compounds which are active against many endoparasites of animals and humans, including
Onchocerca volvulus the agent of "river blindness". The avermectins are also active against arthropod ectoparasites (Fisher et al, 1984) and are effective in controlling numerous agricultural pests (Putter et al., 1981). The semi -synthetic avermectin, ivermectin, is a major compound in use world wide for control of animal parasites. Therefore, it is commercially important to know how many genes are involved in the biosynthesis of the avermectins, how the genes are regulated, and what the genes' functions are. Efficient procedures for transformation of S. avermitilis have been developed (Klapko & MacNeil, 1987) and a variety of plasmid vectors have been identified which replicate in 5. avermitilis (Klapko & MacNeil, 1987; MacNeil, 1988; MacNeil & Gibbons, 1986).
Mutants of 5. avermitilis that have altered pathways of avermectin biosynthesis have been described. These includes a mutant which fails to close the furan ring of avermectin (Gogelman et al., 1983), a mutant which produces avermectin aglycones (Schulman et al., 1985), and mutants which are deficient in O- methylation of avermectin (Ruby et al., 1986; Schulman et al., 1987). Ikeda et al. (1987) reported the isolation of two classes of S. avermitilis mutants. These include nonproducers (NPA mutants), which produce no detectable avermectins; aglycone producers (AGL mutants), which are blocked in the glycosylation avermectin aglycones; OMT mutants which lack the ability to methylate the O at C-5, and GMT mutants which lack the ability to methylate the O at C-3' and C-3" of the oleandrose moiety. Ikeda et al. used a natural fertility system to show linkage between the mutations in these classes, indicating that at least some of the genes for avermectin biosynthesis are clustered. The genes for avermectin 5-keto reductase and avermectin 5 O-methyl transferase have been cloned (Ikeda et al., 1995; Ikeda et al., 1998). A series of overlapping cosmid clones representing 150 kb of genomic DNA were isolated by complementation of C-5 O-methyl transferase mutant and glycosylation deficient mutants. Deletion mapping over a 150 kb region located the avermectin gene cluster to a 100 kb segment (MacNeil et al., (1993). Complementation analysis, using various restriction fragments from one end of the avermectin gene cluster, has identified 3 complementation classes involved in the synthesis and/or attachment of oleandrose to the avermectin aglycone (MacNeil et al., (1992)).
SUMMARY OF THE INVENTION
The present invention extends the genetic analysis of the avermectin genes involved in glycosylation. Through sequencing and analysis of a 10 kb segment of the genome of Streptomyces avermitilis the invention provides polynucleotides of eight ORFs that correlate to seven glycosylation deficiency complementation classes. The invention further provides eight polypeptides encoded by the ORFs. Aspects of this invention are isolated nucleic acid fragments of the 11 kb fragment of the S. avermitilis genome disclosed herein. The fragments preferable encode at least one of the proteins encoded on the genomic fragment. Any such polynucleotide includes but is not necessarily limited to nucleotide substitutions, deletions, additions, amino-terminal truncations and carboxy-terminal truncations such that these mutations encode an ORF that can be expressed as a protein or protein fragment of enzymatic, biochemical, biosynthetic or diagnostic use.
In particular embodiments, the isolated nucleic acid molecule of the present invention can be a deoxyribonucleic acid molecule (DNA), such as genomic DNA and complementary DNA (cDNA), which can be single (coding or noncoding strand) or double stranded, as well as synthetic DNA, such as a synthesized, single stranded polynucleotide. The isolated nucleic acid molecule of the present invention can also be a ribonucleic acid molecule (RNA). In particular embodiments, the nucleic acid can include the entire sequence of the gene cluster, the sequence of any one of the ORFs, a sequence encoding an ORF and an associated promoter, or smaller sequences useful for expressing peptides, polypeptides or full length proteins encoded in the fragment of the S. avermitilis genome disclosed herein. In particular embodiments the nucleic acid can have natural, non-natural or modified nucleotides or internucleotide linkages or mixtures of these. Aspects of the present invention include nucleotide probes and primers derived from the nucleotide disclosed herein. In particular embodiments of the invention, probes and primers are used to identify or isolate polynucleotides encoding the avermectin pathway proteins disclosed herein or mutant or polymorphic forms of the proteins. Probe and primers can be highly specific for the nucleotide sequences disclosed herein.
An aspect of this invention is a substantially purified form of a protein described herein. In preferred embodiments the proteins have the amino acid sequence disclosed herein and set forth in SEQ ID NOs.
Aspects of the present invention include fragments, polymorphs and/or mutants of the polypeptides disclosed herein, including but not necessarily limited to amino acid substitutions, deletions, additions, amino terminal truncations and carboxy-terminal truncations such that these mutations provide for active proteins or active protein fragments or protein fragments of diagnostic use.
Aspects of the present invention include recombinant vectors and recombinant hosts which contain the nucleic acid molecules disclosed throughout this specification In particular embodiments, the vectors and hosts can be prokaryotic or eukaryotic. In particular embodiments the hosts express peptides, polypeptides, protems or fusion proteins of the avermectin pathway polypeptides disclosed herein. In further embodiments the host cells are used as a source of expression products Aspects of the invention are polyclonal and monoclonal antibodies raised in response to either the entirety of a polypeptide disclosed herein, or only a fragment, or a single epitope thereof.
Aspects of this invention include the use of the nucleic acids or proteins disclosed herein, and their active polypeptide fragments, together, individually, or in combination with other enzymatically active polypeptides to perform combinatorial biocatalysis in vitro and in vivo in an appropπate host cell. In preferred embodiments, the nucleic acids or polypeptides disclosed herein are used to perform biotransformations of macrolide compounds, including the glycosylation of avermectin or other macrolide aglycones. In particular embodiments, the nucleic acid and proteins can be used in vivo in a bacteπal host, in vitro in combination with an actinomycete fermentation, or in vitro in combination with enzymatically active polypeptides that are not from the avermectin biosynthetic pathway to effect the synthesis of a pharmaceutically active compound, including but not limited to an antibiotic compound. Each document mentioned in this specification is hereby incorporated herein by reference in its entirety.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1. A map showing the location of the 8 avermectin genes on the 11 kb Pstl fragment and indicating the subclones of the region used m the complementation analysis.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides nucleotide sequences of eight genes of the Streptomyces avermitilis avermectin biosynthesis pathway. The genes are a cluster of genes involved in the synthesis and addition of oleandrose to avermectin aglycone. The invention also provides the polypeptides encoded by these genes. The genes and polypeptides can be used to glycosylate avermectin aglycones, other macro des or other hydroxy compounds. The genes and polypeptides can be used in combination with other biosynthetic genes to produce known or novel compounds. Polynucleotides
A preferred aspect of the present invention is a nucleic acid that encodes at least one polypeptide encoded by the sequence disclosed below. A preferred embodiment is a nucleic acid that encodes at least one polypeptide encoded by the sequence disclosed below and has the same sequence from that segment of the sequence disclosed below follows:
1 GGATCCATCG CCAACGCCTC ACGCGGACTG ATCCCGAAAA ACCCCGCATC
51 GAACTCCGCC GCACCCTCCA GGAAACCGCC CCGGCGCGTA TACGACGAAC
101 CCGCCCGCCC CGGCTCCGGA TCATAGAAAG CCTCCACGTC CCAACCCCGG 151 TCGACCGGAA ACTCCCCCAC CGC ATCCCGA CCCGACGCAA TCAACTCCCA
201 GAAATCCTCC GCCGACTCCA CACCCCCCGG AAAACGGCAC GCCATCCCCA
251 CAATTGCAAT CGGCTCCTGC TCGCCCGATT CAATCTGCTG AAGTCGACGC
301 CGCACATTGA GGAGATCGGC AGTAACGCGC TTGAGATAGT CGCGGAGCTT
351 TTCCTCGTTA GCCATGGACC GGTCTCCTCG ACAAGAGAAA TCGGAAATTA 401 AAAAACACGC ATGGGACTCT CACAGGCTAG AGCGACGAGA GCAGCACAAA
451 TACCCCTAGA TACCCCAGAC CCCTGATGCT CGATGAATGC CGCTATAGCT
501 AGGGGGTATG GCGCCAGACA TGAATTCACA GCGTTTCGGC GGCCGGCTGG
551 CGCTTGTCAC AGGTGCAGGC GGTGGCATCG GGCGGGCGAC CTGCGCTCTC
601 GGATCGGCCG GGGCGCGAGT GGTGTGCGTG GACCGGGACG GCCGCGGCGC 651 CGGGGTGACG CCGACCTGGC CGGAGCGGGG CGCGCGGGCG GCCTGGCCCG 701 AGGTGGCCGA CGTGTCCGAC GGAGCGGCGA TGGAGCGGTT GCCCGAGCGC
751 GTCGCCGAGA CGTACGGGGT CGTGGACCTG CTGGTGAACA ACGCCGGCAT
801 CGGCATGGCG GGGCGTTTTC TCGACACGTC CGTCGAGGAC TGGCAGCGCA
851 CCCTGGGCGT CAACCTCTGG GGTGTCATTC ATGGTTGCCG CCTCATCGGC 901 CGGCAGATGG CGGAGCGCGG GCAGGGCGGG CACATCGTGA CGGTGGCGTC
951 GGCGGCGGCG TTCCAGCCGA CGCGGGCGGT CCCCGCGTAT GCCACCAGCA
1001 AGGCGGCGGT GCTGATGCTG AGCGAGTGCC TGCGCGCGGA GTTCGCGGAG
1051 TTCGGGGTCG GAGTGAGCGT GGTGTGCCCG GGCTTCGTCC GTACGTCGTT
1101 CGCGTCGGCG ATGCATTTCG CCGGTGTGCC CCGGCTGGAG CAGGAGCGGC 1151 TGCGGGCGCT GTTCGCCGGT CGCGGATGCA GCGCGGAGAA GGTGGCCGCG
1201 GCGGTACTGC GGTCGGTGGC GCGCGACTCG GCCGTGGTGA CCGTGACGGC
1251 GGAAGCGCGG CTGTCACGGC TGATGAGCCG CTTCACGCCA CGCCTGCGCG
1301 CCGCGGTGGC GCGGATGGAT CCCCCTTCGT AGGGCTGGCG GGGATCCCCT
1351 CCTTGCCTTC GAACATCTTC CGACGATGGG CAGTGAGAGA TGTCAGATCA 1401 TTTTCTCTTC ATGAGTGCGC CGTTCTGGGG GCATGTGTTC CCCAGTCTCG
1451 CCGTGGCGGA GGAGCTCGTG CACCGGGGCC ACCACGTCAC CTTTGTGACG
1501 GGCGCGGAAA TGGCCGATGC GGTGCGTTCC GTGGGCGCTG ATTTCCTGCG 1551 GTACGAGTCC GCCTTCGAGG GTGTCGACAT GTACCGGCTG ATGACCGAGG
1601 CCGAGCCGAA CGCCATCCCC ATGACGCTGT ACGACGAGGG CATGTCCATG
1651 TTGCGTTCGG TGGAGGAGCA CGTCGGCAAG GACGTTCCGG ACCTGGTGGC
1701 CTACGACATC GCCACCTCCC TCAACGTGGG TCGTGTCCTC GCCGCCTCCT
1751 GGAGCAGGCC GGCCATGACG GTCATTCCCC TGTTCGCGTC CAACGGGCGC
1801 TTCTCCACGA TGCAGTCGGT ATTGGATCCG GATTCCGCTC AGGTCAGTGC
1851 GCCGCCGCCG CGCTTCTCGG AGCAGATGGA GTTGTTCGGC CTCGGGGCGC 1901 TGGTGCCGCG CCTCGCGGAG CTGCTCGTTT CCCGGGGTAT CACGGAACCG
1951 GTCGACGATT TCCTTTCCGG ACCGGAGGAC TTCAACCTGG TGTGTCTGCC
2001 GCGCGCCTTC CAGTACGCGG GCGACACCTT CGACGAGCGG TTCGCCTTCG
2051 TCGGACCATG TCTGGGTAAG CGCAGGGGTC TGGGCGAGTG GACACCACCG
2101 GGCAGCGGGC ATCCAGTGGT GCTCATCTCC CTCGGGACCG TGTTCAACCG 2151 GCAGCTGTCC TTCTTCCGCA CGTTCGTCCG GGCGTTCACC GACGTCCCCG
2201 TGCACGTCGT GATCTCGCTC GGCAAGGGGG TCGACCCCGA TGTGCTGCGG
2251 CCGCTGCCGC CGAATGTCGA GGTGCACCGG TGGGTGCCGC ACCATGCGGT
2301 GCTGGAGCAT GCCAGGGCTC TGGTCACGCA CGGCGGTACC GGCAGTGTGA
2351 TGGAGGCACT GCACGCAGGG TGCCCGGTGC TCGTCATGCC CTTGTCGCGG 2401 GACGCGCAGG TGACCGGCCG GCGGATCGCC GAGCTGGGGC TGGGTCGTAT 2451 GGTGCAGCCG GAGGAGGTCA CGGCGACGAC GCTGCGCCGG CACGTGCTGG
2501 ACATCATCTC CGATGACGCG ATCACCCGAC AGGTCAGGCA GATGCAGCGG
2551 GCCACGGTCG AGGCGGGCGG CGCCCTGCGG GCAGCGGACG AGACCGAGCG
2601 GTTTCTGCGC CGGACGCGCC GTCACTGACC GGCAGCTCGG GCCGGGCGGT 2651 GAGTGGCTCC CACAGGGTTC GGTTCTCCAC GTACCACTGA ACGGTCTGTG
2701 CCAGCCCCTC CTCGAAGGGC ACGCGGGGCG CGTAACCGAG CTCGGCGGAG
2751 ATCTTGCTGA TGTCCAGCGA GTAGCGCCGG TCGTGCCCCT TGCGGTCGGT
2801 CACGGGTTCG ACCATCGACC AGTCCACGCC GAGCAGGTCC AGGAGCCGGG
2851 CGGTGAGCTC ACGGTTGGAC AGCTCCGTCC CGCCTCCGAT GTGGTAGATC 2901 TCGCCGGGCC TGTCGCGTTC GGCGACCAGG GCGATGCCAC GGCAGTGGTC
2951 GTCCACGTGC AGCCAGTCGC GGACGTTTTC GCCGTCGCCG TACAAGGGCA
3001 CCTTCGTGCC GTTCAGCAGA TGGGTGACGA ACCGCGGGAT GAGTTTCTCC
3051 GGGAACTGGT GGGGGCCGTA GTTGTTCGAG CATCGGGTGA TGATCACTGG
3101 TAGGCCGTGC GTGCGGTGGA AGGACCGGGC GAGCAGGTCG GAGGACGCCT 3151 TGGACGCGGA GT AGGGCGAG TTCGGCTCC A GCGGGGCGTC CTCGGTCCAC
3201 GAGCCGGAGT CGATGGAGCC GTAGACCTCG TCCGTCGAGA TGTACACGAA
3251 GCGGTCCACG GCGGCGTCGG TGGCGGCGCG GAGCAGGGTG TGAGTGCCGA 3301 GGACATTGGT GCGTACGAAC TCGGCGGCGT CGGCCACGGA CCGGTCCACG 3351 TGTGACTCCG CCGCGAAGTG GACCACCATG TCGGAGCCGT CCATCAGGTC 3401 CGCGACCAAG GGCCCGTCGC AGATGTCGCC GTGCACGAAG ATCAGGGATG 3451 GGCTTCCCAG GACCGGTGCG AGGTTCTCCA GGCGACCCGC GTAGGTCAGC 3501 TTGTCGAGCA CCACGACCTC GGCACCGGTG AACGCCGGAT ACGCGCCCGT
3551 CAGCAACCGC CGTACGAAAT GGGAACCGAT GAAACCGGCG CCGCCCGTCA
3601 CGAGTAGGCG CATCCCGGGC TCCTCACCGC GGCTTCCGCC GCAATACTCA 3651 TCAGATACTC GCCGTAGCCG GAGCCGGCCA GTTCGACCCC GCGCAGATAG
3701 CAGTCGTCCG CGTCGATCAG ACCCATCCGG AAGGCGATCT CCTCGAGACA
3751 GGCGATCCGT ACTCCCTGGC GCTTCTCCAG GACCTGCACA TACTGCCCGG
3801 CGTGCATCAG CGAGTCGTGC GTCCCCGCAT CGAGCCAGGT GAAGCCCCGG
3851 CCCAGGTCCA CCAGCCGGGC CCGCCCCTCG GCGAGGTAGG CCCTGTTGAC 3901 GTCGGTGATC TCCAGCTCGC CGCGGGCCGA CGAGCGGATG CCCCGGGCCA
3951 CCTCGATCAC GTCGTTGTCG TACAGGTACA GGCCTGTGAT CGCCAGGTTG
4001 GACCGGGGGG CGGTGGGTTT CTCCTCGACG GACAGCAGCT TTCCGGAGGC
4051 GTCGACCTCT CCGACTCCGT ACCGTTCGGG ATCCGTCACC GCGTATCCGA
4101 ACAACACACA GCCGTCGACA TCGCGGGTGT GGCTGCGCAG CAGGTGCGAA 4151 AAGCCCATGC CATGGAAGAT GTTGTCCCCA AGGACAAGGG ACACCTGATC 4201 CTGACCGATG AAATCGGCGC CGATGAGGAA TGCCTCGGCG ATTCCTCCCG
4251 GTCGCTGCTG CGCGGCGTAG TCGATGTTCA GCCCGAGGCG GCTTCCGTCT
4301 CCGAGCAGTC TCCGGAATTG TTCGAGATGA TCGGGTGAGG AAATCACCAG
4351 GATGTCTTTT ATGCCGCCGA GCATCAACAC GGAGAGCGGG TAGTAGATCA 4401 TGGGTTTGTC GTAGACAGGG AGCAGCTGCT TGGAAAGGGC ACGGGTCAAC
4451 GGGTAAAGCC GAGAGCCGGT TCCCCCCGCG AGCACGATTC CCTTCATGTC
4501 GGACTCCCCG CAGTCGACGT TATATATCTC GTGCCGTCTG CCCGACGGTA
4551 CCAAGTGGCG GAAAACGCAC CAGGAATTCG AGCGCCGCTA GGGGGAAGGG
4601 CTCAAGAAGA TAGGGGCCAC CAGATGGGGC GGTTTTCGGT GTGCCCGCCC 4651 CGGCCGACCG GAATACTGAA GAGCATGCTG ACGACTGGGA TGTGCGACCG
4701 ACCGCTGGTC GTCGTACTCG GAGCCTCCGG CTATATCGGG TCGGCCGTCG
4751 CGGCGGAACT CGCCCGGTGG CCGGTCCTGT TGCGGCTGGT GGCCCGGCGA
4801 CCGGGCGTCG TTCCGCCGGG CGGCGCCGCG GAGACCGAGA CGCGTACGGC
4851 CGACCTGACG GCGGCGAGCG AGGTCGCCCT CGCCGTGACG GACGCCGACG 4901 TGGTGATCCA CCTGGTCGCG CGCCTCACCC AGGGAGCGGC ATGGCGGGCG
4951 GCGGAGAGCG ATCCGGTGGC CGAGCGGGTG AACGTCGGGG TGATGCACGA
5001 CGTCGTCGCG GCCCTGCGGT CCGGGCGCCG CGCCGGGCCG CCCCCGGTGG 5051 TGGTGTTCGC CGGGTCGGTC TACCAGGTGG GCCGCCCGGG TCGGGTCGAC
5101 GGCAGTGAGC CGGACGAGCC CGTGACGGCC TATGCCCGTC AGAAACTCGA
5151 CGCCGAACGG ACGTTGAAGT CCGCCACGGT CGAGGGTGTC CTGCGGGGGA
5201 TCTCGCTGCG GCTGCCCACC GTCTACGGCG CGGGGCCGGG CCCGCAGGGC
5251 AACGGCGTCG TGCAGGCGAT GGTGCTCCGG GCGCTCGCCG ACGAGGCCCT
5301 CACCGTGTGG AACGGAAGCG TGGTGGAGCG TGACCTGGTG CATGTGGAGG
5351 ATGTCGCGCA GGCCTTCGTG AGCTGCCTGG CGCACGCGGA TGCGCTCGCC 5401 GGGCGGCACT GGCTGCTCGG CAGCGGTCGT CCTGTGACCG TCCCGCACCT
5451 CTTCGGTGCC ATCGCCGCCG GCGTGTCCGC CCGCACCGGG CGCCCCGCGG
5501 TGCCCGTGAC CGCGGTGGAC CCTCCGGCGA TGGCGACGGC GGCGGACTTC
5551 CACGGGACCG TCGTCGACTC CTCGGCGTTC CGCGCGGTCA CCGGGTGGCG
5601 GCCGCGGCTG TCGCTTCAGG AGGGCCTGGA CCACATGGTG GCGGCTTACG 5651 TGTAGCGCCG GGGTGGCGGC CGGGCCCGGG CGGTGACGGC CCGGATCCGG
5701 GTCGGCCGTC ACAGCTTCTC GTCGAGGGCG GGGCTCGCGC GGTACTCCGG
5751 CAACATGCCG CGTCGCAGGG CCTGCTGGAG AGTCGGCGCG CGCGCCGGTC
5801 CGCGCTCGGA GAGGATCGGT GCCCGCCCGA GGTGGTGGCC GAGGGGCAGG
5851 GCGAGGTCCG GATCCTCGGG CGAGAGGGCG TGTTCGTTCT GCGGAACGTA 5901 GCCGCTCGAC ATCAGGTACA CCATCGCCGT GTCGTCTTCC AGCGCCACGA 5951 ACGCGTGCCC GACCCCGATC GGCAGGTAGA CGGAACGGAA GCGCTCCTGG
6001 TCGAGGAGGA CCGAGTCCCA CTGCCCGAAA GTCGGTGAGC CGGTGCGCAG
6051 GTCGACGACG AAGTCCAGGG CCCGTCCCCG GGCGCAGTGG ACGTACTTGG
6101 CCTGGCCGGG TGGTGTCGCG GTGAAGTGCA CGCCGCGGAC GACGCCGCGG 6151 CGCGAGACGC TCTGGC AGGT CTGCGCGGTG GGAAACCGGT GCCCGACGGC
6201 CTCGCTGAGG ACCGGTTCCT GGTAGGGGGT GACGAAGAGC CCGCGCTCGT
6251 CGGGGAAGAC CGTCGGGGTG AATTCGACGG CGCCCTCGAC GACGAGCCTC
6301 CGGACCGTGA CACCGGCGGC GGTGGCCCGG GCGCCCGCGG GCGGGGCGGG
6351 CCGGTCGGCG GAGCTCCGGC GAGGCCGGCC AAGGGTCATC GCTGCACTCT 6401 CTCTGTCGTG CGGGTTGTCA TACGGGTAGT CGTACGGGCC GGTTCCGGAG
6451 TCACAGCTCG ACGGCGCGGG TGGTGAGCAG GGACAGCAGG GTGCGGGCCT
6501 GCACGTTCAC GTAACGGCCG TACCGCAGCA GCTGGGTCAG CTGGCCCGGG
6551 GTGCACCAGC GGTACCCCGG GGGCGGGTCG TTCGGCGCCT GGCTCTCGTC
6601 GGCCTCGACG AACAGGTAGC GCGCCTGTGC GTGCAGAAAG CGACCGCCCT 6651 CCTCCGAGTG GACCGCCGCG TAGCGGATGC GGTCGGGCGC GGCCTCCAGC
6701 ACCAGGTCGA GGAAGCGCGG CCTGGCCGGT CCCGTGAGGT GGGCGTAGTT
6751 GCGCGGGGTG TACTGGACCG TCGGGCCGAG TTCGATCGTG TCGAGGAAGC 6801 CGCCCTCGAC CCTGCCGTGG GCGAGCAGGT GCGGTACGCC GCCGATCCGC
6851 CGGGTCAGGA AGGCGGTGAT GCCGTGGCCG CACGGTTCGA TCAGGGGCTG
6901 GGTCCAGGCG GCGACCTCCC GGTTGGAGGC CTCGACACGG ACCGCGACCA
6951 CACGGAAGTA CCGGTCCGCG TGGTGGGCGA TGGACTCCGC GCCCGTGGTC
7001 CAGCCGGGGA TGCCGGCCAG GGGCACGCGG CGGGCGTGCA CGGAGTGCCG
7051 GGAGCGTTCG GCGGCGTACC AGGAGAGCAG TTCGGCGTCG CTGTGCAGGG
7101 CCGCGGGCTC GTCGAACGGG GTGGGAAGGC AGGCGAGGAC CGTGCGTGCG 7151 TCCATGTTCA CC AGGTTGTC CCGGTGCATC AGTTCGCCGA TCTGCCCCAG
7201 TGTCAGCCAG CGGAAGTCGT CGTCCAGTGG TACGTCCTCG TCGGTCTCCA
7251 CCACGATGTT GCGGTTGAAC TTCCGGTGGA ACCAGGCTCC GTGCTCGGAC
7301 TGGAGGACGT CGACCACCAC GGTGGCGCGC CGGGGCTGTG TGAAGTACTC
7351 GAGGTACTTC ACGGCGGCGC CCCCGTGGAC CTTGGTGTAG TTGCTGCGCG 7401 TGGCCTGCAC GGTGGGCGAC AGCTGGACCA GGTTGATGTT GCCGGGCTCC
7451 ATCTTGGCCT GCATCAGGAA GTGCAGGACC CCGTCGAACT TCTTGGCGAG
7501 GATGCCGAGG ATGCCGATCT CGGGCTGGTG GATGATGGGC TGCTGCCATT
7551 CCGGGAAGGG CTGTTCACCG CCTCGGACGT GCAGTCCCTC CACGGAGAAG
7601 AACCGGCCGC TGCGGTGGGC CAGATTGCCG GTTCCGGGGT GAAACGACCA 7651 GGCGTCCATC CCGTGGAAGG GGATGCGCTC GACCCGGAAC CGGTGGGCCC 7701 CGGACCGCCG CGTCCACCAG CCGGTGAACG CGTCGAGGGA CGTCCGGGCG
7751 CCGGTGTCGC CCACGGCGGC GGAGCGGGCG AGGCACGCGG GCAGGGCGGC
7801 GTCGTGCCGC GCGGTGAGCG GTGCTGGGCT CGGTGTGGTC GGCATCGGCT
7851 CGTACGCTCA TGCACCCCAC GTCATGTAGA TCACCGGTGG CTCGCGGCCG 7901 GGCAGTTGGC GCAGTGGGGC GTGGTCGAGG CCGAACGCCT CGCTCAGCGC
7951 CCTGGTCTCC CCCGGCCATT TGGGGTGGGT GAGTTCGTCG AAGGCGAGGA
8001 TGCTGCCCCT GGTCAGGTGC GGTGTGATGA CGTCCAGCAG TTCGCGCGTG
8051 GGGCGGTAGA GGTCCAGGTC GAAGTAGGCC AGCGCGATGA CGGTGTGCGG
8101 GTGTTCCGCC AGGTATTGGG GCACCGTTTC GCGTACGTCG CCCTGGACCA 8151 CGAAGGAACG CTGGGTGTGG CCGTAGGGTT CGTTCGCCTC GTGCGCCGCG
8201 AGCACCTGCC GCAGGTGCTC CACTTCGCCG TCCGGCACGG CGAACCGCCC
8251 AGGGACCGCG CTGGTGCTGA CCTCGTCCGC CTCGTCGATG TCGGGGAAGC
8301 CGGTGAACGT GTCGAAGCCG ATGACGCGGC GCAGCGAGTT GTACGGCTCA
8351 TAGATGCTGC GCAGCGCGGT CAGCGTGGCG AGGTGCCGTC CGTGCAGAAC 8401 GCCGAACTCC ATGATGACGC CGGGGACTTC CGGCAGCATG CGGTACAGCG
8451 CGTCCATGGA GAGCAGGTCG GCGAGCTGGT TGCGCCGCAT GTAGACGGAC
8501 AGGTTGTCGA TCAGGTACTT CGGCGGGATC GGGCTGTCGA CGAGGAGCTT 8551 GGTCAGCTGC TCGCGGGCAG CGCGTTCCTG CTCGGACTCG TGCGGCACGA
8601 TCCGGGGATC GGTGAACTCC CGCTCGGTCA TGGAGGCCTT TCCTTTCATG
8651 GGTCGGTACC GGGCGCGCCG GACGTGCCGG TCGTACCGGG CGTGCCGGCG
8701 GGCACGACGC TGTCGGGTCA GGACAGCCAG GCGTCGGGGG CGGATCCGCC
8751 GCGGCCGACC GGGGGGAACA GCTCCTCCAG GCGGGCCAGG ACGGGCTCGG
8801 GCAGCGGGGT GCGCAGGGCG TGCAGTGCCC CGTCCACGTG CTGTTCGGTG
8851 CGCGGCCCGA TGACCAGCCC GGTCACGCCG GGCCGCGACA GCACCCAGGC 8901 CATGCCGACA TGGGCGGGGT CGAGGCCGTG GTCCGCGCAC ACGTCCTCGT
8951 ACGCCGCGAT GGTGGTGCGG TGGTGCTCCA GGGCCTCGAC GGCCCGGCCC
9001 TGTGCCGACT TGACCGCGGT GTTCTCCCGC GTCTTGCGCA GGACACCGCC
9051 GAGCAGGCCG CCGTGCAGTG GCGACCAGAC CAGGACGCCG ACACCGTAGG
9101 CGGACGCGGC GGGGATGACT TCCAGCTCGG CGTGTCGGGT CACGAGGTTG 9151 TAGACGCACT GCTCGGAGGC GAGGCCCAGG GCGTTGCGCC GCCGGGCCGC
9201 CTCCTGGGCG GAAGCGATGT CCCAGCCCGC GAAGTTGGAG GAGCCGACGT
9251 AGCGCACCTT GCCCTGCGTG ATGAGCAGGT CCATCGCCTG CCACACCTCG
9301 TCCCAGCCGG CGCGGCGGTC GATGTGGTGC AGCTGGTACA GGTCGATCCA
9351 GTCGGTGCGC AGTCGGCGCA GCGAGGCGTC GCAGGCGGCC ACGATATTGC 9401 GTACGGACAG TCCGTGATCG TTGGGGCCGC TGCCCATCGG ATCGCCGACC 9451 TTGGTGGCCA GCACCACCTG CTCACGCCGG GCGGGGCGGT CCGCCAGCCA
9501 CCTGCCGATG ACCTCTTCGG TGTACCCCTT GTGGACGCGC CAGCCGTAGG
9551 TGTTGGCGGT GTCGAACAGG GTGATGCCCT GAGCCAGGGC GTGATCCATC
9601 AGTCGGCGCG CTTCGGGCTC CTCCACCCGT CCGCCGATGT TGACCGTTCC
9651 GAGCGCCAGT CGGCTGATCC TCAGCCGGGT CCTGCCCAGT TCGGTGTGGA
9701 GGGGAGCACT GCTGTTGCTG TCGGACTGGA CGGGTGCGGG CTCGGCCGTC
9751 GTAGGCATCA TCGATCAGTC GACACTCCCT CGTGCGTGAG CGGCGGGCGC
9801 TCGAGCAGGA CCCTGACCTG AGGCCCAGGA GGCTACCGGC GATCATGCGA
9851 TACAGGCAGC CGCTCGATGG TGGGACACGG GCTGCCGTCG CCGGGCATAG
9901 GGGCTGATGG GGGTTGTCCG GTGCGGGTCC GGCTGACAGC CTCGTGGACA
9951 CCAAGTTGAT CCAGTTGATC CACTCCGAAA GGCAGAGGCT GCAG (SEQ ID NO: l)
The sequence SEQ ID NO: 1 is characterized by the following open reading frames (ORFs) noted below. Each ORF encodes a protein in the avermectin biosynthetic pathway. Avermectin glycosylation genes AvrB, C, and D were identified by complementation analysis previously (MacNeil et al Gene (1992) 111:61-68 and map to ORF2, ORF3b and ORF3a respectively. Newly identified genes for avermectin glycosylation are designated AvrE, F, G, H, and I.
Mod7-PKS 1-365 - Beginning of mod7 PKS ORF1 508-1332 + 274 aa Macrolide B-keto reductase ORF2 AvrB 1390-2628 + 386 aa Glycosyl transferase ORF3a AvrD 3598-4497 - 300 aa TDP-glucose synthase ORF3b AvrC 3613-2534 - 360 aa TDP-glucose 4,6 dehydrase ORF4 AvrE 4624-5655 + 343 aa Glycosyl reductase ORF5 AvrF 5709-6389 - 226 aa Glycosyl 3,5epimerase ORF6 AvrG 6451-7845 - 464 aa Oleandrose synthesis ORF7 AvrH 7858-8631 - 257 aa Glycosyl methyltransf erase ORF8 Avrl 8718-9761 - 347 aa Oleandrose synthesis
Promoters:
1) Divergent PKS7-ORFl,2 between 365 and 508. 2) Divergent ORF3a,b-ORF4 between 4497 and 4624
3) ORF8,7,6,5 9994 to 9761
An isolated nucleic acid molecule of the present invention can include a deoxyribonucleic acid molecule (DNA), such as genomic DNA and complementary DNA (cDNA), which can be single (coding or noncoding strand) or double stranded, as well as synthetic DNA, such as a synthesized, single stranded polynucleotide. The isolated nucleic acid molecule of the present invention can also include a ribonucleic acid molecule (RNA).
As used herein a "polynucleotide" is a nucleic acid of more than one nucleotide. A polynucleotide can be made up of multiple polynucleotide units that are referred to by description of the unit. For example, a polynucleotide can comprise within its bounds a polynucleotide(s) having a coding sequence(s), a polynucleotide(s) that is a regulatory region(s) and/or other polynucleotide units commonly used in the art. The present invention also relates to recombinant vectors and recombinant hosts, both prokaryotic and eukaryotic, which contain the substantially purified nucleic acid molecules disclosed throughout this specification. The DNA sequences of the present invention encoding a polypeptide disclosed herein, in whole or in part, can be linked with other DNA sequences, i.e., a sequences to which the nucleic acid is not naturally linked, to form "recombinant DNA molecules" a nucleic acid disclosed herein. The novel DNA sequences of the present invention can be inserted into vectors in order to direct recombinant expression of polypeptides disclosed herein. Such vectors may be comprised of DNA or RNA; for most purposes DNA vectors are preferred. Typical vectors include plasmids, modified viruses, bacteriophage, cosmids, yeast artificial chromosomes, and other forms of episomal or integrated DNA that can encode a polypeptide disclosed herein. One skilled in the art can readily determine an appropriate vector for a particular use.
An "expression vector" is a polynucleotide having regulatory regions operably linked to a coding region such that, when in a host cell, the regulatory regions can direct the expression of the coding sequence. The use of expression vectors is well known in the art. Expression vectors can be used in a variety of host cells and, therefore, the regulatory regions are preferably chosen as appropriate for the particular host cell. Preferred expression vectors can be those particularly designed for use in actinomycetes or the particular host chosen for a particular application of a gene or protein disclosed herein.
A "regulatory region" is a polynucleotide that can promote or enhance the initiation or termination of transcription or translation of a coding sequence. A regulatory region includes a sequence that is recognized by the RNA polymerase, ribosome, or associated transcription or translation initiation or termination factors of a host cell. Regulatory regions that direct the initiation of transcription or translation can direct constitutive or inducible expression of a coding sequence. Preferred regulatory regions can be those particularly designed for use in actinomycetes or the particular host chosen for a particular application of a gene or protein disclosed herein. Polynucleotides of this invention contain full length or partial length sequences of ORFs disclosed herein. Polynucleotides of this invention can be single or double stranded. If single stranded, the polynucleotides can be a coding, "sense," strand or a complementary, "antisense," strand. Antisense strands can be useful as modulators of the receptor by interacting with RNA encoding the receptor. Antisense strands are preferably less than full length strands having sequences unique or highly specific for RNA encoding the receptor.
The polynucleotides can include deoxyribonucleotides, ribonucleotides or mixtures of both. The polynucleotides can be produced by cells, in cell-free biochemical reactions or through chemical synthesis. Non-natural or modified nucleotides, including inosine, methyl-cytosine, deaza-guanosine, and others known to those of skill in the art, can be present. Natural phosphodiester internucleotide linkages can be appropriate. However, polynucleotides can have non-natural linkages between the nucleotides. Non-natural linkages are well known in the art and include, without limitation, methylphosphonates, phosphorothioates, phosphorodithionates, phosphoroamidites and phosphate ester linkages. Dephospho-linkages are also known, as bridges between nucleotides. Examples of these include siloxane, carbonate, carboxymethyl ester, acetamidate, carbamate, and thioether bridges. "Plastic DNA," having, for example, N-vinyl, methacryloxytethyl, methacrylamide or ethyleneimine internucleotide linkages, can be used. "Peptide Nucleic Acid" (PNA) is also useful and resists degradation by nucleases. These linkages can be mixed in a polynucleotide.
As used herein, "purified" and "isolated" are utilized interchangeably to stand for the proposition that the polynucleotides, proteins and polypeptides, or respective fragments thereof in question has been removed from its in vivo environment so that it can be manipulated by the skilled artisan, such as but not limited to sequencing, restriction digestion, site-directed mutagenesis, and subcloning into expression vectors for a nucleic acid fragment as well as obtaining the protein or protein fragment in quantities that afford the opportunity to generate polyclonal antibodies, monoclonal antibodies, amino acid sequencing, and peptide digestion. Therefore, the nucleic acids claimed herein can be present in whole cells or in cell lysates or in a partially, substantially or wholly purified form. A polynucleotide is considered purified when it is purified away from environmental contaminants. Thus, a polynucleotide isolated from cells is considered to be substantially purified when purified from cellular components by standard methods while a chemically synthesized nucleic acid sequence is considered to be substantially purified when purified from its chemical precursors.
Included in the present invention are nucleotide sequences that hybridize to the sequences disclosed herein under stringent conditions. By way of example, and not limitation, a procedure using conditions of high stringency is as follows: Prehybridization of filters containing DNA is carried out for 2 hr. to overnight at 65°C in buffer composed of 6X SSC, 5X Denhardt's solution, and 100 μg/ml denatured salmon sperm DNA. Filters are hybridized for 12 to 48 hrs at 65 °C in prehybridization mixture containing 100 μg/ml denatured salmon sperm DNA and 5-20 X lθ6 cpm of 32p_ιabeled probe. Washing of filters is done at 37°C for 1 hr in a solution containing 2X SSC, 0.1% SDS. This is followed by a wash in 0.1X SSC, 0.1% SDS at 50°C for 45 min. before autoradiography.
Other procedures using conditions of high stringency would include either a hybridization step carried out in 5XSSC, 5X Denhardt's solution, 50% formamide at 42°C for 12 to 48 hours or a washing step carried out in 0.2X SSPE, 0.2% SDS at 65°C for 30 to 60 minutes. Reagents mentioned in the foregoing procedures for carrying out high stringency hybridization are well known in the art. Details of the composition of these reagents can be found in, e.g., Sambrook, Fritsch, and Maniatis, 1989, Molecular Cloning: A Laboratory Manual, second edition, Cold Spring Harbor Laboratory Press. In addition to the foregoing, other conditions of high stringency which may be used are well known in the art.
Polypeptides
Preferred aspects of the present invention are substantially purified forms of the polypeptides encoded by the fragment of the S. avermitilis genome disclosed herein. Preferred embodiments of these aspects of the invention proteins that have an amino acid sequence which is set forth in SEQ ID NOs:2-10 and disclosed as follows in single letter code:
Peptide sequences: ORFl polypeptide
1 MAPDMNSQRF GGRLALVTGA GGGIGRATCA LGSAGARVVC VDRDGRGAGV 51 TPTWPERGAR AAWPEVADVS DGAAMERLPE RVAETYGVVD LLVNNAGIGM 101 AGRFLDTSVE DWQRTLGVNL WGVIHGCRLI GRQMAERGQG GHIVTVASAA 151 AFQPTRAVPA YATSKAAVLM LSECLRAEFA EFGVGVSVVC PGFVRTSFAS
201 AMHFAGVPRL EQERLRALFA GRGCSAEKVA AAVLRSVARD SAVVTVTAEA 251 RLSRLMSRFT PRLRAAVARM DPPS SEQ ID NO:2
ORF2 (AvrB) polypeptide 1 MSDHFLFMSA PFWGHVFPSL AVAEELVHRG HHVTFVTGAE MADAVRSVGA
51 DFLRYES AFE GVDMYRLMTE AEPNAIPMTL YDEGMSMLRS VEEHVGKDVP 101 DLVAYDIATS LNVGRVLAAS WSRPAMTVIP LFASNGRFST MQSVLDPDSA 151 QVSAPPPRFS EQMELFGLGA LVPRLAELLV SRGITEPVDD FLSGPEDFNL 201 VCLPRAFQYA GDTFDERFAF VGPCLGKRRG LGEWTPPGSG HPVVLISLGT 251 VFNRQLSFFR TFVRAFTDVP VHVVISLGKG VDPDVLRPLP PNVEVHRWVP
301 HHAVLEHARA LVTHGGTGSV MEALHAGCPV LVMPLSRDAQ VTGRRIAELG 351 LGRMVQPEEV TATTLRRHVL DIISDDAITR QVRQMQRATV EAGGALRAAD 401 ETERFLRRTR RH SEQ ID NO:3 ORF3b (AvrC) polypeptide
1 MRLLVTGGAG FIGSHFVRRL LTGAYPAFTG AEVVVLDKLT YAGRLENLAP 51 VLGSPSLIFV HGDICDGPLV ADLMDGSDMV VHFAAESHVD RS V ADAAEFV 101 RTNVLGTHTL LRAATDAAVD RFVYISTDEV YGSIDSGSWT EDAPLEPNSP 151 YSASKASSDL LARSFHRTHG LPVIITRCSN NYGPHQFPEK LIPRFVTHLL 201 NGTKVPLYGD GENVRDWLHV DDHCRGIALV AERDRPGEIY HIGGGTELSN 251 RELTARLLDL LGVDWSMVEP VTDRKGHDRR YSLDISKIS A ELGY APRVPF 301 EEGLAQTVQW YVENRTLWEP LTARPELPVS DGASGAETAR SRPLPAGRRP 351 PRPWPAASA SEQ ID NO 4
ORF3a (AvrD) polypeptide
1 MKGIVLAGGT GSRLYPLTRA LSKQLLPVYD KPMIYYPLSV LMLGGIKDIL 51 VISSPDHLEQ FRRLLGDGSR LGLNIDY AAQ QRPGGIAEAF LIGADFIGQD 101 QVSLVLGDNI FHGMGFSHLL RSHTRDVDGC VLFGYAVTDP ERYGVGEVDA 151 SGKLLS VEEK PT APRSNLAI TGLYLYDND V IE V ARGIRSS ARGELEITDV
201 NRAYLAEGRA RLVDLGRGFT WLDAGTHDSL MHAGQYVQVL EKRQGVRIAC 251 LEEIAFRMGL IDADDCYLRG VELAGSGYGE YLMSIAAEAA VRSPGCAYS SEQ ID NO 5
ORF4 (AvrE) polypeptide
1 MGRFSVCPPR PTGILKSMLT TGMCDRPLVV VLGASGYIGS AVAAELARWP 51 VLLRLVARRP GVVPPGGAAE TETRTADLTA ASEVALAVTD ADVVIHLVAR 101 LTQGAAWRAA ESDPVAERVN VGVMHDVVAA LRSGRRAGPP PVVVFAGSVY 151 QVGRPGRVDG SEPDEPVTAY ARQKLDAERT LKSATVEGVL RGISLRLPTV 201 YGAGPGPQGN GVVQAMVLRA LADEALTVWN GSVVERDLVH VEDVAQAFVS
251 CLAHADALAG RHWLLGSGRP VTVPHLFGAI AAGVSARTGR PAVPVTAVDP 301 PAMATAADFH GTVVDSSAFR AVTGWRPRLS LQEGLDHMVA AYV SEQ ID NO 6
ORF5 (AvrF) polypeptide 1 MTLGRPRRSS ADRPAPPAGA RATAAGVTVR RLVVEGAVEF TPTVFPDERG
51 LFVTPYQEPV LSEAVGHRFP TAQTCQS VSR RGVVRGVHFT ATPPGQAKYV 101 HCARGRALDF VVDLRTGSPT FGQWDSVLLD QERFRSVYLP IGVGHAFVAL 151 EDDTAMVYLM SSGYVPQNEH ALSPEDPDLA LPLGHHLGRA PILSERGPAR 201 APTLQQALRR GMLPEYRASR ALDEKL SEQ ID NO 7 ORF6 (AvrG) polypeptide
1 MPTTPSPAPL TARHDAALPA CLARSAAVGD TGARTSLDAF TGWWTRRSGA 51 HRFRVERIPF HGMDAWSFHP GTGNLAHRSG RFFS VEGLHV RGGEQPFPEW 101 QQPIIHQPEI GILGILAKKF DGVLHFLMQA KMEPGNINLV QLSPTVQATR 151 SNYTK VHGGA AVKYLEYFTQ PRRAT VV VD V LQSEHG AWFH RKFNRNIVVE
201 TDEDVPLDDD FRWLTLGQIG ELMHRDNLVN MDARTVLACL PTPFDEPAAL 251 HSDAELLSWY AAERSRHSVH ARRVPLAGIP GWTTGAESIA HHADRYFRVV 301 AVRVEASNRE VAAWTQPLIE PCGHGITAFL TRRIGGVPHL LAHGRVEGGF 351 LDTIELGPTV QYTPRNYAHL TGPARPRFLD LVLEAAPDRI RY AAVHSEEG 401 GRFLHAQ AR Y LFVE ADESQ A PNDPPPGYRW CTPGQLTQLL RYGRY VNVQA
451 RTLLSLLTTR AVEL SEQ ID NO 8
ORF7 (AvrH) polypeptide
1 MTEREFTDPR IVPHESEQER AAREQLTKLL VDSPIPPKYL IDNLSVYMRR 51 NQLADLLSMD ALYRMLPE VP GVIMEFGVLH GRHL ATLT AL RSIYEPYNSL
101 RRVIGFDTFT GFPDIDEADE VSTSAVPGRF AVPDGEVEHL RQVLAAHEAN 151 EPYGHTQRSF VVQGDVRETV PQYLAEHPHT VIALAYFDLD LYRPTRELLD 201 VITPHLTRGS ILAFDELTHP KWPGETRALS EAFGLDHAPL RQLPGREPPV 251 IYMTWGA SEQ ID NO 9
ORF8 (Avrl) polypeptide
1 MMPTTAEPAP VQSDSNSSAP LHTELGRTRL RISRLALGTV NIGGRVEEPE 51 ARRLMDHALA QGITLFDT AN TYGWRVHKGY TEEVIGRWLA DRPARREQV V 101 LATKVGDPMG SGPNDHGLSV RNIVAACDAS LRRLRTDWID LYQLHHIDRR 151 AGWDE VWQAM DLLITQGKVR Y VGSSNFAGW DI AS AQE AAR RRNALGLASE
201 QCVYNLVTRH AELEVIPAAS AYGVGVLVWS PLHGGLLGGV LRKTRENTAV 251 KS AQGRAVEA LEHHRTTIAA YEDVCADHGL DPAHVGMAWV LSRPGVTGLV 301 IGPRTEQHVD GALHALRTPL PEPVLARLEE LFPPVGRGGS APDAWLS SEQ ID NO 10
The present invention also relates to fragments and mutant or polymoφhic forms of the protems set forth in SEQ ID NOs:2-10, including but not necessaπly limited to amino acid substitutions, deletions, additions, ammo terminal truncations and carboxy-terminal truncations such that these provide for protems or protein fragments of enzymatic, biocatalytic, biosynthetic or diagnostic use. Using the disclosure of polynucleotide and polypeptide sequences provided herein to isolate polynucleotides encoding naturally occurring forms of the proteins disclosed herein, one of skill in the art can determine whether such naturally occurring forms are mutant or polymoφhic forms by sequence comparison. One can determine whether the mutant or polymoφhic forms, or fragments of any protein disclosed herein, are biologically active by routine testing of the protein or fragment in a in vitro or in vivo assay for the biological activity of the full length version of the protein as encoded by the nucleotide sequence disclosed herein. For example, one can express N-terminal or C-terminal truncations, or internal additions or deletions of a protein in a host cell and test whether the altered form can perform the same enzymatic step as performed by the full-length polypeptide disclosed herein.
It is known that there is a substantial amount of redundancy in the various codons which code for specific amino acids. Therefore, this invention is also directed to those DNA sequences encode RNA comprising alternative codons which code for the eventual translation of the identical amino acid sequence of any of the avermectin pathway proteins disclosed herein. Therefore, the present invention includes nucleic acid sequences that vary because of codon redundancy which can result in differing DNA molecules expressing an identical protein.
As with many enzymes, it is possible to modify many of the amino acids of the proteins disclosed herein, particularly those which are not found in the ligand binding or catalytic domains, and still retain substantially the same biological activity as the original protein. Thus this invention includes modified polypeptides which have amino acid deletions, additions, or substitutions but that still retain substantially the same biological activity as proteins disclosed herein. Also included within the scope of this invention are polypeptides having changes which do not substantially alter the ultimate physical or functional properties of the expressed protein. A "conservative amino acid substitution" refers to the replacement of one amino acid residue by another, chemically similar, amino acid residue. Examples of such conservative substitutions are: substitution of one hydrophobic residue (isoleucine, leucine, valine, or methionine) for another; substitution of one polar residue for another polar residue of the same charge (e.g., arginine for lysine; glutamic acid for aspartic acid). In particular, substitution of valine for leucine, arginine for lysine, or asparagine for glutamine is not expected to cause a change in functionality of the polypeptide. It is known that DNA sequences coding for a peptide can be altered so as to code for a peptide having properties that are different than those of the naturally occurring peptide. Methods of altering the DNA sequences include but are not limited to site directed mutagenesis. Examples of altered properties include but are not limited to changes in the affinity of an enzyme for a substrate or a receptor for a ligand.
For the puφoses of this invention, naturally occurring, or wild-type protein has an amino acid sequence shown as SEQ ID NOs:2-10 and is encoded by the particular nucleic acid sequences disclosed herein. As used herein, a "functional equivalent" of a wild-type protein possesses a biological activity that is substantially the same biological activity of the wild type protein. A polypeptide has "substantially the same biological activity" as a wild-type if that polypeptide has a Krj for a ligand that is no more than 5-fold greater than the Kd of the wild-type for the same ligand. The term "functional derivative" is intended to include those "fragments," "mutants," "variants," "degenerate variants," "analogs," "homologues" or "chemical derivatives" of the wild type protein that exhibit substantially the same biological activity. The term "fragment" is meant to refer to any polypeptide subset of wild-type protein disclosed herein. The term "mutant" is meant to refer to a molecule that may be substantially similar to the wild-type form but possesses distinguishing biological characteristics. Such altered characteristics include but are in no way limited to altered substrate binding, altered substrate affinity and altered sensitivity to chemical compounds affecting biological activity of the wild-type. The term "variant" is meant to refer to a molecule substantially similar in structure and function to either the entire wild-type protein or to a fragment thereof. As used herein in reference to a gene or encoded protein, a
"polymoφhic" form that is naturally found as an allele in the population at large. A polymoφhic form can have a different nucleotide sequence from the particular nucleic acid or protein disclosed herein. However, because of silent mutations, a polymoφhic gene can encode the same or different amino acid sequence as that disclosed herein. Further, some polymoφhic forms will exhibit biological characteristics that distinguish the form from wild-type protein activity, in which case the polymoφhic form is also a mutant. Polymoφhic forms encompass allelic variants.
A protein or fragment thereof is considered purified or isolated when it is obtained at a concentration at least about five-fold to ten-fold higher than that found in nature. A protein or fragment thereof is considered substantially pure if it is obtained at a concentration of at least about 100-fold higher than that found in nature. A protein or fragment thereof is considered essentially pure if it is obtained at a concentration of at least about 1000-fold higher than that found in nature.
Expression of Proteins of this Invention
The present invention also relates to recombinant vectors and recombinant hosts, both prokaryotic and eukaryotic, which contain the substantially purified nucleic acid molecules disclosed throughout this specification. Therefore, the present invention also relates to methods of expressing the proteins and their biological equivalents described herein and reactions employing these recombinantly expressed gene products, including in vivo or in vitro biosynthetic, biocatalytic or biotransformation reactions employing the genes, proteins, vectors and host cells disclosed herein. A variety of expression vectors can be used to express recombinant proteins in host cells. Expression vectors are defined herein as DNA sequences that are arranged for the transcription of cloned DNA and the translation of their mRNAs in an appropriate host. Such vectors can be used to express the nucleotide sequences of this invention in a variety of hosts such as bacteria, blue-green algae, plant cells, insect cells and animal cells. Specifically designed vectors allow the shuttling of
DNA between hosts such as bacteria-yeast or bacteria-animal cells. An appropriately constructed expression vector should contain: an origin of replication for autonomous replication in host cells, selectable markers, a limited number of useful restriction enzyme sites, optionally a potential for high copy number, and promoters. A promoter is defined as a DNA sequence operably linked to a coding region so that it interacts with cellular proteins to direct RNA polymerase to bind to DNA and initiate mRNA synthesis. A strong promoter is one which causes mRNAs to be initiated at high frequency. A promoter can be inducible. Expression vectors can include, but are not limited to, cloning vectors, modified cloning vectors, specifically designed plasmids or viruses.
Commercially available mammalian expression vectors which can be suitable for recombinant protein expression, include but are not limited to, pcDNA3.1 (Invitrogen), pLLTMUS28, pLITMUS29, pLLTMUS38 and pLLTMUS39 (New England Biolabs), pcDNAI, pcDNAIamp (Invitrogen), pcDNA3 (Invitrogen), pMClneo (Stratagene), pXTl (Stratagene), pSG5 (Stratagene), EBO-pSV2-neo (ATCC 37593) pBPV-l(8-2) (ATCC 37110), pdBPV-MMTneo(342-12) (ATCC 37224), pRSVgpt (ATCC 37199), pRSVneo (ATCC 37198), pSV2-dhfr (ATCC 37146), pUCTag (ATCC 37460), and 1ZD35 (ATCC 37565).
A variety of bacterial expression vectors can be used to express recombinant protein in bacterial cells. Commercially available bacterial expression vectors which are suitable for recombinant expression include, but are not limited to pQE (Qiagen), pETl la (Novagen), lambda gtl 1 (Invitrogen), and pKK223-3 (Pharmacia). Preferrred vectors include vectors designed for expression of proteins in actinomycetes including but not limited to the pIJ series developed at the John Innes Institute and described in Hopwood, D.A. et al., 1985. Genetic Manipulation of Streptomyces, A Laboratory Manual. F. Crowe & Sons, Ltd., Norwich, England.) A variety of fungal cell expression vectors can be used to express recombinant protein in fungal cells. Commercially available fungal cell expression vectors which are suitable for recombinant expression include but are not limited to pYES2 (Invitrogen) and Pichia expression vector (Invitrogen).
A variety of insect cell expression vectors can be used to express recombinant receptor in insect cells. Commercially available insect cell expression vectors which are suitable for recombinant expression include but are not limited to pBlueBacHI and pBlueBacHis2 (Invitrogen), and pAcG2T (Pharmingen). An expression vector containing DNA encoding a protein can be used for expression of the protein in a recombinant host cell. Recombinant host cells can be prokaryotic or eukaryotic, including but not limited to bacteria such as E. coli or Streptomycetes, fungal cells such as yeast, mammalian cells including but not limited to cell lines of human, bovine, porcine, monkey and rodent origin, and insect cells including but not limited to Drosophila- and silkworm-derived cell lines. Cell lines derived from mammalian species which can be suitable and which are commercially available, include but are not limited to, L cells L-M(TK") (ATCC CCL 1.3), L cells L-M (ATCC CCL 1.2), Saos-2 (ATCC HTB-85), 293 (ATCC CRL 1573), Raji (ATCC CCL 86), CV-1 (ATCC CCL 70), COS-1 (ATCC CRL 1650), COS-7 (ATCC CRL 1651), CHO-Kl (ATCC CCL 61), 3T3 (ATCC CCL 92), NIH/3T3 (ATCC CRL 1658), HeLa (ATCC CCL 2), C127I (ATCC CRL 1616), BS-C-1 (ATCC CCL 26), MRC-5 (ATCC CCL 171) and CPAE (ATCC CCL 209). The appropriateness of any cell line for any particular puφose can be assessed by simply testing the expression of a protein of this invention in the cell line. The expression vector can be introduced into host cells via any one of a number of techniques including but not limited to transformation, transfection, protoplast fusion, and electroporation. The expression vector-containing cells are analyzed to determine whether they produce protein Identification of expressing cells can be done by several means, including but not limited to immunological reactivity with antibodies, labeled ligand binding and the presence of host cell-associated recombinant protein activity
The cloned DNA obtained through the methods descπbed herein can be recombinantly expressed by molecular cloning into an expression vector containing a suitable promoter and other appropπate transcπption regulatory elements, and transferred into prokaryotic or eukaryotic host cells to produce recombinant protein. Techniques for such manipulations can be found descπbed in Sambrook, et al , supra , and are well known and easily available to the one of ordinary skill in the art
Expression of protein can also be performed using in vitro produced synthetic mRNA. Synthetic mRNA can be efficiently translated in vaπous cell-free systems, including but not limited to wheat germ extracts and reticulocyte extracts
To determine the sequence(s) that yields optimal levels of recombinant protein, molecules including but not limited to the following can be constructed- a DNA fragment containing the full-length open reading frame for a protein as well as vaπous constructs containing portions of the DNA encoding only specific domains of the protein or rearranged domains of the protein. The expression levels and activity of the protein can be determined following the introduction, both singly and in combination, of these constructs into appropπate host cells Following determination of the DNA cassette yielding optimal expression in transient assays, this construct is transferred to a vaπety of expression vectors, including but not limited to those for mammalian cells, plant cells, insect cells, oocytes, bacteπa, and yeast cells where expression is assessed.
Following expression of a recombinant protein in a host cell, the recombinant polypeptides can be recovered Several protein puπfication procedures are available and suitable for use. Protein and polypeptides can be puπfied from cell lysates and extracts, or from conditioned culture medium, by vaπous combinations of, or individual application of methods including ultrafiltration, acid extraction, alcohol precipitation, salt fractionation, ionic exchange chromatography, phosphocellulose chromatography, lecithin chromatography, affinity (e.g., antibody or His-Ni) chromatography, size exclusion chromatography, hydroxylapatite adsoφtion chromatography and chromatography based on hydrophobic or hydrophilic interactions. In some instances, protein denaturation and refolding steps can be employed. High performance liquid chromatography (HPLC) and reversed phase HPLC can also be useful. Dialysis can be used to adjust the final buffer composition.
Antibodies
The present invention also relates to polyclonal and monoclonal antibodies raised in response to a protein disclosed herein, or a fragment thereof. It is preferable to raise antibodies to epitopes which show the least homology to other known proteins.
An antibody is specific for an epitope if one of skill in the art can use standard techniques to determine conditions under which one can detect a polypeptide of this invention in a Western Blot of a sample from a host cell that expresses a protein of this invention. The blot can be of a native or denaturing gel as appropriate for the epitope. An antibody is highly specific for an epitope if no nonspecific background binding is visually detectable. An antibody can also be considered highly specific if the binding of the antibody to the protein can not be competed by non- homologous peptides, polypeptides or proteins, but can be competed by homologous peptides or polypeptides or the full length form of the relevant protein as disclosed herein.
Recombinant protein can be separated from other cellular proteins by use of an immunoaffinity column made with monoclonal or polyclonal antibodies specific for full-length protein, or polypeptide fragments of protein. Additionally, polyclonal or monoclonal antibodies can be raised against a synthetic peptide (usually from about 9 to about 25 amino acids in length) from a portion of a protein disclosed in SEQ ID NOs:2-10. Monospecific antibodies are purified from mammalian antisera containing antibodies reactive against a protein or are prepared as monoclonal antibodies reactive with a protein using the technique of Kohler and Milstein (1975, Nature 256: 495-497). Monospecific antibody as used herein is defined as a single antibody species or multiple antibody species with homogenous binding characteristics for a particular protein. Homogenous binding as used herein refers to the ability of the antibody species to bind to a specific antigen or epitope, such as those associated with a protein described herein. Specific antibodies are raised by immunizing animals such as mice, rats, guinea pigs, rabbits, goats, horses and the like, with an appropriate concentration of a protein described herein or a synthetic peptide generated from a portion of the descπbed proteins with or without an immune adjuvant
Preimmune serum is collected pπor to the first immunization. Each animal receives between about 0.1 mg and about 1000 mg of protein associated with an acceptable immune adjuvant Such acceptable adjuvants include, but are not limited to, Freund's complete, Freund's incomplete, alum-precipitate, water in oil emulsion containing Corynebacterium parvum and tRNA The initial immunization consists of injecting protein or peptide fragment thereof, preferably in Freund's complete adjuvant, at multiple sites either subcutaneously (SC), intrapeπtoneally (IP) or both Each animal is bled at regular intervals, preferably weekly, to determine antibody titer The animals may or may not receive booster injections following the initial immunization Those animals receiving booster injections are generally given an equal amount of protein m Freund's incomplete adjuvant by the same route. Booster injections are given at about three week intervals until maximal titers are obtained At about 7 days after each booster immunization or about weekly after a single immunization, the animals are bled, the serum collected, and ahquots are stored at about -20°C.
Monoclonal antibodies (mAb) reactive with a protein are prepared by immunizing mbred mice, preferably Balb/c, with the protein. The mice are immunized by the IP or SC route with about 1 mg to about 100 mg, preferably about 10 mg, of protein m about 0.5 ml buffer or saline incoφorated an equal volume of an acceptable adjuvant, as discussed herein. Freund's complete adjuvant is preferred. The mice receive an initial immunization on day 0 and are rested for about 3 to about 30 weeks. Immunized mice are given one or more booster immunizations of about 1 to about 100 mg of protein m a buffer solution such as phosphate buffered saline by the intravenous (IV) route. Lymphocytes, from antibody positive mice, preferably splenic lymphocytes, are obtained by removing spleens from immunized mice by standard procedures known in the art. Hybπdoma cells are produced by mixing the splenic lymphocytes with an appropπate fusion partner, preferably myeloma cells, under conditions which will allow the formation of stable hybπdomas.
Fusion partners can include, but are not limited to: mouse myelomas P3/NSl/Ag 4-1; MPC-11; S-194 and Sp 2/0, with Sp 2/0 being preferred. The antibody producing cells and myeloma cells are fused in polyethylene glycol, about 1000 mol. wt., at concentrations from about 30% to about 50%. Fused hybπdoma cells are selected by growth in hypoxanthme, thymidme and ammopteπn supplemented Dulbecco's Modified Eagles Medium (DMEM) by procedures known in the art. Supernatant fluids are collected form growth positive wells on about days 14, 18, and 21 and are screened for antibody production by an immunoassay such as solid phase immunoradioassay (SPIRA) using the protein as the antigen. The culture fluids are also tested in the Ouchterlony precipitation assay to determine the isotype of the mAb. Hybridoma cells from antibody positive wells are cloned by a technique such as the soft agar technique of MacPherson, 1973, Soft Agar Techniques, in Tissue Culture Methods and Applications, Kruse and Paterson, Eds., Academic Press.
Monoclonal antibodies are produced in vivo by injection of pristine primed Balb/c mice, approximately 0.5 ml per mouse, with about 2 x 106 to about 6 x lθ6 hybridoma cells about 4 days after priming. Ascites fluid is collected at approximately 8-12 days after cell transfer and the monoclonal antibodies are purified by techniques known in the art.
In vitro production of mAb is carried out by growing the hybridoma in DMEM containing about 2% fetal calf serum to obtain sufficient quantities of the specific mAb. The mAb are purified by techniques known in the art.
Antibody titers of ascites or hybridoma culture fluids are determined by various serological or immunological assays which include, but are not limited to, precipitation, passive agglutination, enzyme-linked immunosorbent antibody (ELISA) technique and radioimmunoassay (RIA) techniques. Similar assays are used to detect the presence of the protein in a biological sample or in an in vitro biocatalysis reaction.
It is readily apparent to those skilled in the art that the herein described methods for producing monospecific antibodies can be utilized to produce antibodies specific for peptide fragments, or full-length proteins described herein.
Antibody affinity columns are made, for example, by adding the antibodies to Affigel-10 (Biorad), a gel support which is pre-activated with N- hydroxysuccinimide esters such that the antibodies form covalent linkages with the agarose gel bead support. The antibodies are then coupled to the gel via amide bonds with the spacer arm. The remaining activated esters are then quenched with 1M ethanolamine HC1 (pH 8). The column is washed with water followed by 0.23 M glycine HC1 (pH 2.6) to remove any non-conjugated antibody or extraneous protein. The column is then equilibrated in phosphate buffered saline (pH 7.3) and the cell culture supernatants or cell extracts containing full-length protein or protein fragments are slowly passed through the column. The column is then washed with phosphate buffered saline until the optical density (A28O) falls to background, then the protein is eluted with 0.23 M glyc e-HCl (pH 2.6). The puπfied protein is then dialyzed against phosphate buffered saline.
Levels of recombinant protein in host cells is quantified by a vaπety of techniques including, but not limited to, immunoaffinity and/or ligand affinity techniques. Specific-antibody affinity beads or specific antibodies are used to isolate 35S-methιonme labeled or unlabelled recombinant protein. Labeled recombinant protein is analyzed by SDS-PAGE. Unlabelled protein is detected by Western blotting, ELISA or RIA assays employing either protein specific antibodies and/or antiphosphotyrosme antibodies.
Avermectin Glycosylation Genes and Proteins
A cluster of genes involved in the synthesis and/or addition of oleandrose to avermectin aglycone has been cloned. pVE650, a 47.8 kb plasmid was isolated from a library of 5. avermitilis by its ability to complement a mutant producing non-glycosylated avermectins. Five overlapping cosmid clones of S. avermitilis genomic DNA were isolated using a fragment of pVE650 as a probe. Subclones from pVE650 and an overlapping cosmid were used in complementation studies with 23 mutants defective in the glycosylation of avermectin aglycone. Seven complementation classes were identified. A 11-kb Pstl fragment of S. avermitilis genomic DNA complemented all 23 mutants, indicating the genes for avermectin glycosylation were clustered. The 11 kb Pstl fragment can be cloned from a deposited strain, ATCC 67890, which contains plasmid ρVE859
The 11 kb Pstl fragment of the avermectin gene cluster from S. avermitilis was subcloned into an integration vector, pVE1053. The resulting plasmid, pVE1190 could complement all the mutants known to us that are defective in glycosylation. The result indicated that pVEl 190 encoded all the genes for biosynthesis and attachment of oleandrose disacchaπde to avermectin aglycone. Upon sequencing 10 kb region of the fragment, it was discovered that the fragment contained nine open reading frames.
The 11-kb subclone was mutagenized with Tn5 and Tn5seql. Fourteen insertions were transferred to S. avermitilis and used in complementation analysis. An eighth complementation class was identified. Sequencing of an 10-kb region identified nine ORFs and an additional partial ORF. Eight of the nine ORFs were correlated to seven glycosylation complementation classes confirming that these eight genes are involved in the biosynthesis and attachment of oleandrose to avermectin aglycones. Sequence comparison to Genbank data bases identified 6 of the genes as: dTDP-glucase synthase(ORF 3a), dTDP-glucose 4,6 dehydrase(ORF3b), dTDP-4-keto-hexose reductase (ORF4), dTDP-hexose 3,5 epimerase(ORF5), dTDP- hexose 3' O-methylase(ORF7), and an avermectin aglycone-dTDP-oleandrose glycosyltransferase(ORF2). The ninth ORF was essential for biosynthesis of the avermectin aglycones. The partial ORF encoded part of an avermectin polyketide synthase module 7.
The genes from this cluster or the encoded polypeptides could be used to glycosylate avermectin aglycones or other macrolide aglycones. For instance US patent US 5,312,753 describes the glucosylation of the C13 and C14a positions of avermectin derivatives by a S. avermitilis strain. Another use of the polynucleotides and polypeptides would be to use them separately and in combination with other cloned genes or expressed proteins to make and attach known and novel sugars to known and novel macrolides or to other hydroxyl containing compounds.
EXAMPLE 1
Cloning of the Gene Cluster
Bacterial strains and plasmids. Ligation mixtures were used to transform E. coli MM294 (E. coli
Genetic Stock Center, New Haven, CT.) Derivatives of pVE616 were isolated from the triply DNA methylase deficient host ET12567. The isolation of mutants deficient in glycosylation of avermectin aglycones has been described (Ruby, et al., 1990). Some of the glycosylation mutants were isolated from a mutant deficient in C-5 O- methylation of avermectin (Ruby 1986, Ruby et al, 1990). S. avermitilis mutants deficient in 3',3" O-methylation (GMT) have been described (Ruby et a., 1985 ). 5. lividans strain 1326 and its SLP2-SLP3" derivative TK21 (Hopwood et al, 1983) were obtained from D. Hopwood (John Innes Institute, Norwich, UK). pBR322 was obtained from BRL (Bethesda, MD) and pIJ922 was obtained from D. Hopwood (Hopwood et al, 1985). pVE616 is a 4.4 kb AmpR derivative of pBR322 which contains a 1.8 kb BamΑ fragment which expresses thiostrepton-resistance in Streptomyces (Gene). Cultures were preserved by adding 0.1 ml of dimethyl sulfoxide (Aldrich Chemical Co., Milwaukee, WI) to 0.9 ml of culture and quick freezing the mixture at -70°C.
Media, Solutions, and Chemicals Streptomyces were grown as dispersed cultures for the isolation of chromosomal or plasmid DNA in YEME medium (Thompson, et al, 1982) with 30% sucrose and 0.25% glycine. E. coli was grown in LB (Miller, 1972). Solid media containing 1.5% agar included LB for E. coli (Miller, 1972), R2YE for S. lividans (Thompson, et al, 1982), RM14 for S. avermitilis (MacNeil & Klapko, 1987), and YME-TE for S. avermitilis . YME-TE contained per liter: yeast extract 3.0 g, malt extract 10.0 g, dextrose 4.0 g and 4 ml of a trace element solution (per liter: HC1 (37.3%) 49.7 ml, MgSO4-7H2O 61. lg, CaCO3 2.0g, FeCl3-6H2O 5.4 g, ZnSO4-7H2O 1.44 g, MnSθ4-H2θ 1.11 g, CuSO4-5H2O 0.25 g, H3BO3 0.062g, Na2MoO4-2H2O 0.49 g). YME-TE was adjusted to pH 7.0 with NaOH before autoclaving. Fermentation medium A, contained, per liter: glucose 20.0 g, yeast extract 20.0g, Hy-Case SF 20.0 g/ml, MgSO4-7H2O (12.5%), NaCl (12.5%), MnSθ4-H2θ (0.5%), ZnSO4-7H2O (1.0%), CaCl2"2H2θ (2.0%), FeSO4-7H2O 0.025 g, and KNO3 2.0 g. Fermentation medium B, which was adjusted to pH 7.2 with NaOH before autoclaving contained, per liter, peptonized milk 20.0 g, Ardamine pH 4.0 g, glucose 90.0 g, MgSO4-7H2O 0.5 g, CUSO4 5H2O (0.06 mg/ml) 1 ml, ZnSO4-6H O (1 mg/ml) 1 ml, CoCl2-6H2O (0.1 mg/ml) 1ml, and FeCl -6H2O (3 mg/ml) 1 ml. TE buffer (10 mM Tris, pH 7.9, 1 mM EDTA) was used to store and dilute DNA. Polyethylene glycol 1000 (PEG), agarose and ampicillin were obtained from Sigma Chemical Co., St Louis, MO. Formamide was obtained from EBI (New Haven, CT). Thiostrepton (gift from E. R. Squibb & Sons, Princeton, NJ) was added to a final concentration of 5 μg/ml in liquid medium, 10 μg/ml in solid medium, and 15 μg/ml when added as an overlay to select transformants. Ampicillin was added to a final concentration of 100 μg/ml.
Isolation of DNA
Large (500 ml) and small (1.5 ml) scale preparations of plasmid DNA were isolated from E. coli by the alkaline lysis procedure (Maniatis et al. 1982). A modified alkaline lysis procedure was developed for Streptomyces. Small scale plasmid preparations were prepared form cultures grown in 5 ml of YEME and washed as described previously (MacNeil, 1987). Cell pellets were resuspended in 1 ml of 50 mM glucose, 25 mM Tris pH 8, 10 mM EDTA, and 50 μl of a 15 mg ml lysozyme solution in 50 mM glucose, 25 mM Tris pH 8, 10 mM EDTA was added. Following incubation for 15 minutes at 37°C, 1.5 ml of a 0.2 N NaOH, 1% SDS solution was added, the mixture was vortexed for 5 seconds and the mixture was incubated for 15 minutes on ice. Next 150 μl of ice cold pH 4.8 potassium acetate solution (5 M with respect to acetate, 3 M with respect to potassium) was added, the mixture vortexed for 10 seconds, and incubated on ice for 15 minutes. The mixture was centrifuged for 15 minutes at 12,000 x g, at 4°C and the resulting supernatant was transferred to a new tube. 2.0 ml of -20°C isopropanol or isopropanol containing
0.05% diethyl pyrocarbonate was added, mixed, and centrifuged at 12,000 x g for 15 minutes at 4°C. The DNA pellet was dried and the DNA was dissolved in 0.5 ml of 0.3 M ammonium acetate. The solution was transferred to a 1.5 ml Eppendorf tube, mixed with 400 μl of phenol, previously equilibrated with 1 M Tris pH 7.9, and the aqueous phase separated by centrifugation in a microfuge for 3 minutes. The aqueous phase was removed to another Eppendorf tube and extracted with 400 μl of chloroform. The resulting aqueous DNA solution was precipitated with 2 volumes of ethanol, washed with 70% ethanol, and the plasmid DNA resuspended in 100 μl of TE. Large scale plasmid preparations were isolated from 1 1 YEME cultures of Streptomyces by a scaled up alkaline lysis procedure except that the DNA precipitated by isopropanol was resuspended in a CsCl solution and subjected to two bandings. Chromosomal DNA from Streptomyces was prepared as described by Hopwood et al, 1985.
Transformations with plasmid DNA.
The procedures for preparation of protoplasts, storage of protoplasts, polyethylene glycol mediated transformation of protoplasts, regeneration of protoplasts, and selection of transformants has been described for S. lividans (MacNeil, 1987) and S. avermitilis (MacNeil & Klapko, 1987). Transformation of E. coli with plasmid DNA has been described (Maniatis et al, 1982).
Restriction enzyme analysis
Restriction enzymes were obtained from New England Biolabs (Beverly, MA), Bethesda Research Labs (Bethesda, MD), or IBI (New Haven, CT) and were used according to the manufactures directions. Agarose gels were prepared and electrophoresis performed as described (Maniatis et al, 1982).
Construction of subclones from pVE650 and pVE859 Restriction fragments to be used in the construction of subclones from pVE650 and pVE859 were purified from agarose gel slices by electroelution and ligated to CIAP treated vector DNA. Subclones into pVE616 were transformed into MM294 and the appropriate constructs were identified. Plasmid DNA was transformed into ET 12567 (a triply DNA methylation deficient strain), purified by CsCl centrifugation, and 5 μg of the resulting DNA was used to transform S. avermitilis . Subclones into pIJ922 were transformed into S. lividans TK21, analyzed, purified from CsCl gradients, and lOOng of the plasmid DNA was transformed into S. avermitilis .
S. avermitilis fermentations and analysis of avermectin production
Single colonies from transformation plates were picked with a sterile toothpick on to YME-TE medium and subjected to small scale solid fermentations as described MacNeil et al., 1992. After 12-16 days incubation at 27-28°C, the mycelia was extracted with methanol, aliquots of the extract were applied to E. Merck Silica Gel 60 F-254 TLC plates and the avermectins developed for 15 minutes with a dichloromethane: ethylacetate:methanol 9:9: 1 solvent mixture. Avermectins are visualized under UV illumination. Under these conditions 4 glycosylated avermectins are resolved from strains which produce wild type avermectins. OMT- cultures produce predominantly the B avermectins. Mutants unable to glycosylate avermectin aglycones also produce 4 bands, however, since aglycones are a better substrate for the C5-Omethyltransferae, mostly the A- aglycones are produced. In contrast in the OMT- strains, residual C5-O-methyltransferase only methylates about 1/2 the aglycones resulting in 4 bands. The aglycones run faster in the TLC system than the corresponding glycosylated avermectins. As shown previously ( Gene) the order, from fastest to slowest band is, avermectin aglycone Aia+b, avermectin aglycone A2a+b, avermectin Ai a+b and avermectin aglycone Bi a+b, avermectin A2a+b and avermectin aglycone B2a+b, avermectin Bia+b, and avermectin B2a+b.
Colony hybridizations The cosmid library of S. avermitilis was constructed in the 6.7 kb, double lambda cos vector, pVE328, and consists of 2016 cosmid clones stored as individual cultures in 21 microtiter dishes. Replicates of the library were made on LB plates containing ampicillin, colonies were transferred to Biotrans nylon membranes (1.5 μM pore size), and colonies processed to release and fix DNA to the filters (Maniatis et al., ). The resulting 21 filters were individually hybridized with 3 p labeled probes. Preparation of probes, hybridizations and autoradiography were as described above for Southern analysis. Putative hybridizing clones were retested by patching duplicates to LB plates with ampicillin, lifting the colonies to nitrocellulose (Schleicher & Schuell, Keene, NH), fixing the DNA to the filters and hybridizing with the probe. Plasmid DNA was isolated from the cosmid clones which retested positive, restricted with BamKL, and confirmed by a Southern analysis.
Isolation of pVE650 S. avermitilis produces 8 major avermectins which can be separated by
TLC into 4 bands representing, from most polar to least, avermectin Ai a+b, A2a+b, Bia+b and B2a+b. A pIJ922 based library of 5. avermitilis DNA was constructed and screened for complementation of two mutants defective in avermectin biosynthesis (Avr). One mutant was a C-5 O-methyltransferase mutant (OMT), which produces predominantly avermectin Bi a+b and B2a+b. The other mutant was
MA6278, an avermectin aglycone producer. Several overlapping plasmids were isolated which complemented OMT mutants (Streicher et al). When the plasmids which complemented OMT mutants were introduced into several mutants altered in, or defective in, avermectin biosynthesis, no other mutants were complemented (Streicher et al). Approximately 3000 transformants of MA6278 were screened for avermectin production by small scale fermentation and TLC analysis of methanol extracts of each transformant. One transformant complemented the defect in MA6278. A plasmid was isolated from this transformant and designated pVE650. The presence of avermectin glycosylation genes on pVE650 was confirmed by retransforming MA6278 by pVE650 and detecting glycosylated avermectins by TLC. Most aglycone producing mutants (21/26) were complemented by pVE650.
Physical analysis of pVE650
A restriction map was determined for pVE650 see MacNeil et al, 1992. The insert in pVE650 is delimited by BamϊΩ. sites, no sites were found in the 24 kb insert for the following enzymes: Asel, Dral, EcoRV, HindUl, Hpal, Ndel, Nhel, Spel, Sspl, and Xbal. No common restriction bands were found between pVE650 and pATl, a plasmid which complements OMT mutants (Streicher et al).
The insert in pVE650 was found to be colinear with the chromosome of S. avermitilis by Southern analysis. The 9 BamHl fragments greater than 400 bp were used as probes against BamHl and Sstl digestions of genomic DNA from avermectin producing and nonproducing strains. Seven of the nine Z?αmHI fragments hybridized to a band identical in size to the BamHl fragment used as probes. Therefore, the seven BamHl fragments do not appear to have undergone rearrangement to form pVE650. This was confirmed by the Sstl digestions in which adjacent BamHl fragments hybridize to an overlapping Sstl fragment. Two BamHl fragments at the ends of the insert in pVE650, the 2.1 kb and 1.1 kb fragments, hybridized to larger fragments. These results indicate that pVE650 resulted from the ligation of a Sα«3AI fragment into the BamHl site of pIJ922 in such a way that BamHl sites formed at both junctions.
EXAMPLE 2
Identification of the Genes for Avermectin Glycosylation
Identification of three genes for avermectin glycosylation on pVE650 The 26 AGL" mutants were divided into 4 complementation classes by introducing subclones of pVE650 into the AGL" mutants. Complementation tests were performed by introducing subclones into various aglycone producing mutants and testing transformants for the ability to produce avermectins or avermectin aglycones. Fragments form pVE650 were subcloned into pU922, a low copy number Streptomyces vector (Hopwood et al., 1985), or pVE616, an E. coli vector that fails to replicate in Streptomyces but which can integrate by recombination between the chromosome and the cloned fragment. Between 6 and 12 transformants were tested for avermectin production as visualized by TLC analysis of fermentation extracts. Occasionally, an individual transformant failed to produce avermectin aglycones or avermectins. Positive complementation was scored if at least 5/6 transformants produced avermectins. Although S. avermitilis is proficient for recombination, we believe that the production of avermectins was the result of trans complementation rather than recombination. On occasion we have seen results indicative of recombination in which only 1/12 to 3/12 transformants produce avermectins. These putative recombinants were observed with only one or two members of a complementation class and only with a subclone derived from the integration vector. FIG. 1 indicates the subclones which were used to successfully complement AGL" mutants. Table 1 identifies the mutants in each complementation class and presents the complementation results with key subclones. Twenty-one aglycone producing mutants, representing complementation Classes I, π, and HI, were complemented after introduction of pVE650, but 5 Agl" mutants and two GMT- mutants were not. Class I mutants were complemented when they contained pVE650, or subclone pVE908 (2.4 EcoRl-BglU fragment). Class II mutants were complemented by pVE650 or subclone pVE807 (2.6 kb Bgl l fragment), but not by pVE908. Class HI mutants were not complemented by pVE807 or pVE908. Although we can not exclude the occurrence of intragenic complementation, it is likely that each complementation class represents at least one gene for avermectin glycosylation. We have designated three genes to represent the loci defective within the mutants of complementation Classes I, π, and HI, avrB, avrD, and avrC, respectively.
Isolation of cosmid clones which overlap pVE650 sequences Since two avermectin genes were located to a 6.6 kb region at one end of pVE650, it was possible that the AGL" mutants which were not complemented by pVE650 might contain mutations in the DNA that maps adjacent to pVE650 in the S. avermitilis genome. To test this hypothesis the 1.1 kb BamHl fragment from the end of the insert in pVE650 was used in a chromosome walk experiment to isolate overlapping clones from a S. avermitilis cosmid library. Colony hybridization to 2016 cosmid clones identified 5 cosmids. One cosmid, pVE855, contained all the DNA represented by pVE650 and additional DNA from each end. Collectively the cosmids represent 60 kb of S. avermitilis DNA. None of the cosmids overlapped sequences on pATl. From one cosmid, pVE859, we identified a 15 kb BglR fragment which contained the 470 bp EcoRl to BamHl fragment near the end of pVE650.
Thus, this 15 kb fragment represents the chromosomal BglR fragment that is adjacent to the 140 bp BglR fragment of pVE650 and extends 13 kb beyond the DNA contained on pVE650. This fragment was cloned into pIJ922 to yield pVE941. pVE941 contains all the S. avermitilis DNA on pVE807 and, as expected, complements Class II aglycone producers. pVE941 also complemented all 5 AGL" mutants not complemented by pVE650 and two GMT" strains. Thus, the genes for glycosylation of avermectin are clustered since all the mutants defective in synthesis or addition of oleandrose to avermectin aglycone are complemented by pVE650 and/or pVE941.
Localization of additional genes for glycosylation of avermectin aglycone
Additional subclones were prepared from pVE855 and used in complementation tests. pVEllll (4.1 EcoRI fragment of pVE650 plus the 1.8 kb EcσRI fragment of pVE941) complemented Class I, II and Class HI mutants. Thus the mutants in Class HI are be defective in a gene, designated αvrC, located between avrB and avrD. MA6057 and MA6622 were complemented by only pVE941 and pVEl 115 and are designated class IV. pVE1019, which contained the 3.5 kb BamHl fragment from pVE941, complemented the defects in the two GMT mutants and AGL" strain MA6590. This later mutant was designated Class V. Two mutants complemented by pVE941 and pVE1018, but not by pVE650 or pVE1019, were designated Class VI. Table 1 summarizes the complementation results which have defined the 7 classes of mutants involved in glycosylation of the avermectin aglycone.
Subcloning a region which complements all AGL" mutants An 12 kb Pstl fragment, which overlaps both pVE650 and p VE941 , was subcloned onto pVE1043 to yield pVEl 115. Mutants from all the complementation classes were complemented by pVEl 115. Thus, it appears that all the genes for glycosylation of avermectin have been cloned on pVEl 115. The 7 complementation classes define the minimum number of genes involved in avermectin glycosylation. Each complementation class may represent more than one gene. FIG. 1 shows the location of the 7 identified genes involved in glycosylation of avermectin.
Only AGL" mutants are complemented by pVE650 We tested pVE650 for the presence of other genes by complementation analysis. pVE650 was introduced into mutants representing each phenotypic class of S. avermitilis defective or altered in avermectin biosynthesis. No complementation was observed in MA6238 (C-22, C-23 dehydrase [DH"]), MA5218 (C-6, C-8' furan ring formation [FUR"]), MA6316 (C-3', C-3" O-methyltransferase or glycosyl O- methyltransferase [GMT"]), MA6262, (nonproducer of avermectin [NPA ]), or MA6233 (OMT").
The complementation results with pVEl 115 clearly show that the genes for glycosylation of avermectin are tightly clustered. pVEl 115, which contains a 12 kb Pstl fragment from S. avermitilis , complemented all 26 mutants which fail to glycosylate avermectin and 2 mutants which fail to methylate hydroxyls at the C-3', C- 3" positions. This suggests that pVE1115 may contain all the genes for synthesis and for attachment of oleandrose to avermectin aglycone. However, it is possible our collection of mutants does not include defects in all the genes involved m avermectin glycosylation. If this is so, then pVEl 115 may not contain all the glycosylation genes.
Sequence of the glycosylation region.
BamHl, EcoRI, and Pstl-BamHI fragments from pVEl 101 were subcloned and sequenced on both strands using a pπmer walking strategy. DNA was sequenced manually using Sequenase (US Biochemicals) and an ABI 373A automated sequencer (Perkin Elmer) according to the manufacture's recommendations. The resulting 9994 nt sequence is shown as SEQ ID NO:l. And was analyzed by the GCG software suite (Genetics Computer Group). 9 complete ORF were identified and the genes involved in glycosylation designated AvrB through Avrl as shown on FIG. 1. In this region there are two sets of overlapping genes. The AvrB and AvrC genes are convergently transcπbed and their coding regions overlap for 95 nt. The AvrD and AvrC genes are co-transcπbed but encode proteins in different reading frames and overlap for 16 nts. A compaπson of the open reading frames in the sequence to the clones used in complementation analysis results in the identification of 8 genes essential for avermectin glycosylation.
TFASTA compaπson of the ORFs to Genbank resulted in highly significant similaπties to several known genes. ORF1 showed similaπty to keto-reductases. ORF2 showed greater than 30% identity to glycosyl-transferases. ORF3a was greater than 60% identical to TDP-glucose-4,6-dehydratases, ORF3b was greater than 60% identical to several TDP-glucose synthases, and ORF4 showed weak homology to keto reductases. ORF5 had greater than 50% identity to hexose 3,5 epimerases. ORF7 was identified as a glycosyl methyltransferase since that ORF could complement the GMT- mutants. Macrohdes contain many unusual sugars (Omura, S Macrolide Antibiotics, Academic Press, 1984). A biochemical study of the mutants and cloned genes will help elucidate the biochemical pathway for synthesis of oleandrose. The cloned genes for synthesis and addition of oleandrose to avermectin aglycone can be useful in mtergenic complementation studies to identify genes involved in glycosylation of other macrohdes Alternatively, the cloned DNA can be useful as a probe to identify genes involved in the synthesis and/or addition of other sugar moieties to other macrohdes. For example, the acil gene of S. coehcolor, which is required for synthesis of actmorhodin, has been useful as a probe to identify putative polyketide synthetases from other species (Bergh and Uhlen, 1992)
The genes for glycosylation of the avermectin aglycone can be useful m the production of novel antibiotics. Since avermectins are much more potent antiparasitic agents than avermectin aglycones (Campbell, W. Ivermectm and Abamect , Spπnger-Verlag, 1989) or the non-glycosylated, but similar milbemycms (Omura, S Macrolide Antibiotics, Academic Press, 1984), it is evident that the oleandrose disacchaπde moiety enhances the potency of avermectin. The genes descπbed herein for synthesis and attachment of oleandrose to avermectin aglycone can be useful for the construction of hybπd antibiotics. For example, the introduction of a plasmid containing at least one gene of the present invention into strains that produce antibiotics with a hydroxyl group may result in hybπd glycosylated antibiotics. Potentially useful substrates for glycosylation are other macrohdes (Omura, 1984).
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Table 1. Complementation of S. avermitilis aglycone producing mutants
Class Mutants pVE650 pVE908 pVE807 pVE941 pVE1019 pVE1018 pVE1115
I GG900. MA6595. + + MA6580. MA6593, MA6056.MA6624
π MA6582, GG898, + -
MA6579. MA6581 , MA6589, MA6591.
MA5872 in MA0278, MA6580, +
MA6583, MA6584, MA658S, MA6587, MA6588. MA6060
rv MA6057, MA6622 _
V MA6590 -
VI MA6592, MA6594 GMT MA6316, MA6323 . . . + + - +
Plasmids used are shown in FIG. 1. PVE650 has been described (MacNeil et al., 1992), pVEl 115 contains the 11 kb Pstl fragment which complements all avermectin aglycone producing mutants. In most cases, at least 6 transformants of each plasmid into each mutant were tested for avermectin production by Microferm and TLC analysis.

Claims

WHAT IS CLAIMED:
1. An isolated polynucleotide selected from the group consisting of: (a) a polynucleotide encoding a polypeptide having an amino acid sequence selected from the group consisting of the eight amino acid sequences encoded by the polynucleotide sequence SEQ ID NO:l.
(b) a polynucleotide which is complementary to a polynucleotide of (a), (c) a polynucleotide representing a polymoφhic form of (a), and
(d) a polynucleotide comprising at least 20 nucleotides of the polynucleotide of (a), (b) or (c), said 20 nucleotides being highly specific for polynucleotide of (a).
2. The polynucleotide of claim 1 wherein the polynucleotide comprises nucleotides selected from the group consisting of natural, non-natural and modified nucleotides.
3. The polynucleotide of claim 1 wherein the internucleotide linkages are selected from the group consisting of natural and non-natural linkages.
4. The polynucleotide of claim 1 that includes the entire nucleotide sequence of SEQ ID NO:l.
5. The polynucleotide of claim 1 that includes at least a nucleotide sequence of the one of the open reading frames of SEQ ID NO: 1.
6. The polynucleotide of claim 5 having a sequence of Streptomycete genomic DNA.
7. The polynucleotide of claim 5 having a sequence of an RNA.
8. An expression vector comprising a polynucleotide of claim 1.
9. A host cell comprising the expression vector of claim 8.
10. A process for expressing a protein encoded by a nucleic acid having the sequence of SEQ HD NO: 1 in a recombinant host cell, comprising:
(a) introducing an expression vector of claim 9 into a suitable host cell; and,
(b) culturing the host cells of step (a) under conditions which allow expression of said protein from said expression vector.
11. A substantially purified polypeptide having an amino acid sequence selected from the group consisting of
(a) a polypeptide having an amino acid sequence of encoded for by a nucleic acid having the sequence of SEQ ID NO: 1, and
(b) a polypeptide representing a polymoφhic form of (a).
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US6864073B1 (en) 1999-02-24 2005-03-08 The Kitasato Institute Avermectin aglycon synthase genes
US7371548B2 (en) 1999-02-24 2008-05-13 The Kitasato Institute Avermectin aglycon synthase genes
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WO2002088176A3 (en) * 2001-04-26 2003-05-30 Ecopia Biosciences Inc Genes and proteins for the biosynthesis of polyketides
EP1524318A1 (en) * 2001-04-26 2005-04-20 Ecopia Biosciences Inc. Genes and proteins for the biosynthesis of polyketides
EP1262562A3 (en) * 2001-05-30 2004-06-09 The Kitasato Institute Actinomycetes polynucleotides
EP1852508A3 (en) * 2001-05-30 2009-02-18 The Kitasato Institute Actinomycetes polynucleotides
US7630836B2 (en) 2001-05-30 2009-12-08 The Kitasato Institute Polynucleotides
EP1477563A3 (en) * 2003-05-16 2004-11-24 Wyeth Cloning genes from streptomyces cyaneogriseus subsp.noncyanogenus for biosynthesis of antibiotics and methods of use
US7396660B2 (en) 2003-05-16 2008-07-08 Wyeth Cloning genes from Streptomyces cyaneogriseus subsp. Noncyanogenus for biosynthesis of antibiotics and methods of use

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