SAM operon

Abstract

The invention provides isolated nucleic acid compounds encoding a novel SAM synthetase of Streptomyces fradiae. Also provided are vectors and transformed heterologous host cells for expressing the SAM synthetase and a method for preparing S-adenosylmethionine from recombinant host cells transformed with the SAM synthetase gene.

Description

[0001] We hereby claim the benefit under Title 35, United States Code, §119(e) of U.S. provisional patent application No. 60/030,898 filed Nov. 13, 1996

BACKGROUND OF THE INVENTION

[0002] This invention relates to recombinant DNA technology. In particular the invention pertains to the cloning of the SAM operon genes from Streptomyces fradiae and the use of said genes and their encoded proteins to produce S-adenosylmethionine (SAM) in a recombinant host.

[0003] S-adenosylmethionine is a product of natural origin found in all living organisms. SAM is a product of considerable importance for its role in biological reactions such as transmethylations. While the enzymes that catalyze these reactions are varied in their substrate specificity they are practically universal in their requirement of S-adenosylmethionine as the ultimate methyl group donor. Some methyl transfer reactions are important in the synthesis of certain antibiotics, such as tylosin.

[0004] Tylosin is a macrolide antibiotic composed of a 16-membered branched lactone, tylactone, and residues of three attached sugars, mycaminose, mycarose, and mycinose. Tylosin is produced commercially by Streptomyces fradiae (ATCC 19609; NRRL 2702) and is used as an animal growth promotant and veterinary antibiotic. The multi-step biosynthesis of tylosin has been studied both physiologically and genetically (See generally, R. H. Baltz and E. T. Seno, “Genetics of Streptomyces fradiae and tylosin biosynthesis. Ann. Rev. Microbiol. 42, 547-74 (1988)). At least 13 biosynthetic genes and 2 regulatory genes are necessary for normal production of tylosin. Tylosin synthesis requires multiple methylation reactions, the last two of which are rate-limiting. In the last step a specific methyltransferase catalyzes the transfer of a methyl group from SAM to the tylosin precursor molecule, macrocin. Thus, the availability of SAM as the methyl group donor is essential in the synthesis of tylosin.

[0005] S-adenosylmethionine is produced when an adenosyl group is transferred from ATP to methionine. SAM is synthesized in the cell by the action of three enzymes encoded by the SAM operon—SAM synthetase, methyl transferase (MT), and methylene tetrahydrofolate reductase (MTHR).

[0006] SUMMARY OF INVENTION

[0007] The present invention provides, inter alia, isolated nucleic acid molecules comprising the SAM operon from Streptomiyces fradiae. The invention also provides the protein products encoded by the SAM operon, in substantially purified form.

[0008] Having the cloned SAM operon of Streptomyces fradiae enables the production of S-adenosylmethionine in recombinant host cells.

[0009] In one embodiment the present invention relates to an isolated nucleic acid that encodes SAM synthetase from Streptomiyces fradiae, said nucleic acid comprising nucleotide residues 986 through 2209 of the nucleotide sequence identified as SEQ ID NO. 1.

[0010] In another embodiment the present invention relates to an isolated nucleic acid that encodes MT from Streptomyces fradiae, said nucleic acid comprising nucleotide residues 2241 through 3341 of SEQ ID NO.1.

[0011] In another embodiment the present invention relates to an isolated nucleic acid that encodes MTHR from Streptomyces fradiae, said nucleic acid comprising nucleotide residues 3338 through 4255 of SEQ ID NO.1.

[0012] In another embodiment the present invention relates to a novel SAM synthetase from Sreptomyces fradiae in substantially purified form comprising the sequence identified as SEQ ID NO. 2.

[0013] In still another embodiment the present invention relates to a novel MT from Streptomyces fradiae in substantially purified form comprising the sequence identified as SEQ ID NO. 3.

[0014] In yet another embodiment the present invention relates to a novel MTHR from Streptomyces fradiae in substantially purified form comprising the sequence identified as SEQ ID NO.5.

[0015] In a further embodiment the present invention relates to a ribonucleic acid molecule encoding SAM synthetase, said ribonucleic acid molecule comprising residues 986 through 2209 of the sequence identified as SEQ ID NO. 6:

[0016] In yet another embodiment, the present invention relates to a recombinant DNA vector that incorporates the Streptomyces fradiae SAM operon genes in operable linkage to gene expression sequences enabling said genes to be transcribed and translated in a host cell.

[0017] In still another embodiment the present invention relates to homologous or heterologous host cells which have been transformed or transfected with one or more of the cloned SAM operon genes from Streptomyces fradiae such that said gene(s) is/are expressed in the host cell.

[0018] In a still further embodiment, the present invention relates to a method for producing S-adenosylmethionine in recombinant host cells transformed with the S. fradiae SAM synthetase gene.

DESCRIPTION OF THE DRAWING

[0019] FIG. 1. Plasmid pRBD26, useful for expression of the Streptomyces fradiae SAM synthetase gene of the present invention in the homologous host cell or other actinomycete.

[0020] FIG. 2. Activated methyl cycle.

DEFINITIONS

[0021] The term “operon” as used herein refers to a genetic unit comprising a region of a chromosome having one or more structural genes said unit producing a messenger RNA molecule that may or may not be polycistronic (i.e. encoding more than one protein). Transcription of said RNA is under the control of a single promoter.

[0022] “SAM” refers to S-adenosylmethionine.

[0023] “MT” refers to methyltransferase.

[0024] “THF” refers to tetrahydrofolate.

[0025] “MTHR” refers to methylene tetrahydrofolate reductase.

[0026] “ATP” refers to adenosine triphosphate.

[0027] The terms “cleavage” or “restriction” of DNA refers to the catalytic cleavage of the DNA with a restriction enzyme that acts only at certain sequences in the DNA (viz. sequence-specific endonucleases). The various restriction enzymes used herein are commercially available and their reaction conditions, cofactors, and other requirements are used in the manner well known to one of ordinary skill in the art. Appropriate buffers and substrate amounts for particular restriction enzymes are specified by the manufacturer or can readily be found in the literature.

[0028] The term “plasmid” refers to an extrachromosomal genetic element. The starting plasmids herein are either commercially available, publicly available on an unrestricted basis, or can be constructed from available plasmids in accordance with published procedures. In addition, equivalent plasmids to those described are known in the art and will be apparent to the ordinarily skilled artisan.

[0029] “Recombinant DNA cloning vector” as used herein refers to any autonomously replicating agent, including, but not limited to, plasmids and phages, comprising a DNA molecule to which one or more additional DNA segments can or have been added.

[0030] The term “recombinant DNA expression vector” as used herein refers to any recombinant DNA cloning vector, for example a plasmid or phage, in which a promoter and other regulatory elements are present to enable transcription of the inserted DNA.

[0031] The term “vector” as used herein refers to a nucleic acid compound used for introducing exogenous DNA into host cells. A vector comprises a nucleotide sequence which may encode one or more protein molecules. Plasmids, cosmids, viruses, and bacteriophages, in the natural state or which have undergone recombinant engineering, are examples of commonly used vectors.

[0032] The terms “complementary” or “complementarity” as used herein refers to the capacity of purine and pyrimidine nucleotides to associate through hydrogen bonding in double stranded nucleic acid molecules. The following base pairs are complementary: guanine and cytosine; adenine and thymine; and adenine and uracil.

[0033] “Isolated nucleic acid compound” refers to any RNA or DNA sequence, however constructed or synthesized, which is locationally distinct from its natural location.

[0034] A “primer” is a nucleic acid fragment which functions as an initiating substrate for enzymatic or synthetic elongation of, for example, a nucleic acid molecule.

[0035] The term “promoter” refers to a DNA sequence which directs transcription of DNA to RNA.

[0036] A “probe” as used herein is a labeled nucleic acid compound which hybridizes with a complementary nucleic acid compound.

[0037] The term “hybridization” as used herein refers to a process in which a single-stranded nucleic acid molecule joins with a complementary strand through nucleotide base pairing. “Selective hybridization” refers to hybridization under conditions of high stringency. The degree of hybridization depends upon, for example, the degree of complementarity, the stringency of hybridization, and the length of hybridizing strands.

[0038] The term “stringency” refers to hybridization conditions. High stringency conditions disfavor non-homologous basepairing. Low stringency conditions have the opposite effect. Stringency may be altered, for example, by temperature and salt concentration.

DETAILED DESCRIPTION

[0039] The SAM operon of the present invention comprises three genes encoding SAM synthetase, MT, and MTHR. Together these enzymes comprise the so-called “activated methyl cycle” which produces S-adenosylmethionine (see FIG. 2). The “activated methyl cycle” provides the methyl groups required for the final steps in tylosin production.

[0040] The SAM synthetase gene disclosed herein comprises a DNA sequence of 1224 nucleotide base pairs (residues 986 through 2209 of SEQ ID NO. 1). The MT gene disclosed herein comprises a DNA sequence of 1101 nucleotide base pairs (residues 2241 through 3341 of SEQ ID NO.1). The MTHR gene disclosed herein comprises a DNA sequence of 918 nucleotide base pairs (residues 3338 through 4255 of SEQ ID NO.4). There are no intervening sequences in the SAM operon. The 5′ end of the MTHR gene overlaps (in another reading frame) with four nucleotide residues at the 3′ end of the MT gene. Specifically, the “TGA” stop codon of the MT gene ends at residue 3341 of SEQ ID NO.4, while the “GTG” start codon of the MTHR gene begins at residue 3338 of SEQ ID NO.4. Those skilled in the art will recognize that owing to the degeneracy of the genetic code (i.e. 64 codons which encode 20 amino acids), numerous “silent” substitutions of nucleotide base pairs could be introduced into this sequence without altering the identity of the encoded amino acid(s) or protein product. All such substitutions are intended to be within the scope of the invention.

[0041] The SAM synthetase of the present invention, designated SEQ ID NO.2, comprises a protein of 407 amino acid residues. The MT of the present invention, designated SEQ ID NO.3, comprises a protein of 366 amino acid residues. The MTHR of the present invention, designated SEQ ID NO.5, comprises a protein of 305 amino acid residues.

[0042] Gene Isolation Procedures

[0043] Those skilled in the art will recogize that the gene of the present invention may be obtained by a plurality of applicable genetic and recombinant DNA techniques including, for example, polymerase chain reaction (PCR) amplification, or de novo DNA synthesis.(See e.g., J. Sambrook et al. Molecular Cloning, 2d Ed. Chap. 14 (1989)).

[0044] Methods for constructing gene libraries in a suitable vector such as a plasmid or phage for propagation in procaryotic or eucaryotic cells are well known to those skilled in the art. [See e.g. J. Sambrook et al. Supra]. Suitable cloning vectors are widely available.

[0045] Skilled artisans will recognize that the SAM operon genes of Streptomyces fradiae comprising the present invention or fragments thereof could be isolated by PCR amplification of Streptomyces fradiae genomic DNA or cDNA using oligonucleotide primers targeted to a suitable region of SEQ ID NO. 1. The coding regions of the MT and MTHR genes are, respectively, 2241 through 3341, and 3338 through 4255 of SEQ ID NO.1. Methods for PCR amplification are widely known in the art. See e.g. PCR Protocols: A Guide to Method and Application, Ed. M. Innis et al., Academic Press (1990). The amplification reaction comprises genomic DNA, suitable enzymes, primers, and buffers, and is conveniently carried out in a DNA Thermal Cycler (Perkin Elmer Cetus, Norwalk, Conn.). Amplification of a DNA fragment of the correct size can be detected most conveniently by agarose gel electrophoresis.

[0046] Protein Production Methods

[0047] One embodiment of the present invention relates to the substantially purified SAM operon proteins or fragments thereof encoded by the genes disclosed herein.

[0048] Skilled artisans will recognize that the proteins of the present invention can be synthesized by any number of different methods. The amino acid compounds of the invention can be made by chemical methods well known in the art, including solid phase peptide synthesis or recombinant methods. Both methods are described in U.S. Pat. No. 4,617,149, incorporated herein by reference.

[0049] The principles of solid phase chemical synthesis of polypeptides are well known in the art and may be found in general texts in the area. See, e.g., H. Dugas and C. Penney, Bioorganic Chemistry (1981) Springer-Verlag, New York, 54-92. For example, peptides may be synthesized by solid-phase methodology utilizing an Applied Biosystems 430A peptide synthesizer (Applied Biosystems, Foster City, Calif.) and synthesis cycles supplied by Applied Biosystems. Protected amino acids, such as t-butoxycarbonyl-protected amino acids, and other reagents are commercially available from many chemical supply houses.

[0050] The proteins of the present invention can also be produced by recombinant DNA methods using the cloned SAM operon genes of Streptomyces fradiae disclosed herein. Recombinant methods are preferred if a high yield is desired. Expression of said cloned genes can be carried out in a variety of suitable host cells well known to those skilled in the art. In a recombinant method, a gene is introduced into a host cell by any suitable means, well known to those skilled in the art. While chromosomal integration of the cloned SAM operon genes is within the scope of the present invention, it is preferred that the genes be cloned into a suitable extra-chromosomally maintained expression vector, the coding region of the genes in operable linkage to a constitutive or inducible promoter.

[0051] The basic steps in the recombinant production of the SAM operon enzymes, SAM synthetase, MT, or MTHR of the present invention are:

[0052] a) constructing a natural, synthetic or semi-synthetic DNA encoding said enzyme(s);

[0053] b) integrating said DNA into an expression vector in a manner suitable for expressing said enzyme(s), as the natural protein product or as a fusion protein;

[0054] c) transforming or otherwise introducing said vector into an appropriate eucaryotic or prokaryotic host cell forming a recombinant host cell,

[0055] d) culturing said recombinant host cell in a manner to express said enzyme(s); and

[0056] e) recovering and substantially purifying said enzyme(s) by any suitable means, well known to those skilled in the art.

[0057] Expressing Recombinant S. fradiae SAM Operon in a Procaryotic or Eucaryotic Host Cell

[0058] In general, procaryotes are used for cloning DNA sequences and for constructing the vectors of the present invention. Procaryotes may also be employed in the production of the protein of the present invention. For example, the Escherichia coli K12 strain 294 (ATCC No. 31446) is particularly useful for the prokaryotic expression of foreign proteins. Other strains of E. coli, bacilli such as Bacillus subtilis, enterobacteriaceae such as Salmonella typhimurium or Serratia marcescans, various Pseudomonas species and Actinomycetes, such as Streptomyces fradiae , Streptomyces coelicolor, and Streptomyces lividans, may also be employed as host cells in the cloning and expression of the recombinant proteins of this invention.

[0059] Promoter sequences suitable for driving the expression of genes in procaryotes include b-lactamase [e.g. vector pGX2907, ATCC 39344, contains a replicon and b-lactamase gene], lactose systems [Chang et al., Nature (London), 275:615 (1978); Goeddel et al., Nature (London), 281:544 (1979)], alkaline phosphatase, and the tryptophan (trp) promoter system [vector pATH1 (ATCC 37695) which is designed to facilitate expression of an open reading frame as a trpE fusion protein under the control of the trp promoter]. Hybrid promoters such as the tac promoter (isolatable from plasmid pDR540, ATCC-37282) are also suitable. Useful promoters for driving gene expression in a Streptomyces host are known. For example, the snpR promoter is useful for gene expression in Streptomyces (See e.g. 6th Conference on the Genetics and Molecular Biology of Industrial Microorganisms, Oct. 20-24, 1996, Bloomington, Ind., Abstract P37). Still other bacterial promoters, whose nucleotide sequences are generally known, enable one of skill in the art to ligate such promoter sequences to DNA encoding the proteins of the instant invention using linkers or adapters to supply any required restriction sites. Promoters for use in bacterial systems also will contain a Shine-Dalgarno sequence operably linked to the DNA encoding the desired polypeptides. The cloned DNA comprising the SAM operon of the present invention carries the endogenous promoter in the region comprising nucleotide residues 1 through 985 of SEQ ID NO.1. This promoter will function in other Actinomycetes. These examples are illustrative rather than limiting.

[0060] The protein of this invention may be synthesized either by direct expression or as a fusion protein comprising the protein of interest as a translational fusion with another protein or peptide which may be removable by enzymatic or chemical cleavage. Expression as a fusion protein may prolong the half-life, increase the yield of the desired peptide, or provide a convenient means of purifying the protein. A variety of peptidases (e.g. enterokinase and thrombin) cleave a polypeptide at specific sites or digest peptides from the amino or carboxy termini (e.g. diaminopeptidase). Furthermore, particular chemicals (e.g. cyanogen bromide) will cleave a polypeptide chain at specific sites. The skilled artisan will appreciate the modifications necessary to the amino acid sequence (and synthetic or semi-synthetic coding sequence if recombinant means are employed) to incorporate site-specific internal cleavage sites. See e.g., P. Carter, “Site Specific Proteolysis of Fusion Proteins”, Chapter 13, in Protein Purification: From Molecular Mechanisms to Large Scale Processes, American Chemical Society, Washington, D.C. (1990).

[0061] In addition to procaryotes, a variety of mammalian cell systems and eucaryotic microorganisms such as yeast are suitable host cells. The yeast Saccharomyces cerevisiae is the most commonly used eucaryotic microorganism. A number of other yeasts such as Kluyveromyces lactis are also suitable. For expression in Saccharomyces, the plasmid YRp7 (ATCC-40053), for example, may be used. See, e.g., L. Stinchcomb, et al., Nature, 282:39 (1979); J. Kingsman et al., Gene, 7:141 (1979); S. Tschemper et al., Gene, 10:157 (1980). Plasmid YRp7 contains the TRP1 gene which provides a selectable marker for use in a trp1 auxotrophic mutant.

[0062] Purification of Recombinantly-Produced SAM Operon Enzymes

[0063] An expression vector carrying any of the cloned SAM operon genes as claimed herein is transformed or transfected into a suitable host cell using standard methods. Cells which contain the vector are propagated under conditions suitable for expression of the SAM operon enzyme(s) encoded by the vector. As an example, a vector-bound SAM synthetase gene is placed under the control of an inducible promoter. Suitable growth conditions would incorporate an appropriate inducer. Recombinantly-produced SAM synthetase may then be purified from cellular extracts of transformed cells by any suitable means, well known to those skilled in the art.

[0064] In a preferred process for protein purification the gene encoding the SAM operon enzyme of the present invention is modified at the 5′ end to incorporate several histidine residues at the amino terminus of the encoded protein product. The “His-tag” enables a single-step protein purification method referred to as “immobilized metal ion affinity chromatography” (IMAC), essentially as described in U.S. Pat. No. 4,569,794 which hereby is incorporated by reference. The IMAC method enables rapid isolation of substantially pure protein starting from a crude cellular extract.

[0065] Other embodiments of the present invention comprise isolated nucleic acid sequences which encode SEQ ID NO:2, SEQ ID NO.3, and SEQ ID NO.5. As skilled artisans will recognize, the amino acid compounds of the invention can be encoded by a multitude of different nucleic acid sequences because most of the amino acids are encoded by more than one codon due to the degeneracy of the genetic code. Because these alternative nucleic acid sequences would encode the same amino acid sequences, the present invention further comprises these alternate nucleic acid sequences.

[0066] Nucleic acid sequences encoding the proteins of the invention may be produced using synthetic methodology. The synthesis of nucleic acids is well known in the art. See, e.g., E. L. Brown, R. Belagaje, M. J. Ryan, and H. G. Khorana, Methods in Enzymology, 68:109-151 (1979). The DNA segments could be generated using a conventional DNA synthesizing apparatus, such as the Applied Biosystems Model 380A or 380B DNA synthesizers (Applied Biosystems, Inc., 850 Lincoln Center Drive, Foster City, Calif. 94404) which employ phosphoramidite chemistry. Alternatively, phosphotriester chemistry may be employed to synthesize the nucleic acids of this invention. [See, e.g., M. J. Gait, ed., Oligonucleotide Synthesis, A Practical Approach, (1984).]

[0067] In an alternative and preferred methodology, namely PCR, the DNA sequence comprising a portion or all of SEQ ID NO:1 can be generated from Streptomyces fradiae genomic DNA using suitable oligonucleotide primers complementary to SEQ ID NO:1 or region therein, as described in U.S. Pat. No. 4,889,818, which hereby is incorporated by reference. Suitable protocols for performing the PCR are widely known and are disclosed in, for example, PCR Protocols: A Guide to Method and Applications, Ed. Michael A. Innis et al., Academic Press, Inc. (1990).

[0068] The ribonucleic acids of the present invention may be prepared using the polynucleotide synthetic methods discussed supra, or they may be prepared enzymatically using RNA polymerase to transcribe a suitable DNA template.

[0069] The most preferred systems for preparing the ribonucleic acids of the present invention employ the RNA polymerase from the bacteriophage T7 or the bacteriophage SP6. These RNA polymerases are highly specific, requiring the insertion of bacteriophage-specific sequences at the 5′ end of the template to be transcribed. See, J. Sambrook, et al., supra, at 18.82-18.84.

[0070] This invention also provides nucleic acids, RNA or DNA, which are complementary to SEQ ID NO:1 or SEQ ID NO:6.

[0071] The present invention also provides probes and primers useful for a variety of molecular biology techniques including, for example, hybridization screens of genomic or subgenomic libraries. A nucleic acid compound comprising SEQ ID NO:1, SEQ ID NO:6 or a complementary sequence thereof, or a fragment thereof, and which is at least 18 base pairs in length, and which will selectively hybridize to Streptomiyces fradiae DNA or mRNA encoding the SAM operon of the present invention, is provided. Preferably, the 18 or more base pair compound is DNA. These probes and primers can be prepared by enzymatic methods well known to those skilled in the art (See e.g. Sambrook et al. supra). In a most preferred embodiment these probes and primers are synthesized using chemical means as described above.

[0072] Another aspect of the present invention relates to recombinant DNA cloning vectors and expression vectors comprising the nucleic acids of the present invention. Many of the vectors encompassed within this invention are described above. The preferred nucleic acid vectors are those which comprise DNA. The most preferred recombinant DNA vectors comprise the isolated DNA sequence, SEQ ID NO:1 or suitable region therof. Plasmid pRBD26 is an especially preferred DNA vector of the present invention.

[0073] The skilled artisan understands that choosing the most appropriate cloning vector or expression vector depends upon a number of factors including the availability of restriction enzyme sites, the type of host cell into which the vector is to be transfected or transformed, the purpose of the transfection or transformation (e.g., stable transformation as an extrachromosomal element, or integration into the host chromosome), the presence or absence of readily assayable or selectable markers (e.g., antibiotic resistance and metabolic markers of one type and another), and the number of copies of the gene to be present in the host cell.

[0074] Vectors suitable to carry the nucleic acids of the present invention comprise RNA viruses, DNA viruses, lytic bacteriophages, lysogenic bacteriophages, stable bacteriophages, plasmids, viroids, and the like. The most preferred vectors are plasmids.

[0075] When preparing an expression vector the skilled artisan understands that there are many variables to be considered, for example, whether to use a constitutive or inducible promoter. Inducible promoters are preferred because they enable high level, regulatable expression of the operably-linked genes of the present invention. The skilled artisan will recognize a number of inducible promoters which respond to a variety of inducers, for example, carbon source, metal ions, heat, and others. The practitioner also understands that the amount of nucleic acid or protein to be produced dictates, in part, the selection of the expression system. The addition of certain nucleotide sequences is useful for directing the localization of a recombinant protein. For example, a sequence encoding a signal peptide preceding the coding region of a gene, is useful for directing extra-cellular export of the resulting polypeptide.

[0076] Host cells harboring the nucleic acids disclosed herein are also provided by the present invention. A preferred host is E. coli which has been transfected or transformed with a vector which comprises a nucleic acid of the present invention. Another preferred host is any member of the Actinomycetes.

[0077] The present invention also provides a method for constructing a recombinant host cell capable of expressing SEQ ID NO:2, SEQ ID NO.3, and/or SEQ ID NO.5, said method comprising transforming or otherwise introducing into a host cell a recombinant DNA vector that comprises an isolated DNA sequence which encodes said SEQs or fragments thereof. Preferred vectors for expression are those which comprise SEQ ID NO:1. An especially preferred expression vector for use in E. coli is pRBD26, which comprises SEQ ID NO:1. (See FIG. 1). Transformed host cells may be cultured under conditions well known to skilled artisans such that the vector-encoded SAM operon enzymes are expressed in the recombinant host cell.

[0078] Regulating Production of Tylosin and Other Methylated Compounds

[0079] The SAM operon maps within the tylosin biosynthetic gene cluster, a region of the S. fradiae chromosome that encodes the structural and regulatory genes needed for tylosin biosynthesis. The SAM operon enzymes comprise the so-called “activated methyl cycle.” This series of enzymatic reactions produces the methyl group donor molecule, SAM. The enzymatic activities encoded by the SAM operon as well as the chromosomal location of the SAM operon suggests that the SAM operon may participate in the regulation of tylosin synthesis within the cell. Thus, controlling the expression of the SAM operon genes may lead to controlled expression and production of certain methylated compounds within the cell, such as tylosin or other methylated antibiotic. Altering the levels of production of methylated compounds might be possible by altering the copy number and/or expression of the SAM operon genes.

[0080] The following examples more fully describe the present invention. Those skilled in the art will recognize that the particular reagents, equipment, and procedures described below are merely illustrative and are not intended to limit the present invention in any manner.

EXAMPLE 1

Construction of a DNA Vector Carrying the Streptomyces fradiae SAM Operon Genes

[0081] Plasmid pRBD26 (See FIG. 1) is an approximately 10.3 kilobase pair vector which can be propagated in E. coli and used for targeted integration of the SAM operon into the S. fradiae chromosome. This plasmid contains an origin of replication (OriT), an ampicillin resistance gene (Amp), the intØC31 site, useful for targeting integration into the bacteriophage ØC31 attachment site on the S. fradiae chromosome.

[0082] Plasmid pRBD26 carries a 4.8 kilobase pair fragment of S. fradiae genomic DNA (SEQ ID NO.1) that encodes the SAM operon. The S. fradiae genomic DNA was ligated into the blunt-ended EcoRV site of pSET152 (Bierman et al. Gene, 116, 43-49, 1992) using standard cloning methods. The resulting plasmid was designated pRBD26.

EXAMPLE 2

[0083] Construction of a Vector for Expressing the Streptomyces fradiae SAM Synthetase Gene in a Heterologous Host

[0084] The DNA sequence coding for S. fradiae SAM synthetase is isolated from S. fradiae genomic DNA or from pRBD26, most conveniently by PCR using oligonucleotide primers complementary to the 5′ and 3′ terminal regions of the gene (viz. nucleotide residues 986 through 2209 of SEQ ID NO.1). For ease of cloning the SAM synthetase gene, the oligonucleotide primers are synthesized to contain one or more restriction enzyme cloning sites. Also for convenience in purifying the encoded SAM synthetase protein, the SAM synthetase gene is modified at the 5′ end (viz. amino terminus of encoded protein) by adding an oligonucleotide encoding 8 histidine residues and a factor Xa cleavage site after the ATG start codon at nucleotide positions 988 of SEQ ID NO: 1. Placement of the histidine residues at the amino terminus of the encoded SAM synthetase protein enables the IMAC one-step protein purification procedure (See below).

[0085] The PCR amplified SAM synthetase gene is then inserted into an appropriate expression vector in which the SAM synthetase gene is operably-linked with a high expression promoter, for example the T7 promoter or the lambda pL promoter (See e.g. U.S. Pat. No. 4,874,703, incorporated by reference). Any suitable plasmid may be used for this purpose. A particularly useful plasmid for this purpose is pET11A, which is available commercially from Novogen (Madison, Wis.).

EXAMPLE 3

[0086] Expression of SAM Synthetase Gene in Echerichia coli

[0087] A plasmid capable of expressing SAM synthetase (see e.g. Example 2) is transformed into E. Coli BL21 (DE3) (hsdS gal lcIts857 ind1Sam7nin5lacUV5-T7gene 1) using standard methods (See e.g. Sambrook et al. Supra).

EXAMPLE 4

[0088] Purification of SAM Synthetase

[0089] Transformants selected as in Example 3 are chosen at random and tested for the presence of the transforming vector by agarose gel electrophoresis using quick plasmid preparations. Id. Colonies that contain the vector are grown, processed, and the SAM synthetase, produced by the vector-bound SAM synthetase gene, purified by immobilized metal ion affinity chromatography (IMAC), essentially as described in U.S. Pat. No. 4,569,794, the entire contents of which is hereby incorporated by reference.

[0090] Briefly, the IMAC column is prepared as follows. A metal-free chelating resin (e.g. SEPHAROSE 6B IDA, Pharmacia) is washed in distilled water to remove preservative substances and infused with a suitable metal ion [e.g. Ni(II), Co(II), or Cu(II)] by adding a 50 mM metal chloride or metal sulfate aqueous solution until about 75% of the interstitial spaces of the resin are saturated with colored metal ion. The column is then ready to receive a crude cellular extract prepared from a recombinant host transformed or transfected with a vector encoding the SAM synthetase of this invention.

[0091] After washing the column with a suitable buffer, pH 7.5 to remove unbound proteins and other materials, the bound protein is eluted in a buffer at pH 4.3, essentially as described in U.S. Pat. No.4,569,794.

EXAMPLE 5

[0092] Production of S-adenosylmethionine in a Recombinant E.coli Host Cell that Overexpresses the S. fradiae SAM Synthetase Gene

[0093] A recombinant vector carrying the S. fradiae SAM synthetase gene in operable linkage with a T7 promoter (see e.g. Example 2) is transformed or otherwise introduced into a suitable strain of E. coli. The recombinant cells are grown under conditions that induce gene expression from the vector-borne T7 promoter.

[0094] A crude extract is prepared from the induced culture by any suitable method and the extract contacted with an activated polysaccharide material, for example, as described in U.S. Pat. No. 4,028,183 the entire contents of which is incorporated by reference. The polysaccharide material is activated by a reagent suitable for bonding proteins, for example cyanogen bromide. This reaction is conveniently carried out in a column. A solution of ATP and methionine in a suitable buffer is passed through the column and the eluate, which is enriched in SAM, is collected. The SAM is precipitated with picrolonic acid (See e.g. Anal. Biochem. 4, 16-28, 1971).

EXAMPLE 6

Expression of the S. Fradiae SAM Operon in a Recombinant Actinomycete

[0095] Plasmid pRBD26 is transformed into E. coli BL21 as in Example 3. Transformants harboring the plasmid are used for conjugal transfer of pRBD26 to Streptomyces spp. essentially as described in Bierman et al. “Plasmid cloning vectors for the conjugal transfer of DNA from Escherichia coli to Streptomyces spp. ” Gene, 116, 43-49 (1992). Briefly, one ml of a frozen mycelial culture of S. fradiae was diluted into 9 ml of TS broth and grown for 18 h aerobically at 29_ C. The culture was homogenized and 2 ml was transferred into 18 ml of fresh TS broth and grown for 16 h at 29_ C. to obtain a late log-phase culture. This culture was homogenized and fragmented with ultrasound, and 1 ml was transferred to 9 ml TS broth. The culture was incubated aerobically at 37_ C. for 3 h. The mycelium was recovered by centrifugation, washed once in TS broth and resuspended in 2 ml TS broth (recipient culture). The E. coli donor was grown at 37_ C. overnight in TY broth plus 100 ug apramycin (Am)/ml, subcultured 1:100 and grown for 3 h at 37_ C. The cells were pelleted, washed once in TS broth and resuspended in 2 ml TS broth (donor culture). Equal volumes of the donor culture and ten-fold dilutions of the recipient culture were mixed, and 100 ul were plated to AS1 (Streptomyces medium) supplemented with 10 mM MgCl2. Plates were incubated at 37_ C. for 16 h, and then covered with 3 ml to 4 ml of soft R2 agar containing 1.5 mg nalidixic acid and 1.5 mg of Am. Incubation at 37_ C. was continued for about a week to allow outgrowth of the exconjugants.

Claims

1. A substantially pure SAM symthetase from Streptomyces fradiae having the amino acid sequence;

1 Met Ser Arg Arg Leu Phe Thr Ser Glu Ser Val Thr Glu Gly His Pro   1               5                  10                 15 Asp Lys Ile Ala Asp Arg Ile Ser Asp Thr Val Leu Asp Ala Leu Leu              20                  25                  30 Ala Arg Asp Pro Arg Ala Arg Val Ala Val Glu Thr Leu Ile Thr Thr          35                  40                  45 Gly Gln Val His Ile Ala Gly Glu Val Thr Thr Thr Ala Tyr Ala Pro      50                  55                  60 Ile Ala Gln Leu Val Arg Asp Thr Val Leu Ser Ile Gly Tyr Asp Ser  65                  70                  75                  80 Ser Ala Lys Gly Phe Asp Gly Ala Ser Cys Gly Val Ser Val Ser Ile                  85                  90                  95 Gly Ala Gln Ser Pro Asp Ile Ala Arg Gly Val Asp Thr Ala Tyr Glu             100                 105                 110 Arg Arg Gly Gly Gly Thr Ala Pro Gly Gly Pro Gly Asp Glu Leu Asp         115                 120                 125 Arg Gln Gly Ala Gly Asp Gln Gly Leu Met Phe Gly Tyr Ala Cys Asp     130                 135                 140 Glu Thr Pro Glu Leu Met Pro Leu Pro Ile Asn Leu Ala His Arg Leu 145                 150                 155                 160 Ser Arg Arg Leu Ser Glu Val Arg Lys Asn Gly Thr Ile Pro Tyr Leu                 165                 170                 175 Arg Pro Asp Gly Lys Thr Gln Val Thr Ile Glu Tyr Asp Gly Asp Lys             180                 185                 190 Ala Val Arg Leu Asp Thr Val Val Val Ser Ser Gln His Ala Ser Gly         195                 200                 205 Ile Asp Leu Asp Ser Leu Leu Ala Pro Asp Ile Arg Arg His Val Val     210                 215                 220 Glu Pro Val Leu Ala Gly Leu Ala Glu Asp Gly Ile Lys Leu Asp Thr 225                 230                 235                 240 Ala Gly Tyr Arg Leu Leu Val Asn Pro Thr Gly Arg Phe Glu Ile Gly                 245                 250                 255 Gly Pro Met Gly Asp Ala Gly Leu Thr Gly Arg Lys Ile Ile Ile Asp             260                 265                 270 Thr Tyr Gly Gly Met Ala Arg His Gly Gly Gly Ala Phe Ser Gly Lys         275                 280                 285 Asp Pro Ser Lys Val Asp Arg Ser Ala Ala Tyr Ala Met Arg Trp Val     290                 295                 300 Ala Lys Asn Val Val Ala Ala Gly Leu Ala Ser Arg Cys Glu Val Gln 305                 310                 315                 320 Val Ala Tyr Ala Ile Gly Lys Ala Glu Pro Val Gly Leu Phe Val Glu                 325                 330                 335 Thr Phe Gly Thr Ala Thr Val Asp Val Glu Arg Ile Glu Gln Ala Ile             340                 345                 350 Gly Glu Val Phe Asp Leu Arg Pro Ala Ala Ile Ile Arg Asp Leu Asp         355                 360                 365 Leu Leu Arg Pro Ile Tyr Ala Lys Thr Ala Ala Tyr Gly His Phe Gly     370                 375                 380 Arg Glu Leu Pro Glu Phe Thr Trp Glu Arg Thr Asp Arg Thr Glu Gln 385                 390                 395                 400 Leu Ile Ala Ala Ala Gly Leu  *                 405

which is SEQ ID NO 2.

2. A substantially pure methyltransferase from Streptomyces fradiae having the amino acid sequence:

2 Met Arg Ile Ala Val Thr Gly Ser Ile Ala Thr Asp His Leu Met Ala   1               5                  10                  15 Phe Pro Gly Arg Phe Gly Asp Gln Leu Ile Pro Asp Gln Leu Ala Arg              20                  25                  30 Val Ser Leu Ser Phe Leu Val Asp Gly Leu Glu Val Arg Arg Gly Gly          35                  40                  45 Val Ala Val Gly Ile Ala Phe Gly Leu Gly Arg Pro Gly Pro Thr Pro      50                  55                  60 Leu Leu Val Gly Ala Val Gly Asn Asp Phe Ala Asp Tyr Gly Thr Trp  65                  70                  75                  80 Pro Lys Glu His Gly Val Asp Thr Gly Gly Val Leu Val Pro Thr Glu                  85                  90                  95 His Gln Thr Ala Arg Phe Leu Cys Ile Thr Asp Arg Asp Ala Asn Gln             100                 105                 110 Ile Ala Ala Ser Tyr Thr Gly Ala Met Arg Glu Ala Arg Asp Ile Gly         115                 120                 125 Leu Arg Arg Thr Gly Ala Leu Pro Ala Pro Arg His Gly Leu Val Leu     130                 135                 140 Ile Cys Pro Asp Asp Pro Ala Ala Met Val Arg His Thr Ala Gln Cys 145                 150                 155                 160 Arg Glu Pro Gly Leu Pro Phe Val Ala Asp Pro Ser Gln Gln Leu Ala                 165                 170                 175 Arg Leu Glu Thr Asp Glu Val Arg Ala Leu Val His Gly Ala His Trp             180                 185                 190 Val Phe Thr Asn Glu Tyr Glu Ala Ala Leu Leu Leu Glu His Ser Gly         195                 200                 205 Trp Lys His Ser Glu Thr Leu Glu Arg Val Gly Ala Trp Val Thr Thr     210                 215                 220 Leu Gly Gly Ala Gly Val Arg Ile Glu Arg Ala Gly Glu Pro Pro Leu 225                 230                 235                 240 Thr Val Pro Ala Val Pro Asp Val Pro Val Val Asp Pro Thr Gly Ile                 245                 250                 255 Gly Ala Ala Phe Arg Ala Gly Phe Leu Ala Gly Ala Gly Arg Gly Leu             260                 265                 270 Ser Ile Val Ser Ala Ala Arg Leu Gly Cys Val Leu Ala Ala Arg Ala         275                 280                 285 Leu Gly Thr Val Gly Pro Ala Asp Leu Pro Asp Arg Ser Gly Gly Ser     290                 295                 300 Ala Arg His Gly Glu Gly Arg Val Arg Arg Gly Arg Gly Gly Ala Ala 305                 310                 315                 320 Arg Pro Arg Ala Gly Arg Pro His Met Thr Arg Pro Cys Pro Gly Ser                 325                 330                 335 Arg Arg Glu Pro Pro Ala Gly Arg Pro Ala Arg Ala Ala Ala Val Ile             340                 345                 350 Arg Arg Pro Gly Ala Gly Gly Pro Thr Ala Gly Gly Cys Arg  *         355                 360                 365

which is SEQ ID NO 3.

3. A substantially pure MTHR from Streptomyces fradiae having the amino acid sequence:

3 Met Arg Thr Thr Leu Arg Glu Ile Leu Gly Ser Gly Arg Leu Ser Phe   1               5                  10                  15 Ser His Glu Phe Phe Pro Pro Arg Thr Glu Ala Gly Thr Arg Thr Leu              20                  25                  30 Trp Asn Ala Ile Arg Arg Ile Glu Pro Leu Ala Pro Thr Phe Val Ser          35                  40                  45 Val Thr Tyr Gly Ala Gly Gly Ser Ser Arg Asp Arg Thr Val Glu Val      50                  55                  60 Thr Lys Arg Ile Ala Thr Asp Thr Thr Leu Arg Pro Val Ala His Leu  65                  70                  75                  80 Thr Ala Val Gly His Ser Val Ala Glu Leu Arg Arg Ile Ile Gly Gln                  85                  90                  95 Tyr Ala Asp Ala Gly Val Arg Asp Val Leu Ala Leu Arg Gly Asp Pro             100                 105                 110 Pro Gly Asp Pro Asn Ala Pro Trp Val Pro His Pro Glu Gly Leu Thr         115                 120                 125 His Ala His Glu Leu Val Ser Leu Val Arg Gly Ser Gly Gly Phe Gly     130                 135                 140 Val Gly Val Ala Ala Phe Pro Glu Arg His Pro Arg Ser Pro Asp Trp 145                 150                 155                 160 Asp Ser Glu Ile Arg His Phe Val Arg Lys Cys Arg Ala Gly Ala Asp                 165                 170                 175 Tyr Ala Ile Thr Gln Met Phe Phe Arg Val Glu Asp Tyr Leu Arg Leu             180                 185                 190 Arg Asp Arg Val Ala Ala Ala Gly Cys Cys Thr Pro Val Ile Pro Gly         195                 200                 205 Ile Met Pro Ala Thr Asp Val Arg Gln Ile Ala Arg Phe Ala Glu Leu     210                 215                 220 Ser His Ala Thr Phe Pro Glu Gly Leu Ala Arg Arg Leu Glu Ala Ala 225                 230                 235                 240 Arg Gly Asn Pro Ala Glu Gly His Arg Ile Gly Val Glu Tyr Ala Thr                 245                 250                 255 Ala Met Ala Gly Arg Leu Leu Ala Glu Gly Ala Pro Gly Leu His Tyr             260                 265                 270 Ile Thr Leu Asn Arg Ser Thr Ala Thr Leu Glu Ile His Arg Asn Ile         275                 280                 285 Leu Gly Thr Pro Ala Pro Gly Ser Ala Arg Gln Val Leu Ala Ala Pro     290                 295                 300 Leu  * 305

which is SEQ ID NO 5.

4. An isolated nucleic acid compound encoding the protein of claim 1.

5. An isolated nucleic acid compound comprising a sequence encoding the protein of claim 1 or fragment thereof wherein said compound has a sequence selected from the group consisting of:

(a) residues 986 through 2209 of SEQ ID NO:1;

(b) residues 986 through 2209 of SEQ ID NO:6

(c) a nucleic acid compound complementary to (a) or (b); and

(d) a fragment of (a), (b), or (c) that is at least 18 base pairs in length and which will selectively hybridize to genomic DNA encoding SEQ ID NO. 2.

6. An isolated nucleic acid compound of claim 5 wherein the sequence is (a) or a sequence complementary to (a).

7. An isolated nucleic acid compound of claim 5 wherein the sequence of said compound is (b) or a sequence complementary to (b).

8. An isolated nucleic acid compound encoding the protein of claim 2.

9. An isolated nucleic acid compound comprising a sequence encoding the protein of claim 2 or fragment thereof wherein said compound has a sequence selected from the group consisting of:

(a) residues 2241 through 3341 of SEQ ID NO:1;

(b) residues 2241 through 3341 of SEQ ID NO:6

(c) a nucleic acid compound complementary to (a) or (b); and

(d) a fragment of (a), (b), or (c) that is at least 18 base pairs in length and which will selectively hybridize to genomic DNA encoding SEQ ID NO. 3.

10. An isolated nucleic acid compound of claim 9 wherein the sequence is (a) or a sequence complementary to (a).

11. An isolated nucleic acid compound of claim 9 wherein the sequence of said compound is (b) or a sequence complementary to (b).

12. An isolated nucleic acid compound encoding the protein of claim 3.

13. An isolated nucleic acid compound comprising a sequence encoding the protein of claim 3 or fragment thereof wherein said compound has a sequence selected from the group consisting of:

(a) residues 3338 through 4255 of SEQ ID NO:4;

(b) residues 3338 through 4255 of SEQ ID NO:6

(c) a nucleic acid compound complementary to (a) or (b); and

(d) a fragment of (a), (b), or (c) that is at least 18 base pairs in length and which will selectively hybridize to genomic DNA encoding SEQ ID NO. 5.

14. An isolated nucleic acid compound of claim 13 wherein the sequence is (a) or a sequence complementary to (a).

15. An isolated nucleic acid compound of claim 13 wherein the sequence of said compound is (b) or a sequence complementary to (b).

16. An isolated nucleic acid compound encoding the SAM operon from Streptomyces fradiae, wherein said compound is SEQ ID NO.1 or SEQ ID NO.6.

17. A vector comprising an isolated nucleic acid compound of claim 16 wherein said compound is SEQ ID NO.1.

18. A vector comprising an isolated nucleic acid compound of claim 5 in operable linkage to a promoter sequence.

19. A vector comprising an isolated nucleic acid compound of claim 9 in operable linkage to a promoter sequence.

20. A vector comprising an isolated nucleic acid compound of claim 13 in operable linkage to a promoter sequence.

21. A host cell containing a vector of claim 17.

22. A host cell containing a vector of claim 18.

23. A host cell containing a vector of claim 19.

24. A host cell containing a vector of claim 20.

25. A method for constructing a recombinant host cell having the potential to express SEQ ID NO:2, SEQ ID NO.3, and SEQ ID NO.5 said method comprising introducing into a host cell by any suitable means a vector of claim 16.

26. A method as in claim 25 wherein said host cell is an Actinomycete.

27. A method for expressing SEQ ID NO:2, SEQ ID NO.3 and SEQ ID NO.5 in the recombinant host cell of claim 26, said method comprising culturing said recombinant host cell under conditions suitable for gene expression.

28. A method for synthesizing S-adenosylmethionine, comprising the steps of:

a) admixing a suitable amount of the following reagents:

i) a substantially pure SAM synthetase as claimed in claim 1,

ii) ATP;

iii) methionine; and

b) purifying said S-adenosylmethionine by any suitable means.

29. A method for producing S-adenosylmethionine comprising the steps of:

a) preparing a crude extract from the recombinant cells of claim 22;

b) adding to said extract suitable amount of ATP and methionine; and

c) recovering said S-adenosylmethionine by any suitable means.

30. A method for producing S-adenosylmethionine comprising the steps of:

a) culturing the host cell of claim 21 under conditions suitable for synthesis of S-adenosylmethionine, and

b) recovering S-adenosylmethionine from said host cell by any suitable means.