Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/10—Transferases (2.)
  • C12N9/1003—Transferases (2.) transferring one-carbon groups (2.1)
  • C12N9/1018—Carboxy- and carbamoyl transferases (2.1.3)
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P35/00—Preparation of compounds having a 5-thia-1-azabicyclo [4.2.0] octane ring system, e.g. cephalosporin
  • C12P35/06—Cephalosporin C; Derivatives thereof

Definitions

• Cephamycin C is a cephalosporin produced by Nocardia lactamdurans (Stapley et aL. 1972), Streptomyces clavuligerus (Brown et ah, 1979) and several other actinomycetes (see review by Martin and Liras, 1989).
• Cephamycin C is synthesized from the precursor amino acids L- ⁇ -aminoadipic acid, L-cysteine and L-valine by the multienzyme -aminoadipyl-cysteinyl-valine synthetase (Martin et ah, 1992; Aharonowitz et aL, 1993) which is encoded by the pcbAB gene (Coque et aL, 1991a).
• -Aminoadipic acid is formed from L-lysine by the lysine-6- aminotransferase, encoded by the ]at gene (Coque et al., 1991b; Madduri et al., 1991).
• the tripeptide is later cyclized to form isopenicillin N and this intermediate is epimerized to form penicillin N which is later converted to deacetoxycephalosporin C (DAOC) by the deacetoxy- cephalosporin C synthase (expandase).
• DAOC deacetoxycephalosporin C
• expandase deacetoxy- cephalosporin C synthase
• Cephalosporin C is the end product of the biosynthetic pathway in Cephalosporium acremonium. However in cephamycin - producing actinomycetes further reactions are involved in the synthesis of the C-7-methoxyl group and in the attachment of the carbamoyl group at C-3' (Fig. 1). Little information is available however about the so-called "late”genes, which convert deacetoxycephalosporin C into cephamycin C. The deacetoxycephalosporin C is known to be hydroxylated in S.
• deacetylcephalosporin C (DAC) by an oc- ketoglutarate-requiring dioxygenase (Turner et al., 1979; Baker et aL, 1991 ), but the enzyme has not been described in N. lactamdurans.
• the deacetylcephalosporin C is enzymatically converted into O- carbamoyldeacetylcephalosporin C by an O-carbamoyltransferase that transfers a carbamoyl group from carbamoylphosphate (Brewer et aL, 1980).
• the methoxyl group at C-7 in the cephamycins derives from molecular oxygen and methionine (Whitney et al., 1972) by the action of a monooxygenase and a methyltransferase (O'Sullivan et aL, 1979).
• oxygenases there are at least two types of oxygenases involved in hydroxylations of microbial metabolites.
• the first class are oc-keto- glutarate-dependent and require Fe ⁇ + ions to introduce one of the oxygen atom as from 02 into the substrate (Abbot and Lindstedt, 1974).
• a second class of flavin monooxygenases require pyridine nucleotides as electron donors and 02-
• One of the best known flavin monooxygenases is the p-hydroxyphenylacetate-3-hydroxylase of Pseudomonas putida. which is a two protein component enzyme (Arunachalan et aL, 1992).
• the carbamoyl group is present in some intermediates of primary metabolism such as citrulline and carbamoylaspartate. These molecules are formed from ornithine or aspartate and carbamoyl phosphate by the ornithine carbamoyl transferase or the aspartate carbamoyl transferase, during the biosynthesis of arginine or pyrimidines, respectively.
• the carbamoyl group is present in a variety of antibiotics and other metabolities. It is found in venturicidin A produced by Streptomyces aureofaciens Duggar and Streptomyces hvgroscopicus A- 130 (Brufani et aL 1971; 1968), in the 3'-0- carbamoyl- 2-deoxy- ⁇ -D-rhamose moiety of the antifungal antibiotic irumamycin, produced by Streptomyces subflavus (Nakagawa et aL, 1985) and the related macrolide antibiotic X- 149523 from Streptomyces sp_.
• DAC biosynthetic intermediate deacetylcephalosporin C
• the genes encoding the C-7 hydroxylase and the C-7 O-methyltransferase (cmcl, crncJ) have never been described from N. lactamdurans.
• the carbamoylation at the C-3'-hydroxymethyl side chain of DAC occurs after methoxylation, or perhaps in parallel forming a metabolic grid (Fig. 1).
• a preliminary description of an ATP-dependent carbamoyl transferase that uses carbamoylphosphate as the carbamoyl donor has been described by Brewer et aL, (1980).
• the sequence of one of the latter proteins resembles both cholesterol hydroxylases and methyltransferases of different origins acting on hydroxyl groups present in aromatic or quinone-type compounds; both proteins are required for the hydroxylation at C-7 and the transfer of the methyl group from S- adenosylmethionine to the 7-hydroxycephem intermediate.
• the isolation, nucleotide sequence, and the characterization of a gene encoding a 3'-hydroxymethylcephem 0-carbamoyltransferase of Nocardia is disclosed.
• crncH The gene is named crncH according to the standard nomenclature of genes involved in the biosynthesis of ⁇ -lactam antibiotics (cef for genes common to cephalosporin and cephamycin producers; cmc for genes specific for cephamycin biosynthesis) (Martin et aL, 1991 ; Aharonowitz et al., 1992).
• FIG. 1 A diagram of the late steps of the cephamycin C biosynthetic pathway is shown; carbamoylation may also proceed prior to the introduction of the methoxyl group at C7.
• FIG. 2 Restriction map of the 5.4 kb BamHI DNA fragment of N. lactamdurans containing ORF7, ORF8, ORF9 and ORF10. Black bars (below) indicate the DNA fragments subcloned to give the plJ702-derived plasmids.
• Figure 3 Sequence of 2672 bp internal to the 5.4 kb BamHI DNA fragment.
• the first 69 nt correspond to the 3' end of the pcbC gene.
• the deduced amino acid sequences encoded by ORF7, ORF8 and ORF9 are indicated on the right.
• the translation initiation and termination codons, and the putative ribo some-binding sites are underlined.
• a sequence (nt 2734-2765) downstream of ORF9 forming a stem and loop structure in the RNA that may correspond to a transcription terminator is indicated with arrows.
• Figure 4 Nucleotide and deduced amino acid sequence of the crncH gene (ORF10) of N. lactamdurans. Note the short intergenic region with the inverted repeat and the GGAGGA (putative ribosome binding) sequence preceding the first in frame ATG.
• FIG. 5 Fragment of the S. clavuligerus cephamycin cluster carrying the cefF and the cmcH genes.
• the plasmids pULFJP62 and pULFJ30 are indicated by solid bars.
• FIG. 6 Panels A and B - HPLC analysis of the reaction products of a 3' cephem hydroxylase (DAOC hydroxylase) assay using desalted ammonium sulfate fractions (30-70%) of extracts of S. lividans plJ702-58a as indicated in Materials and Methods.
• Panel A Time zero.
• Panel B After two hours of reaction.
• Figure 7 Panels A, B, C and D - HPLC of the reaction products of 7 cephem hydroxylase (Panels A, B) and 7-hydroxy cephem- methyltransferase assays (Panels C, D) using desalted ammonium sulfate fractions of extracts of S. lividans pUL702-55a.
• A, C Time zero.
• B, D After two hours of reaction.
• FIG 8 Panels A, B and C - NADH oxidation by extracts of Panel A S. lividans pUL702-56a, Panel B S. lividans pUL702-57a and Panel C S. lividans plJ702 in the presence (n) and absence (s) of cephalosporin C (50 ⁇ g/ml). At zero time 50 ⁇ g NADH were added to the reaction.
• the present invention is drawn to the isolation, purification and characterization of DNA molecules which encode enzymes involved in the late steps of biosynthesis of cephamycins.
• the present invention is also drawn to the use of these DNA molecules for expression in recombinant host cells. Recombinant expression of the DNA molecules of the present invention is useful for the production of cephamycin antibiotics. Recombinant expression will also facilitate the production, purification and characterization of the recombinant proteins, and use of the recombinant proteins for antibiotic production.
• the present invention relates to DNA encoding novel enzymes for cephamycin biosynthesis termed cmcH, cmcl and cmcJ.
• the present invention is also related to recombinant host cells which express the cloned enzyme-encoding DNA contained in a recombinant expression plasmid.
• the DNA of the present invention is isolated from cephamycin producing cells.
• the cephamycin-producing cells suitable for the isolation of DNA encoding these enzymes include but are not limited to Nocardia lactamdurans, Streptomyces clavuligerus. Streptomyces lipmanii. Streptomyces panayensis, Streptomyces cattleya. Streptomyces griseus. Streptomyces wadavamensis. Streptomyces todorominensis. Streptomyces filipinensis cephamycini and Streptomyces heteromorphus.
• the most preferred cephamycin producing cells are of the genus Nocardia.
• cells and cell lines may also be suitable for use to isolate DNA encoding the enzymes of the present invention. Selection of suitable cells may be done by screening for enzymatic activity in the cells. Methods for detecting the enzymatic activity are well known in the art and are described below. Cells which possess the enzymatic activity in these assays may be suitable for the isolation of DNA encoding the enzymes.
• Any of a variety of procedures may be used to clone DNA. These methods include, but are not limited to, direct functional expression of the DNA following the construction of an enzyme-containing DNA library in an appropriate expression vector system.
• Another method is to screen an enzyme activity- containing DNA library constructed in a bacteriophage or plasmid shuttle vector with a labelled oligonucleotide probe designed from the amino acid sequence of the specific protein.
• the preferred method consists of screening an enzyme-containing DNA library constructed in a bacteriophage or plasmid shuttle vector with a partial DNA encoding the specific protein. This partial DNA is obtained by the specific PCR amplification of DNA fragments through the design of degenerate oligonucleotide primers from the amino acid sequence known for the particular enzyme or other enzymes which are related to the enzymes of the present invention.
• libraries as well as libraries constructed from other cells or cell types, may be useful for isolating enzyme-encoding DNA.
• Other types of libraries include, but are not limited to, DNA libraries derived from other cells or cell lines other than Nocardia cells, and genomic DNA libraries.
• suitable DNA libraries may be prepared from cells or cell lines which have the particular enzymatic activity.
• the selection of cells or cell lines for use in preparing a DNA library to isolate enzyme- encoding DNA may be done by first measuring cell associated enzymatic activity using the known assays used herein.
• DNA libraries can be performed by standard techniques well known in the art. Well known DNA library construction techniques can be found for example, in Maniatis, T., Fritsch, E.F., Sambrook, J., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1982).
• DNA encoding the enzymes of the present invention may also be isolated from a suitable genomic DNA library.
• genomic DNA libraries can be performed by standard techniques well known in the art. Well known genomic DNA library construction techniques can be found in Maniatis, T., Fritsch, E.F., Sambrook, J. in Molecular Cloning: A Laboratory Manuel (Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1982).
• the amino acid sequence or DNA sequence of the particular enzyme or a related enzyme from another organism is necessary.
• the particular enzyme or a related enzyme may be purified and partial amino acid sequence determined by automated sequenators. It is not necessary to determine the entire amino acid sequence, but the linear sequence of two regions of 6 to 8 amino acids can be determined for the PCR amplification of a partial DNA fragment.
• the DNA sequences capable of encoding them are synthesized. Because the genetic code is degenerate, more than one codon may be used to encode a particular amino acid, and therefore, the amino acid sequence can be encoded by any of a set of similar DNA oligonucleotides. Only one member of the set will be identical to the enzyme sequence but others in the set will be capable of hybridizing to the DNA even in the presence of DNA oligonucleotides with mismatches. The mismatched DNA oligonucleotides may still sufficiently hybridize to the DNA to permit identification and isolation of enzyme-encoding DNA.
• Another method for obtaining DNA encoding the enzymes of the present invention is to utilize DNA sequences encoding a separate and distinct protein, but one which is known or suspected to possibly have at least some degree of homology.
• DNA encoding the separate and distinct protein may have partial homology or share a region of homology with the DNA encoding the enzymes sought.
• a library as described above, can be screened to identify DNA fragments which hybridize with the probe. Hybridizing DNA fragments identified by this means are further characterized to determine whether they encode the sought enzymes.
• DNA clones encoding the enzymes are isolated in a two-stage approach employing polymerase chain reaction (PCR) based technology and DNA library screening.
• PCR polymerase chain reaction
• NH2-terminal and internal amino acid sequence information from the purified enzyme or a homologous protein is used to design degenerate oligonucleotide primers for the amplification of enzyme-specific DNA fragments.
• these fragments are cloned to serve as probes for the isolation of full length DNA from a DNA library derived from Nocardia or other cephamycin producing cells.
• the cloned DNA obta ed through the methods describ e d above may be recombinantly expressed by molecular cloning into an expression vector containing a suitable promoter and other appropriate transcription regulatory elements, and transferred into prokaryotic or eukaryotic host cells to produce recombinant enzyme.
• Techniques for such manipulations can be found described in Maniatis, T, et aL, supra, and are well known in the art.
• Expression vectors are defined herein as DNA sequences that are required for the transcription of cloned DNA and the translation of their mRNAs in an appropriate host. Such vectors can be used to express eukaryotic DNA in a variety of hosts such as bacteria, bluegreen algae, plant cells, insect cells fungal cells including yeast and filamentous fungi and animal cells.
• RNA-fungal cells e.g., Bacillus subtilis
• 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, a potential for high copy number, and active promoters.
• a promoter is defined as a DNA sequence that directs RNA polymerase to bind to DNA and initiate RNA synthesis.
• a strong promoter is one which causes mRNAs to be - 10
• Expression vectors may include, but are not limited to, cloning vectors, modified cloning vectors, specifically designed plasmids or viruses.
• a variety of expression vectors may be used to express the recombinant cephamycin biosynthesis enzymes of the present invention in fungal cells.
• Commercially available expression vectors which may be suitable for recombinant enzyme expression, include but are not limited to, pIJ702 (ATCC 35287), pVEI (ATCC 14585), and pULJL43 (University of Leon) .
• Host cells may be prokaryotic or eukaryotic, including but not limited to bacteria, mammalian cells, insect cells, fungal cells including yeast and filamentous fungi.
• Cells derived from fungal species include but are not limited to, Cephalosporium acremonium (Acremonium chrysogenum . Aspergillus nidulans. Penicillium chrvsogenum. and Penicillium notarum.
• the expression vector may 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 individually analyzed to determine whether they produce the recombinant protein. Identification of enzyme-expressing cells may be done by several means, including but not limited to immunological reactivity with anti-enzyme antibodies, and the presence of host cell-associated enzymatic activity.
• DNA molecules including but not limited to the following can be constructed: the full-length open reading frame of the DNA and various constructs containing portions of the DNA encoding only specific domains of the protein or rearranged domains of the protein. All constructs can be designed to contain none, all or portions of the 5' and/or 3' untranslated region of th eenzyme- encoding DNA. Enzymatic activity and levels of protein expression can be determined following the introduction, both singly and in combination, of these constructs into appropriate host cells. Following determination of the DNA cassette yielding optimal expression in transient assays, this DNA construct is transferred to a variety of expression vectors (including recombinant viruses), including but not limited to those for insect cells, bacteria and fungal cells including yeast and filamentous fungi.
• expression vectors including recombinant viruses
• Levels of the specific recombinant protein in host cells is quantitated by a variety of techniques including, but not limited to, immunoaffinity and/or enzymatic activity techniques.
• Enzyme-specific affinity beads or enzyme-specific antibodies are used to isolate -"S -methionine labelled or unlabelled recombinant enzyme.
• Labelled recombinant enzyme is analyzed by SDS- PAGE.
• Unlabelled recombinant protein is detected by Western blotting, ELISA or RIA assays employing EP3 specific antibodies.
• Enzymatic activity of the recombinant enzyme is also detected and measured as described below.
• the enzyme may be recovered to provide the enzyme in active form, capable of carrying out its specific activity.
• Several recombinant enzyme purification procedures are available and suitable for use.
• Recombinant enzyme may be purified from cell lysates and extracts, or from conditioned culture medium, by various combinations of, or individual application of salt fractionation, ion exchange chromatography, size exclusion chromatography, hydroxylapatite adsorption chromatography and hydrophobic interaction chromatography.
• recombinant enzyme can be separated from other cellular proteins by use of an immuno-affinity column made with monoclonal or polyclonal antibodies specific for full length enzyme, or polypeptide fragments of the enzyme.
• Monospecific antibodies to the enzyme are purified from mammalian antisera containing antibodies reactive against the specific enzyme or are prepared as monoclonal antibodies reactive with the enzyme using the technique of Kohler and Milstein, Nature 256: 495-497 (1975).
• Monospecific antibody as used herein is defined as a single antibody species or multiple antibody species with homogenous binding characteristics for the enzyme.
• Homogenous binding refers to the ability of the antibody species to bind to a specific antigen or epitope, such as those associated with the enzyme, as described above.
• Recombinant enzyme-specific antibodies are raised by immunizing animals such as mice, rats, guinea pigs, rabbits, goats, horses and the like, with an appropriate concentration of the enzyme either with or without an immune adjuvant.
• Enzyme-specific antibody affinity columns are made 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 recombinant enzyme or fragments of it are slowly passed through the column.
• the column is then washed with phosphate buffered saline until the optical density (A280) f a ⁇ s t0 background, then the protein is eluted with 0.23 M glycine-HCl (pH 2.6).
• the purified protein is then dialyzed against phosphate buffered saline.
• N. lactamdurans LC41 1 an improved cephamycin C producer strain, was used as the source of DNA and RNA.
• Streptomyces lividans 1326 (Hopwood et al., 1985) a strain unable to synthesize ⁇ - lactam antibiotics, was used as a host for transformation and for expression experiments.
• E. coli DH5 ⁇ was used for high frequency transformation and E. coli WK6 with the helper phage M13K07 to obtain single-strand DNA.
• the genes of the cephamycin C biosynthetic cluster were isolated from phages lambda EMBL-C2 and C-8 (Coque et al., 1991a), and subcloned into plasmids pBluescript KS( + ) or plJ2921. To express the N. lactamdurans genes in J>. lividans they were subcloned into plJ702 (Katz et aL, 1983).
• Fragments to be sequenced were subcloned in pBluescript KS(+) in both orientations. Ordered sets of nested DNA fragments were generated by sequential deletions using the Erase-a-Base system (Promega, Madison, Wis). The DNA was sequenced in both orientations by the dideoxynucleotide method (Sanger et al., 1977) using Taq polymerase (Promega) and 7-deaza-dGTP to avoid compressions. Isolation of plasmid DNA, digestion with endonucleases, labelling and Southern hybridizations were carried out according to standard procedures (Sambrook et al., 1989). Transformation of S. lividans was done as described previously (Hopwood et al. 1985; Garc ⁇ a-Dominguez et al., 1991).
• ORFs Open reading frames in DNA were identified using the GENEPLOT Program. Inverted repeated sequences were located with the STEM&LOOP Program. Protein comparisons were made with the AALIGN program using the EMBL Swiss Prot data bank. Dot Plot analysis was made with the DOT-PLOT Program using a window size of 25 amino acids and a percent match of 30%.
• ORF open reading frames
• lactamdurans cephamycin C gene cluster were digested with BamHI and hybridized with a 503 bp Avail DNA fragment internal to the cefE gene from N. lactamdurans.
• a 5.4 kb BamHI DNA fragment (Fig. 2) [known to contain the pcbC gene but not the cefE gene (Coque et al., 1991; 1993)] gave a strong positive hybridation. The entire region of this 5.4 kb DNA fragment downstream from pcbC was sequenced.
• Three ORFs, ORF7, ORF8 and ORF9 were found and a fourth complete ORF (ORF10) was present dowstream of ORF9.
• the four ORFs showed clearly, using the GENEPLOT program (DNASTAR), a high frequency of GC in the third position of the codons as expected in actinomycetes genes.
• ORF9 Characterization of ORF9 as the cefF gene.
• the ORF9 is located 1.6 kb dowstream from pcbC and has a size of 933 nucleotides (Fig. 3) and a G + C content of 68.1 %. It encodes a protein with a deduced Mr of 34,366 and a predicted pl of 4.65.
• the gene is separated from the upstream ORF (ORF8) by 23 nucleotides.
• a putative ribosome binding site (RBS) GAGGAGCA is present in the intergenic region 7 bp upstream from the ATG translation initiation triplet.
• a secondary structure Downstream of the TAG termination codon, a secondary structure (nt 2734-2767) forms a stem and loop structure that may correspond to a terminator with a calculated free energy of -31 Kcal/mol.
• Computer comparison of the amino acid and nucleotide sequence of ORF9 with expandases and hydroxylases involved in cephalosporin or cephamycin biosynthesis showed 80.8% identity in nucleotides and 77.5% in amino acids with the cefF encoded protein of S clavuligerus.
• the protein encoded by ORF9 showed also a high similary to the bifunctional expandase/hydroxylase of A. chrvsogenum (encoded by cefEF) and to the expandases of N. lactamdurans and S. clavuligerus (encoded by cefE).
• ORF7 Downstream from the pcbC gene and separated by only 13 nucleotides, starts an ORF (ORF7) of 71 1 nucleotides encoding a protein (named P7) of 236 amino/acids (27364 daltons), with a deduced pi of 4.89.
• ORF8 a sequence of 876 nucleotides (Fig. 3) encoding a deduced protein (P8) of 292 amino acids (32,090 daltons), with a pi of 5.02.
• the G + C content of both ORF7 and ORF8 was 67.5 and 71.9% respectively.
• ORF7 A comparative DOTPLOT analysis of the predicted protein encoded by ORF7 with other proteins present in databanks indicates that the ORF7 product shows homology to 0-methyltransferases involved in chemotaxis (che genes) from S. typhimurium and E. coli. hydroxyindoil, cathecol and caffeic acid O-methyltransf erases, and lower homology to tylosin methyltransferases from Streptomyces fradiae (tcmP, tcmO) and methyltransferases for the methylation of oligosaccharides involved in nodulation in Azorhizobium (nodS).
• a typical S-adenosylmethionine binding motif is present in the N-te ⁇ ninal region (amino/acids 10 to 26) (Ingrosso et aL, 1989). Since all these proteins catalyze the O- methylation of phenolic or heterocyclic hydroxyl groups, ORF7 seems to encode the C-7 hydroxycephem methyltransferase. However, in addition, the protein exhibits a 30.5% identity in 59 amino/acids with human and rat cholesterol 7- ⁇ -monooxygenase, an enzyme which introduces oxygen at C-7 position in the cholesterol nucleus.
• the ORFS protein showed no significant homology with any protein present in the EMBO and Swiss-Prot data banks and it behaves as a coupling protein.
• ORF 10 an incomplete ORF (ORF 10) was found. This ORF was located downstream of cefF.
• ORF was located downstream of cefF.
• a 3.6 kb BamHI DNA fragment of the cephamycin C gene cluster [known to be adjacent to the 5.4 kb BamHI fragment downstream from ORF9 (cefF) (Coque et al., 1993)] was subcloned and sequenced in both orientations.
• This 3.6 kb BamHI fragment contains the bja gene (Coque et aL, 1993), the 3' region of ORF10, and the 5' end of ORF14.
• a 3.4 kb NotI DNA fragment was isolated from the recombinant phage lambda EMBL-C2 and subcloned in pBluescript KS(+). The fragment was recovered, the ends filled with Klenow polymerase, ligated to a synthetic S-mer Bglll linker and subcloned in Bglll digested pLT2921. From this plasmid the 3.4 kb fragment with Bglll ends was subcloned in the Streptomyces plasmid plJ702 in the same orientation of the mel gene and downstream from the mel promoter to give plasmid pUL702-37a (Fig. 2).
• the translation initiator ATG codon of ORF 10 is located 74 bp downstream from cefF. It was preceeded by a GGAGGA sequence that resembles the Shine Delgarno ribosome binding sequences.
• An inverted repeat of 15 bp that may form, if transcribed, a stem and loop structure with a calculated free energy of -31 kcal/mol, is present in the intergenic region. If this structure is a functional terminator, ORF10 should be expressed from its own promoter; alternatively ORF10 may be expressed from an upstream promoter and the inverted repeat may work as a terminator regulated by an antitermination mechanism.
• ORF10 contains 1563 nt (Fig.
• the protein encoded by ORF10 showed 32.1 % and 30.2% identity (in 287 and 281 amino acids respectively) with the C-terminal end of the nodU genes from both Rhizobium fredii and Bradyrhizobium japonicum.
• the nodU genes encode O-carbamoyl transferases for the biosynthesis of carbamoylated polysaccharides required for nodulation (Lewin et al., 1990).
• the gene corresponding to ORF10 encodes, therefore the cephem-carbamoyltransferase and was named cmcH, according to the proposal of Martin et aL, (1991) and Aharonowitz et aL, (1992) for designation of the ⁇ -lactam biosynthesis genes.
• cmcH encoded protein showed little overall homology with aspartate carbamoyl transferases and ornithine carbamoyl transferase of E. coli. Aspergillus nidulans and a variety of other microorganisms.
• GEM12 the cmcH and the adjacent cefF gene were subcloned into pIJ702 as a 6.2 kb BamHI DNA fragment originating from plasmid pULFJP62.
• lambda GEM 12 phages containing DNA fragments of the cephamycin cluster were hybridized with the cefF or the cmcH genes of N. lactamdurans. positive hybridizations were found in phage EMBL-C5 and in the 6.2 kb BamHI DNA fragment.
• the 6.2 kb DNA fragment of S clavuligerus contains, therefore, the cefF (Kovacevic et al., 1991) and the cmcH genes.
• the hybridizing J>. clavuligerus DNA sequence was mapped more precisely within a 3.0 kb Kpnl DNA fragment containing also the cefF gene (Fig. 5).
• the 3.0 kb Kpnl DNA fragment was subcloned in pBluescript KS(+) giving plasmid pULFJP30 and 160 bp at the Kpnl site of the distal end (downstream from cefF ) were sequenced.
• the 160 nt sequence matched almost perfectly the sequence of the cmcH gene of N. lactamdurans (nt 1479- 1639 in Fig. 3 A) with a 80% identity in nucleotides and 81 % identity in the deduced amino acids.
• Cell free extracts were obtained from S. lividans transformants grown for 48 hours in minimal medium with glucose and lysine (Madduri et aL, 1991). The cells were washed and suspended in 100 mM, MOPS pH 7.5, containing DTT (1 mM), PMSF (1 mM) and DNAse (20 ⁇ g/ml). The cell suspension was sonicated in a Branson B-12 sonifier. Nucleic acids were removed from the cell free extracts by treatment with protamine sulphate (0.1 %) and the protein in the supernatant was precipitated with ammonium sulphate (0-80%). The protein precipitate was dissolved in the same MOPS buffer and passed through a PD-10 column (Pharmacia).
• 3'-Hydroxymethylcephem O-carbamoyltransferase activity was assayed by the following three different methods, i) By using decarbamoylcefur-oxime and as substrates and measuring the ethyl acetate-extractable carbamoylated cefuroxime radioactivity in a Phillips PW4700 scintillation counter, as reported by Brewer et al., (1980).
• cmcH genes of N. lactamdurans and £. clavuligerus encode a functional 3'-hydroxymethylcephem O-carbamoyltransferase activity.
• This activity (also known as DAOC hydroxylase) was measured after precipitation of the crude extracts with ammonium sulphate (30-70%) and desalting the preparation through a Sephadex G- 25 column (Pharmacia PD-2).
• the assay based on the conversion of DAOC to DAC, was carried out as described by Kovacevic et al., (1989) and incubated for 120 min at 30°C. The reaction was stopped with methanol (200 ⁇ l); the precipitated proteins were removed by centrifugation at 14,000 rpm for 10 min and the reaction product was quantified in the supernatant.
• DAOC hydroxylation of DAOC to DAC was followed by HPLC using a Waters ⁇ Bondapak CIS column (300 x 3.8 mm) equilibrated and eluted with NaH2P ⁇ 4 200 mM pH 4.0 at a flow of 1.5 ml/min. The eluted fractions were monitored at 254 nm. Under these conditions DAOC eluted with a retention time of 10.7 minutes and DAC at 3.7 minutes.
• the cefF gene encodes a functional cephem-3-hydroxylase without cephem-7-hydroxylase activity.
• plJ702 [Katz, E. et aL, 1983, J.Gen.MicrobioL, 129, pp.2703-2714] to give plasmid plJ702-54a.
• a 1.4 kb Notl-Mlul DNA fragment (internal to the 5.4 kb fragment) was end-filled and subcloned into the polylinker of plJ2921 , recovered with Bglll and subcloned into the B i ⁇ site of plJ702 to give plJ702-58a (Fig. 2).
• the OFR9 was subcloned downstream from and in the same orientation as the mel promoter. Additionally the 1.4 kb Notl-Mlul fragment was subcloned in the BamHI site of plJ699 [Kieser, D., and Melton, R.D., 1988, Gene, 65, pp.83-91] to give plasmid plJ699-58a.
• the enzymes were measured in the 30-70% ammonium sulphate precipitate of crude extracts after desalting through a Sephadex G-25 column as indicated above.
• the assays are based on the conversion of cephalosporin C to 7 -hydroxy cephalosporin C and 7-methoxycephalo- sporin C (Xiao and Demain, 1991 ).
• the reactions were carried out in a water bath with shaking to favor the oxygen transfer required for the reaction. After incubation for 120 minutes at 30°C the reaction was stopped by addition of acetic acid (10 ⁇ l); the proteins were eliminated by centrifugation at 14,000 rpm for 5 minutes.
• the supernatant was applied to a QAE-Sephadex column (1 ml bed volume) equilibrated with 50 mM Tris-HCl pH 6.0. After washing the column with 600 ⁇ l of the same buffer, the products of the reaction were eluted with 3N NaCl (600 ⁇ l). Chromatography of the products of the reaction was performed in a Waters ⁇ Bondapack 8 column as indicated previously except that the elution was done with a flow of 1.5 ml and a gradient of methanol as follow: Time 0-20 minutes, 0% methanol; 30 minutes, 5% methanol; 40 minutes, 10% methanol.
• ORF7 and ORFS might correspond to genes encoding enzymes for the methoxylation at C-7 in the cephem nucleus. Therefore a 3061 bp Pstl DNA fragment from plJ702-54a (containing ORF7 and ORFS) was subcloned into plJ2921 , rescued with Bglll and subcloned in plJ702, downstream from and in the same orientation as the mel promoter to give expression plasmid plJ702-55a. Cell free extracts of 48 hour cultures of S.
• the C-7 hydroxylase and methyltransferase activities of the transformants during the fermentation was determined.
• Two types of transformants were used: S. lividans [plJ702-55a], which contains the three genes downstream of the mel promoter and constructions in which either the cefF gene or the fragment containing ORF7-ORF8 were subcloned in plJ699 a plasmid with two transcriptional terminators in which the ORF7-ORF8 should be expressed from its own promoters.
• the time course of methyltransferase activity during the fermentation overlaps with that of the 7-hydroxylase activity, suggesting that both activities are expressed coordinately.
• ORF 7 encodes a 7-hydroxylase activity but the two proteins encoded by ORF7 and ORF8 are required for 7-methoxylase activity.
• ORF7 and ORFS were subcloned individually.
• DNA fragments Pstl- Xhol (2056 bp) and Kpnl (1048 bp) containing ORF7 and ORFS respectively (Fig. 2) were cloned in plJ2921, rescued with Bglll and subcloned in the Bglll site of plJ702 downstream from and in the orientation of the mel promoter to give plasmids pUL702-56a and pUL702-57a.
• the 7-hydroxylase shows a cephalosporin-dependent NADH-oxidase activity.
• ORF7 and ORFS behave as a two protein component system.
• Cell-free extracts of S. lividans [plJ702-56a] expressing ORF7 showed a strong cephalosporin-dependent NADH oxidase activity (Fig. 8).
• the protein encoded by ORFS did not show a significant NADH-oxidase activity but was absolutely essential for productive 7-methoxylation.
• hydroxylation at C-7 is mediated by a hydroxylase that uses NADH as an electron donor for reduction of molecular oxygen to introduce the hydroxyl group.
• the protein encoded by ORF8 is strictly required for introduction of the methyl group to form 7-methoxy CPC derivatives.
• MOLECULE TYPE cDNA
• SEQUENCE DESCRIPTION SEQ ID NO: 1 :
• CTTTCCCCAC CACCGGCGGG AAACCGCGGC TGAACGACGC CGGGAAGCTG ATGGCGCTGG 660
• AGCACCGCCC AGGTCACCAA CACCGGCACC TACACCGACT ACTCCATGTC GTACTCGATG 300
• GACGTCAGCC TGGACATGGA GAAGGCGACC TTCGGCGACT GGATCGGGAC CAACTACGTC 900 ACGATGCACG CGGTCACCTC GTAGCCACCG TGCCCGCGAC CCCCGGCCCA CGAGGCCGGG 960
• MOLECULE TYPE cDNA
• SEQUENCE DESCRIPTION SEQ ID NO:3 :
• TCGGCATCGA CCGGGACCTG TCCCGCTGCC AGATCCCCGA GTCCGAGATG AAGAACATCT 480 CGCTGCGCGA GGCCGACTGC AGCCTGGACC GGTGGAAGCT CGTGGACGCG CTGGACGGCG 540

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