CA2188257A1 - Dna encoding cephamycin biosynthesis late enzymes - Google Patents

Abstract

DNA encoding the late enzymes involved in the synthesis of the antibiotic cephamycin have been isolated and purified. The particular enzymes are involved in the late steps of cephamycin biosynthesis. These DNA's have been sequenced and cloned into recombinant expression vectors for their recombinant expression in host cells. The DNA's, vectors containing them and recombinant host cells which express them are useful for the production of antibiotics.

Description

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TITLE OF THE INVENTIQN
DNA ENCODING CEPHAMYC~N BIOSYNTHESIS LATE
ENZYMES

Cephamycin C is a cephalosporin produced by NQcardia lact~mrlllr~ns (Stapley et al.. 1972), Streptomyces clavuligerus (Brown et al., 1979) and several other actinomycetes (see review by Martin and Liras, 1989). Cephamycin C is synthesized from the precursor amino acids L-a-~mino~iric acid, L-cysteine and L-valine by the multienzyme a-aminoadipyl-cysteinyl-valine synthetase (Martfn et al., 1992;
Aharonowitz et ~., 1993) which is encoded by the ~AB gene (Co4ue et L, 1991a). a-~min~ iric acid is formed from L-lysine by the Iysine-6-aminotransferase, encoded by the lat gene (Coque etL., 1991b; Madduri 5 et al., 1991). The tripeptide is later cyclized to form isopenicillin N and this intPrm~rli~t~ is ~pi lleli,ed to form penicillin N which is later converted to deacetoxycephalosporin C (DAOC) by the deacetoxy-cephalosporin C synthase (~p~n~l~s~). The genes encoding these three enzymatic step.s, ~C, cefD and ~E in N. l~t~m~llrans are known to 20 be clustered with lat and DcbAB (Coque et al., 1993a,b).
Cephalosporin C is the end product of the biosynthetic pathway in Ce~h~losporll-m ~rn~mn~illm However in cephamycin-producing actinomycetes further reactions are involved in the synthesis of the C-7-methoxyl group and in the ~tt~ hm~nt of the carbamoyl group at 25 C-3' (Fig. 1). Little il~lllld~iOII is available however about the so-called "late"genes, which convert deacetoxycephalosporin C into cephamycin C. The deacetoxycephalosporin C i~ known to be hydroxylated irl S.
clavuli~erus to form deacetylcephalosporin C (DAC) by an a-ketoglutarate-requiring dioxygenase (Turner ~ j~.L, 1~79; Baker et al., 3 1 99 1 ), but the enzyme has not been described in N. Iac~:~m~--r~ns In parallel, the deacetylcephalosporin C is enzymatically converted into O-carbamoyldeacetylcephalosporin C by an O-carbamo)~l~,dll~rc~,dse that transfers a ~arbamoyl group from carbamoylphosphate (Brewer et al., 19R0). T~le methoxyl group at C-7 in the cephamycins derives from W0 9~129253 , ~ ~
2~882~7 molecular oxygen and methionine (Whitney et al., 1972) by the action of a monooxygenase and a methyltran.sferase (O'Sullivan et ~1-, 1979).
There are at least two types of oxygenases involved in hydroxylations of microbial metabolite.~. The first class are a-keto-5 glutarate-dependent and require Fe2+ ions to introduce one of the oxygen atom as from O2 into the substrate (Abbot and I inrl.stt~lt, 1974). A
second class of flavin monooxygenases require pyridine nucleotides as electron donors and O2- One of the best known flavin monooxygenases is the p-hydroxyphenylacetate-3-hydroxylase of Pseudomonas ~, which is a two protein component enzyme (Arlln~h~l~n et al., 1992).
For many years it has been unclear whether the C-7 hydroxylase was different from the C-3' hydroxylase which converts DAOC into DAC.
Demain and coworkers [Xiao et al., 1991] have purified the 7-hydroxylase activity of S. clavuli~erus and the sequenced amino acid 5 terminal end of the protein is very similar to that of the previously cloned C-3 hydroxylase (Xiao et al., 1991).
The carbamoyl group is present in some int~rrnr~ t~s of primary metabolism such as citrulline and carbamoylaspartate. These molecules are formed from ornithine or aspartate and carbamoyl 20 phosphate by the ornithine carbamoyl ~ .,r~la.se or the aspartate carbamoyl ~ se, du~ing the biosynthesis of arginine or pyrimidines, respectively.
In secondary metabolism, the carbamoyl group is present in a variety of antibiotics and other metabolities. It is found in venturicidin 25 A produced by Stre~tomvces aureofaciens Duggar and Streptomyces h~vgroscopicu~ A-130 (Brufani et al. 1971; 1968~, in the 3'-O- carbamoyl-2-deoxy-~-D-rhamose moiety of the antifungal antibiotic irumamycin, produced by Streptomyces subflavus (Nakagawa et al., 19~5) and the related macrolide antibiotic X-149523 from Streptomvces sp. (Omura et 30 al., 1985), in the antitumoral antibiotics mitomycins and porfiromycins produced by Streptomyces caespitosus. Streptomyces ordus and Streptomyces verticillatu~ (Glasby 1979), and in novobiocin, an inhibitor of the DNA gyrase, produced by Streptomyces niveus (Kominek 1972).
The presence of carbamoyl groups in oligos~ h~ri~1~s of E~hi7nbium ~.

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involved in nodulation is also well docl~m~ntl~-l (Price et al., 1992;
Holsters çt ~1., 1993).
In the ,~-lactam antibiotics of the cephamycin family, a carbamoyl group is attached to the C-3' hydroxymethyl side chain of 5 cephamycin C. A good kno~ledge exists at present on the biochemistry (Jensen 19~6; Martin and Liras 19R9) and genetics (Kovacevic et al., 1990; Kovacevic and Miller 1991; Coque et al., l991a,b;) of cephamycin C biosynthesis (see review by Aharonowitz et al., 1992). Genes encoding enzymes for the early (1~, ~AB, ~C) (Coque et al., 1991; Madduri et al., 1991), and intermediate steps of the pathway (~D cefE, cefF) have been cloned for S~ ollly~s clavuli~erus (Kovacevic et al., 1990;
Kovacevic et al., 1989) and Nocardia l~t~m~ rans (Coque et al., 1993).
The late steps of cephamycin C biosynthesis are poorly understood. The biosynthetic intermediate deacetylcephalosporin C
5 (DAC) is the substrate for a two step methoxylation at C-7. The genes encoding the C-7 hydroxylase and the C-7 O-methyltransferase (ç~l, cmcJ) have never been described from N. I~rt~m~ rans. The carbamoylation at the C-3'-hydroxymethyl side chain of DAC occurs after methoxylation, or perhaps in parallel forming a metabolic grid (Fig.
20 1). A preliminary description of an ATp-flpppn~l~nt carbamoyl ~lall:~r~laSe that uses carbamoylphosphate as the carbamoyl donor has been described by Brewer et ~1-, (1980). However nothing is known about the gene encoding such an enzyme or any other gene encoding enzymes that carry out carbamoylation reactions in the biosynthesis of 25 different antibiotics. Moreover, it is unknown whether the genes for cephem-carbamoyl lldll~r~,la~S and ornithine (or aspartate) carbamoyl transferdses have any similarity.
The identification, isolation and purification of the DNA
encoding the enzymes which catalyze the late step.s of cephamycin 30 biosynthesis would be extremely useful to produce these antibiotics.
These DNA'.s would be useful to establish re~ l,bi,ldlll host cells to produce these antibiotics on an industrial scale.

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Three genes located in the cluster of cephamycin C
biosynthesis in _. Iact~nnr~urans which encode the deacetoxycephalo-sporin C hydroxylase and two other proteinsl which introduce the methoxyl group at C-7 have been isolated and spqllenrcA The se4uence 5 of one of the latter proteins resembles both cholesterol hydroxylases and meth~ ,.sr~las~s 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 intermf rli~te. In addition, the isolation, nucleotide sequence, and the ~llala~ àtiOn of a gene encoding a 3'-hydroxymethylcephem O-carbamuyl~ relase of Nocardia is ~ clnsçrl The gene is named crncH according to the standard nnmPnrl~hire of genes involved in the biu~yll~ is of ~-lactam antibiotics (ç~f for genes comrnon to cephalosporin and cephamycin 5 producers; cmc for genes specific for cephamycin biosynthesis) (Martin et al., 1991; Aharonowitz et al., 1992).
BRlEiF DESCRIPTION OF THE DRAWINGS
Figure I - 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.
Figure 2 - Restriction map of the 5.4 kb'~mHI DNA
fragment of N. Iacr~m(11lrans containing ORF7, ORF8, ORF9 and 2s 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 ~amHI
DNA fragment. The first 69 nt correspond to the 3' end of the ~C gene.
3 The deduced amino acid scqllenr~ encoded by ORF7, ORF8 and ORF9 are indicated on the right. The translation initiation and termin~ltinn codons, and the putative ribosome-binding sites are underlined. A
sequence (nt 2734-~765) downstream of ORF9 forming a ~tem and loop ~W095/29253 2~ ~8257 ~ 111 cr:~ I

structure in the RNA that may correspond to a tran.scription terminator is indicated with arrows.
Figure 4 - Nucleotide and deduced amino acid sequence of 5 the cmcH gene (ORF10) of N. I~ct;~mdurans. Note the short intergenic region with the inverted repeat and the GGAGGA (putative ribosome binding) sequence preceding the first in frame ATG.
Figure S - Fragment of the S. clavuligerus cephamycin cluster carrying the cefF and the cmcH genes. The plasmids pULFJP62 and pULFJ30 are indicated by solid bars.
Figure 6 - Panels A and B - HPLC analysis of the reaction products of a 3' cephem hydroxylase (DAOC hydroxylase) assay using 15 desalted allll,lo~ ll sulfate fractions (30-70%) of extracts of S. Iividans plJ702-S~a 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 20 products of 7 cephem hydroxylase (Panels A, B) and 7-hydroxy cephem-methylll~l~r~,lase assays (Panels C, D) using desalted ammonium sulfate fractions of extracts of S. Iivi~l~n~ pUL702-SSa. A, C) Time ~ero. B, D) After two hours of reaction.
Figure ~ - Panels A, B and C - NADH oxidation by extracts of Panel A S. Iividans pUL702-56a, Panel B S. Iividans pUL702-57a and Panel C S. Iividan.s plJ702 in the presence (n) and absence (s) of cephalosporin C (50 llg/ml). At zero time 50 !lg NADH were added to the reaction.

DETAILED DESCRIPTION OF THE INVENTI(~N
The present invention is drawn to the isolation, purification and characterization of DNA molecules which encode erlzymes involved in the late steps of biosynthesis of cephamycins. The present invention is wo gsn92s3 2 1 8 8 2 ~ 7 E~ 5~

also drawn to the use of these DNA molecules for expression in recombinant host cell.~. Recombinant expression of the DNA molecule~s of the present invention is useful for the production of cephamycin antibiotics. Recombinant expression will also facilitate the production, 5 purification and characterization of the recombinant proteins, and u~e 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 It~COIIIbillalll host cell.s 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. In particular, the cephamycin-producing cells suitable for the isolation of DNA encoding these enzymes include but are not 5 lirnited to Nocardia lactamrl~ ns Streptomvce~ clavuli~erus.
Streptomyces lipmanii, Streptomyces panayensis~ Streptomyce.s ~1~, Streptomyces ~, Streptomyces wadayarnensis, Streptomyces tod~ ?l"i"~"~is Streptomyces filipinensi.s ce~h~nlycini and Streptomyces heteromorphus. The most 20 preferred cephamycin producing cells are of the genus Nocardia.
Other cells arld 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 25 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 mclude, but are not limited to, direct 30 functional expression of the DNA following the construction of an enzyme-containing DNA library in an ~ 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 WO9!i/29253 r~ . C~\4 - the arnino 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 5 obtdined by the specific PCR amplification of DNA fragments through the design of degenerate oligonucleotide primers from the arnino acid sequence known for the particular enzyme or other enzymes which are related to the enzymes of the present invention.
It is readily apparent to those skilled in the art that other types of 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
librdries derived from other cells or cell lines other than Nocardia cells, and genomic DNA libraries.
It is readily apparent to those skilled in the art that 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 20 enzymatic activity using the known assays used herein.
Preparation of DNA libraries can be perforrned by standard ~ s well known in the art. Well known DNA
library construction t~rhniqu~s can be found for example, in Maniatis, T., Fritsch, E.F., Sambrook, J., Molecular Cloning: A
25 Laboratory Manual (Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1982).
It is also readily apparent to those skilled in the art that DNA encoding the enzymes of the present invention may also - be isolated from a suitable genomic DNA library.
Construction of genomic DNA libraries can be performed by standard t~ hniqll~s well known in the art. Well known genomic DNA library construction techniques can be found in Maniatis, T., Fritsch, E.F., Sarnbrook, J. in Molecular Cloning:

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A Laboratory Manuel (Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1982).
In order to clone the enzyme-encoding DNA gene by one of the above methods, the amino acid sequence or DNA
5 sequence of the particular enzyme or a related enzyme from another organism is necessary. To accomplish this, the particular enzyme or a related enzyme may be purified and partial amino acid sequence determined by automated s~ r~ . It is not necessary to determine the entire amino acid sequence, but the linear sequence of two regions of 6 to 8 arnino acids can be determined for the PCR amplification of a partial DNA fragment.
Once suitable amino acid sequences have been j~1PntifiP~, the DNA SPq~lPncPs capable of encoding them are sy~ lPci~e~l Because the genetic code is ~ PnPrA~tP~ more than 5 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 llli~ lP~ The micmAtrhPd DNA
oligonucleotides may still sufficiently hybridize to the DNA to permit if iPntifil Ation and isolation of enzyme-encoding DNA.
Another method for obtaining DNA encodrng the enzymes of the present invention i.s to utilize DNA sP~Pn~PS
25 encoding a separate and distinct protein, but one which is known or suspected to possibly h~ve at least some degree of homology.
DNA encoding the ~eparate and distinct protein may have partial homology or share a region of homology with the DNA encoding the enzymes sought. By using the DNA encoding a protein 3 suspected of sharing some degree of homology as a hybridization probe, 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 fur~er characterized to determine whether they encode the sought enzy-mes.

WO9!il29253 2 1 ~ 8 ~ ~ 7 P.111J,.,5~ ~4~ 1 9 _ Using one of the above methods, DNA clones encoding the enzymes are isolated in a two-stage approach employing polymerase chain reaction (PCR) based technology and DNA library screening. In the first stage, NH2-terminal and internal amino acid sequence information from the purified enzyme or a homologous protein is used to design de~ d~
oligonucleotide primers for the amplification of enzyme-specific DNA fragments. In the second stage, 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 producmg cells.
The cloned DNA obtdined through the methods described above may be recombinantly expressed by molecular cloning into an ~,ul~,s~iol~ vector cont~inin~ a suitable promoter andother~p~luplid~ s~ lionregulatoryelements,and transferred into prokaryotic or eukaryotic host cells to produce recombinant enzyme. Te, l " ,i~ 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 ll~ls~ u~ion of cloned DNA
and the translation of their mRNAs in an a~lulululidl~ 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 fil~lm~nt~ fungi and animal cells.
Specifically designed vectors allow the shuttling of DNA between hosts such as bacteria-fungal cells or bacteria-animal cells. An ~lu,u~ululiately 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 W0 95129253 ~ 237 P~1/L ~

initiated at high frequency. Expression vectors may include, but are not limited to, cloning vectors, modifled cloning vectors, specifically designed plasmids or viruses.
A variety of e~pression vectors may be used to express the recombinant cephamycin bio.~ynthe~i~ enzymes of the present invention in fungal cells. Commercially available expression vectors which may be suitabie for recombinant enzyme expression, include but are not limited to, pi'J702 (ATCC 35287), pVEI (ATCC 14585). and pULJL43 (University of Leon) .
o DNA encoding the enzymes of the present invention may be cloned into an expression vector for expression in a host cell. Host cells may be prokaryotic or eukaryotic, imcluding but not limited to bacteria, m~mm~ n cells, insect cells, fungal cells including yeast and filamentous furlgi. Cells derived from fungal species which may be suitable and which are commercially available, include but are not limited to, Cephalosporium acremnni-lm (Acremnnil~m chrysogenum), Aspergillu~ nidulans, Penicillium chrysogenum. and Penic;lli--m notatum.
The expression vector may be introduced into host cells via any one of a number of tP~hniq~ 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. I~lentific~rion 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.
To determine the DNA sequence(s) that yields optimal levels of enzymatic activity and/or protein, 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 encodirlg oniy specific domains of the protein or rearranged domains of the protein. All constructs can be designed to contaim none, all or .. . .. .. . .. . . . . .. . . .. . . . ... . . . ..

VO 95/29253 ~ 1 ~ 8~5 7 . ~
portions of the 5' and/or 3' untranslated region of th eenzyme-encoding DNA. Enzymatic activity and levels of protein expression can be d~ ed following the introduction, both smgly and in combination, of these constructs into ~~ ial~ host 5 cell.s. Following d~te, ,I,i, ,~tion 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.
Levels of the specific rec~llll,~l~ll protein in host cells is ~ ntit~t~d by a variety of techniques including, but not limited to, immllno~ffinity and/or enzymatic activity techni4ues.
Enzyme-specific affinity beads or enzyme-specific antibodies are 15 used to isolate 3~S-methionine labelled or unlabelled recombinant enzyme. Labelled rec-)mhin~nt enzyme is analyzed by SDS-PAGE. Unlabelled recombinant protein is detected by Western blotting, ELISA or RIA assay.s employing EP3 specific antibodies.
Enzymatic activity of the recombinant enzyme is also detected and 20 measured as described below-Following expression of the enzyme in a host cell, theenzyme may be recovered to provide the erlzyme in active form, capable of carrymg out its specific activity. Several recombinant enzyme purification procedures are available and suitable for use.
25 R~cu.llbi~ lll enzyme may be purified from cell Iysates 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 30 interaction chromatography.
In addition, rec~,llll,illall~ 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 fr~m~nts of the enzyme.

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Monospecific antibodies to the enzyme are purified from m~nnm~ n antisera cnnt~inin~ 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: 495~97 (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 as used herein refers to the ability of the antibody species to bind to a specific antigen or epitope, o such as those associated with the enzyme, as described above.
Re~u,lll,illalll enzyme-specific antibodies are raised by il""""~i~;"~
animals such as mice, rats, guinea pigs, rabbits, goats, horses and the like, with an a,ululu,u.ia~ concentration of the enzyme either with or without an immune adjuvant.
It is readily apparent to those skilled in the art that the above described methods for producing monospecific antibodies may be utilized to produce antibodies specific for polypeptide fragments,orfull-length polypeptide.
Enzyme-specific antibody affinity columns are made by adding the antibodies to Afflgel-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 bûnds with the spacer arm. The remaining activated esters are then quenched with I M ethanolamine HCl (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 ~ fr:~n~ollc protein. The column is then equilibrated in phosphate buffered saline (pH 7.3) and the cell culture supernatants or cell extract~ containing recombinant enzyme or fragment.s of it are slowly passed through the column. The column is then washed with phosphate buffered saline until the optical density (A280) falls to background, then the protein is eluted with 0.23 M glycine-HCI (pH 2.6). The purified protein is then dialyzed against phosphate buffered saline.

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- The following ~xamples are provided as illustrative of the present invention without, however, limiting the same thereto.
EXAMPLE I

Bacterial strains and plasmid.s N. Iactamdurans LC411, an improved cephalnycin C
producer strain, was used as the source of DNA and RNA. Streptomyces lividans 1326, (Hopwood et al., 1985) a strain unable to synthesize 13-lactam antibiotics, was used as a host for l~ roll,lation and for expression ~elilllclll~. _. coli DH51x was used for high frequency transfor~nation and E. coli WK6 with the helper phage M13K07 to obtain single-strand DNA.
The genes of the cephamycin C biosynthetic cluster were 15 isolated from phages lambda EMBL-C2 and C-8 (Coque et al., 1991a), and subcloned into plasmids pBluescript KS( + ) or plJ292 1. To express the N. l~et~m~llrans genes in S. Iividans they were subcloned into plJ702 (Katzetal., 1983).

r~.lllelll~liulls con~litions For preparation of seed cultures S. Iividans transformant.s containing recnmhin~nt plasmids were grown in YEME medium with 25 34% sucrose (Hopwood et al., 1985). After 48 hours of growth in YEME
25 ml of this culture was used to inoculate 500 ml triple baffled flasks containing 100 ml of minimal medium with glucose and Iysine (Coque et al., 1991b) and grown at 30C in an orbital shaker at 250 rpm. All the inoculum and fermentation media used to grow cultures of transformant 30 strains contained thioestrepton (5 ,ug/ml).
Cell free extracts were prepared from washed mycelium suspended in MOPS buffer 100 mM pH 7.5 containing 20 llg/ml DNAse and 1 mM PMSF. The cells were broken by sonication with a Branson Sonifier B-12 or altematively with a French Press (Aminco).
.

DNA isolati--n sequencin,~ and manipulation.
Fragments to be se~ nced were subcloned in pBluescript 5 KS(+) in both orientations. Ordered sets of nested DNA fragment.s were generated by seq~enti~l deletions using the Erase-a-Base system (Promega, Madison, Wis). The DNA was se~ pnred in both orientation~s by the dideoxynucleotide method (Sanger et al., 1977) using Ta~l polymerase (Promega) and 7-deaza-dGTP to avoid compressions.
lsolation of plasmid D~NA, digestion with endonucleases, labelling and Southern hybridizations were carried out according to standard procedures (Sambrook et al., 1989). T.~l~rul...dtion of S. IiYi~n~ was done as described previously (Hopwood ~ ~,1- 1985; Garcia-Dominguez et al., 1991).
DNA ;~nl1 protein sequence analvsis.
Open reading frames (ORFs) in DNA were identified using the GENEPLOT Program. Inverted repeated seq~ nres were located with the STEM&LOOP Program. Protein col.l,ual isulls were made with 20 the AALIGN prograrn using the EMBL Swiss Prot data bank. Dot Plot analysi~ was made with the DOT-PLOT Program using a window size of 25 amino acids and a percent match of 30%.
()ne of three open reading frames (ORF) located dc~ from 25 the ncbC gene llyb~ s to ~E probes-During the cloning of ~fE gene from ~ f~mtlllrans usinga 734 bp Sacll internal probe of the A. chr,v.~ogenum gene ~EF, two positive hybridization bands were found in the total DN~ of N.
Iactarndurans: one of tllem was characterized (~E gene) and shown to 30 encode the DAOC synthase (expar~dase) (Coque et al., 1993a). The identity of the second hybridizing band, was unknown but could be a closely related gene or a duplicate ~E gene. Therefore, in order to fle~rmin~ what the DNA band encoded, phages containing DNA
fragments of the N. I~rr~m~ n~ cephamycin C gene cluster were W095~29253 2~B25~ S lI
,5 digested with BamHI and hybridized with a 503 bp ~11 DNA fragment internal to the cefE gene from N. Iaçtamdurans. A 5.4 kb ~HI DNA
fragment (Fig. 2) [known to contain the pcbC gene but not the cefE gene (Coque et al.,l991; 1993)] gave a strong positive hybridation. The entire 5 region of this 5.4 kb DNA fragment downstream from ~LC was sequenced. Three ORFs, ORF7, ORF8 and ORF9 (Fig 2) 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.
By subcloning and deletion t~ S it was shown that the cefE probe hybridizes specifically to ORF9.
Chd~d~ ,dtion 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 20 intergenic region 7 bp upstream from the ATG translation initiation triplet. Downstream of the TAG termination codon, a secondary structure (nt 2734-2767) forms a stem and loop structure that may correspond to a ~ Ol with a r~ rPd free energy of -31 Kcal/mol.
Computer comparison of the amino acid and nucleotide 25 se~uence of ORF9 with exr~n~ s and hydroxylases involved im cephalosporin or cepharnycin biosynthe.~is showed 80.8% identity in nucleotides and 77.5% in amino acids with the cefF encoded protein of S.
clavuli~erus. The protein encoded by ORF9 showed also a high similary to the bifunctional expandase/hydroxylase of A. chryso~enum (encoded 30 by cefEF) and to the exr~n~ of N. l~ m-lllrans and S. clavulis~erus (encoded by cefE).
Cllar.~ DC of ORF7 and ORF8.
Downstream from the ~C gene and separated by only 13 nucleotides, starts an ORF (ORF7) of 711 nucleotides encoding a protein WO 9S/29253 2 1 8 8 2 5 7 P~~ b ~o, ~ I

(named P7) of 236 amino/acid~ (27364 dalton.s), with a deduced pl of 4.~9. lmmr(li~ft~ly downstream from ORF7, with a separation of only 7 nucleotides begins ORF8, a sequence of 876 nucleotides (Fig. 3) encoding a deduced protein (P~) of 292 arnino acids (32,090 daltons), with a pl of 5.02. The G + C content of both ORF7 and ORF8 wa~ 67.5 and 71.9% respectively. Thirteen nucleotides upstream from the ATG of ORF7 a putat;ve RBS sequence (GAGGAGCA) was observed, identical to that found upstream of ORF9. The small separation between ORF7 and ORF8 and the presence of a putative RBS, GA~GG, before the end ORF7 (inside the coding sequence), suggest that both genes are co-translated without release of the ribosomes.
A co~ d,d~ive DOTPLOT analysis of the predicted protein encoded by ORF7 with other proteins present in 11~f~h:~nk~ indicates that the ORF7 product shows homology to O-methyl~-a,l~rclascs involved in chemotaxis (Çh~ genes) from S. typhimurium and E. ~Q~ hydroxyindoil, cathecol and caffeic acid O-methyl~ldu~r~,a~s, and lower homology to tylosin meth~l~,dll~r~,ases from Streptomyces fradiae (~mP, tcmO) and methyltransferases for the methylation of oligosaccharides involved in nodulation in A~u,l,i,-,l,iulll (~S). A typical S-adenosylmethionine binding motif, is present in the N-terminal region (arnino/acids 10 to 26) (Ingrosso ç~ al., 1989). Since all these proteins catalyze the O-methylation of phenolic or heterocyclic hydroxyl groups, ORF7 seems to encode the C-7 hydroxycephem meth~d~,dl,~r~,a~. However, in addition, the protein exhibits a 30.5% identity in 59 arnino/acid~ with human and rat cholesterol 7-oc-monooxygenase, an enzyme which introduces oxygen at C-7 position in the cholesterol nucleus.
The ORF8 protein showed no si~nifir~nt homology with any protein present in the EMBO and Swiss-Prot data banks and it behaves a~
a coupling protein.
Location of ORF10 of 1~ rf~mril~r:ln~.
During the characterization of the genes ~fF, cmcl and cmcJ present in a 5 4kb ~EaHI DNA fragment, an incomplete ORF
(ORFIO) was found. This ORF was located downstream of ~fF. In WO 9S/29253 2 1 8 8 ~ 5 7 P~

order to obtain the complete sequence of this gene, a 3.6 kb ~mHI DNA
fragment of the cephamycin C gene cluster [known to be adjacent to the 5.4 kb BamHI fragment downstream from ORF9 (~çfF) (Coque et al., 1993)] was subcloned and Sçq~ nre(i in both orientations. This 3.6 kb ~aHI fragm~nt contains the bla gene (Coque et al., 1993), the 3' region of ORF10, and the 5' end of ORF14. In order to obtain ORF10 im a single DNA fragment, 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 8-mer B~III linker and subcloned in BglII digested plJ2921. From this plasmid the 3.4 kb fragment with BglII 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).
Ch.~ t~. ln of OI~F10 The translation initiator ATG codon of ORF10 is located 74 bp downstream from cefF. It was preceeded by a GGAGGA sequence that resembles the Shine Delgarno ribosome binding ~eq~'nrç~. An inverted repeat of 15 bp that may form, if transcribed, a stem and loop structure with a calculated free energy of -31 kcaVmol, is present in the intergenic region. If this structure is a functional ~ lulalu-, ORF10 should be expressed from its own ~lolllul~l; alternatively ORF10 may be expressed from an upstream promoter and the inverted repeat may work as a ~elllluld~ul regulated by an ~ntit~rmin~tion mechanism. ORF10 contains 1563 nt (Fig. 4) and has a G + C content of 68.8% which is similar to the average G + C content of the N. l~ mdllrans s~qll~n~d genome (70.4%) (Coque et al., 1 993a). It encodes a protein of 520 amino acids with a deduced Mr of 57149 and a pI of 5.2.
When amino acid comparison was made using the Aalign Program 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 Bradvllli~ubiull-japonicum. The nodU genes encode O-carbamoyl transferases for the biosynthesis of carbamoylated polysaccharides required for nodulation WO95/29253 2 ~ ~8~ I/U~.'01 (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. The cmcH
5 encoded protein showed little overall homology with aspartate carbamoyl L~ r~,ldses and ornithine carbamoyl l.a,~ ,se of E. ~QIi, Asper~illus nidulans and a variety of other microo,~a~ ls.
Location of the cmcH gene of S. clavl~l;c~. .,.
From a genomic library of S. clavuli~erus DNA in lambda-GEM12 the cmcH and the adjacent cefF gene were subcloned into plJ702 as a 6.2 kb BamHI DNA fragment originating from plasmid pULFJP62.
When lambda GEM12 phages c~,"l;,;"i"~ DNA fragments of the cepharnycin cluster were hybridized with the cefF or the cmcH genes of 5 N. Iactamdurans. 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. clavuli~erus conta~ns, therefore, the cefF (Kovacevic et al., 1991) and the cmcH genesJ
The hybridizing S. clavuli~eerus DNA sçquence was mapped 20 more precisely within a 3.0 kb KpnI DNA fragment con~inin~ also the cefF gene (Fig. 5). The 3.0 kb ~I DNA fragment was subcloned in pBluescript KS(+) giving plasmid pULFJP30 and 160 bp at the KpnI site of the distal end (downstream from cefF ) were s~qllpnre~i The 160 nt sequence matched almost perfectly the sequence of the cmcH gene of N.
25 lactamdurans (nt 1472-1639 in Fig. 3A) with a X0% identity in nucleotides and ~1% identity in the deduced amino acids. These results established that the relative location of cefF and cmcH is identical in the cephamycin cluster of both cephamycin C producers S. clavuli~erus and N. Iactarndurans. although the overall organization of the clusters is 30 different (Martin et al., 1992, and Martin and Gutiérrez 1994).

WO 95~29253 2 1 8 ~ 2 ~ 7 r~

F~AM~LE 4 Cell growth ~n-1 preparation of cell-free extracts.
Cell free extracts were obtained from S. Iividans 5 ~ r~ ants grown for 48 hours im mmimal medium with glucose and Iysine (Madduri et ~1., 1991). The cells were washed and suspended in 100 mM, MOPS pH 7.5, containing DTT (I mM), PMSF (1 mM) and DNAse (20 yg/ml). The cell S~ rl~ ll 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 ~ulJe~ l~lt was plc~ iL~l~d with ~mmonillrn sulphate (0-80%). The protein ~ iL~l~ was dissolved in the same MOPS buffer and passed through a PD-10 column (Pharmacia).
EXAMPLE ~
3'-Hvdroxymethylcephem O-carbamo~ r~ldse (CCT) assays.
3'-Hydroxymethylcephem O-carbamoylLI~l~r~l~se activity was assayed by the fol~owing three different methods. i) By using 20 decarbamoylcefur-oxime and [14C]carbamoylphosphate as s~hstr~t~s and measuring the ethyl acetate-extractdble carbarnoylated cefuroxime radioactivity in a Phillips PW4700 sCin~ ti~-n counter, as reported by Brewer et al., (1980). ii) Alternatively, forqualitative llrl~ inn of the carbamoylation TLC chromatography of the reaction products was 25 performed on Silica gel 60 plates (20 x 20) using n-propanol:glacial acetic acid:water (5:1:2) and the compounds containing the cephem ring were detected by spraying the plates with a) 12/K15 mM solution contdining 1% w/v sodium azide and b) starch 1% solution.
cmcH genes of 1~ mrl--ran5 and S. rl~v,.lh;~ encode a f ~ 3'-hy~ JAylll~ ly~ O~ yltransferase activity.
Cell free extracts of cultures (4~ hours in minimal medium) of S.l~aZ~ [pLT702], S. !ividans [pUL702-37a] (cont~inin~ the cmcH

:
w0 9s/2g2s3 ~ 2 ~ 7 r~ o gene from N. lact~m~ rans) and S. Iividans [pULFP62] (containing the cmcH gene from S. clavuligerus) were obtained and the CCT activity was ntifi~l Table I shows that the CCT specific activity in S. Iividans strains cnnr~inin~ the N. Iactamdurans or S. clavuligerus genes are 7.~-5 and 7.7-fold higher, respectively, than the background level of S. Iividans transformed with pU702. The 3'-hydroxymethylcephem O-carbamoyl-transferase was found to precipitate after treatment of the cell free extracts with ,.. ~ sulphate in the 45-6~ % fraction and has been purifled for further biochemical characterization. This demonstrates that ORF10 (cmcH) encodes a functional 3'-hydroxymethylcephem O-carbamoyltransferase .
Table I
carbamoylLdll~r~la~
STRAIN [14C]carbamoyl~ alwdi~lle Protein* S . activit formed (cpm*/ml) (mg/ml) (cp3mg of protein) Experiment I
S. Iividans plJ702 13.285 20.5 641 S. Iividans pUL702-37a 29.515 6 0 4919 Experiment 2 S. Iividans pIJ702 3.550 8.5 410 S. Iividans pULFP62 14.025 5-5 2550 *Radioactive carbamoylated cefuroxime extracted by the Brewer as a m after speed-vac dessication of I ml of the organic phase. s y, cp 3'-Methylcephem-Hydroxylase 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-W0 95l29253 2 ~ ~ 8~ 5 ~ r~ Q~ , - 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 30C. The reaction was stopped with methanol (200 !11); the precipitated proteins were removed by centrifugation at 14,000 rpm for 10 min and the reaction product was quantified in the supernatant. The hydroxylation of DAOC to DAC was followed by HPLC using a Waters IlBondapak C18 column (300 x 3.8 mm) equilibrated and eluted with NaH2P04 200 mM pH 4.0 at a flow of 1.5 ml/min. The eluted fractions were monitored at 254 mn. Under these c~n~ ns DAOC eluted with a retention tirne of 10.7 minutes and DAC
at 3.7 minutes.
The ~fF gene encodes a ru~ ' cephem-3-hydroxylase without cephem-7-hydroxylase activity.
In order to fully characterize the product of ORF9, the 5.4 kb HI fragment was subcloned in pU702 [Katz, E. et al., 1983, J.Gen.Microbiol., 129, pp.2703-2714] to give plasmid plJ702-54a.
Ad~itionally, a 1.4 kb Notl-~llll DNA fragment (internal to the 5.4 kb fragment) was end-filled and subcloned into the polylinker of pU2921, 20 recovered with ~ and subcloned into the BglII site of plJ702 to give pU702-58a (Fig. 2). In both ca~es the OFR9 was subcloned downstream from and in the same orientation as the ~I promoter. Additionally the 1.4 kb Notl-Mlul fragment was subcloned in the ~nHI site of plJ699 [Kieser, D., and Melton, R.D., 1988, Gene, 65, pp.~3-91~ to give plasmid 25 pU699-58a.
Cell free extracts from a 48 hour culture of ~. Iividans transformed by the expression plasmid [pU702-~4a] were assayed for DAOC hydroxylase activity. HPLC analysis of the products of the reaction indicated the formation of a product ~vith a retention time of 3.7 30 minutes (Fig. 6B) which co-eluted with pure deacetylcephalosporin C
(Fig. 6A) demonstrating DAOC hydroxylase activity. The same DAOC
hydroxylase activity was observed im cultures of S. Iividans [plJ702-58a].
Cultures of S. Iividall~ [plJ702-58a] and S. lis~i~ [pU699-58a] were tested for C-7 hydroxylase activity at different tirnes during the .

wo gsn9253 ~ 2 18 ~ 2 5 7 . ~,l/L__ '0~ I
~e.~ lla~ion. No activity C-7 hydroxylase could be detected at any time, in-lic~in~; that ORF9 encodes the cefF gene of N. Iactamdurans~ which hydroxylates the cephem nucleus at the C-3' position but not in the C-7 position. These results shows that two different hydroxylases are 5 required in the late reactions of cephamycin biosynthesis.
EX~MPLl~ 7 7-Cephem-hydroxylase and 7-Hvdroxvcephem methvltransferase assay.s.
The enz~nes were measured in the 30-70% ammonium sulphate ~IC~ o~ 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-hydroxycephalosporin C and 7-rnethoxycephalo-sporin C (Xiao and Demain, 1991). The reactions were carried out in a 15 water bath with shaking to favor the oxy~en transfer required for the reaction. After i~ for 120 minutes at 30C the reaction was stopped by addition of acetic acid (10 ,ul); the proteins were ~limin~tPd by centrifugation at 14,000 rpm for 5 minutes.
The ~ul~el Il~ mI was applied to a QAE-Sephadex column ( I
20 ml bed volume) equilibrated with 50 mM Tris-HCI pH 6Ø After washing the column with 600 111 of the same buffer, the products of the reaction were eluted with 3N NaCI (600 ~1l). Chromatography of the products of the reaction was performed in a Waters llBondapack C18 column as indicated previously except that the elution was done with a 2s 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.
Under these conditions the retention times of the substrate and products were as follows: cephalosporin C 29.2 min; 7-hydroxycephalosporin C
14.2 min; 7-methoxy-cephalosporin C 17.7 minute~. Alternatively, S-3 adenosyl-L-[methyl-14C] methionine (25 ~Ci/ml) was u.sed in the assay (250 nCi per reaction) and the formation of labeled 7-methoxycephalosporin C was monitored after HPLC chromatography using a radioisotope detector (Beckman 171).

WO9!;/29253 2 ~ 8~2rJ7 r ~

E~IJr~J~ n of ORF7 and ORF8 in S. Iividarls result in C-7 snethoxylating activity.
The similarity of the protein encoded by ORF7 with methyl~lall~r.,las~s suggest that ORF7 (and ORF8) might correspond to 5 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 ORF8) was subcloned into plJ2921, rescued with ~1~1 and subcloned in plJ702, downstream from and in the same orientation as the mel promoter to give expression plasmid plJ702-55â.
Cell free extracts of 48 hour cultures of ~ ~n [plJ702-55a] were precipitated with ~ ;l l l l l sulphate (30-70%) and tested for C-7 hydroxylase and I~ yl~ las~ activity. A product with a retention time of 14.2 minutes (Fig. 7B) is formed during the C-7 hydroxylation reaction. This peak decreases when s-adenosyimPthionin~ (SAM) is 5 added to the assay (Fig. 7D), with conrnmit~nt formation of a second product with a retention time of 17.7 minute.s showing that both C-7 hydroxylase and methyltransferase activities (identified as 7-methoxyCPC) are present in the l,dn~r~",lld"l~, none of the two activities was found in control S. lividans [plJ702] cell free extracts (Figs. 7A and 20 7C). To identify the product with retention time 17.7 minutes labeled SAM was used in the assay and the radioactivity in the reaction products were observed by HPLC and by TLC followed by autoradiography.
Three peaks of radioactivity were found in the HPLC eluted fractions with retention times of 10.7, 14.0 and 17.7 minutes. For nation of the 25 three labeled peaks was fi~perlri~nt upon addition of CPC substrate to the reaction. One of the peaks (retention time 17.7) minutes was identified âS
7-methoxycephalosporin C, whereas the others probably correspond to hydrolysis products of the 7-methoxycephalosporin C due to the acid treatment to stop the reaction.
The C-7 hydroxylase and methyllld,~r~,àse activities of the transformants during the fe""~"l~lion was determined. Two types of L.d~l~ro""ants were used: S. Iividdns ~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 W09~1~9253 ?~8~7 .~

subcloned in plJ699 a plasmid with two transcriptional terminators in which the ORF7-ORF~ should be expressed from its own promoters.
The time course of methyltransferase activity during the fermentation overlapc with that of the 7-hydroxylase activity, suggesting that both activities are expressed coordinately.
ORI~ 7 encodes a 7-hydroxylase activity but the two proteins encoded by ORF7 and ORF8 are required for 7-methoxylase activity.
To define which enzyme activity corresponds to each gene, ORF7 and ORF~ were subcloned individually. DNA fragments Pstl-Xhol (2056 bp) and K~l (1048 bp) conf~inin~ ORF7 and ORF8 respectively (Fig. 2) were cloned in plJ2921, rescued with B~III and subcloned in the B~III site of plJ702 downstream from and in the orientation of the mel promoter to give plasmids pUL702-56a and pUL702-57a C-7 hydroxylase activity was found in cultures of S. Iividans [pUL702-56a] in repeated experirnents. No 7-hydroxymethyltransferase activity was observed unless S. Iividans was ~.al.~r~ d with DNA
fragments carrying both ORF7 and ORF8 (i.e. pUL702-55a).
Therefore in vitro compl~m~nl~fi~-n of both proteins was tested. Cell free extracts of S. Iividans [pUL702-56a] and S. Iividans [pUL702-57a] were incubated together for 30 minutes in the presence of the cofactors required for i) the C-7 hydroxylase activity; and ii) the C-7 hydroxylase and methyltransferase activities. Under these conditions a 25 peak of C-7 hydroxycephalosporin C was observed showing that a C-7 hydroxylase activity was illdeed encoded by ORF7. No formation of 7-methoxy CPC was observed in vitro when the ORF7 and ORF8 proteinc were mixed together, s~ ecfin~ that expression levels of ORF8 are low but sufficient to detect methoxy CPC. It is likely that proteins from both 3 ORF7 and ORF8 become associated in vivo but such an association in vitro is difficult due to the low concentration of the proteins in cell free extracts.

WO 9S/292S3 2 ~ 8 ~ 2 5 7 r~ o~ I
The 7-hydroxylase shows a ~ sporin-d~p~nd~ ..1 NADH-oxidase activity.
- The proteins encoded by ORF7 and ORF8 behave as a two protein c~ system. Cell-free extracts of S. Iividarls [pU702-56a]
5 expressing OR~7 showed a strong cephalosporin-dependent NADH
oxidase activity (Fig. 8). The protein encoded by ORF8 did not show a signif1cant NADH-oxidase activity but was absolutely essential for productive 7-methoxylation. These results indicate that hydroxylation at C-7 is mediated by a hydroxylase that uses NADH as an electron donor for reduction of mo]ecular 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.

W09~il29253 2~ ~8~57 P~
.
- 26 - .
SEQUENCE LISTING : -(1) GENERAL II~FORMATION:
(i) APPLICANT: Martin, Juan F.
Coque, Juan R.
Enguita, Francisco J.
Fuente, ~Tuan L.
Llarena, Francisco J.
Liras, PalPma (ii) TITLE OF IN\7ENTION: DNA ENCODING CEPHAMYCIN BIOSYNTHESIS
LATE GENES
10 (iii) NUMBER O~ SEQUENCES: 8 (iV) ~UKK~ Ul~ ADDRESS:
(A) ADDRESSEE: John W. Wallen III
(B) STREET: P.O. Box 2000 ( C ) CITY: Rahway (D) STATE: New Jersey (E) COUNTRY: USA
15 (F) ZIP: 07065 (v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release ,YI.0, Version ~1.25 20 (vi) CURRENT APPLICATION DATA:
(A) APPLICATION NOMBER:
(B) FILING DATE:
(C) CLASSIFICATION:
(viii) ATTOFNEY/Ak:ENT INFOR~ATION:
(A) NAME: Wallen III, John W.
(B) RFr~C~R~l'Tn¦~ NOMBER: 35, 403 25 (C) REFERENCE/DOCE~ET NUMBER: 19179 (iX) T~T~FrnMMrTl~Trl~l~IoN INFORMATION:
(A) TELEPHONE: (90a) 594-3905 (B) TELEE~X: (908~ 594-4720 ( 2 ) INFORMATION FOR SEQ ID NO :1:
( i ) SEQUENCE CllARACTERISTICS:
(A) ~ENCTH: 170~ base pairs (B) TYPE: nucleic ~rid (C) STR~ l)Tnl~T~ : single (D) TOPOI.OGY~ eor (ii) MOLECULE ~YPE: cDNA

WO 9S/292S3 . _l/L~0 1 ~r I
~1 8~7 (xi ) SEQUENCE DESCRIPTION: SEQ ID NO :1:
ACGATGCACG CGGTCACCTC GTAGCCACCG TGCCCGCGAC L:~LLLLr_--~ CGAGGCCGGG

blj'l'~L CGACCCTCGT CACGCGTTGG Pr~PrrP~rr GCTCATCGTC GCGTTCAPAC

CGGGGCACGA ~jr~ GCCGCGATCG GCGATCGCCG GTTGCTCTAC TCGCTCGAAT

rr~PrPPrrP ~ rir~ r. CGGTACTCGC CGATCCTGGC CACCACCGTG CTCGACCTCG

rrr~ rrT GGGCGAGGTG CCGGACGTGG i..;.r_.L. j~, rr-r.rTrrPrr GACCTGCWC

CCAACCGCAT CTCCTACACC ~;~.[ r:r ~ ~Jrj~., ACTCGGGCAT rr.PPr.PPrr~r ACCGTGACCA

L5 ~1W~Lj~11 CTTCWCAAG GAWTGAAGT TCTTCAGCTC CACGCACGAA CGTTCGCACA

TCTACATGGC CCTWGCATG ~rrrrr~ :r.ri Prr.PrPrrrr GGTCC~GACG ~ ri WGAGGGTGA r~ .rjr~ L TTCTACGTGA TCGACGGGCA CCAGCWATC Prrr~rPPr.r TCCAWTGAT ~;1~L~X;r1~C ~rj~b~j~1 ACTCGTTCCT ~1L~r~--~1-- GCCGACCCCA

CTTTCCCCAC rPrrr,rjrr_r~ PPPrrr.rrjrr TGAACGACGC CGGGAAGCTG ATGGCGCTGG

~b~ ; CGACTCCGCC r7~rrrr~: CW~LCATCAC GCACGTGGTC GAGCGGATCC

TCAAGCAGGA CTCGATGTAC ~ AWGTGAATA CCWGATTCG GTGCTGTACA

ACGCCGGGGT CGAGTCGCCG GAGTGCAAGA ~ ~ j~rjr_Lrj~ CCTGCTCACC GAACGCCTCT:

TCGAGACCTT CGCCGAGGTC rrrPrir~r pr~ AGATGCCCGA Ar~rPrrrrr- CTCTACATCT

~ ;rj~rj~;r_1~; CGGGCTGAPC TGCGACTGGA ACAGCCTGTG GGCGCAGCTC GGCCACTTCT

~1'~1~;1"1' ~1~1~r~ ~ TGCACCAACG ACTCCGGTTC ~:rj~G~1rj~jrjC ACCGCCATCG

WO 95/29253 2 1 8 ~ 2 5 7 r~

ACGCGCTCAC CACCTTC~CC GGTGACCCGC ACGTCGACTG GAGCGTCTAC AGCGGACTGG

AATTCGTCAC rr.~rP~rrr~r rrrnl~rrrnr. CCAGGTGGAC r_L~r r~ ; CTCGAGCACG
ACGAGCTCTC ~jL-~jC~ L~ r l LL~ L- ~ ~ L~ ~ CTGGGTGCAG I

AGATCGGTCC ~i~L-WW~L- TGCAACCGCT CGCTGCTGGC CGAGCCGTTC L~ ;~r.~ A
rr~rnr~rrr. GCTCAACGAG ATCAAGCAGC GWAGGACTA CCGCCCGATC ~;~r"..~;,L1 ;r_ ~;, LLA r~A~ AAGGTCTTCC ACGAGGACTT CGAAGACCCG TACATGCTCT
ACTTCCGGCG rrTnrr~rr,An TCCAGCGGCC LL---~ ;W ~ GACCC CGTG GACWTTCW

15 ~_ Llj1~j~A GACCGTGCGG GATTcr~GGcA ~rrrnr~rl~T GCACCGGCTG 1L'1'~W-r_'1`
;~r LL~ r~ 1LL GCAACACCTC GCTGAACTTC A~rr,r~rr~nr GGTTCATCAA CCGCATGTCG GACCTGGTGC TCTACTGCGA A;.Lr~ W~ ATCTCWACA

1LL I ~L~. rnZ~Tl~rrTrr, TACCAGCGTG CCGAGGGCTG ~rrrrr~nrrr rnnArrnrrr~

TGAGCGCGAG .r, 17 0 0 r~ LLl W
(2~ INFORMATION FOR SEQ ID NO 2 (i) SEQUENCE rTT~R~r~RTSTI
~A) LENGT~ 972 base pa;rs (B~ TYPE nucleic ~cid (C) STR~ c':: sinr.~1e ( D ~ TOPOI OGY 1 inear ( ii ) ~OLECULE r~PE CDNA

(Xi ) SEQr~ENCE DESCRIPTION SEQ ID NO 2 AAGACGGTAC CGGTCTTCAG CATGGCCGAA CTGCGCGACG L-.1~L~.r~ GWACGAGTTC

CGCGAGTWG l'~ r GGTCTTCTAC CTCACCGWT ACGGCGCCAC CGAA~GAGAC

W0 95/29253 ~ 8 ~ 5 7 r~ ,s~c - I

CACCGGGTGG CCACCGACAC CGCGA-T~GAC ~ AAGGCACGGC rnArnAnAAr.

cAr~nrrr~r~A CCACGAAGGT CCCGACCATG ~ u~ ~l ACTCGGCGCT rr.Ar.rrCr.AA

AGCACCGCCC AGGTCACCAA rArrnr.~Arr TACACCGACT ACTCC~TGTC GTACTCGATG -~

GGCATCGGCG GCAACCTGTT CCCGTC~AAG GAGTTwAGT CGGTCTGGAC GGACTACTTC
r~rA~rr~GT ArrGrnrrn,r ~ rAr~Ar.Arr ~ ~Cl~i~i TGCTGACCGC CGCGGGCACC

TACGACGGCG AGGACCTCGA CACCCTGCTC GACTGCGACC b(~ iW CCTGCGGTAC

TTCCCGGAGG TCCwGAGCA ~ GAGTACGAGC CACGCCGGAT GGCCCCGCAC

TACGACCTGT CCATCATCAC CTTCATCCAC CAGACCCCGT nrrrrD~rr.r TTTCGTCAGC

CTGCAGGCCG AAGTGGACGG TGAGATGGTG AGCCTGCCGC ACGTCGAGGA ~ ilV

GCGCGATCGC ~i~i~Lii'l'~ ArrrArr,nrr. CGGTGCCCGC çrrrAArrAr CACGTGGTCT rrrrrrArrr GAGCATGCTC i~ArrGrArrr. ACCGCI~CC~C GAGCGTGTTC

11~,--'l'(iW--~ wTCGACCGA TTTCACCTTC ~ rrrrArnAA GTACGGCCTC

GACGTCAGCC TGGACATGGA r~AArr~rnArr TTCGGCGACT GGATCGGGAC CAACTACGTC

ACGATGCACG CGGTCACCTC r~Ar.rrArrr. TGCCCGCGAC ~ J~ ( ~ A CrArnrrrrG

rl ~ CG - -( 2 ) INF~RMATION FOR SEQ ID NO: 3:
3 0 ~ i ) SEQUENCB CHARACTERISTICS:
(A~ LENGTH: 900 l~ase pairs (B) TYPE: nucleic ~cid (C) S~RANr)EnN~CS 3ingle (D) TOPOLOGY: linear ( i i ) MO~ECULB TYPE - cDNA

~o 95~2g2s3 2 ~ 5 ~ / C ~

(xi1 SEQUENCE DESCRIPTIO~: SEQ ID NO:3:
CGTGCCCACC GTGTCGAGTC CGCTGTACTA l -l ( - - I ' GACCCCGG Arr~rr~r-CGGGGACIGG TGCATCGACG CCGTGACCCG rrrrrrrr~ ACTTCCGCAA

bb ~ b'l~i r~Ar~i~rr~rr~ TCACCGACCT rrr.rnz~rr.rr TCGATCCGGC rrnrrr~r~rD~

rn~ rnrnn TTCGAGA~-G-G TCACCGCGCC CACCGGCGCG TCCCACCGGG b~ 'lWA

CAGCGAGGAA 1- .l 1!. 11~. AGCAGTACCG GCGGGAAACC GGTGAGCTGC TCCGCTCGCT

CACCGGCGCG W~bl~iii~i AGTTCTTCGA CGCCACCCTG rr~rr~r~r.r ~rr.rnrrrn~

rr.Arrrr.~ rr GCCCAGTCCC CGCACCAGCG GGTGCACGTG GACCAGAGCC CGGGCAGCGC

rrr~r.rr~rr. rrrr~r~rrr~C ACCTCGGCCC CGGCCGGGAG 1l~bb-~ TCCAGATCAT

CAACGTCTGG I: ~ . TCGAGCCGGT GCGCAACTTC [ ~ TGTGCGACTA

bbl~i~lV GACCTGTCCG CCGACCTGGT crrr~'rrr~ CTGGACTTCC~ CGG~CTGGCT
r~7~rr~rrC GAGAACTACT ~ ~bi~ C~ACCCCGCG A~--bl l~ ACTTCTGGGA

CTCGCTGACA CCGGCCGAAG ~ ;bl~ll CAAGTGCT~C r.~rl~rrrrr.~ ~ ~b~7~i~I

25 GGCC~TGGCC GGTGGCGAGC cr~r-~rr~rrr~ CGAACTGCGC GACGTGGCGG~(,l.l.l.,~
rr~r~rrnrr TTCTTCGi~CG ~n~rr~nrr GTCGACCGGC CACCTGCGCA ~ll.

ACTGCGCGCG l~ll.C ACGAATGAAC GACGAACGAG GAGCAGGGGA; AATGTCGGAC

( 21 INFORMATION FOR SEQ ID NO: 4:
( i ) SEQUENCE C~ARACTERISTICS:
(A~ LENGT~: 800 b~se pairs (B) TYPE: nucleic ~cid ( C ) sTR7~ Rn~\TRc~ s ingle (D) TOPOLOGY: lirl~r WO 95/29253 ;;~ 2 5 7 ( i i ) ~OLECULE TYPE cDNA
(xi~ SEQUENCE DESCRIPTION SEQ ID NO 4 ;~ TACGGCGACT ACCTCAACCA CGGCCTGCAC TCGCTGATCG TGAAGAACGG

CC~GACCTGA TCGAGGAGCA CGCATGACTG ArArrArrrr CCAGGACTTC CTGGACCTCA

ACCTGTTCCG ~i(ib~i~'L~W~i GAGGACCCGG TCTACCACCC ;~ rrrrArrrr~-CGCGCGACTG GCCGCTCGAC ~ AGGCCCCGCG CGATCTCGGG TTCTCCGACT

~ G~ L~ CCAGTGGCGC ~ A TGCTGAAGAA rrrr~7~rZ~rr CAGGCCGCCT

ACCACGACCT GATwTCGAA ~lV~ w~,--~ GCACGGTGAT CGAGCTGwC GTGTACAGCG

CGGGACATGG CCGAGCTGAT GwCTTCGAC TGCCAGGTGC

TCGGCATCGA CCGGGACCTG i~-b~L~ AGATCCCCGA GTCCGAGATG AAGAACATCT

~ ~ GGCCGACTGC AGCCTGGACC GGTwAAGCT CGTwACGCG CTGGACGGCG

TGGGCGACCA CAAGTAGCTG ~ b~ GCTTGTGGAA GTTGTAGGAC GCCACCAGCC

TGGACCACCT CCTGCACGAA wCGACTACT TCATCATCGA GGACATGATC CCGTACTGGT

ACCGGTACAG CCCCAAGCTG CTC~LCCGAGT ACCTCGCCGC ~ilL.~W~; GAGCTGAGCA

TwACATGGT CTACGCCAAC GCCAGTTCAC AACTGGAACG ~ ~w l ~G

CACCGAAGGC GTAGGTGGAT

( 2 ) INFORI~ATION FOR SEQ ID NO 5 ( i ) SEQUENCE CHARACTERISTICS
(A) LENGTH 520 amino acids (B) TYPE ~mino acid (C~ S'l`RANn~nN~: single (D) TOPOLOGY linear woss/2s2s3 2~882~7 r~ oiO~I

(ii) MOLECULE IYPE: protein (xi) SEQUENCE DESCP~IPTION: SEQ ID NO:5:
Met Leu Ile Val Ala Phe Lys Pro Gly His Asp Gly Ala Val Ala Ala Ile Gly Asp Arg Arg Leu Leu Tyr Ser Leu Glu Ser Glu Lvs Asp Ser 20 25 : 30 Arg Pro Arg Tyr Ser Pro Ile Leu Ala Thr Thr Val Leu Asp Leu Ala Glu Arg Leu Gly Glu Val Pro Asp Val Val Ala Leu Gly Gly Trp Ser Asp Leu Arg Pro Asn Arg Ile Ser Tyr Thr Gly Ala Gly T~r Ser Gl Ile Glu Glu Pro Thr Val Thr Thr Ser Arg Phe Phe Gly Lys Glu Val Lys Phe Phe Ser Ser Thr His Glu Arg Ser His Ile Tyr Met Ala Leu 100 105 : 110 Gly Met Ala Pro Arg Asp Asp Ser Pro Val Gln Thr Val Leu Val Trp Glu Gly Asp Val Gly Ala Phe Tyr Val Ile Asp Gly His Gln Arg Ile 130 == 135 ~ 140 Thr Arg Lys V~1 Gln Val l~et Ser Gly Pro Gly Ala Arg Tyr Ser Phe 10.5 150 155 160 Leu Phe Gly Leu Ala Asp Pro Thr Phe Pro Thr Thr Gly Gly Lys Pro 165 170 ~ 175 Arg Leu Asn Asp Ala Gly Lys Leu Met Ala Leu Ala Ald Phe Gly Asp Ser Ala Asp AIa Asp Ala Asp Ile Thr His Val Val Glu Arg Ile Leu Lys Gln Asp Ser Met Tyr Pro Ala Pro Lys Gly Glu Tyr Arg Asp Ser 210 215 2;!0 ~
Val Leu Tyr A~n Ala Gly Val Glu Ser Pro Glu Cys Lys Ile Ala Ala 225 :~: 230 235 240 Ala Leu Leu Thr Glu Arg Leu Phe Glu Thr Phe Ala Glu Val Ala Arg Gln Glu Met Pro Glu Gly Ser Pro Leu Tyr Ile Ser Gly Gly Cys Gly WO95/29253 2188257 r~"l c ,~ I

260 265 2~0 Leu Asn Cys Asp Trp Asn Ser Leu Trp Ala Gln Leu Gly His Phe Ser Ser Val Phe VA1 Ala Pro Cys Thr Asn Asp Ser Gly Ser Ala Leu Gl Thr Ala Ile Asp Ala Leu Thr Thr Phe Thr Gly Asp Pro His Val As Trp Ser Val Tyr Ser Gly Leu Glu Phe Val Thr Asp Thr Gln Pro As Pro Ala Arg Trp Thr Ser Arg Pro Leu Glu His Asp Glu Leu Ser Gly Ala Leu Ala Gly Gly Arg Val Val Ala Trp Val Gln Gly Arg Trp Glu Ile Gly Pro Arg Ala Leu Cys Asn Arg Ser Leu Leu Ala Glu Pro :2he Gly Ala Val Thr Arg Asp Arg Leu Asn Glu Ile Lys Gln Arg Glu As Tyr Arg Pro Ile Ala Pro Val Cys Arg Val Glu Asp Leu Gly Lys Val 405 0.10 415 ~ --Phe His Glu Asp Phe Glu Asp Pro Tyr Met Leu Tyr Phe Arg Arg Val 420 425 ~30 Arg Glu Ser Ser Gly Leu Arg Ala Val Thr His Val Asp Gly Ser Ala Arg Val Gln Thr Val Arg Asp Ser Gly Asn Pro Gln Met His Ar 450 455 460 g Leu Leu Ser Ala Phe Ala Ala Gln Ar Gl 465 470 g y Val Gly Val Leu Cys Asn Thr Ser Leu Asn Phe Asn Gly Glu Gly Phe Ile Asn Arg Met Ser Asp Leu Val Leu Tyr Cys Glu Ser Arg Gly Ile Ser Asp Met Val Val Gly As 30 Thr Trp Tyr Gln Arg Ala Glu Gly ( 2 ) INFORMATION FOR SEQ ID NO: 6:
(i) SEQUENCE rHARAr'rRR~CTICS:
(A) LENGTH: 310 a~ino aclds ( B ) TYPE: a~ino acid (C) S'rRAl~)Rr~CC: single W0 95/29253 r~
71882~7 ( D ) TOPOLOGY: 1 inear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
Met Ser Asp Lys Thr Val Pro Val Phe Ser Met Ala Glu Leu Arg Asp 5 10 : 15 Gly Ser Arg Gln Asp Glu Phe Arg Glu Trp Ala Arg Arg Gly Val Phe Tyr Leu Thr Gly Tyr Gly Ala Thr Glu Arg Asp His Arg Val Ala Thr Asp Thr Ala Met Asp Phe Phe Ala Gln Gly Thr Ala Glu Glu Lys Gln Ala Val Thr Thr Lys Val Pro Thr Met Arg Arg Gly Tyr Ser Ala Leu 65 . 70 75 . 80 Glu Ala Glu Ser Thr Ala Gln Val Thr Asn Thr Gly Thr Tyr Thr As 85 90 : 95 Tyr Ser Met Ser Tyr Ser Met Gly Ile Gly Gly Asn Leu Phe Pro Ser 100 105 . 110 Lys Glu Phe Glu Ser V~l Trp Thr Asp Tyr Phe Asp Ser Leu Tyr Arg 115 120 . 125 A1A Ala Gln GIu Thr Ala Arg Leu Val Leu Thr A1A Ala Gly Thr Tyr 130 135 140 ~
Asp Gly Glu Asp Leu Asp Thr Leu Leu Asp Cys Asp Pro Val Leu Ar 145 : 150 155 160 Leu Arg Tyr Phe Pro Glu Val Pro Glu His Arg Ala Ala Glu Tyr Glu 165 170 : 175 Pro Arg Arg Met Ala Pro His Tyr Asp Leu Ser Ile Ile Thr Phe Ile la0 185 190 His Gln Thr Pro Cys Ala Asn Gly Phe Val Ser Leu Gln Ala Glu Val 195 200 2~5 Asp Gly Glu ~et Val 3er Leu Pro::His Val Glu Asp Ala Va V
210 215 220 1 al Val Leu Cys Gly Ala Ile Ala Pro Leu Va1 Thr Gln Gly Ala Val Pro Ala 225 230 ~= 235 : 240 Pro Asn His Hi3 Val Val Ser Pro Asp Ala Ser Met Leu Lys Gly Ser WO 95l29253 r~ s 2~

Asp Arg Thr Ser Ser Val Phe Phe Leu Arg Pro Ser Thr Asp Phe Thr Phe Ser Val Pro Asp Ala Arg Lys Tyr Gly Leu Asp Val Ser Leu As Met Glu Lys Ala Thr Phe Gly Asp Trp Ile Gly Thr Asn Tyr Val Th~-Met His Ala Val Thr Ser (2) INFORMATION FOR SEQ ID NO:7:
( i ) SEQUENCE CHAE~ACTERISTICS:
(A) LENGTH: 288 amino acids (B) TYPE: amino aoid ( C ) sTRAl~nRnt~Ec c: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCP~IPTION: SEQ ID No:7:
Val Pro Thr Val Ser Ser Pro Leu Tyr Tyr Ala Ala Pro Leu Thr Pro Asp Gly Gly Asp Gly Asp Trp Cys Ile Asp Ala Val Thr Arg Pro Pro Glu Val Leu Phe Asn Phe Arg Lys Val Gly Val Glu Thr Thr Ile Thr Asp Leu Arg Glu Gly Ser Ile Arg Pro Ala Leu Asp Glu Thr Gly Phe Glu Lys Val Thr Ala Pro Thr Gly Ala Ser Gln Arg Gly Leu Leu As Ser Glu Glu Ala Ala Leu Glu Gln Tyr Arg Arg Glu Thr Gly Glu Leu Leu Arg Ser Leu Thr Gly Ala Asp Val Val Glu Phe Phe Asp Ala Th Leu Arg Arg Gln Asp Ala Ala Asp Asp Pro Ala Ala Gln Ser Pro His Gln Arg Val His Val Asp Gln Ser Pro Gly Ser Ala Arg Ala Arg Ala Glu Arg His Leu Gly Pro Gly Arg Glu Phe Arg Arg Phe Gln Ile Ile WO95/29253 2 1 882~ 7 J.~

Asn Val Trp Arg Pro Leu Leu Glu Pro Val Ar A
165 ~ 170 g sn Phe Pro Leu Ala Leu Cys Asp Tyr Arg Ser Leu Asp Leu Ser Ala Asp Leu Val Pro Thr 18~ 185 190 Arg Leu Asp Phe Pro Asp Trp Leu Lys Asp Arg Glu Asn l9S 200 Tyr Ser Val Arg His Asn Pro Ala His Arg Trp Tyr Phe Trp Asp Ser Leu Thr Pro 210 215 : 220 Ala Glu Ala Leu Val Phe Lys Cys r Asp Ser Ala Ser Ar 225 .~:: 2~0 Ty 235 : 240 Ala Met ALa Gly Gly Glu Pro Asp Gly Gly Glu Leu Arg Asp VA1 Ala Gly Leu Cys Pro His Thr Ala Phe Phe Asp Glu Asn G

Gly His Leu Arg Thr Ser Leu Glu Leu Ar 2'75 280 g Ala Leu Ala Phe Hls Glu ( 2 ) INFORMATION FOR SEQ ID NO: 8:
( i ) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 236 amino acids (B) TYPE: ~nino acid (C~ S'rBANnFn1~ single ~D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
Met Thr Asp Thr Thr Arg Gln Asp Phe Leu Asp Leu Asn Leu Phe Ar Gly Leu Gly G1u Asp Pro Val Tyr His Pro Pro Val Le,u Ala Asp A
20 25 . 30 Pro Arg Asp Trp Pro Leu Asp Arg Trp Ala Glu Ala Pro Arg Asp Leu Gly Phe Ser Asp Phe Ala Arg Tyr Gln Trp Arg G1y Leu Arg Met L
50 SS 60 eu Lys Asn Pro As~Thr Gln Ala Ala Tyr His Asp Leu Met Val Glu Leu Arg Pro Arg Thr Val Ile Glu Leu Gly Val r Ser Gl Gl Ser Leu 8 S Ty Y Y

WO9~/29253 21 882~7 ~ e,'~
.

Ala Arg Phe Arg Asp Met Ala Glu Leu Met Gly Phe Asp Cys Gln Val 100 105 ~ 110 Leu Gly Ile Asp Arg Asp Leu Ser Arg Cys Gln Ile Pro Glu Ser Glu ~et Lys Asn Ile Ser Leu Arg Glu Ala Asp Cys Ser Asp Leu Ala Thr Phe Glu His Leu Arg Asp Leu Pro His Pro Leu Val Phe Ile Asp Asp Ala His Ala Asn Thr Phe Asn Ile Leu Arg Trp Ser Val Asp His Leu Leu His Glu Gly Asp Tyr Phe Ile Ile Glu Asp Met Ile Pro Tyr Trp Tyr Arg Tyr Ser Pro Lys Leu Leu Thr Glu Tyr Leu Ala Ala Phe Ala Gly Glu Leu Ser Met Asp Met Val Tyr Ala Asn Ala Ser Ser Gln Leu Glu Arg Gly Val Leu Arg Arg Ser Ala Pro Lys Ala

Claims (20)

WHAT IS CLAIMED IS:

1. An isolated and purified DNA molecule encoding a late enzyme of cephamycin biosynthesis.

2. The isolated and purified DNA molecule of claim 1, wherein said DNA encodes a protein having 3'-hydroxymethylcephem O-carbamoyltransferase activity.

3. The DNA molecule of claim 2 wherein said DNA
comprises a nucleotide sequence:

TGCTCTACTGCGAATCCCGCGGCATCTCGGACATGGTCGTCGGCGATACCTGGTACCAGCGTGCCGAGG
GCTGACCCCGGCGCCGGACGGCCGTGAGCGCGAGCGTCCGGCGG (SEQ.ID.NO.:1) or functional derIvative thereof.

4. The isolated and purified DNA molecule of claim 1, wherein said DNA encodes a protein having 3'-methylcephem-hydroxylase activity.

5. The DNA molecule of claim 4 wherein said DNA
comprises a nucleotide sequence:

(SEQ.ID.NO.:2) or functional derivative thereof.

6. The isolated and purified DNA molecule of claim 1, wherein said DNA encodes a protein having C-7 hydroxycephem methyltransferase activity.

7. The DNA molecule of claim 6 wherein said DNA
comprises a nucleotide sequence:
CGTGCCGACCGTGTCGAGTCCGCTGTACTACGCCGCCCCGCTGACCCCGGACGGCGGGGACGGGGACTG
GTGCATCGACGCCGTGACCCGGCCGCCGGAGGTGCTGTTCAACTTCCGCAAGGTGGGCGTGGAGACGAC
CATCACCGACCTGCGCGAGGGCTCGATCCGGCCGGCGCTAGACGAAACGGGGTTCGAGAAGGTCACCGC

(SEQ.ID.NO.:3) or functional derivative thereof.

8. The DNA molecule of claim 6 wherein said DNA
comprises a nucleotide sequence:

(SEQ.ID.NO.:4) or functional derivative thereof.

9. A process for the production of cephamycin antibiotics by a cell culture wherein said cells contain one or more recombinant genes encoding one or more late enzymes of cephamycin biosynthesis.

10. The process of claim 9 wherein the late enzymes of cephamycin biosynthesis are selected from the group consisting of 3'-hydroxymethylcephem O-carbamolytransferase, 3'-methylcephem-hydroxylase, and C-7 hydroxycephem methyltransferase.

11. The process of claim 9 wherein the late enzymes of cephamycin biosynthesis are selected from the group consisting of 3'-hydroxymethylcephem O-carbamolytransferase, 3'-methylcephem-hydroxylase, and C-7 hydroxycephem methyltransferase and wherein said cell culture is capable of cephamycin antiobiotic biosynthesis.

12. The process of claim 11 wherein said cells capable of cephamycin biosynthesis are a species of Nocardia.

13. An isolated and purified protein wherein said protein is a late enzyme of cephamycin biosynthesis.

14. The isolated and purified protein of claim 13, wherein said protein has 3'-hydroxymethylcephem O-carbamoyltransferase activity.

15. The protein of claim 14 wherein said protein comprises an amino acid sequence:

S G N P Q M R R L L S A F A A Q R G V G V L C N T S L N F N G E G F I
N R M S D L V L Y C E S R G I S D M V V G D T W Y Q R A E G
(SEQ.ID.NO.:5) or functional derivative thereof.

16. The isolated and purified protein of claim 13, wherein said protein has 3'-methylcephem-hydroxylase activity.

17. The protein of claim 16 wherein said protein comprises an amino acid sequence:

(SEQ.ID.NO.:6) or functional derivative thereof.

18. The isolated and purified protein of claim 13, wherein said protein has C-7 hydroxycephem methyltransferase activity.

19. The protein of claim 18 wherein said protein comprises an amino acid sequence:

thereof.

20. The protein of claim 18 wherein said protein comprises an amino acid sequence:

or functional derivative thereof.