Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
1992-10-29
2001-11-27
Guzo, David (Department: 1636)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S455000, C435S456000, C435S320100, C435S471000, C536S023100, C536S023200, C536S023500, C536S023700
Reexamination Certificate
active
06322998
ABSTRACT:
BACKGROUND OF THE INVENTION
Beta-lactam antibiotics are among the most important clinically, and the isolation of novel &bgr;-lactam compounds continues six decades after the discovery of the penicillins by Fleming. The common structural feature of the penicillins and cephalosporins (including cephamycins) is the &bgr;-lactam ring.
These antibiotics are produced by a variety of prokaryotes and lower eukaryotes. The penicillins, exemplified by the compounds penicillin G (benzyl-penicillin) or penicillin V, are produced by filamentous fungi, most notably
Penicillium chrysogenum
. The cephalosporins, first isolated as a product from the lower eukaryote,
Cephalosporium acremonium
(syn.
Acremonium chrysogenum
), are also metabolites of many prokaryotes, especially
Streptomyces clavuligerus, S. lipmanii
and
S. cattleya
, that also produce cephamycins and other &bgr;-lactams such as oxypenams (clavulanic acid) and carbapenems (thienamycin).
The development of cell-free systems from &bgr;-lactam-producing organisms has allowed the establishment of the biosynthetic steps in the pathway of the sulfur-containing &bgr;-lactams (penicillins and cephalosporins).
The initial steps in the formation of penicillins in filamentous fungi (e.g.,
P. chrysogenum
), and the cephalosporins produced by both prokaryotes (e.g.,
S. clavuligerus
) and lower eukaryotes (e.g.,
C. acremonium
), are identical. ACV synthetase catalyzes the condensation of the amino acid precursors L-&agr;-aminoadipate, L-cysteine, and L-valine to the tripeptide LLD-ACV. The next step forms the first &bgr;-lactam in the pathway by the cyclization of the tripeptide yielding isopenicillin N (IPN), a precursor to all penicillins, cephalosporins and cephamycins.
After synthesis of IPN, the pathways to cephalosporins and penicillins diverge. In
P. chrysogenum
, for example, the &agr;-aminoadipyl side chain of IPN can be exchanged for one of many (nearly 100 to date) hydrophobic side chains derived from the corresponding acyl CoA. One of the most familiar examples is the formation of penicillin G (benzylpenicillin) from phenylacetyl CoA and IPN. However, in the fungus
C. acremonium
, the &agr;-aminoadipyl side chain is isomerized to produce penicillin N. The five-membered thiazolidine ring of the penicillin is then “expanded” to the six-membered dihydrothiazine ring that is characteristic of the cephalosporins. This reaction is catalyzed by deacetoxycephalosporin C synthetase (DAOCS) and produces the first cephalosporin in the pathway, deacetoxycephalosporin C (DAOC).
The present invention provides DNA compounds comprising an isolated DNA sequence encoding an activity involved in the production of penicillins and cephalosporins, said activity being referred to as &dgr;-(L-&agr;-aminoadipyl)-L-cysteinyl-D-valine (ACV) synthetase. The present invention expands the repertoire of beta-lactam biosynthetic enzymes which can be overproduced. This ability facilitates the bioconversion of substrate analogs to novel beta-lactams and strain improvement by increased gene dosage.
SUMMARY OF THE INVENTION
The present invention comprises DNA sequences that encode the enzyme ACV synthetase of
Cephalosporium acremonium
. The enzyme catalyzes the condensation of the amino acid precursors L-&agr;-aminoadipate, L-cysteine, and L-valine to the tripeptide &dgr;-(L-&agr;-aminoadipyl)-L-cysteinyl-D-valine (ACV). The reaction is a critical step in the biosynthesis of the beta-lactam antibiotics. This pathway is depicted in FIG.
1
.
The DNA compounds of the present invention encode the ACV synthetase enzyme of
Cephalosporium acremonium
. A preferred DNA compound of the invention that encodes the ACV synthetase activity was isolated from
Cephalosporium acremonium
genomic DNA. The cloned ACV synthetase gene is useful for increasing the yield of penicillins and cephalosporins in fungi, particularly if the reaction catalyzed by ACV synthetase is the rate-limiting step. This is accomplished by increasing the gene dosage or placement of the coding region behind a strong promoter.
The DNA compound that encodes the ACV synthetase activity can be used to construct recombinant DNA expression vectors. Four types of these expression vectors are especially useful: the first drives high-level expression of the ACV synthetase protein in
E. coli
; the second in Penicillium; the third in Aspergillus; and the fourth in Cephalosporium.
The following section provides a more detailed description of the present invention. For purposes of clarity and as an aid in the understanding of the invention the following items are defined below.
AmR—the apramycin resistance-conferring gene.
ApR—the ampicillin resistance-conferring gene; also used to denote the ampicillin-resistant phenotype.
bp—a base pair of double-stranded DNA.
cLEU2p—Cephalosporium LEU2 promoter.
cpcbCp—the Cephalosporium IPNS promoter.
HPT—hygromycin B phosphotransferase coding region.
IPNS—isopenicillin N synthetase.
Isopenicillin N synthetase—an enzyme, also known as cyclase or IPN cyclase, that catalyzes the formation of isopenicillin N from &dgr;-(L-&agr;-aminoadipyl)-L-cysteinyl-D-valine.
Isolated DNA compound—Any DNA sequence, however constructed or synthesized, which is locationally distinct from its natural location in genomic DNA. The definition includes the isolated DNA sequence in all its forms other than the natural state. For example, the DNA sequence may be inserted into a plasmid or phage vector or inserted into the genome of the organism from which it came or any other organism to increase the gene dosage.
lacZ&agr;—the promoter and &bgr;-galactosidase (lacZ) &agr;-fragment derived from the
E. coli
lac operon.
ORI—a plasmid or vector origin of replication, the DNA sequence that serves as an attachment or start site for DNA polymerase.
pcbc—the IPNS gene.
phlR—the phleomycin resistance gene.
ppcbC ORF—the IPNS coding sequence.
ppcbCp—the Penicillium IPNS promoter.
REFERENCES:
patent: 4885252 (1989-12-01), Ingolia et al.
patent: 0320272 (1989-06-01), None
Smith et al. EMBO J. 9:741 (1990).
Cantwell Cathleen A.
Hodges Roland L.
Hoskins JoAnn
Queener Stephen W.
Skatrud Paul L.
Cantrell Paul R.
Cohen Charles E.
Eli Lilly and Company
Guzo David
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