Chemistry: molecular biology and microbiology – Vector – per se
Reexamination Certificate
2000-01-10
2004-03-23
Achutamurthy, Ponnathapu (Department: 1652)
Chemistry: molecular biology and microbiology
Vector, per se
C435S252300, C435S252350, C536S023200
Reexamination Certificate
active
06709859
ABSTRACT:
The present invention relates to recombinant DNA molecules, and in particular to recombinant vectors for use in the transformation of a microbial host which contain inserted DNA fragments carrying one or more genes coding for one or more enzymes involved in the biosynthesis of P-Lactam antibiotics, especially penicillins and cephalosporins.
Progress in understanding the biosynthesis of &bgr;-lactam antibiotics produced by micro-organisms such as
Streptomyces clavuligerus
has been slow. Nevertheless it has been established that the biosynthetic pathways of certain penicillins and cephalosporins (including cephamycins) are closely related.
Isopenicillin N is an intermediate in the biosynthesis of both groups of compounds and is formed by the action of a ‘cyclase’ enzyme on the tripeptide &dgr;(L-&agr;-aminoadipyl)-L-cysteinyl-D-valine (sometimes referred to as LLD-ACV or, more simply, ACV as used hereinbelow). The intermediate isopenicillin N may be converted either into penicillin G or, by the action of an ‘epimerase’ enzyme, into penicillin N and it is from the latter that various cephalosporins and cephamycins may be derived by a multi-step pathway following an initial ring-expansion with an ‘expandase’ enzyme. A recent summary of the state of the art is given by J. F. Martin and P. Liras in
Trends in Biotechnology,
1985, 3, 39-44. Thus, in the biosynthesis of Cephamycin C, penicillin N is converted into deacetoxycephalosporin C which is then converted by a dioxygenase enzyme into desacetylcephalosporin C.
The latter is O-carbamoylated to give O-carbamoyldesacetylcephalosporin C, which is then converted into Cephamycin C. It is probable, in the light of worK by J. D. Hood et al. (
J. Chem.Soc., Chem. Commun,
1983, pages 1187-1188 and references therein) that the
7
&agr;-methoxy group in Cephamycin C is introduced in two steps, i.e. via the action of a 7-hydroxylase enzyme on O-carbamoyldesacetylcephalosporin C to give the 7&agr;-hydroxy derivative, followed by subsequent methylation.
As is now well known, by means of recombinant DNA techniques, it is possible to insert into a host cell DNA carried on a vector with the result that the transformed host may become endowed with the capacity to synthesise whatever protein(s) or enzyme(s) the gene(s) carried on the insert DNA may encode. (For a full discussion of recombinant DNA methodology, and a glossary of the terms used therein, see ‘Principles of Gene Manipulation’ by R. W. Old and S. B. Primrose, 3rd Edition, Blackwell Scientific Publications, 1985).
The isolation and expression in
E. coli
of the isopenicillin N synthetase (cyclase) gene from
C. acremonium
has recently been reported by S. M. Samson et al (
Nature,
1985, 318, 191-194).
In order to clearly define the invention reference is made to the accompanying drawings in which:
FIG.
1
(
a
) is an endonuclease restriction map of
S. clavuligerus
ATCC 27064 chromosomal DNA (I) coding for genes involved in penicillin and cephalosporin biosynthesis;
FIG.
1
(
b
) is an endonuclease restriction map of the portion of the DNA (I) contained in a plasmid designated pBROC 138;
FIG.
1
(
c
) is an endonuclease restriction map of the portion of the DNA (I) contained in a plasmid designated pBROC 137;
FIG.
1
(
d
) is an endonuclease restriction map of a portion of the DNA (I) contained in a plasmid designated pBROC 303;
FIG. 2
is an endonuclease restriction map of plasmids pBROC 137 and pBROC 138; and
FIG. 3
is an endonuclease restriction map of plasmid pBROC 303.
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Burnham Martin Karl Rossel
Hodgson John Edward
Normansell Ian David
Achutamurthy Ponnathapu
Beecham Group p.l.c.
Kinzig Charles M.
Lockenour Andrea V.
Moore William W.
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