DNA encoding ACV synthetase

Details DNA encoding ACV synthetase DNA encoding ACV synthetase

1997-08-11

2001-07-10

Guzo, David (Department: 1636)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Preparing compound having a 1-thia-4-aza-bicyclo

C435S047000, C435S069100, C435S252300, C435S254100, C435S254110, C435S254300, C435S254500, C435S320100, C435S471000, C435S476000, C435S484000, C536S023200, C536S023700, C536S023740

Reexamination Certificate

active

06258555

ABSTRACT:

The present invention relates to DNA molecules, and to recombinant vectors for use in the transformation of a microbial host. In particular, the invention relates to biosynthetic genes for enzymes involved in penicillin biosynthesis, vectors comprising such genes, host cells transformed with the vectors, and the use of such host cells in penicillin production.
It has been established that the biosynthetic pathways of 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 to penicillin G or, by the action of an ‘epimerase’ enzyme, to 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.
The generation of the intermediate tripeptide ACV from its constituent amino acids is the least well understood and most difficult step to study of the entire pathway as reported by Adlington et al (
Biochem. J.
, 1983, 213, 573-576) and reviewed by S. E. Jensen in CRC
Critical Reviews in Biotechnology
, 1986, Vol. 3, Part 3, pages 277-310, and Nuesch et al (
Ann. Rev. Microbiol
, 1987, 41, 54).
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 been reported by S. M. Samson et al (Nature, 1985, 318, 191-194).
In addition, the isopenicillin N synthetase (IPNS) gene of
Penicillin chrysogenum
has been isolated and sequenced by Carr et al. (
Gene
, 1986, 48, 257-266).
The isolation and expression of certain genes of
S. clavuligerus
ATCC 27064 which are involved in the biosynthesis of &bgr;-lactams has been disclosed in European Patent Application Publication No. 0 233 715.
Hitherto, however, no DNA has been specifically identified as being of use in the production of an enzyme involved in the synthesis of ACV.
The present invention provides DNA comprising a gene encoding ACV synthetase.
As used herein the term ‘gene encoding ACV synthetase’ or ‘ACV synthetase gene’ is used to describe DNA which codes for an enzyme involved in the biosynthesis of ACV from its precursors.
The DNA of the invention may be isolated as described hereinbelow, from total or chromosomal DNA of organisms well known in the art which produce penicillins and/or cephalasporins, for example Pencillium, Aspergillus, Flavobacterium, Cephalosporium, and Streptomyces species. It will, of course, be understood that the DNA of the invention has been separated from the majority of such chromosomal DNA and is not in its ‘natural’ state, i.e. the form in which it occurs in nature. In one aspect, the DNA of the invention is in isolated and substantially purified form and/or consists essentially of the ACV synthetase gene.
In addition to the ACV synthetase gene, the DNA of the invention may additionally comprise further genes involved in the biosynthesis of penicillin and cephalosporin &bgr;-lactams, in particular the isopenicillin N synthetase (IPNS) gene and/or the acyltransferase (ACT) gene. The DNA of the invention may also comprise regulatory elements or regulatory genes involved in the biosynthesis of penicillin and cephalosporin &bgr;-lactams or may contain flanking DNA which has no particular or known function.
In a particular aspect, the DNA of the invention comprises the entire biosynthetic gene cluster for the production of a penicillin when the gene cluster is expressed in a suitable host organism, especially a fungal host. It will be appreciated that the said gene cluster has been separated from the majority of flanking chromosomal DNA and is not in its natural state.
As used herein the term “penicillin” includes isopenicillin N and also encompasses penicillins such as penicillins V and G, which may be formed when the host organism is cultured in the presence of a suitable side-chain precursor, for example phenoxyacetic acid or phenylacetic acid.
The present invention further provides a recombinant vector capable of transforming a host cell, which vector comprises the DNA of the invention. Preferably the said vector is a high expression vector capable of expressing high levels of gene transcript.
In another aspect of the invention there is provided a host cell, especially a fungal host, transformed with the recombinant vector of the invention.
The invention further provides a process for transforming a host cell with a recombinant vector according to the invention which comprises mixing together the host and recombinant vector under conventional transformation conditions.


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Smith et al.,EMBO Journal, vol. 9, No. 9, pp. 2743-2750 (1990).

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