Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound having a 1-thia-5-aza-bicyclo
Reexamination Certificate
1999-01-15
2002-04-09
Nashed, Nashaat T. (Department: 1651)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound having a 1-thia-5-aza-bicyclo
C435S254110
Reexamination Certificate
active
06368820
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to the field of fermentative production of acylated cephalosporins and their conversion into modified and/or deacylated cephalosporins.
BACKGROUND OF THE INVENTION
&bgr;-Lactam antibiotics constitute the most important group of antibiotic compounds, with a long history of clinical use. Among this group, the prominent ones are the penicillins and cephalosporins. These compounds are naturally produced by the filamentous fungi
Penicillium chrysogenum
and
Acremonium chrysogenum
, respectively.
As a result of classical strain improvement techniques, the production levels of the antibiotics in
Penicillium chrysogenum
and
Acremonium chrysogenum
have increased dramatically over the past decades. With the increasing knowledge of the biosynthetic pathways leading to penicillins and cephalosporins, and the advent of recombinant DNA technology, new tools for the improvement of production strains and for the in vivo derivatization of the compounds have become available.
Most enzymes involved in &bgr;-lactam biosynthesis have been identified and their corresponding genes been cloned, as can be found in Ingolia and Queener, Med. Res. Rev. 9 (1489), 245-264 (biosynthesis route and enzymes), and Aharonowitz et al., Ann. Rev. Microbiol. 46 (1992), 461-495 (gene cloning).
The first two steps in the biosynthesis of &bgr;-lactam compounds are the condensation of the three amino acids L-5-amino-5-carboxypentanoic acid (L-&agr;-aminoadipic acid) (A), L-cysteine (C) and L-valine (V) into the tripeptide LLD-ACV, followed by cyclization of this tripeptide to form isopenicillin N, a penicillin having an a-aminoadipyl side chain. The latter compound contains the typical, &bgr;-lactam structure. These first two steps are common in penicillin, cephamycin and cephalosporin producing fungi and bacteria.
In penicillin-producing fungi, like
P. chrysogenum
, the third step involves the exchange of the hydrophilic &agr;-aminoadipyl side chain of isopenicillin N for a hydrophobic, aromatic side chain by the action of the enzyme acyltransferase. The enzymatic exchange reaction mediated by acyltransferase takes place inside a cellular organelle, the microbody, as has been described in EP448180.
In cephalosporin-producing organisms, the third step is the isomerization of isopenicillin N to penicillin N by an epimerase, whereupon the five-membered ring structure characteristic of penicillins is expanded by the enzyme expandase to the six-membered ring characteristic of cephalosporins.
Currently, there is an increasing need for the fermentative production of &bgr;-lactam compounds, especially with regard to the cephalosporin intermediates 7-aminodeacetoxycephalosporanic acid (7-ADCA), 7-aminodeacetylcephalosporanic acid (7-ADAC) and 7-aminocephalosporanic acid (7-ACA). Commercial production of these compounds currently requires extensive chemical synthesis steps, which are expensive and noxious to the environment. Fermentative routes to these compounds are described using recombinant
P. chrysogenum
strains (EP 532341 and EP 540210).
P. chrysogenum
generally is thought to be more suitable than A. chrysogenum for the fermentative production of cephalosporin intermediates, mainly because the &bgr;-lactam biosynthetic capacity of
P. chrysogenum
is higher than of
A. chrysogenum
, due to extensive strain improvement. For the cephalosporin C producer
A. chrysogenum
, strain improvement has started much later than for
P. chrysogenum
, and additionally no amplification of cephalosporin biosynthetic genes was observed (Smith et al. Curr. Genet. 19 (1991), 235-237), contrary to the penicillin biosynthetic genes which are amplified indeed (Smith et al. Mol. Gen. Genet. 216 (1 989), 492-497; Barredo et al. Curr. Genet. 16 (1989), 453-459; Fierro et al., Proc. Natl. Acad. So;. 92 (1995), 6200-6204).
Recently, it was observed that desacetoxy-cephalosporin was formed in a
P. chrysogenum
strain expressing expandase, implicating that
P. chrysogenum
might contain an epimerase activity as well (Alvi et al., J. Antibiotics 48 (1995), 338-340). This phenomenon diminishes the supposed advantage of
P. chrysogenum
above
A. chrysogenum
as a production organism for the fermentative production of extractable cephalosporins.
A chrysogenum
strains which express the Penicillium acyltransferase gene have been described (European Patent EP 357119, Gutiérrez et al., Mol. Gen. Genet. 225 (1991), 56-64), but only the production of penicillin G with said recombinant Acremonium strains is disclosed.
Crawford et al. (Bio/Technology 13 (1995), 58-62) suggest the production of adipyl-cephalosporins by feeding adipic acid to an acyltransferase expressing Acremonium strain. However, it is also indicated that this approach would result in a mixture of cephalosporins, with adipyl as well as aminoadipyl side chains, which would present difficulties for downstream processing.
SUMMARY OF THE INVENTION
The present invention discloses a fermentative process for the production of an N-acylated cephalosporin derivative.
Specifically, the process of the invention comprises the fermentation of an Acremonium strain in the presence of a suitable acyl side chain precursor, wherein said Acremonium strain is transformed with an expression cassette comprising an acyltransferase coding sequence and transformants are selected wherein said acyltransferase coding sequence is expressed to a level which leads to a production level of said N-acylated cephalosporin derivative which is similar to or higher than the production level of &agr;-aminoadipyl-7-cephalosporin derivatives.
The process of the invention further comprises the fermentation of an acyltransferase-expressing Acremonium strain in the presence of a suitable acyl side chain precursor, wherein said strain does not express hydroxylase and/or acetyltransferase activity.
The resulting N-acylated cephalosporin derivative is subsequently recovered from the culture fluid.
The N-acylated cephalosporin derivative produced according to the invention is used for the preparation of semisynthetic cephalosporins. Alternatively, the N-acylated cephalosporin derivative is deacylated by chemical or enzymatical means, to produce 7-ADCA, 7-ADAC or 7-ACA.
DETAILED DESCRIPTION OF THE INVENTION
The present invention discloses a fermentative process for the production of N-acylated cephalosporin derivatives, with the proviso that said N-attached acyl group is not the naturally occurring &agr;-aminoadipyl group. Specifically, the present invention discloses a process for the production of N-acylated cephalosporin derivatives using Acremonium as the production organism, wherein Acremonium expresses acyltransferase activity and is cultured in the presence of a suitable N-acyl side chain precursor.
A “suitable” N-acyl side chain precursor is understood to be a side chain precursor which is acceptable to the enzyme acyltransferase as well as to be a side chain precursor which produces a penicillin derivative which is amenable to ring expansion by the enzyme expandase. The &agr;-aminoadipyl side chain present in natural penicillin N-derived cephalosporin compounds is understood not to be covered by the term “suitable” N-acyl side chain.
Examples of such suitable acyl side chain precursors are adipic acid, thiodipropionic acid and carboxymethylthiopropionic acid.
In the process of the invention, an Acremonium strain is used which expresses an acyltransferase gene to a high level. A high expression level is important to minimize the production of &agr;-aminoadipyl-cephalosporin derivative relative to the production of N-acyl-cephalosporin derivative.
To obtain Acremonium strains with a high production level of N-acyl-cephalosporin derivatives relative to the level of &agr;-aminoadipyl-cephalosporins, said strains should have a sufficiently high acyltransferase expression level. A sufficiently high acyltransferase expression level is obtained by using for instance a strong promoter to direct expression of the enzyme. A sufficiently high acyltransferase expression level is fu
Bovenberg Roelof Ary Lans
Kerkman Richard
Schipper Dirk
DSM N.V.
Morrison & Foerster / LLP
Nashed Nashaat T.
Walicka Malgorzata A.
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