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
1999-05-17
2003-02-11
Marx, Irene (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
C435S043000, C435S045000, C435S047000, C435S046000, C435S051000
Reexamination Certificate
active
06518039
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to the field of fermentative production of N-deacylated cephalosporin compounds, such as 7-ADCA.
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 is described by Ingolia and Queener, Med. Res. Rev. 9 (1989), 245-264 (biosynthesis route and enzymes), and Aharonowitz, Cohen, and Martin, Ann. Rev. Microbiol. 46 (1992), 461-495 (gene cloning).
The first two steps in the biosynthesis of penicillin in
P. chrysogenum
are the condensation of the three amino acids L-5-amino-5-carboxypentanoic acid (L-&bgr;-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. This compound contains the typical &bgr;-lactam structure.
These first two steps in the biosynthesis of penicillins are common in penicillin, cephamycin and cephalosporin producing fungi and bacteria.
The third step involves the exchange of the hydrophilic D&bgr;-aminoadipic acid side chain of isopenicillin N by L-5-amino-5-carboxypentanoic acid by the action of the enzyme acyltransferase (AT). The enzymatic exchange reaction mediated by AT takes place inside a cellular organelle, the microbody, as has been described in EP-A-0448180.
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.
The only directly fermented penicillins of industrial importance are penicillin V and penicillin G, produced by adding the hydrophobic side chain precursors phenoxyacetic acid or phenylacetic acid, respectively, during fermentation of
P. chrysogenum,
thereby replacing the side chains of the natural &bgr;-lactams with phenoxyacetic acid or phenylacetic acid.
Cephalosporins are much more expensive than penicillins. One reason is that some cephalosporins (e.g. cephalexin) are made from penicillins by a number of chemical conversions. Cephalosporin C, by far the most important starting material in this respect, is very soluble in water at any pH, thus implying lengthy and costly isolation processes using cumbersome and expensive column technology. Cephalosporin C obtained in this way has to be converted into therapeutically used cephalosporins by a number of chemical and enzymatic conversions.
The cephalosporin intermediate 7-ADCA is currently produced by chemical derivatization of penicillin G. The necessary chemical steps to produce 7-ADCA involve the expansion of the 5-membered penicillin ring structure to a 6-membered cephalosporin ring structure.
Recently, fermentative processes have been disclosed to obtain 7-ADCA.
In EP-A-0532341 the application of an adipate (5-carboxypentanoate) feedstock was shown to result in formation of a penicillin derivative with an adipyl side chain, viz. adipyl-6-aminopenicillanic acid. This incorporation is due to the fact that the acyltransferase has a proven wide substrate specificity (Behrens et al., J. Biol. Chem. 175 (1948), 751-809; Cole, Process. Biochem. 1 (1966), 334-338; Ballio et al., Nature 185 (1960), 97-99). In addition, when adipate is fed to a recombinant
P. chrysogenum
strain expressing an expandase, the adipyl-6-APA is expanded to its corresponding cephalosporin derivative. Finally, the removal of the adipyl side chain is suggested, yielding 7-ADCA as a final product.
The patent application EP-A-0540210 describes a similar process for the preparation of 7-ACA, including the extra steps of converting the 3-methyl group of the ADCA ring into the 3-acetoxymethyl group of ACA.
WO95/04148 and WO95/04149 disclose a feedstock of certain thiogroup-containing dicarboxylic acids with a chain length of 6 or 7 atoms to an expandase-expressing
P. chrysogenum
strain, resulting in the incorporation of these precursors into the penicillin backbone and subsequent expansion to the corresponding 7-ADCA derivatives.
In general, it is however thought that an expandase that may provide the crucial link between penicillin N and cephalosporin biosynthesis has a narrow specificity (Maea et al.,Enzyme and Microbial Technology (1995) 17: 231-234; Baldwin et al., J. Chem. Soc. Chem. Commun. 374-375, 1987), preventing the possibility for catalysing the oxidative ring expansion of penicillin N with unnatural side chains.
It now surprisingly is found that a feedstock of dicarboxylic acids with a chain length which is longer than 7 carbon atoms produce &bgr;-lactam derivatives incorporating a side chain with a chain length of either 6 or 7 atoms.
SUMMARY OF THE INVENTION
The present invention discloses a process for the production of an N-deacylated cephalosporin compound comprising the steps of:
fermenting a microbial strain capable of &bgr;-lactam production and expressing acyltransferase as well as expandase activity, and optionally acetyltransferase and/or hydroxylase activity, in the presence of a side chain precursor according to formula (1)
HOOC—x—(CH
2
)
n
—COOH (1)
wherein
n is an even number of at least 2, and
X is (CH
2
)
p
-A-(CH
2
)
q
, wherein
p and q each individually are 0, 1, 2, 3 or 4, and
A is CH═CH, C≡C, CHB, C═O, O, S, NH, the nitrogen optionally being substituted or the sulfur optionally being oxidized, and B is hydrogen, halogen, C
1-3
alkoxy, hydroxyl, or optionally substituted methyl, with the proviso that p+q should be 2 or 3, when A is CH═CH or CO≡C, or p+q should be 3 or 4, when A is CHB, C═O, O, S or NH,
or a salt, ester or amide thereof, said side chain precursor yielding a acyl-6-APA derivative, the acyl group having a structure according to formula (2)
HOOC—X—CO— (2)
wherein X is defined as above,
said acyl-6-APA derivative being in situ expanded to the corresponding acyl-7-ADCA derivative, and optionally further reacted to the acyl-7-ADAC or acyl-7-ACA derivative,
recovering the acyl-7-cephalosporin derivative from the fermentation broth
deacylating said acyl-7-cephalosporin derivative, and
recovering the crystalline 7-cephalosporin compound.
DETAILED DESCRIPTION OF THE INVENTION
The present invention discloses a process for the production of N-deacylated cephalosporin compounds (7-ADCA, 7-ADAC or 7-ACA) via the fermentative production of their acylated counterparts, applying a feed of novel side chain precursors.
The present invention surprisingly shows that fermentation of a microbial strain capable of &bgr;-lactam production and expressing acyltransferase as well as expandase activity in the presence of a dicarboxylic acid having a chain length which is longer than 7 atoms results in the formation of an acyl-7-ADAC derivative incorporating an acyl group with a chain length of 6 or 7 atoms, respectively.
According to the invention, additional 7-acylated cephalosporin derivatives than acyl-7-ADCA, i.e. acyl-7-ADAC or acyl-7-ACA, respectively, are produced by a microbial strain capable of &bgr;-lactam production and expressing acyltransferase as well expandase, if said microbial strain additionally expresses hydroxylase or hydroxylase plus acetyltra
Bovenberg Roelof Ary Lans
De Vroom Erik
Lugtenburg Johannis
Nieboer Maarten
Schipper Dirk
DSM N.V.
Marx Irene
Morrison & Foerster / LLP
LandOfFree
Process for the fermentative production of deacylated... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Process for the fermentative production of deacylated..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Process for the fermentative production of deacylated... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3177834