Process for the fermentative production of deacylated...

Details Process for the fermentative production of deacylated... Process for the fermentative production of deacylated...

1999-08-26

2002-06-25

Saidha, Tekchand (Department: 1652)

Chemistry: molecular biology and microbiology

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

Preparing compound having a 1-thia-5-aza-bicyclo

C435S049000, C435S051000, C435S183000, C435S230000, C435S252300, C435S320100, C435S935000, C435S193000, C435S197000, C536S023200, C536S023740

Reexamination Certificate

active

06410259

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 decribed 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-&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. 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-&agr;-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 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.
The present invention discloses a process for the fermentative production of cephalosporin compounds using novel side chain precursors, which has several advantages above existing processes, advantages with respect to yield and with respect to a decreased level of byproducts.
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—COOH  (1)
 wherein
X is (CH
2
)
m
—CH═A—(CH
2
)
n
or (CH
2
)
m
—C≡C—(CH
2
)
n
, wherein
m and n each individually are 0, 1, 2 or 3 and m+n =2 or 3, and
A is CH or N, or
X is (CH
2
)
p
—CH═CH—CH═C—(CH
2
)
q
, wherein
p and q each individually are 0 or 1 and p+q=0 or 1,
 or a salt, ester or amide thereof, said side chain precursor yielding an acyl-6-APA derivative incorporating said precursor, said acyl-6-APA derivative being in situ expanded to the corresponding acyl-7-ADCA derivative, optionally further reacted to the acyl-7-ADAC or acyl-7-ACA derivative, and
recovering the acyl-7-cephalosporin derivative from the fermentation broth
deacylating said acyl-7-cephalosporin derivative, and
recovering the crystalline N-deacylated cephalosporin compound.
DETAILED DESCRIPTION OF THE INVENTION
The present invention discloses a process for the production of N-deacylated cephalosporin derivatives (7-ADCA, 7-ADAC or 7-ACA) via the fermentative production of their N-acylated counterparts, applying a feed of novel side chain precursors. Using these precursors, novel N-acylated cephalosporin derivatives are formed.
According to the invention, the fermentation of a microbial strain capable of &bgr;-lactam production and expressing acyltransferase as well as expandase activity, and optionally hydroxylase or hydroxylase as well as acetyltransferase activity, in the presence of a dicarboxylic acid having one or two unsaturated bonds leads to an improved incorporation of said side chain precursor into the cephalosporin backbone. As a consequence, low levels of undesired acyl-6-APA derivatives are detectable in the process of the invention. In addition, the present invention shows that an improved yield of N-acylated cephalosporin derivative is obtained on an unsaturated precursor as compared to the yield on adipic acid.
The side chain precursor according to the invention has a structure according to formula (1):
HOOC—X—COOH  (1)
wherein
X is (CH
2
)
m
—CH═A—(CH
2
)
n
or (CH
2
)
m
—C≡C—(CH
2
)
n
, wherein m and n

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