Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound having a 1-thia-5-aza-bicyclo
Patent
1998-02-18
2000-02-01
Achutamurthy, Ponnathapu
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound having a 1-thia-5-aza-bicyclo
435 43, 435 46, 435 51, 4352545, 435218, C12P 3702, C12P 3502, C07D50100
Patent
active
060201517
DESCRIPTION:
BRIEF SUMMARY
FIELD OF THE INVENTION AND BRIEF DESCRIPTION OF THE PRIOR ART
The present invention concerns a biosynthetic process for preparation and recovery of 7-aminodesacetoxycephalosporanic acid (7-ADCA).
.beta.-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 .beta.-lactam biosynthesis have been identified and their corresponding genes been cloned, as can be found in 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-.alpha.-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 .beta.-lactam structure.
The third step involves the exchange of the hydrophillic side chain of L-5-amino-5-carboxypentanoic acid by a hydrophobic side chain 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.
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. Another reason is that, so far, only cephalosporins with a D-5-amino-5-carboxypentanoyl side chain could be fermented. 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 methods currently favoured in industry to prepare the intermediate 7-ADCA involve complex chemical steps leading to the expansion and derivatization of penicillin G. One of the necessary chemical steps to produce 7-ADCA involves the expansion of the 5-membered penicillin ring structure to a 6-membered cephalosporin ring structure (see for instance U.S. Pat. No. 4,003,894). This complex chemical processing is both expensive and noxious to the environment.
Consequently, there is a great desire to replace such chemical processes with enzymatical reactions such as enzymatic catalysis, preferably during fermentation. A key to the replacement of the chemical expansion process by a biological process is the central enzyme in the cephalosporin biosynthetic pathway, deacetoxycephalosporin C synthetase, or expandase.
The expandase enzyme from the bacterium Streptomyces clavuligerus was found to carry out in vitro, in some cases, penicillin ring expansions (Baldwin et al., Tetrahedron 43(13), 3009 (1987)). In Cantwell et al. (Current Genetics, 17, 213-221 (1990)), expression of S. clavuligerus expandase in P. chrysogenum is described. Espression of the expandase did not result in formation of cephalosporins in a fermentation as suggested in the publications. Only when introduced into P. chrysogenum together with the isopenicillin N epimerase gene of S. clavuligerus, conversion of the
REFERENCES:
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patent: 5731165 (1998-03-01), Bovenberg et al.
patent: 5795733 (1998-08-01), Bovenberg et al.
Demain A. Enzymatic 7-ADCA: I said it couldn't be done. Biotechnology, Jan. 1995, vol. 3, No. 1, pp. 23-24.
Cantwell, C.A., et al., "Cloning and expression of a hybrid Streptomycesclavuligerus cef E gene in Penicillium chrysogenum," Curr Genet (1990) 17:213-221.
Crawford, L., et al., "Production of Cephalosporin Intermediates by Feeding Adipic Acid to Recombinant Penicillium chrysogenum Strains Expressing Ring Expansion Activity," Bio/Technology (1995) 13:58-62.
Baldwin, J.E., et al., "The Enzymatic Ring Expansion of Penicillins to Cephalosporins: Side Chain Specificity," Tetrahedron (1987) 43(13):3009-3014.
Bovenberg Roelof Ary Lans
Koekman Bertus Pieter
Schipper Dirk
Vollebregt Adrianus Wilhelmus Hermanus
Achutamurthy Ponnathapu
DSM N.V.
Mayhew Bradley S.
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