Penicillin conversion

Details Penicillin conversion Penicillin conversion

1999-04-22

2002-05-07

Saucier, Sandra E. (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

C435S048000, C435S049000

Reexamination Certificate

active

06383773

ABSTRACT:

BACKGROUND OF THE INVENTION
Penicillin began the antibiotic revolution. Providing the first real weapon against microbial infections, penicillin (see
FIG. 1
) first appeared to be a “magic bullet” that would cure all of man's ills. Infectious microbes soon developed resistance to penicillins, however. Great efforts in the pharmaceutical industry have focussed and still focus on the development of alternative antibiotics. One of the most useful families of agents is the cephalosporins (see FIG.
2
).
The first cephalosporin, cephalosporin C, was isolated from
Cephalosporium acremonium
(also known as
Acremonium chrysogenum
) in 1954.
C. acremonium
produces cephalosporin C by first synthesizing penicillin N, and then converting this penicillin into cephalosporin C according to the pathway presented in FIG.
3
. As shown in
FIG. 3
, penicillin N is first converted to deacetoxycephalosporin C (DAOC) through oxidative expansion catalyzed by an enzyme known as “DAOC synthase” (DAOCS), or “expandase”. A hydroxylase activity, which in
C. acremonium
is part of the same DAOCS enzyme, then converts the DAOC to deacetylcephalosporin C (DAC). In the final step of the conversion, an acetyl transferase substitutes an acetoxy group for the DAC hydroxyl and thereby produces cephalosporin C.
Further study revealed that
C. acremonium
is not the only organism that produces cephalosporins from penicillin N. In particular,
S. clavuligerus
also has both expandase and hydroxylase activities, which activities are separable from one another in this organism. Unfortunately, however, no organism has been identified that naturally produces any commercially useful cephalosporin. Commercially useful cephalosporins (see, for example,
FIG. 2B
) are typically produced by chemical ring expansion of, for example, penicillin G to yield deacetoxycephalosporin G. Other cephalosporins can then be produced through enzymatic removal of the deacetoxycephalosporin G side chain (phenylacetyl) and substitution of a different side chain. The multi-step chemical ring expansion process is time consuming, expensive, and polluting.
Alternatively, commercially useful cephalosporins could be produced by isolating either the DAOC or the DAC intermediate from
C. acremonium
or
S. clavuligerus
fermentations, and chemically treating the isolate to eliminate the D-&agr;-aminoadipyl side chain and produce a substrate (7-aminodeacetoxycephalosporanic acid [7-ADCA] or 7-aminodeacetylcephalosporanic acid [7-ADAC]) that can subsequently be chemically treated to generate a medically useful cephalosporin (see FIG.
4
). Although it avoids the chemical ring expansion step, this strategy is also expensive, since the levels of DAOC or DAC that naturally accumulate are small. There is a need for an improved system for producing cephalosporins.
In particular, there is a need to develop a system that allows cephalosporin production from a penicillin other than penicillin N. Preferably, the system would allow cephalosporin production from an inexpensive penicillin such as penicillin G or penicillin V. As shown in
FIG. 5
, penicillin G conversion would produce intermediates (deacetoxycephalosporin G [DAOG], deacetylcephalosporin G [DAG]) that could be treated with penicillin acylase to produce the same 7-ADCA or 7-ADAC substrates mentioned above.
Various efforts have been made to utilize the
C. acremonium
or
S. clavuligerus
expandase enzyme either alone or with a hydroxylase enzyme to convert penicillins other than penicillin N into a cephalosporin or cephalosporin intermediate or substrate. Such efforts have almost uniformly failed. Many researchers have reported that the
C. acremonium
and
S. clavuligerus
expandase enzymes have very narrow specificity and fails to expand penicillins other than penicillin N and certain very close relatives.
For example, Kohsaka and Demain, the original discoverers of
C. acremonium
expandase, have reported that only penicillin N, and not penicillin G or 6-aminopenicillanic acid (6-APA), are substrates for expandase activity in crude extracts (Kohsaka et al.,
Biochem. Biophys. Res. Commun.
70(2):1976:465-473, 1976; Demain et al., U.S. Pat. No. 4,178,210, issued Dec. 11, 1979). Further work by this group has demonstrated that partially purified enzyme does not expand adipyl-6-APA, ampicillin, or penicillin G (Kupka et al.,
FEMS Microbiol. Lett.
16:1-6, 1983).
Similarly, researchers have reported that the
S. clavuligerus
expandase expands the ring of penicillin N, but not that of at least twenty other penicillins, including penicillin G, penicillin V, penicillin K, penicillin dihydroF, adipyl-6-APA, m-carboxyphenylacetyl-6-APA, ampicillin, butyryl-6-APA, D-glutamyl-6-APA, and ampicillin (Jensen et al.,
J. Antibiot.
35:1351-1360, 1982; Dotzlaf et al.,
J. Biol. Chem.
264:10219-10227, 1989; Yeh et al. in 50
Years of Penicillin: History and Trends
[Kleinkauf et al., eds.], Public, Prague, pp. 208-223, 1994; Maeda et al.,
Enzyme Microb. Technol.
17:231-234, 1995).
One group has reported that
Penicillium chrysogenum
cells that have been engineered to express the
S. clavuligerus
expandase gene can produce adipyl-7-aminodeacetoxycephalosporanic acid (adipyl-7-ADCA) when grown in the presence of adipic acid (Conder et al., U.S. Pat. No. 5,318,896, issued Jun. 7, 1995; Crawford et al.,
Bio/Technol.
13:58-62, 1995).
P. chrysogenum
cells are capable of converting adipic acid to adipyl-6-APA; the observation of adipyl-7-ADCA production by the recombinant cells therefore suggests that the
S. clavuligerus
expandase, when expressed in
P. chrysogenum
cells, may be able to expand the endogenous adipyl-6-APA.
A small number of other studies have reported some ability of
S. clavuligerus
or
C. acremonium
expandase enzymes to expand D-carboxymethylcysteinyl-6-APA, a very close relative to penicillin N (Bowers et al.,
Biochem. Biophys. Res. Commun.
120:607-614, 1984) and adipyl-6-APA (Baldwin et al.,
J. Chem. Soc. Chem. Commun.
1466:374-375, 1987; Shibata et al.,
Bioorg. Med. Chem. Lett.
6:1579-1584, 1996), in vitro. One group (Baldwin et al.,
J. Chem. Soc. Chem. Commun.
1466:374-375, 1987) has also suggested that m-carboxyphenylacetyl-6-APA, D-glutamyl-6-APA, and glutamyl-6-APA might also serve as in vitro substrates, albeit at very low levels. Subsequent work failed to confirm these reports, however (Yeh et al., in 50
Years of Penicillin: History and Trends [Kleinkauf et al, eds], Public, Prague, pp.
208-223, 1994).
One brief abstract reported that a recombinant form of
S. clavuligerus
expandase, when expressed in and purified from
Escherichia coli,
might be able to expand penicillin G (Baldwin et al., Abstract P-262, Abstracts of the 7th International Symposium on Genetics of Industrial Microorganisms, Montreal, Jun. 26-Jul. 1, 1994, pg. 184). Unfortunately, the report did not contain sufficient detail to allow ready duplication of the results and no subsequent work has confirmed the finding.
Thus, the prior art attempts to develop an improved system for producing cephalosporins from penicillins other than penicillin N have generally failed. In particular, efforts to develop a system that utilizes penicillin G as a substrate have been unsuccessful. There remains a need for development of improved systems for converting penicillins other than penicillin N. Particularly desirable systems would utilize exogenously-added penicillins rather than relying on in vivo microbial penicillin production. Particularly preferred systems would obviate the need for multi-step chemical ring expansion methods.
SUMMARY OF THE INVENTION
The present invention provides techniques and reagents for the bioconversion of penicillins other than penicillin N into cephalosporins or cephalosporin precursors. The inventive conversion system allows biological ring expansion of penicillin substrates such as penicillin G, and replaces the multi-step chemical ring expansion process currently performed in industry. The inventive system can

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