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Penicillin conversion

 

Abstract

The present invention provides a biological system for expanding the dethiazolidine ring of penicillins into the dehydrothiazine ring of cephalosporins or cephalosporin precursors. In particular, the invention defines reaction conditions under which expandase enzyme can convert penicillin substrates other than penicillin N into cephalosporins. The invention therefore provides a relatively inexpensive, uncomplicated, and environmentally friendly biological system for cephalosporin production from penicillins, which system can replace the synthetic chemical approaches currently utilized. In particular, the invention provides a system for producing DOAG and/or DAG, which can be enzymatically converted into 7-ADCA and 7-ADAC, which, in turn, can be enzymatically or chemically converted into valuable cephalosporins of commerce.

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References Cited [Referenced By]

U.S. Patent Documents

 

Other References

Baldwin et al., "The enzymatic expansion of penicillins to cephalosporins: side chain specificity", Tetrahedron 43(13) : 3009-14 (1987).* Roy et al., "Characterization of Streptomyces sp. strain DRS-1 and its ampicillin transformation product", Folia Microbiol. 42(4) : 333-6 (1997).* Svec et al., "Engineering aspects of carriers for immobilized biocatalysts", Biotechnology and Genetic Engineering Reviews 13 : 217-35 (1996).* Baldwin et al., "Enzymatic Ring Expansion of Penicillins to Cephalosporins: Side Chain Specificity" J. Chem. Soc. Chem. Commun. 1466:374-375, 1987. Baldwin et al., "Genetic Engineering of Cephalosporin Biosynthesis" Abstract P-262 in Program and Adstracts of 7th International Symposium on Genetics of Industrial Microorganisms, Montreal, p. 184, Jun. 26-Jul. 1, 1994. Bowers et al., "Enzymatic Synthesis of the Penicillin and Cephalosporin Nuclei from an Acyclic Peptide Containing Carboxymethylcysteine" Biochem. Biophys. Res. Commun. 120(2):607-613, Apr. 30, 1984. Cortes et al., "Regulation of the biosynthesis of cephamycin C by Streptomyces lactamdurans" Biochem. Soc. Trans. 12:863-864, 1972. Crawford et al., "Production of Cephalosporin Intermediates by Feeding Adipic Acid to Recombinant Penicillium chrysogenum Strains Expressing Ring Expansion Activity" Bio/Tech., 13:58-62, Jan. 13, 1995. Demain, "Production of Beta-Lactum Antibiotics and its Regulation" Proc. Natl. Sci. Council, ROC, Part B: Life Sciences, 15(4):251-265, 1991. Demain, "Biosynthesis of .beta.-Lactam Antibiotics" In Antibiotics Containing Beta-Lactam Structure (Demian and Solomon, eds.), Springer-Verlag, Berlin, 189-228, 1983. Dotzlaf et al., "Purification and Properties of Deacetoxycephalosporin C Synthase from Recombinant Escherichia coli and Its Comparison with the Native Enzyme Purified from Streptomyces clavuligerus" J. Biol. Chem. 264(17):10219-10227, Jun. 15, 1989. Dotzlaf et al., "Copurification and Characterization of Deacetoxycephalosporin C Synthase/Hydroxylase from Cephalosporium acremonium" J. Bacteriol., 164(4):1611-1618, Apr., 1987. Durckheimer et al., "Recent Developments in the Field of Cephem Antibiotics" Adv. Drug Res. 17:61-234, 1988. Jensen et al., "Analysis of Penicillin N Ring Expansion Activity from Streptomyces clavuligerus by Ion-Pair High-Pressure Liquid Chromatography" Antimicrobial. Agents Chemo. 24(3):307-312, Sep. 1983. Jensen et al., "Beta Lactams" Genetics and Biochem. of Antibio. Prod., 239-268, 1995. Jensen et al., "Cephalosporin Formation by Cell-Free Extracts from Streptomyces clavuligerus" J. Antibiot. 35(10):1351-1360, 1982. Kirrstetter et al., "Development of new .beta.-lactam antibiotics derived from natural and synthetic sources" Die Pharmazie 44:178-184, Mar., 1989. Kohsaka et al., "Conversion of Penicillin N to Cephalosporin(s) By Cell-Free Extracts of Cephalosporium acremonium" Biochem. Biophys. Res. Commun. 70(2):465-473, 1976. Kovacevic et al., "Cloning, Characterization, and Expression in Escherichia coli of the Streptomyces clavuligerus Gene Encoding Deacetoxycephalosporin C Synthetase" J. Bacteriol. 171(2):754-760, 1989. Kupka et al., "Partial purification and properties of the .alpha.-ketoglutarate-linked ring-expansion enzyme of .beta.-lactam biosynthesis of Cephalosporium acremonium" FEMS Microbiol. Lett. 16:1-6, 1983. Maeda et al., "The substrate specificity of deaetoxycephalosporin C synthase ("expandase") of Streptomyces clavuligerus is extremely narrow" Enzyme and Microb. Tech. 17:231-234, 1995. Mahro et al., "In vivo conversion of penicillin N into a cephalosporin type antibiotic by a non-producing mutant of Streptomyces clavuligerus" Appl. Microbiol. Biotech. 27:272-275, 1987. Martin et al., ".beta.-Lactams" In Fungal Biotechnology (Anke, ed.), Chapman & Hall, Weinheim, 91-127, 1997. Shen et al., "Desacetoxycephalosporin C synthetase: importance of order of cofactor/reactant addition" Enzyme Microb. Technol. 6:402-404, 1984. Shibata et al., "Adipoyl-6-Aminopenicillinic Acid is a Substitute for Deacetoxycephalosporin C Synthase (DAOCS)" Bioorg. & Med. Chem. Lett. 6(13):1579-1584, 1996. Stapley et al., "Cephamycins, a New Family of .beta.-Lactam Antibiotics" Antimicrob. Ag. Chemother. 2(3):122-131, 1972. Yeh et al., "Biochemical characterization and evolutionary implication of .beta.-lactam expandase/hydroxylase, expandase, and hydroxylase." In 50 Years of Penicillin: History and Trends (Kleinhauf and von Doehren, eds.), Public, Prague, 208-223, 1994. Yoshida et al., "Cell-free ring expansion of penicillin N to deacetoxycephalosporin by Cephalosporium acremonium CW-19 and its mutants" Proc. Natl. Acad. Sci. USA 75(12):6253-6257, Dec. 1978.

Primary Examiner: Saucier; Sandra E.
Attorney, Agent or Firm: Choate, Hall & Stewart, Jarrell; Brenda Herschbach
 

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Parent Case Text

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The present application claims priority to U.S. Ser. No. 60/082,800, filed Apr. 23, 1998, the entire contents of which are incorporated herein by reference.

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Claims

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What is claimed is:

1. A method of converting a penicillin other than penicillin N or ampicillin to a cephalosporin, the method comprising steps of:

providing a source of naturally occurring expandase enzyme, which expandase enzyme is not produced in a cell that also naturally produces the penicillin substrate;

providing exogenously a penicillin substrate other than penicillin N, ampicillin, or a penicillin having the formula: ##STR1##

wherein R is 3-carboxybenzyl or a hydrocarbon containing 1-4 carbon atoms terminally substituted with a radical selected from the group consisting of carboxy, amino, and combinations thereof;

contacting the expandase with the penicillin substrate under conditions that allow the expandase to expand the penicillin substrate.

2. The method of claim 1 wherein the step of providing a source of expandase comprises providing resting cells, growing cells, or immobilized cells that have produced expandase.

3. The method of claim 2 wherein the cells are cells that naturally express the expandase.

4. The method of claim 3 wherein the cells are selected from the group consisting of Xanthomonas lactamgena, Lysobacter lactamgenus, Flavobacterium sp., Flavobacterium chitinovorum, Streptomyces organanensis, Nocardia lactamdurans, Streptomyces lipmanii, Streptomyces jumonjinensis, Streptomyces wadayamensis, Streptomyces cattleya, Streptomyces lactamgens, Streptomyces fradiae, Streptomyces griseus, Streptomyces olivaceus, Streptomyces sp., and Cephalosporum acremonium cells.

5. The method of claim 2 wherein the step of providing a source of expandase enzyme comprises providing cells that produce S. clavuligerus expandase.

6. The method of claim 2 wherein the step of providing a source of expandase comprises providing cells that express an expandase gene that is foreign to the cells.

7. The method of claim 6 wherein the expandase gene is selected from the group consisting of the S clavuligerus expandase gene and the C. acremonium expandase gene.

8. The method of claim 6 wherein the step of providing a source of expandase enzyme comprises providing S. clavuligerus cells.

9. The method of claim 6 wherein the step of providing a source of expandase enzyme comprises providing cells other than S. clavuligerus cells, which provided cells have been engineered to express the S. clavuligerus expandase gene.

10. The method of claim 1 wherein the step of providing a source of expandase comprises providing extract of a cell that produces expandase.

11. The method of claim 1 wherein the step of providing a source of expandase comprises providing purified expandase.

12. The method of claim 11 wherein the step of providing a source of expandase comprises providing pure expandase.

13. The method of claim 11 wherein the step of providing a source of expandase comprises providing an immobilized pure enzyme.

14. The method of claim 1 wherein the step of providing a source of expandase enzyme comprises providing a source of an enzyme selected from the group consisting of S. clavuligerus expandase and C. acremonium expandase.

15. The method of claim 1 wherein the step of providing a penicillin substrate comprises providing a substrate selected from the group consisting of: amoxicillin, butyryl-6-APA, decanoyl-6-APA, heptanoyl-6-APA, hexanoyl-6-APA, nonanoyl-6-APA, octanoyl-6-APA, penicillin F, penicillin G, penicillin V, penicillin mX, penicillin X, 2-thiophenylacetyl-6-APA, and valeryl-6-APA.

16. The method of claim 1 wherein the step of providing a penicillin substrate comprises providing a substrate selected from the group consisting of: penicillin V, penicillin G, penicillin mK, penicillin X, 2-thiophenylacetyl-6-APA, and amoxicillin.

17. The method of claim 1 wherein the penicillin substrate is different from whatever substrate the expandase expands in nature.

18. The method of claim 1 wherein the step of providing a penicillin substrate comprises providing a penicillin other than a penicillin naturally produced by the organism from which the provided expandase originated.

19. A method of converting a penicillin other than penicillin N to a cephalosporin, the method comprising the steps of:

providing a source of expandase enzyme;

providing exogenously a penicillin substrate other than penicillin N, ampicillin, or a penicillin having the formula: ##STR2##

wherein R is 3-carboxybenzyl or a hydrocarbon containing 1-4 carbon atoms terminally substituted with a radical selected from the group consisting of carboxy, amino, and combinations thereof;

contacting the expandase with the penicillin substrate under conditions including:

a ferrous ion (Fe.sup.+2) concentration within the range of 0-4 mM;

a concentration of .alpha.-ketoglutarate with the range of 0-4 mM; and

isolating a cephalosporin.

20. A method of converting a penicillin other than penicillin N to a cephalosporin, the method comprising steps of:

providing a source of expandase enzyme, which expandase enzyme is substantially identical to that naturally found in S. clavuligerus;

providing exogenously a penicillin substrate other than penicillin N, ampicillin, or a penicillin having the formula: ##STR3##

wherein R is 3-carboxybenzyl or a hydrocarbon containing 1-4 carbon atoms terminally substituted with a radical selected from the group consisting of carboxy, amino, and combinations thereof; and

contacting the expandase with the penicillin substrate under conditions that allow the expandase to expand the penicillin substrate.

21. The method of claim 1, 19, or 20 wherein the penicillin substrate is penicillin G.

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Description

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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-.alpha.-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 utilize growing or resting cells (free or immobilized), or isolated expandase (crude or purified), and is capable of converting exogenously-added penicillins. The inventive system can be applied to any penicillin substrate, including natural penicillins (e.g., penicillin G), biosynthetic penicillins (e.g., penicillin V), semisynthetic penicillins (e.g., ampicillin), and/or synthetic penicillins.

Definitions

"Cephalosporin precursor"--The term "cephalosporin precursor", as used herein, refers to a compound that, through one or more chemical reactions not relying on an expandase, can be converted into a cephalosporin. This term is intended to encompass many compounds that are also cephalosporins, so long as they are convertible into other cephalosporins. Preferred cephalosporin precursors have the structure depicted in FIG. 2A, and include 7-ADCA, 7-ADAC, 7-ACA, DAOG, DAG, cephalosporin G, and cephamycin G. DAOG and DAG are particularly preferred.

"Exogenous substrate"--The term "exogenous substrate", as used herein, refers to a substrate that is added to a reaction and is not produced internally by a cell producing expandase. That is, when the expansion reaction occurs inside a cell that produces both the expandase and the penicillin substrate on which the expandase acts, that substrate is an "endogenous" substrate. By contrast, if the penicillin substrate is added e.g., to cells producing the expandase, that substrate is exogenous, even if it is the same chemical compound that is being (or could be) produced by the cell.

"Isolated"--The term "isolated", when applied to a compound that exists in nature, means (i) separated from at least some of the components with which it is normally associated in nature; and/or (ii) produced or prepared through a process (e.g., involving in vitro synthetic chemistry) that does not occur in nature.

"Purified"--A compound is considered "purified" when it is at least about 50% pure, preferably at least 70-80% pure, more preferably at least about 90% pure, yet more preferably at least 95% pure, and most preferably at least 99% pure.

"Recombinant"--The term "recombinant", as used herein, means produced through a method relying on techniques of recombinant DNA technology. For example, an expandase gene is separated from DNA with which is normally associated in nature and is introduced into an expression vector, the gene in the context of the vector is a "recombinant" gene. Similarly, the protein expressed from the gene is a "recombinant" protein.

DESCRIPTION OF THE DRAWINGS

FIG. 1A presents a generalized structure of penicillins; FIG. 1B shows R.sub.1 groups present in different penicillins.

FIG. 2A presents a generalized structure of cephalosporins; FIG. 2B shows R.sub.1 and R.sub.2 groups present in certain different cephalosporins.

FIG. 3 presents a portion of the cephalosporin C biosynthetic pathway utilized by C. acremonium to produce cephalosporin C from endogenous penicillin N.

FIG. 4, Panels A and B depict chemical treatment of the intermediates DAOC and DAC, respectively, to produce cephalosporin precursors 7-ADCA and 7-ADAC.

FIG. 5 depicts production of 7-ADCA and 7-ADAC through a penicillin G biological ring expansion pathway, followed by enzymatic removal of the phenylacetyl side chain.

FIG. 6 shows a time course of the ring expansion of penicillin G by resting cells. (.circle-solid.) indicates the successful inventive reaction conditions; (.box-solid.) indicates reactions that were unsuccessful when utilized in the prior art with cell-free extracts.

FIG. 7 shows the effect of .alpha.-ketoglutarate concentration on ring expansion of penicillin G by resting cells. Reactions contained 50 mM Tris-HCl pH 7.4, 8 mM KCl, 8 mM MgSO.sub.4, 4 mM ascorbic acid, 1.8 mM FeSO.sub.4, and 2 mg/ml penicillin G. Dry cell weight was 12 mg/ml. Samples were taken at 2 hour and centrifuged at 12K rpm for 5 minutes. 200 .mu.l were used in the bioassay.

FIG. 8 shows the effect of Fe.sup.2+ concentration on ring expansion of penicillin G by resting cells. Reactions contained 50 mM Tris-HCl pH 7.4, 8 mM KCl, 8 mM MgSO.sub.4, 4 mM ascorbic acid, 1.28 mM .alpha.-ketoglutarate, and 2 mg/ml penicillin G. Dry cell weight was 10 mg/ml. Samples were taken at 2 hour and centrifuged at 12K rpm for 5 minutes. 200 .mu.l were used in the bioassay.

FIG. 9 shows the effect of cell mass concentration on penicillin G ring expansion by resting cells. The reaction contained 50 mM Tris-HCl pH 7.4, 8 mM KCl, 8 mM MgSO.sub.4, 4 mM ascorbic acid, 1.8 mM FeSO.sub.4, 1.28 mM .alpha.-ketoglutarate, and 2 mg/ml penicillin G. Samples were taken at 2 hour and centrifuged at 12K rpm for 5 minutes. 200 .mu.l were used in the bioassay.

FIG. 10 shows the effect of cell-free extract protein concentration on penicillin G ring expansion by cell-free extracts. The reactions contained 50 mM Tris-HCl pH 7.4, 8 mM KCl, 8 mM MgSO.sub.4, 4 mM ascorbic acid, 1.8 mM FeSO.sub.4, 1.28 mM .alpha.-ketoglutarate, a4 mM DDT and 2 mg/ml penicillin G. Protein concentration was 0.2 mg/ml (.quadrature.), 1 mg/ml (.circle-solid.), 2 mg/ml (.tangle-solidup.), 4 mg/ml (.smallcircle.), or 6 mg/ml (.box-solid.). Samples were taken at 2 hour and centrifuged at 14K.times.g rpm for 5 minutes. 200 .mu.l of the supernatant were used in the bioassay.

FIG. 11 shows the effect of penicillin G concentration on penicillin G ring expansion by cell-free extracts. The reactions contained 50 mM Tris-HCl pH 7.4, 8 mM KCl, 8 mM MgSO.sub.4, 4 mM ascorbic acid, 1.8 mM FeSO.sub.4, 14 mM DDT, and 1.28 mM .alpha.-ketoglutarate. (.box-solid.) No penicillin G; (.circle-solid.), 0.5 mg/ml; (.tangle-solidup.), 1 mg/ml; (.DELTA.) 2 mg/ml; (.quadrature.) 4 mg/ml; (.smallcircle.) 5 mg/ml. Samples were taken at 2 hour and centrifuged at 14K.times.g for 5 minutes. 200 .mu.l were used in the bioassay.

FIG. 12 shows an HPLC analysis of penicillin G ring expansion to DAOG by resting S. clavuligerus NP1 cells. Sensitivity was 0.12 absorbance units of full sensitivity (AUFS). Cells were grown in MT+1% ethanol.

FIG. 13 shows the effect of buffer selection on penicillin G conversion by resting S. clavuligerus NP1 cells. (.circle-solid.) 0.05 M MOPS, pH 6.5, (.smallcircle.) 0.05 M HEPES, pH 6.5, (.DELTA.) 0.05 M Tris-HCl, pH 7.4.

FIG. 14 shows the effect on penicillin G conversion of preincubating resting S. clavuligerus NP1 cells with one or more components of the reaction mixture prior to ring expansion. Preincubations: (.circle-solid.) none, (.smallcircle.) Fe.sup.2+, (.tangle-solidup.) ascorbic acid, (.times.) .alpha.-ketoglutarate, (.box-solid.) ascorbic acid+Fe.sup.2+, (.DELTA.) ascorbic acid+Fe.sup.2+ +.alpha.-ketoglutarate.

FIG. 15 shows the effect of substrate concentration on product formation by resting cells. Substrate was present at: (.circle-solid.) 0.063 mg/ml, (+) 0.125 mg/ml, (.box-solid.) 0.25 mg/ml, (.smallcircle.) 0.5 mg/ml, (.tangle-solidup.) 1.0 mg/ml, (.quadrature.) 2.0 mg/ml, ( ) 4.0 mg/ml, (.DELTA.) 6.0 mg/ml, and (.times.) 8.0 mg/ml.

FIG. 16 shows yields of penicillin G conversion by resting S. clavuligerus NP1 cells, based on amount of substrate changed, at different concentrations of penicillin G.

FIG. 17 shows the effect on penicillin G conversion yields of different biomass levels of resting cells (gram wet weight in 10 ml of reaction mixture) at two different concentrations, 0.063 mg/ml (.box-solid.) and 2 mg/ml (.quadrature.), of penicillin G.

FIG. 18 compares the penicillin G expansion activity of S. clavuligerus NP1 resting cells entrapped in PEI-barium alginate (.circle-solid.) as compared with free resting cells (.smallcircle.).

FIG. 19 shows the effect of biomass concentration on penicillin G conversion activity of entrapped S. clavuligerus NP1 cells. A constant amount of beads (3.4 g wet weight/10 ml reaction mixture) was used. (.box-solid.) 2 g cells (wet weight), (.quadrature.) 4 g cells, (.tangle-solidup.) 6 g cells.

FIG. 20 shows plasmids utilized to study homologous recombination.

FIG. 21 shows the sequences of crossover junctions of hybrid expandase genes obtained in DH5.alpha. cells.

FIG. 22 shows HPLC profiles of ring expansion reactions.

DESCRIPTION OF CERTAIN PREFERRED EMBODIMENTS

As mentioned above, the present invention provides an improved system for the conversion of penicillins into cephalosporins or cephalosporin precursors. In particular, the invention defines reaction conditions under which expandase-producing cells, or extracts or enzymes thereof, convert penicillins other than penicillin N or its close relatives into cephalosporins.

One aspect of the invention involves the definition of reaction conditions that allow such conversion, and the concomitant identification of factors that affect the success and/or efficiency of such conversion. Another aspect of the invention provides compositions and/or methods for achieving such conversion. In general, the invention utilizes (i) an expandase source; (ii) a penicillin substrate; and (iii) reaction conditions that allow conversion of the penicillin substrate into a cephalosporin or cephalosporin precursor. Each of these inventive components is discussed in more detail below.

Expandase Source

The present invention demonstrates that, under appropriate reaction conditions, S. clavuligerus cells (whether they be growing, resting, or immobilized) or cell extracts (free or immobilized) are an appropriate source of expandase activity for converting penicillin substrates other than penicillin N to cephalosporins (see Examples). In light of these teachings, those of ordinary skill in the art will appreciate that S. clavuligerus expandase is an appropriate enzyme for use in accordance with the present invention, regardless of its form or mode of preparation.

For example, S. clavuligerus expandase may be purified from S. clavuligerus cells according to known techniques (see, for example, Jensen et al., Antimicrob. Agents Chemother. 24:307-312, 1983; Dotzlaf, et al., J. Biol. Chem.264:10219-10227, 1983 incorporated herein by reference) and utilized in the practice of the present invention. Moreover, the gene for the S. clavuligerus expandase has been cloned (Kovacevic, et al., J. Bacteriol. 177:754-760, 1989), and may be introduced into an expression construct that allows expandase production in any of a variety of host cells from which it can be used as a cellular product, prepared as an extract, or purified for use in inventive reactions.

Techniques for introducing cloned genes into expression constructs are well known in the art (see, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989, incorporated herein by reference). So long as the expandase protein is produced in the host cell, the expression construct, its mode of preparation (e.g., the selection of regulatory sequences such as the gene promoter, upstream regulatory elements, splicing signals, RNA processing signals, etc.), and its manner of introduction into the host cell (e.g., by transformation, transfection, infection, injection, electroporation, etc.) are appropriate according to the present invention. In certain embodiments of the invention, the expression construct may be engineered to allow secretion of the expandase protein into the cell supernatant.

Preferred host cells in which S. clavuligerus expandase is preferably expressed include, but are not limited to bacterial cells, fungal cells, insect cells, plant cells or vertebrate (including mammalian) cells. Those of ordinary skill in the art will recognize that expression vectors that direct production of a desired protein in a particular kin of host cell are readily available for a wide range of host cells (see, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories Press, Cold Spring Harbor, N.Y., 1989, incorporated herein by reference).

Moreover, the expressed expandase need not be isolated or purified from the host cell in order to be used in accordance with the present invention. That is, as discussed above and in the Examples, S. clavuligerus cells themselves, whether growing, resting, or immobilized in certain polymeric matrices, are appropriate sources of expandase for use in the practice of the present invention. Host cells expressing a recombinant S. clavuligerus gene can readily be assayed as described herein to identify those that are useful sources of expandase for use in the practice of the present invention.

Those of ordinary skill in the art will readily appreciate that S. clavuligerus expandase is not the only expandase that is useful in accordance with the present invention. For example, a wide variety of organisms such as unicellular bacteria (e.g., Xanthomonas lactamgena, Lysobacter lactamgenus, Flavobacterium sp., Flavobacterium chitinovorum, etc.), filamentous bacteria (e.g., Streptomyces organanensis, Nocardia lactamdurans, Streptomyces lipmanii, Streptomyces jumonjinensis, Streptomyces wadayamensis, Streptomyces cattleya, Streptomyces lactamgens, Streptomyces fradiae, Streptomyces griseus, Streptomyces olivaceus, Streptomyces sp.), and filamentous fungi (e.g., C. acremonium) are expected to produce proteins with expandase activity, at least when assayed on penicillin N (see, for example, Caswell et al., in 50 Years of Penicillin: History and Trends, [Kleinkauf et al., eds]., Public, Prague, p.135, 1994; Kohsaka, et al, Biochem. Biophys. Res. Comm., 70:465-473; Stapley et al. Antimicrob. Agents Chemother. 2:122-131, 1972; Cortes et al., Biochem. Soc. Transac. 12:863-864, 1984). Any such cells, or extracts or expandases prepared from such cells, may be screened according to the procedures described herein to identify those with an ability to expand non-penicillin N substrates. Furthermore, desirable expandases defined in such cells may be expressed in host cells as described above with respect to S. clavuligerus expandase, and the host cells, host cell extracts, or isolated or purified host-cell-expressed expandase, may be employed in the practice of the present invention.

According to the present invention, an expandase-producing cell is a suitable source of expandase if, when provided with an exogenous penicillin substrate, preferably penicillin G or penicillin V, it produces a compound that creates a zone of inhibition when tested in paper disc-agar diffusion assay containing penicillinase as described herein. Alternatively or additionally, an expandase-producing cell is a suitable source of expandase if, when tested as described herein, it accomplishes sufficient expansion of a penicillin substrate, preferably penicillin G or penicillin V, that at least one peak corresponding to a cephalosporin precursor (preferably DAOG, DAG, cephalosporin G, and/or cephamycin G) is observed in HPLC.

In general, where intact cells are utilized in the inventive system, they may be growing, they may be resting, and they may be either free or immobilized (e.g., in a polymeric matrix). Those of ordinary skill in the art will appreciate that any of a variety of matrices or immobilization techniques may be employed to immobilize cells (see, for example, Enzymes and Immobilized Cells In Biotechnology [Laskin, ed], Benjamin Cummings Publishing Company, Menlo Park, Calif., 1985, incorporated herein by reference), so long as the expandase remains sufficiently active after immobilization. PEI-barium alginate is particularly preferred, especially for use with S. clavuligerus cells (see Example 5). Of course, the utility of a particular polymeric matrix in any given application may depend to a certain extent on the nature and characteristics of the cells to be embedded therein. It is well within the province of one of ordinary skill in the art to screen a variety of polymeric matrices as described herein to identify one that is suitable for use with cells other than S. clavuligerus cells.

Although the above discussion has focussed on expandases produced by cellular sources, and it is expected that such will be the most common form of expandase utilized in the present invention, it will be understood that any expandase source, including, for example, protein wholly or partially synthesized through in vitro chemical or biochemical methods is also acceptable.

Furthermore, those of ordinary skill in the art will appreciate that, once an expandase gene is cloned, various modifications or alterations to gene sequence, resulting in modifications or alterations of protein sequence, can readily be made using standard recombinant techniques such as, for example, site-directed mutagenesis, polymerase chain reaction mutagenesis, exo- or endo-nuclease digestion, gene shuffling (i.e., equal homologous recombination) (see, for example, Example 6), etc. Such modifications or alterations include, for example, production of fusion proteins; addition, deletion, or substitution of one or more amino acids; etc. Alternatively or additionally, the chemical structure of a particular expandase protein may be altered through modification of the protein after it is made, e.g., through proteolytic cleavage or chemical modification. Expandase enzymes with altered structure as compared with expandases that occur in nature are useful in the practice of the present invention so long as they retain the ability to expand penicillin substrates as described herein.

Penicillins

As discussed above, the present invention provides a system for expansion of penicillins other than penicillin N. Penicillin substrates for use in the practice of the present invention include all natural penicillins (i.e., penicillins that are naturally produced by P. chrysogenum--e.g., penicillin G), biosynthetic penicillins (i.e., penicillins that are produced by P. chrysogenum through directed biosynthesis when a side chain acid is added to the medium--e.g., penicillin V), semi-synthetic penicillins (i.e., penicillins that are made by chemical means from natural or biosynthetic penicillins--e.g., ampicillin), and synthetic penicillins (i.e., penicillins that are made wholly synthetically), other than penicillin N. For example, preferred penicillin substrates include, but are in no way limited to, adipyl-6-APA, amoxicillin, ampicillin, butyryl-6-APA, decanoyl-6-APA, heptanoyl-6-APA, hexanoyl-6-APA, nonanoyl-6-APA, octanoyl-6-APA, penicillin F, penicillin G, penicillin V, penicillin mX, penicillin X, 2-thiopheynlacetyl-6-APA, and valeryl-6-APA. Particularly preferred penicillins include penicillin G, penicillin mK, penicillin X, penicillin V, ampicillin, amoxicillin, and 2-thiophenylacetyl-6-APA. Most preferred are penicillins G and V, which are articles of commerce and are therefore inexpensive.

Reaction Conditions

As described herein, one important aspect of the present invention is the definition of reaction conditions under which expandases that naturally operate on penicillin N will act on other penicillins, preferably when those penicillins are added as exogenous substrates (i.e., are not produced by the cells producing the expandase). Standard reaction conditions for expansion of penicillin N are well known in the art (see, for example, Maeda et al., Enzyme Microb. Technol. 17:231-34, 1995; Cortes et al., Biochem. Soc. Transac. 12:863-864, 1984; Jensen et al., J. Antibiot. 35:1351-1360, 1982; Shen et al., Enzyme Microb. Technol. 6:402-404, 1984; Dotzlaf et al., J. Biol. Chem. 264:10219-10227, 1989). As the present invention demonstrates, however, such conditions are likely not to be useful for conversion of substrates other than penicillin N.

According to the present invention, optimal reaction conditions for a particular expandase source and penicillin substrate can be identified by varying the presence or amount of one or more of: .alpha.-ketoglutarate, Fe.sup.2+, ascorbate, reducing agents (such as, for example, dithiothreitol (DDT) or .beta.-mercaptoethanol (.beta.ME)), and ATP. Also, the characteristics and amount of the expandase source can be varied. For example, expandase can be provided from cells grown to different densities or collected at different stages of growth. Alternatively or additionally, the purity or concentration of the expandase preparation may be varied. Also, variations can be made in the concentration of the substrate and the temperature and pH of the reaction. Buffer selection may also be adjusted. Alternatively or additionally, where the expandase source is cells or is extract or purified protein prepared from cells, the conditions under which the cells are grown (e.g., carbon source, nitrogen source, source of phosphorus and other minerals, availability of oxygen, etc) can be adjusted. As discussed herein, the ideal reaction conditions may vary depending, for example, on the nature (e.g., resting cells, growing cells, immobilized cells, purified enzyme, immobilized enzyme, etc.) of the expandase source.

Preferred buffers for use in inventive expandase reactions include, but are not limited to, Tris, MOPS, HEPES, phosphate buffers, etc. Buffers are preferably employed at concentrations within the rage of about 50-200 mM, and pHs within the range of about 5.0-9.0, depending on the particular buffer. Tris is preferably employed at a pH within the range of 6.5-9.0; HEPES within the range of 6.0-8.5; MOPS within the range of 5.5-8.0; and phosphate within the range of 5.0-7.5. Particularly preferred reactions utilize, 50 mM Tris-HCl at pH 7.4, 50 mM MOPS at pH 6.5, or 50 mM HEPES at pH 6.5 are preferred, with 50 mM MOPS at pH 6.5 or 50 mM HEPES at pH 6.5 being especially preferred.

Ascorbic acid, when it is utilized, is preferably provided in a concentration within the range of 0.8-50 mM, preferably 2-8 mM, and most preferably about 4 mM. As demonstrated herein, ascorbic acid is not required in inventive reactions, particularly in reactions employing resting cells.

.alpha.-Ketoglutarate is preferably provided in a concentration within the range of 0-4 mM, preferably about 0.5-2.0 mM, and most preferably about 1.28-1.5 mM.

Preferred reducing agents include DTT and .beta.ME. As demonstrated herein, such reagents are not essential to expandase reactions, and may actually inhibit reactions with resting cells. Thus, reducing agents are preferably left out of inventive expandase reactions, particularly those employing resting cells. Alternatively, they may be provided at concentrations within the range of about 0.1-14 mM, preferably 14 mM.

Iron concentration in the inventive expandase reactions is preferably maintained within the range of 0-4 mM, preferably about 0.5-2.5 mM, and most preferably 1.8-2.2 mM.

ATP need not be provided in preferred reactions according to the present invention. Where it is provided, it is preferably provided at a concentration less than about 3.5 mM, preferably within the range of about 0-3 mM, and most preferably within the range of about 0.14-2.4 mM.

The penicillin substrate is preferably provided at a high concentration (e.g., more than about 2 mg/ml, preferably more than about 5 mg/ml) in order to produce the largest possible amount of cephalosporin, or at a low concentration (e.g., less than about 2 mg/ml, preferably less than about 1 mg/ml, and most preferably less than about 0.25 mg/ml) in order to achieve a higher efficiency of conversion.

Other salts or reagents that can be employed in certain expandase reactions in accordance with the present invention include, for example, KCl (preferably at a concentration within the range of 0-8 mM, preferably being excluded from reactions with resting cells); MgSO.sub.4 or other Mg.sup.2+ source (preferably at a concentration within the range of 0-8 mM, preferably being excluded from reactions with resting cells), etc.

Expandase is preferably present at the highest concentration possible without inhibiting the reaction (e.g., due to contaminants--including whole cells--in the expandase preparation). Where expandase is provided in the form of cells, the cells are preferably utilized at the lowest possible biomass (e.g., less than about 6 g, wet weight/10 ml solution, preferably within the range of about 0.5-4.0 g wet weight/10 ml solution, and most preferably about 1-3 g, wet weight/10 ml solution).

Also, where the expandase is provided from a cellular source (whether it is provided in cellular form or in isolated or purified form), the cells producing the expandase are preferably grown under conditions of nutrient imbalance and/or of low growth rate, as such conditions are expected to maximize antibiotic production. In particular, the cells are preferably grown in the presence of a stressing agent such as an alcohol (e.g., methanol or ethanol, preferably in the range of 1-2%) or heat (i.e., under conditions of heat shock).

Those of ordinary skill in the art will readily recognize that any of a variety of other reaction and/or preparation conditions can readily be varied and tested according to the procedures set forth herein without undue experimentation. According to the present invention, reaction conditions that produce a zone of inhibition in the growth assays described herein, or produce one or more cephalosporin precursor peaks on HPLC, are desirable for use in accordance with the present invention.

Diversification of Cephalosporin Precursor

A wide variety of chemical reactions are known in the art that can be employed to diversify a cephalosporin precursor, produced as described herein, to provide a desirable cephalosporin. In particular, many approaches have been established for introducing different chemical groups at the 3- and 7-positions of the cephalosporin ring system (see, for example, Durckheimer et al., Adv. Drug. Res. 17:61, 1988, and references cited therein; Drugs 34 (Suppl. 2), 1987, each of which is incorporated herein by reference).

Several particularly useful third- and fourth-generation cephalosporins (e.g., cefotaxime) include a 2-aminothiazol-4-yl-acetamido side chain combined with a syn-alkoxyimino group. A wide variety of modifying reactions are known that can be performed on such structures, or that can generate related compounds from a cephalosporin precursor such as 7-ACA (see summary in Kirrstetter et al., Die Pharmazie, 44:177-184, 1989, incorporated herein by reference).

Other useful cephalosporins include those with polar pyridino or quaternary amino substitutes at C-3' and a neutral or an acidic oxime group in the 7-side chain. Once again, syntheses have been worked out for a wide variety of related compounds (see, for example, Durckheimer et al., Adv. Drug. Res. 17:61, 1988; Drugs of the Future 13:271, 1988).

Those of ordinary skill in the art will recognize that any available technique for generating cephalosporins from cephalosporin precursors provided as described herein is useful in the practice of the present invention. The reactions employed to generate the final cephalosporins are not intended to limit the scope of the invention.

EXAMPLES

Example 1

Penicillin Ring Expansion by S. clavuligerus Resting Cells and Cell-Free Extracts

Materials and Methods

Microorganism: We utilized a known S. clavuligerus mutant, known as "NP1", that does not naturally produce significant levels of cephalosporins, but is known to produce cephalosporin C when fed exogenous penicillin N (see Mahro et al., Appl. Microbiol. Biotechnol. 27:272-275, 1987). The ability of this mutant to produce cephalosporin C under these conditions indicates that the strain's expandase is functional.

Media and Culture Conditions: Mycelia were obtained using 250 ml baffled flasks containing 40 ml of MST medium: 1% soluble starch (Sigma Chemical Co., St. Louis, Mo.); 3% Trypticase Soy Broth Without Dextrose (BBL, Cockeysville, Md.); 90 mM MOPS buffer, pH adjusted to 7.0 before autoclaving. Each flask was inoculated with 50 .mu.l of a spore suspension (prepared and stored at -80.degree. C. in 20% glycerol) and incubated at 30.degree. C., 250 rpm for 48 h.

Materials: Penicillin G, ascorbic acid and .alpha.-ketoglutaric acid were from Sigma Chemical Company (St. Louis, Mo.). Deacetoxycephalosporin G was from Antibiotics, S.A. (Leon, Spain) and Bacto-Penase from Difco Laboratories (Detroit, Mich.).

Preparation of Cell-free Extracts: Fermentation broths were centrifuged at 8,000.times.g and 4.degree. C. for 10 min. Pellets were washed twice using 50 mM Tris-HCI supplemented with 0.1 mM dithiothreitol (DTT). The cells were resuspended in the same buffer and disrupted by four 25-sec. sonication treatments (power setting 5 and duty cycle 50%), in an ice-water bath using a Branson 350 sonifier (Branson Sonic Power Co., Danbury, Conn.). Cell debris was removed by centrifugation (14,000.times.g, 30 min., 4.degree. C.). The resulting extracts containing 8-10 mg protein/ml were placed on ice and utilized immediately. Protein concentrations were measured using the Bio-Rad protein assay (Bio-Rad, Hercules, Calif.). Bovine serum albumin was used as standard.

Resting Cells: From a seed culture (in MST), 0.5 ml was transferred to new flasks containing 40 ml of the same medium. Cells were grown at 30.degree. C., 250 rpm for 24 h. Mycelia from each flask were washed twice, and finally, resuspended in 10 ml of distilled water. Four ml of this cell suspension were used in the reaction mixture.

Ring Expansion Reaction: We defined ring expansion reaction conditions as modifications of the standard reaction mixture for expandase reactions described by Maeda et al. (Maeda et al., Enzyme Microb. Technol. 17:231-34, 1995) except that penicillin G was used a substrate instead of penicillin N. Additions were made following the order established by Shen et al. (Shen et al., Enzyme Microb. Technol. 6:402-404, 1984. Reaction mixtures were incubated in test tubes (cell-free extract) or 250 ml baffled flasks (resting cells) at 220 rpm, 30.degree. C. Reactions containing the protein extract were stopped at various times (see Table 1 and Figure Legends) by mixing 0.5 ml of assay solution with 0.5 ml of methanol. In the case of resting cells, samples were centrifuged to remove cells and supernatants were transferred to new tubes. Expandase activity was detected by paper disc-agar diffusion bioassay.

Detection of Expandase Activity: As mentioned above, expandase activity was detected by assaying production of a growth-inhibitory zone in a paper disc-agar diffusion bioassay. Paper discs were saturated with 200 .mu.l of the reaction mixture (cell-free extracts or supernatant (resting cells) as follows. Two discs were superimposed and four 50 .mu.l samples were applied. After each application, the discs were allowed to dry for 20 min at 37.degree. C. under a laminar hood and, finally, they were placed on LB (1% tryptone, 0.5% NaCl, 0.5% yeast extract, 0.1% glucose) 0.8% agar medium seeded with E. coli ESS (a .beta.-lactam supersensitive mutant), and the plates were incubated overnight at 37.degree. C. The formation of DAOG and/or other cephalosporin(s) was determined by including 50,000 IU/ml of penicillinase (Difco Bacto penase concentrate, Difco Laboratories, Detroit, Mich.) in the assay plates. This penicillinase is a narrow spectrum .beta.-lactamase that attacks penicillins but not cephalosporins. The diameters of zones of growth inhibition were measured and quantified with a calibration curve using DAOG as standard.

Test Substrates: All penicillins used in this work, except penicillin G and ampicillin (Sigma Chemical Co, Mo.), were provided by Saul Wolfe (Simon Fraser University, Canada) or Jose M. Luengo (University of Leon, Spain), and were synthesized as previously described (Maeda et al., Enzyme Microb. Technol. 17:231-34, 1995). DAOG was provided to us by Antibioticos, S. A. (Madrid, Spain).

Results

Penicillin G Ring Expansion by Resting Cells: Prior work had indicated that the thiazolidine ring of penicillin G could not be expanded with cell-free extracts of S. clavuligerus (Maeda et al., Enzyme Microb. Technol. 17:231-234, 1995). We nonetheless endeavored to define reaction conditions that would allow ring expansion. We began our studies using resting cells instead of cell extracts.

In order to identify useful reaction conditions, we varied the concentrations of FeSO.sub.4, .alpha.-ketoglutarate, ascorbate, and ATP in our mixtures. We also tested the effect of cell mass in our reactions.

The unsuccessful Maeda et al. reactions, which were performed with cell-free extracts and were assayed in a growth inhibition assay that used smaller amount of sample than we utilized in our assays, contained 50 mM Tris-HCl pH 7.4, 8 mM KCl, 8 mM MgSO.sub.4, 14 mM DTT, 4 mM ascorbic acid, 0.04 mM FeSO.sub.4, 0.64 mM .alpha.-ketoglutarate, and 0.28 mM penicillin G. We found that resting cell reaction mixtures containing 45 times as much Fe.sup.2+ (i.e., 1.8 mM FeSO.sub.4) and twice as much .alpha.-ketoglutarate (i.e., 1.28 mM .alpha.-ketoglutarate) allowed successful expansion of penicillin G by resting cells (see FIG. 6). Using our version of the growth inhibition assay, we found that even the Maeda et al. reaction conditions (.box-solid.) produced some DAOG (about 6 .mu.g/ml after 3-6 hours of reaction) when employed with resting cells. Resting cells reacted under the inventive conditions (.circle-solid.) produced at least three times as much (about 19 .mu.g/ml after 3-6 hours of reaction).

We further found, as shown below in Table 1, that omission of Fe.sup.2+, .alpha.-ketoglutarate, or ascorbic acid reduced the amount of DAOG produced after two hours of reaction to about 30% of that produced in a complete reaction. On the other hand, omission of ATP, MgSO.sub.4, KCl, or DTT did not have a marked negative effect. In fact, omission of DTT actually increased DAOG production approximately 50%. All reactions contained 50 mM Tris-HCl pH 7.4, 13 mg/ml dry cell weight cells, and 2 mg/ml of penicillin G. One ml of sample was taken from each reaction and was centrifuged at 12K rpm for 5 minutes. The supernatants were then transferred to new tubes and 200 .mu.l were used in the bioassay (in other experiments, 100 .mu.l or 150 .mu.l were used).

 

                             TABLE 1
             Effect of Cofactors of Penicillin G Ring
                    Expansion by Resting Cells
              Cofactor Omitted           .mu.g DAOG/ml
              None                           10.5
              DTT                            15.5
              (16 mM)
              .alpha.-ketoglutarate                 3.7
              (1.28 mM)
              FeSO.sub.4.7H.sub.2 O                 3.2
              (1.8 mM)
              MgSO.sub.4.7H.sub.2 O                10.1
              (8 mM)
              KCl                            11.5
              (8 mM)
              Ascorbic acid                   3.0
              (4 mM)
              ATP                            10.0
              (0.7 mM)

When we varied the concentration of individual reaction components in the context of our successful conditions, we found that increasing the .alpha.-ketoglutarate concentration from 0.64 mM to 1.28 mM doubled the amount of DAOG produced (see FIG. 7); increasing Fe.sup.2+ concentration (FIG. 8) or ascorbate concentration (Table 2) also increased DAOG production until optimal reagent levels (about 1.8 mM for Fe.sup.2+ and 4-8 mM for ascorbate) were reached, but ATP had little effect until high concentrations (around 3.5 mM) began inhibiting the reaction (Table 2). Interestingly, studies of expansion of the penicillin N ring had indicated that ATP stimulated that reaction.

 

                             TABLE 2
    Effect of Modifications in Ascorbate or ATP Concentration on Penicillin
                G Ring Expansion by Resting Cells
          Cofactor       Concentration      .mu.g DAOG/ml
         Ascorbate             0                 2.4
                               0.8               4.0
                               2                 6.0
                               4                 6.4
                               8                 6.4
            ATP                0                 6.6
                               0.14              6.4
                               0.35              6.4
                               0.7               6.6
                               2.4               6.6
                               3.5               4.7

We further found that increasing cell mass enhanced the formation of DAOG until an optimum concentration of about 19 mg/ml dry cell weight was reached; higher concentrations inhibited DAOG production, probably due to limited oxygen supply (FIG. 9).

Penicillin G ring expansion by cell-free extracts: Having defined successful reaction conditions for penicillin G expansion by resting cells, we tested the same conditions using cell-free extracts. As shown in FIG. 10, cell-free extracts were active under these conditions, and higher protein concentration in the reactions gave more DAOG production.

We explored the effect of substrate (i.e., penicillin G) concentration on the cell-free extract reaction and found increased substrate concentration gave increased product formation (FIG. 11). We used substrate concentrations up to 15 times higher than those previously used by Maeda et al. in their attempts to expand penicillin G.

We varied the concentrations of cell-free protein, penicillin G, FeSO.sub.4, and .alpha.-ketoglutarate in our cell-free reactions and found that, consistent with our above results, higher concentrations (within the limits that we tested) tended to yield more product (Table 3).

 

                             TABLE 3
    Effect of Modifications in Concentration of Cell-free protein, Fe.sup.2+,
     .alpha.-
    Ketoglutarate, and Penicillin G on Penicillin G Ring Expansion by Cell-
                          free Extracts
                             .alpha.-
       Protein    FeSO.sub.4   ketoglutarate  Penicillin G    DAOG
       (mg/ml)     (mM)        (mM)         (mg/ml)   (.mu.g/ml)
          4          0.036      0.64            1           3.4
          4          0.036      0.64            0.3         2.6
          4          0.036      0.64            0.1         2.3
          2          0.036      0.64            1           2.6
          2          0.036      0.64            0.3         2.1
          2          0.036      0.64            0.1       <2
          1          0.036      0.64            1           2.1
          1          0.036      0.64            0.3       <2
          1          0.036      0.64            0.1       <2
          4          1.8       1.28            1           5.6
          4          1.8       1.28            0.3         3.4
          4          1.8       1.28            0.1         2.8
          2          1.8       1.28            1           3.4
          2          1.8       1.28            0.3         2.3
          2          1.8       1.28            0.1         2.1
          1          1.8       1.28            1           2.1
          1          1.8       1.28            0.3       <2
          1          1.8       1.28            0.1       <2

Ring Expansion of Other Penicillins by Cell-free Extracts: We tested our reaction conditions for their ability to support ring expansion on other penicillin substrates and found detectable expansion with each of the 15 substrates that we tested. Large zones of inhibition were observed for penicillin G, penicillin X, penicillin mX, and 2-thiophenylacetyl-6-APA; intermediate size zones were observed for adipyl-6-APA, ampicillin, butyryl-6-APA, heptanoyl-6-APA, hexanoyl-6-APA, octanoyl-6-APA, penicillin F, and valeryl-6-APA; smaller zones were observed for decanoyl-6-APA, nonanoyl-6-APA, and penicillin V (Table 4). The reactions contained 4 mg/ml extract (protein concentration), 50 mM Tris-HCl pH 7.4, 1.8 mM FeSO.sub.4, 1.28 mM .alpha.-ketoglutarate, 8 mM MgSO.sub.4, 8 mM KCl, 4 mM ascorbic acid, 14 mM DTT, and 2 mg/ml substrate (except for penicillin mX and octanoyl-6-APA, which were used at 3 mg/ml). Samples were centrifuged at 12K rpm for 5 minutes after 2 hours of reaction. 250 .mu.l of reaction mixture was used in each bioassay.

 

                             TABLE 4
           Expansion of Penicillins Using Cell-Free S.
                 clavuligerus Expandase Extracts
                                        Inhibition Zone
              Substrate                  Diameter (mm)
              None                             0
              Adipyl-6-APA                     20
              Ampicillin                       17
              Butyryl-6-APA                   17.5
              Decanoyl-6-APA                   7
              Heptanoyl-6-APA                 16.5
              Hexanoyl-6-APA                  19.5
              Nonanoyl-6-APA                  10.5
              Octanoyl-6-APA                   16
              Penicillin F                     21
              Penicillin G                     29
              Penicillin V                     7.5
              Penicillin mX                   30.5
              Penicillin X                    30.5
              2-Thiophenylacetyl-6-               32
              APA
              Valeryl-6-APA                    15

Example 2

Conversion of Penicillin G into DAOG by Growing Cells of S. clavuligerus NP1

Materials and Methods

Microorganisms: For expandase production, we utilized the NP1 mutant described in Example 1. For our bioassay, we utilized E. coli strain ESS, a mutant that is hypersensitive to .beta.-lactam antibiotics.

Media: Seed culture was prepared using 250 ml baffled flasks containing 40 ml MST medium (90 mM MOPS, 3% Trypticase Soy Broth without Dextrose, 1% soluble starch) adjusted to pH 7.0 before autoclaving. Fermentation was carried out in the same medium or in the defined medium described by Mahro and Demain (1987) containing, per liter, 5 g MOPS; 3.5 g K.sub.2 HPO.sub.4 ; 1 ml trace solution containing 1 mg FeSO.sub.4.7H.sub.2 O, 1 mg, MnCl.sub.2.4H.sub.2 O, 1 mg ZnSO.sub.4.H.sub.2 O and 1 mg CaCl.sub.2 ; 2 g L-asparagine; 0.6 g MgSO.sub.4.7H.sub.2 O; 10 g glycerol; initial pH 7.0.

Culture Conditions: For the seed culture, each flask was inoculated with 50 .mu.l of a spore suspension (prepared and stored in 20% glycerol at -80.degree. C.) and incubated at 30.degree. C. for 48 h. Fermentation were conducted in 250 ml baffled flasks containing 30 ml of medium at 30.degree. C. at 250 rpm. Fermentations were started by transferring 1.5 ml of unwashed mycelium from the seed culture and their duration was 5 days. Once a day, samples were taken for pH, biomass and antibiotic analyses. Growth was measured as dry cell weight.

Bioassay: Cephalosporin type antibiotic(s) were detected by bioassay using Escherichia coli Ess seeded in LB (1% Tryptone, 0.5% NaCl, 0.5% Yeast Extract, 0.1% Glucose) 0.8% agar medium in the presence of penicillinase (Difco Laboratories, Detroit, Mich.). This is a narrow spectrum .beta.-lactamase that destroys all kinds of penicillins but not cephalosporins. Assays were conducted with filter paper discs saturated with 100 .mu.l of standard (deacetoxycephalosporin G) or supernatants from the fermentation flask. The diameters of zones of growth inhibition were measured after overnight incubation at 37.degree. C.

HPLC Analysis: We were interested in identifying which cephalosporin(s) was produced by growing cells. For that purpose, we developed a rapid system for the separation of penicillin G and deacetoxycephalosporin G using HPLC. The equipment consisted of a Waters LC Module I, 486M1 detector and W600 pump, and a .mu.Bondapack C18 column (30 cm.times.3.9 mm). The mobile phase was 10 mM KH.sub.2 PO.sub.4 (adjusted to pH 3 with concentrated phosphoric acid)-methanol (60:40 v/v). Samples (20 .mu.l) of the fermentation broths were analyzed at a flow rate of 1 ml/min with detection at 225 nm.

Results

We found that growing S. clavuligerus NP1 cells converted penicillin G to DAOG. Specifically, cells grown in the absence of added penicillin G produced only very low levels (in MST medium) of cephalosporin, or no detectable cephalosporin at all (defined medium). Cells grown in the presence of penicillin G produced strong zones of inhibition. In MST supplemented with 50 .mu.g penicillin G/ml, approximately 21% conversion (i.e., 10.5 .mu.g cephalosporin/ml) was observed after 72 hours of incubation; in MST supplemented with 100 .mu.g penicillin G/ml, the same level of conversion was observed after 96 hours. In defined medium, the conversion rates were 9.1% (4.55 .mu.g cephalosporin/ml) for 50 .mu.g/ml penicillin G and 10.5% (10.5 .mu.g cephalosporin/ml) for 100 .mu.g/ml penicillin G after 48 hours.

When the extracellular culture fluids were analyzed by HPLC, a penicillin G peak (observed at 0 h of growth to be eluting with a retention time of 5.6 min) decreased markedly over the 24-120 h time period, and a new peak, corresponding to DAOG and eluting at 4.6 min, appeared. Two additional peaks of unknown origin, having retention times shorter than 4.6 min, were also observed as the reaction progressed.

Example 3

Effect of Alcohols on Penicillin G Conversion by Resting S. clavuligerus NP1 Cells

Materials and Methods

Microorganisms, Media, and Culture Conditions: All experiments were done using S. clavuligerus NP1. Seed cultures were developed using 250 ml baffled flasks containing 40 ml of MST medium: 1% soluble starch (Sigma Chemical Co., St. Louis, Mo.); 3% Trypticase Soy Broth Without Dextrose (BBL, Cockeysville, Md.); 90 mM MOPS buffer, pH adjusted to 7.0 before autoclaving. Each flask was inoculated with 50 .mu.l of a spore suspension (stored in 20% glycerol at -80.degree. C.) and incubated at 30.degree. C., 250 rpm for 48 h.

From a seed culture, 4 ml were transferred to 500 ml baffled flasks containing 80 ml of MT medium (3% Trypticase Soy Broth Without Dextrose; 90 mM MOPS buffer, pH adjusted to 7.0 before autoclaving) with or without 1-2% ethanol or methanol. Alcohols were added just before inoculation. Cells were grown at 30.degree. C., 250 rpm for 24 h. Mycelia from each flask were washed twice and, finally, resuspended in 10 ml of distilled water. Four ml of this cell suspension were used in the ring-expanding biotransformation.

Ring Expansion: The ring expansion mixture contained 1.8 mM FeSO.sub.4, 1.28 mM .alpha.-ketoglutarate, 4 ml cell suspension, 5.6 mM penicillin G and 50 mM MOPS (pH 6.5) in a final volume of 10 ml contained in 250 ml baffled Erlenmeyer flasks. Additions were made in the order established by Shen et al. (Shen et al., Enzyme Microb. Technol. 17:231-234, 1984). Incubation was at 220 rpm and 30.degree. C. for 1 to 3 h. Samples were collected and centrifuged. Biotransformation activity was detected by paper disc-agar diffusion bioassay.

Detection of Expandase Activity: Expandase activity was detected by paper disc-agar diffusion bioassay. Two superimposed paper discs (1/4 inch; Schleicher & Schuell, Keene, N.H.) were saturated with 100 .mu.l of each supernatant or standard. After each application, the discs were allowed to dry for 30 min in a laminar hood and then placed on Petri plates containing 10 ml of LB (1% tryptone, 0.5% NaCl, 0.5% yeast extract, 0.1% glucose) 0.8% agar medium containing 50,000 IU/ml of penicillinase (Difco Bacto penase concentrate, Difco Laboratories, Detroit, Mich.) seeded with E. coli ESS (a .beta.-lactam-supersensitive mutant). The plates were incubated overnight at 37.degree. C. The penicillinase used is a narrow spectrum .beta.-lactamase that destroys the substrate penicillin G but not cephalosporins. The diameters of zones of growth inhibition were measured and quantified with calibration curves using pure DAOG as standard.

HPLC Analysis: The equipment used for HPLC consisted of a Waters LC Module I with a 486M1 detector, W600 pump and a .mu.Bondapack C18 column (30 cm.times.3.9 mm) (Waters Associates, Milford, Mass.). Samples (20 .mu.l) from the biotransformation mixtures were analyzed at a flow rate of 1 ml/min with detection at 260 nm. The elution was done with 10 mM KH.sub.2 PO.sub.4 (adjusted to pH 3 with concentrated phosphoric acid)-methanol (80:20 v/v) in the isocratic mode during the first 5 min followed by a 15 mn linear gradient from 100% of the initial solvent (KH.sub.2 PO.sub.4 -methanol) to 100% methanol.

Dry Cell Weight (DCW) Assay: Two samples of 1 ml were taken from each cell suspension prepared in distilled water (10 ml), centrifuged (14,000.times.g, 10 min) and dried to constant weight at 65.degree. C. The weights listed are those in the reaction mixture.

Preparation of DAG: DAG was provided by Saul Wolfe, who prepared it as follows: A solution of 7-aminodeacetylcephalosporanic acid (801 mg, 3.48 mmoles) and sodium bicarbonate (980 mg, 11.7 mmoles), in acetone (26 ml) and water (32 ml), was treated during 10 min at 0.degree. C. with a solution of phenylacetyl chloride (530 .mu.l, 620 mg. 4.01 mmoles) in acetone (3.2 ml). The reaction mixture was stirred for 1.5 h, diluted with ethyl acetate (2.times.20 ml) and the phases were separated. The organic layer was discarded, and the aqueous layer was acidified to pH 3-4 using 1 M hydrochloric acid and then extracted with ethyl acetate (2.times.20 ml). This extract was washed with saturated sodium chloride (40 ml), dried over anhydrous magnesium sulfate, and evaporated to give a white solid. This solid was triturated with diethyl ether, cooled to -20.degree. C., and filtered to give the product (643 mg, 53%).

.sup.1 HMR (acetone-d6, .delta.): 7.98 (1H, d, 8.7 Hz, NH), 7.34 (4H, t. Ar), 7.24 (1H, d, Ar), 5.78 (1H, dd, 4.8, 8.7 Hz, .beta.-lactam CH), 4.43 (1H, d, 13.5 Hz, PhCHH), 4.36 (1H, d, 13.5 Hz, PhCHH), 3.67 (1H, d, 14.3 Hz, SCHH), 3.62 (1H, d, 14.3 Hz, SCHH), 3.68 (1H, d, 18.4 Hz, CHHOH), 3.61 (1H, d, 18.4 Hz, CHHOH). IR (Kbr): 3401, 3237, 1765, 1723, 1649 cm.sup.-1.

Calcd. for C.sub.16 H.sub.16 N.sub.2 O.sub.5 S.0.25H.sub.2 O: C, 54.46; H, 4.71; N, 7.94 Found: C, 54.90; H, 4.89; N, 7.76.

Results

We found that specific conversion of penicillin G to DAOG by growing S. clavuligerus cells could be stimulated by exposing the cells to stress in the form of alcohol added to the growth medium. We used specific production as a measure of conversion in these experiments rather than volumetric production in order to normalize the effect of inhibition of S. clavuligerus growth by the alcohol. As shown in Table 5, after 3 hours of reaction, cells that had been grown in MST medium produced the lowest specific amount of cephalosporins; cells that had been grown in MT medium (identical to MST medium except that MT medium lacks starch) produced moderately more, and cells that had been grown on MT medium supplemented with 1% ethanol or 2% ethanol produced substantially (up to 6- to 7-fold) more. A modest increase in production was also observed with cells that had been grown on MT medium supplemented with 1% methanol.

 

                             TABLE 5
        Effect of Growth on Alcohol for Penicillin G Ring
                    Expansion by Resting Cells
                                   Cephalosporin Production
                          DCW       Volumetric      Specific
          Medium        (mg/ml)     (.mu.g/ml)     (.mu.g/mg)
            MST           5.8            5.8           1.0
            MT            3.6            5.3           1.5
         MT + 1% E        3.4           12.3           3.6
         MT + 2% E        0.9            6.3           7.0
         MT + 1% M        3.0            5.2           1.7
         MT + 2% M        2.9            4.3           1.5

We noted that, if alcohol addition was delayed to later times in cell growth (2 h, 6 h, or 12 h), there was no stimulatory effect on cephalosporin production. Also, no stimulation of cephalosporin production was observed when MST medium, instead of MT medium, was supplemented with an alcohol. Indeed, addition of an alcohol to MST medium completely inhibited penicillin G expansion as detected in our assays.

We also note that supplementation with alcohol had modest negative effects on cell growth. Specifically, while cells grown in MST or MT formed typical masses of tangled hyphae, cells grown in alcohol-supplemented cultures demonstrated different morphologies. In 1% ethanol, the hyphae were more dispersed; in 2% ethanol, the hyphae were extensively fragmented and dispersed. Also, DCW assays indicated that growth was reproducibly diminished in 1% ethanol or 1-2% methanol as compared with 0% alcohol in MT; 2% ethanol severely restricted growth. Concentrations of alcohol higher than 2% completely inhibited growth.

We used HPLC analysis to track the bioconversion reactions stimulated by our resting cells. Chromatograms taken before the start of the reaction (see FIG. 12A) showed peaks at 3.1, 3.6, and 17 min, corresponding to FeSO.sub.4, .alpha.-ketoglutarate, and penicillin G, respectively. After 1 hour of incubation, two new peaks, at 3.65 and 15.3 mins, appeared on the chromatogram (FIG. 12B). The 15.3 min peak was DAOG but the 3.65 min peak (*) remains unidentified. During the subsequent 2 hours of incubation (FIGS. 12C and D), the 15.3 min and 3.65 min peaks increased in size. No peak corresponding to DAG was detected during the reaction; standards tested indicated that a DAG peak would have eluted at 8 min.

Example 4

Effect of B