U.S. patent application number 09/296903 was filed with the patent office on 2001-07-05 for penicillin conversion.
Invention is credited to ADRIO, JOSE L., BAEZ-VASQUEZ, MARCO A., CHO, HIROSHI, DEMAIN, ARNOLD L., FERNANDEZ, MARIA-JOSEFA E., HINTERMANN, GILBERTO, PIRET, JACQUELINE M..
Application Number | 20010006795 09/296903 |
Document ID | / |
Family ID | 22173545 |
Filed Date | 2001-07-05 |
United States Patent
Application |
20010006795 |
Kind Code |
A1 |
DEMAIN, ARNOLD L. ; et
al. |
July 5, 2001 |
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.
Inventors: |
DEMAIN, ARNOLD L.;
(WELLESLEY, MA) ; CHO, HIROSHI; (TOKYO, JP)
; PIRET, JACQUELINE M.; (CAMBRIDGE, MA) ; ADRIO,
JOSE L.; (LEON, ES) ; FERNANDEZ, MARIA-JOSEFA E.;
(MADRID, ES) ; BAEZ-VASQUEZ, MARCO A.; (MONTERREY
N.L., MX) ; HINTERMANN, GILBERTO; (CAMBRIDGE,
MA) |
Correspondence
Address: |
C. HUNTER BAKER, M.D., PH.D.
CHOATE HALL & STEWART
EXCHANGE PLACE
53 STATE STREET
BOSTON
MA
02109-2891
US
|
Family ID: |
22173545 |
Appl. No.: |
09/296903 |
Filed: |
April 22, 1999 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60082800 |
Apr 23, 1998 |
|
|
|
Current U.S.
Class: |
435/49 ;
435/47 |
Current CPC
Class: |
C07K 2319/00 20130101;
C12P 35/00 20130101; C12N 9/0071 20130101 |
Class at
Publication: |
435/49 ;
435/47 |
International
Class: |
C12P 035/06; C12P
035/00 |
Claims
What is claimed is:
1. A method of converting a penicillin other than penicillin N to a
cephalosporin, the method comprising steps of: providing a source
of expandase enzyme; providing a penicillin substrate other than
penicillin N; 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 1 wherein the step of providing a source of
expandase comprises providing extract of a cell that produces
expandase.
4. The method of claim 1 wherein the step of providing a source of
expandase comprises providing purified expandase.
5. The method of claim 4 wherein the step of providing a source of
expandase comprises providing pure expandase.
6. The method of claim 4 wherein the step of providing a source of
expandase comprises providing an immobilized pure enzyme.
7. The method of claim 2 wherein the cells are cells that naturally
express the expandase.
8. The method of claim 7 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.
9. 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.
10. The method of claim 9 wherein the expandase gene is selected
from the group consisting of the S clavuligerus expandase gene and
the C. acremonium expandase gene.
11. 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.
12. The method of claim 2 wherein the step of providing a source of
expandase enzyme comprises providing cells that produce S.
clavuligerus expandase.
13. The method of claim 9 wherein the step of providing a source of
expandase enzyme comprises providing S. clavuligerus cells.
14. The method of claim 9 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.
15. The method of claim 1 wherein the step of providing a
penicillin substrate comprises providing a substrate selected from
the group consisting of: 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-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, ampicillin, 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 identifying a microorganism that is capable of
converting a penicillin substrate other than penicillin N to a
cephalosporin, the method comprising steps of: providing a
plurality of test microorganisms; providing a penicillin substrate
other than penicillin N at a concentration above about 2 mg/ml;
exposing the microorganism to the substrate under reaction
conditions including a concentration of Fe.sup.2+ within the range
of about 0-4 mM; detecting production of a cephalosporin; and
identifying the test microorganism as one that is capable of
converting the penicillin substrate into a cephalosporin.
20. The method of claim 19 wherein the reaction conditions further
include an expandase source comprising cells producing expandase,
which cells are provided at a biomass of less than about 6 g wet
weight/10 ml solution.
21. The method of claim 20 wherein the cells were grown under
stressing conditions.
22. The method of claim 20 wherein the stressing conditions are
selected from the group consisting of: the presence of 1-2% ethanol
and the presence of 1% methanol.
23. The method of claim 20 wherein the reaction conditions are
characterized by an absence of one or more of: a reducing agent;
.alpha.-ketoglutarate; and ascorbic acid.
24. The method of claim 20 wherein the step of identifying
comprises identifying those microorganisms that, when exposed to
the substrate, produce a compound that creates a zone of inhibition
in the presence of penicillinase.
25. The method of claim 20 wherein the step of identifying
comprises identifying those microorganisms that, when exposed to
the substrate, produce a cephalosporin precursor detectable by
HPLC.
26. A method of preparing a cephalosporin, the method comprising
steps of: providing a penicillin substrate other than penicillin N;
contacting the penicillin substrate with a source of an expandase
enzyme, which expandase enzyme is not naturally produced in a cell
that also naturally produces the penicillin substrate, the
contacting being performed under reaction conditions including: an
iron concentration within the range of 0-4 mM; and a concentration
of .alpha.-ketoglutarate within the range of 0-4 mM; and isolating
a cephalosporin having a chemical structure depicted in FIG. 2.
Description
[0001] 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.
BACKGROUND OF THE INVENTION
[0002] 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).
[0003] 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.
[0004] 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.
[0005] 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-aminodeacetylcephalos- poranic 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.
[0006] 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.
[0007] 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.
[0008] 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).
[0009] 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).
[0010] 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.
[0011] 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 glutaryl-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).
[0012] 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.
[0013] 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
[0014] 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
[0015] "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.
[0016] "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.
[0017] "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.
[0018] "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.
[0019] "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
[0020] FIG. 1A presents a generalized structure of penicillins;
FIG. 1B shows R.sub.1 groups present in different penicillins.
[0021] FIG. 2A presents a generalized structure of cephalosporins;
FIG. 2B shows R.sub.1 and R.sub.2 groups present in certain
different cephalosporins.
[0022] FIG. 3 presents a portion of the cephalosporin C
biosynthetic pathway utilized by C. acremonium to produce
cephalosporin C from endogenous penicillin N.
[0023] 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.
[0024] 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.
[0025] 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.
[0026] 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.
[0027] 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.
[0028] 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.
[0029] 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.
[0030] 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.
[0031] 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.
[0032] FIG. 13 shows the effect of buffer selection on penicillin G
conversion by resting S. clavuligerus NP1 cells. (.tangle-solidup.)
0.05 M MOPS, pH 6.5, (.smallcircle.) 0.05 M HEPES, pH 6.5,
(.DELTA.) 0.05 M Tris-HCl, pH 7.4.
[0033] 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, (x) .alpha.-ketoglutarate,
(.box-solid.) ascorbic acid+Fe.sup.2+, (.DELTA.) ascorbic
acid+Fe.sup.2++.alpha.-ketoglutarate.
[0034] 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
(x) 8.0 mg/ml.
[0035] 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.
[0036] 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.
[0037] 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.).
[0038] 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.
[0039] FIG. 20 shows plasmidls utilized to study homologous
recombination.
[0040] FIG. 21 shows the sequences of crossover junctions of hybrid
expandase genes obtained in DH5.alpha. cells.
[0041] FIG. 22 shows HPLC profiles of ring expansion reactions.
DESCRIPTION OF CERTAIN PREFERRED EMBODIMENTS
[0042] 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.
[0043] 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.
[0044] Expandase Source
[0045] 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.
[0046] 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.
[0047] 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.
[0048] 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).
[0049] 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.
[0050] 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; Corts 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.
[0051] 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.
[0052] 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.
[0053] 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.
[0054] 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.
[0055] Penicillins
[0056] 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.
[0057] Reaction Conditions
[0058] 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; Corts 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.
[0059] 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.
[0060] 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.
[0061] 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.
[0062] .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.
[0063] 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.
[0064] 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.
[0065] 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.
[0066] 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.
[0067] 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.
[0068] 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).
[0069] 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).
[0070] 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.
[0071] Diversification of Cephalosporin Precursor
[0072] 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).
[0073] Several particularly useful third- and fourth-generation
cephalosporins (e.g., cefotaxime) include a
2-aminothiazol-4-yl-acetamido side chain combined with a
synalkoxyimino 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).
[0074] 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).
[0075] 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
[0076] Materials and Methods
[0077] 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.
[0078] 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.
[0079] MATERIALS: Penicillin G, ascorbic acid and
.alpha.-ketoglutaric acid were from Sigma Chemical Company (St.
Louis, Mo.). Deacetoxycephalosporin G was from Antibiotics, S.A.
(Len, Spain) and Bacto-Penase from Difco Laboratories (Detroit,
Mich.).
[0080] 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.cndot.HCl 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.
[0081] 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.
[0082] 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.
[0083] 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.
[0084] 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 Len, Spain), and were synthesized as previously
described (Maeda et al., Enzyme Microb. Technol. 17:231-34, 1995).
DAOG was provided to us by Antibiticos, S.A. (Madrid, Spain).
[0085] Results
[0086] 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.
[0087] 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.
[0088] 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).
[0089] 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).
1TABLE 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 .multidot.
7H.sub.2O 3.2 (1.8 mM) MgSO.sub.4 .multidot. 7H.sub.2O 10.1 (8 mM)
KCl 11.5 (8 mM) Ascorbic acid 3.0 (4 mM) ATP 10.0 (0.7 mM)
[0090] 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.
2TABLE 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
[0091] 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).
[0092] 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.
[0093] 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.
[0094] 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).
3TABLE 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
[0095] 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.
4TABLE 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
[0096] Materials and Methods
[0097] 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.
[0098] 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.2HPO.sub.4; 1 ml trace solution
containing 1 mg FeSO.sub.4.7H.sub.2O, 1 mg, MnCl.sub.2.4H.sub.2O, 1
mg ZnSO.sub.4.H.sub.2O and 1 mg CaCl.sub.2; 2 g L-asparagine; 0.6 g
MgSO.sub.4.7H.sub.2O; 10 g glycerol; initial pH 7.0.
[0099] 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.
[0100] 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.
[0101] 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.2PO.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.
[0102] Results
[0103] 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.
[0104] 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
[0105] Materials and Methods
[0106] 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.
[0107] 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.
[0108] 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.
[0109] 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.
[0110] 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.2PO.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.2PO.sub.4-methanol) to 100% methanol.
[0111] 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.
[0112] 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.1HMR (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.16H.sub.16N.sub.2O.sub.5S.0.25H.sub.2O: C 54.46; H 4.71; N
7.94 Found: C 54.90; H 4.89; N 7.76.
[0113] Results
[0114] 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.
5TABLE 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
[0115] 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.
[0116] 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.
[0117] 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 Buffer Selection, Cell Treatment, and Substrate
Concentration on Penicillin G Expansion by Resting S. clavuligerus
NP1 Cells
[0118] Materials and Methods
[0119] MICROORGANISMS, MEDIA, AND CULTURE CONDITIONS: S.
clavuligerus mutant NP-1, described in Example 1 was used for these
studies. A seed culture was made by inoculating a spore suspension
(40 .mu.l) into 40 ml MST medium in 250 ml baffled flasks, and
incubating for 2 days at 30.degree. C. and 250 rpm. One ml of the
seed culture was used as inoculum for the main culture which
contained 80 ml of MST medium in 500 ml unbaffled flasks. The
flasks were incubated for 24 h at 30.degree. C. and 250 rpm.
[0120] PREPARATION OF RESTING CELLS: Cells were harvested by
centrifugation at 14,000.times.g, for 15 min at 4.degree. C. and
washed twice with cold deionized water. They were resuspended in 10
ml of water.
[0121] RING EXPANSION REACTION: The standard reaction mixture (10
ml) contained 0.05 M Tris-HCl pH 7.4, 8.0 mM KCl, 8.0 mM
MgSO.sub.4.7H.sub.2O, 4.0 mM ascorbic acid, 1.8 mM
FeSO.sub.4.7H.sub.2O, 1.28 mM .alpha.-ketoglutaric acid, 20 mg/ml
penicillin G and 4.0 ml cell suspension. The order of addition of
the components were as previously described (Shen et al., Enzyme
Microb. Technol. 6:402-404, 1984). The reaction started when
penicillin G was added to the reaction mixture which was incubated
at 30.degree. C. and 220 rpm.
[0122] EXPANDASE ASSAY: Product formation was estimated by the
paper disk-agar diffusion bioassay as described in example 1, using
deacetoxycephalosporin G as standard. Escherichia coli strain Ess,
a .beta.-lactam supersensitive, mutant was used as the assay
microorganism.
[0123] Results
[0124] In an effort to expand the discovery reported in Example 1,
we analyzed the effects of buffer selection, treatment of resting
cells, substrate concentration, and cell biomass on the extent of
penicillin G conversion by S. clavuligerus NP1 resting cells.
[0125] FIG. 13 demonstrates that the use of either 0.05 M MOPS
buffer at pH 6.5 or 0.05 M HEPES buffer at pH 6.5 increases the
production of cephalosporin more than two-fold as compared with
reactions performed in 0.05 M Tris-HCl pH 7.4. Subsequent reactions
were therefore performed in 0.05 M MOPS, pH 6.5.
[0126] FIG. 13 also demonstrates that, in all buffers,
cephalosporin production proceeds rapidly for about 1 hour and then
stops altogether or proceeds at a significantly reduced rate. We
asked whether one of the reaction component might be inhibiting the
reaction, perhaps by inactivating the expandase after the
approximately 1 hour period. Specifically, we preincubated the
resting cells for 3 hours in the presence of one or more of the
reaction components; at the end of the preincubation period, all
missing components were added.
[0127] FIG. 14 shows that, when cells were preincubated with either
buffer, Fe.sup.2+, ascorbic acid, or .alpha.-ketoglutarate, product
formation occurred more slowly and total production was reduced by
about 50%. Preincubation with a combination of Fe.sup.2+ and
ascorbic acid, with or without .alpha.-ketoglutarate, virtually
eliminated all penicillin G conversion. These experiments
demonstrate that the flattening of the product formation curve over
time is most likely due to enzyme destruction by Fe.sup.2+ in
combination with ascorbate and/or .alpha.-ketoglutarate.
[0128] In addition to testing the effects of buffer selection and
component preincubation on the penicillin G conversion reaction, we
tested the effect of increases or decreases in substrate
concentration. As shown in FIGS. 15 and 16, larger amounts of
product were produced when more substrate was added (FIG. 15), but
the percent conversion was dramatically reduced (FIG. 16).
Specifically, at 8 mg/ml of penicillin G, about 0.4% was converted;
at 2 mg/ml, about 1.0% was converted, and at 0.063 mg/ml, about
9.0% was converted. Subsequent experiments revealed conversions as
high as 16.5% for 0.015 mg/ml substrate.
[0129] Finally, we tested the effects on percent conversion of
increasing the concentration of biomass in our resting cell
reactions. As is shown in FIG. 17, we found that, for two different
concentrations of penicillin G (0.063 mg/ml and 2 mg/ml),
conversion was markedly enhanced at lower cell concentrations.
Example 5
Conversion of Penicillin G to DAOG by Immobilized S. clavuligerus
NP1 Cells
[0130] Materials and Methods
[0131] CULTURE CONDITIONS: A seed culture of S. clavuligerus mutant
NP-1 was made by thawing a frozen preparation of spores and
inoculating 40 .mu.l into 40 ml MST medium containing 90 mM MOPS
buffer, pH 7.0, 1% starch and 3% trypticase soy broth medium
without dextrose (BBL Becton Dickinson Microbiology Systems,
Cockeysville, Md.) in 250 ml baffled flasks. The flasks were
incubated for 2 days at 30.degree. C. and 250 rpm. One ml of the
seed culture was used as inoculum for the main culture which
contained 80 ml of MST medium in 500 ml unbaffled flasks. The
flasks were incubated for 24 h at 30.degree. C. and 250 rpm.
[0132] PREPARATION OF RESTING CELLS: Cells were harvested by
centrifugation at 14,000.times.g for 15 min at 4.degree. C. and
washed twice with cold deionized water. Washed cells were
resuspended in 10 ml of water giving a concentration (wet weight)
of about 0.31 g/ml.
[0133] PREPARATION OF POLYETHYLENEIMINE (PEI)-BARIUM ALGINATE
ENTRAPPED CELLS: Resting cells from a one-day old culture of S.
clavuligerus NP-1 (2 g wet weight) were directly resuspended in 20
ml of 1.5% (w/v) sodium alginate solution. The resulting sodium
alginate-cell suspension was placed into a 10 ml plastic syringe
containing a 26.6G needle. The suspension was added drop by drop
into a slowly stirring hardening solution of 2% (w/v) barium
chloride containing 1% (w/v) PEI, giving beads of about 1.5-2.0 mm
in diameter. The beads were filtered on a plastic net, washed twice
with water and suspended in 0.05M MOPS buffer, pH 6.5 and stored at
4.degree. C. until used.
[0134] RING EXPANSION REACTION: The standard reaction mixture (10
ml) contained 0.05 M Tris-HCl buffer (pH 7.4), 8.0 mM KCl, 8.0 mM
MgSO.sub.4.7H.sub.2O, 4.0 mM ascorbic acid, 1.8 mM
FeSO.sub.4.7H.sub.2O, 1.28 mM .alpha.-ketoglutaric acid, 200 mg
penicillin G and 4.0 ml of free cell suspension, or 3.4 g (wet
weight) of cells in barium alginate beads. The order of addition of
the components were as described (Shen et al., Enzyme Microb.
Technol. 6:402-404, 1984). The reaction started when penicillin G
was added to the reaction mixture and was incubated at 30.degree.
C. and 220 rpm for 1-12h. Product formation was estimated by the
paper disk-agar diffusion bioassay using deacetoxycephalosporin G
as standard and penicillinase to destroy the substrate penicillin
G. Escherichia coli Ess, a .beta.-lactam supersensitive mutant, was
used as assay microorganism.
[0135] EXPANDASE ASSAY: Product formation was estimated by the
paper disk-agar diffusion bioassay using deacetoxycephalosporin G
as standard and penicillinase to destroy the substrate penicillin
G. Escherichia coli Ess (Kohsaka et al., Biochem. Biophys. Res.
Commun. 70:465-573, 1976), a .beta.-lactam supersensitive mutant,
was used as assay microorganism.
[0136] MATERIALS: Alginic acid sodium salt was from Aldrich
Chemical Company, Inc., Milwaukee, Wis., Agarose Type VII low
gelling temperature, polyethyleneimine 50% (PEI), penicillin G,
ascorbic acid and .alpha.-ketoglutaric acid were from Sigma
Chemical Company (St. Louis, Mo.). Deacetoxycephalosporin G was a
gift from Antibiotics, S.A. (Len, Spain) and Bactopenase was from
Difco Laboratories (Detroit, Mich.).
[0137] Results
[0138] This Example demonstrates that S. clavuligerus NP1 cells
immobilized by entrapment on a polymeric matrix are able to perform
oxidative ring expansion of penicillin G into DAOG. Specifically,
resting cells entrapped in PEI-barium alginate (1.5%) were shown to
be able to expand penicillin G (FIG. 18). Because the weights of
entrapped resting cells could not be normalized to those of free
resting cells, the curves presented in FIG. 18 cannot be directly
compared to one another. However, we find that increases in
entrapped biomass concentration resulted in increased product
formation (FIG. 19).
[0139] In the reactions depicted in FIGS. 18 and 19, both free and
immobilized cells lost a considerable amount of their activity two
hours. However, we found that the cells were able to re-initiate
expandase reactions after being centrifuged (3500.times.g for 5 min
at 4.degree. C.) and washed in 0.05 M MOPS, pH 6.5 (FIG. 20).
Interestingly, the free cells lost 60% of their activity after a
single wash cycle and were completely inactivated after two cycles.
Immobilized cells, by contrast, retained a good portion of their
activity through at least four cycles.
[0140] We tested the activity of cells immobilized in other
polymeric matrices, specifically agarose (4% w/v) and k-carrageenan
(3% w/v), but did not observe cephalosporin production in either
case, possibly due to cell and/or enzyme injury during
immobilization.
[0141] Although the entrapped cells described here exhibited lower
oxidative ring expansion activity than free resting cells, their
immobilization may offer storage stability, recyclability, and
operational stability for biotransformation of penicillins into
cephalosporins.
Example 6
Construction of Hybrid Expandases Through Gene Shuffling by
Homologous Recombination
[0142] Materials and Methods
[0143] STRAINS, MEDIA AND CULTURE CONDITIONS: Escherichia coli
DH5.alpha. (Gibco BRL, Gaithersburg, Md.) and ER1447 (New England
Biolabs, Beverly, Mass.; Palmer et al., Gene 143:1-12, 1994) were
used as host strains for transformations and recombination
experiments. The E. coli ESS mutant described above was used as the
indicator in the bioassay. Both E. coli strains were grown in LB
(1% Tryptone, 0.5% NaCl, 0.5% Yeast Extract, 0.1% glucose) at
37.degree. C.
[0144] Streptomyces lividans 1326 was grown on CG medium (0.4%
Yeast Extract, 1% Malt Extract, 0.4% glucose; adjusted to pH 7.3
with KOH) or R2YE (Hopwood et al., Meth. Enzymol. 153:116-167,
1987) at 30.degree. C.
[0145] For ring expansion activity, cells were grown in MST (1%
soluble starch, 3% Trypticase Soy Broth without Dextrose [BBL,
Cockeysville, Md.], 90 mM MOPS; pH adjusted to 7.0 with KOH before
autoclaving.
[0146] DNA MANIPULATION AND TRANSFORMATION: Plasmid isolation and
transformation into E. coli were carried out using standard
protocols (Sambrook et al., Molecular Cloning: A Laboratory Manual,
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,
1989). Protoplast preparation and transformation of Streptomyces
were performed following the methods described by Hopwood et
al.(Hopwood et al., Manipulation of Streptomyces: a Laboratory
Manual, Norwich: John Innes Foundation, 1985). For restriction
enzyme digestions, the DNA was purified using QIAprep Spin Miniprep
Kit or QIAGEN plasmid Kit (QIAgen Inc., Valencia, Calif.).
Restriction enzyme digestions and ligations were carried out as
recommended by the manufacturers (Boehringer Mannheim and New
England Biolabs).
[0147] PLASMID CONSTRUCTIONS (see FIG. 21):pJALC: A 3 kb BamHI
fragment from pULFJ32 (J. F. Martin, University of Len, Spain)
containing the cefE gene (and part of the cefD gene) from S.
clavuligerus NRRL3585 was cloned into pCYB160 (derived from pCYB1,
New England Biolabs), which already contained the cefE gene from N.
lactamdurans.
[0148] pJALTC: A 2.3 kb KpnI fragment from pMEA4 (Hintermann,
Aspects of the Genomic Organisation in Streptomyces glaucescens,
Ph.D. dissertation, ETH, Zurich, 1984) containing the tyrosinase
gene from S. glaucescens was cloned into pSP73 (Promega, Madison,
Wis.) to yield pJA73T-SP6. A 2.3 kb EcoRI-HindIII fragment from
this plasmid was then ligated with pIJ487 (Hopwood et al., Meth.
Enzymol. 153:116-167, 1987) to yield plasmid pJA487T. This plasmid
was then digested with BclI and the largest fragment (about 3 kb)
was extracted from the agarose gel using GeneCleanII (Bio101,
Vista, Calif.) and ligated into the BglII site of pJALC to give
pJALTC.
[0149] pJA680: pJALTC was digested with EcoRV and NsiI and the 8.6
kb fragment, containing both cefE genes and the tyrosinase gene,
was extracted as mentioned above and inserted into the
PstI-Ecl136II digested Streptomyces vector pIJ680 to create
pJA680.
[0150] RECOMBINATION IN E. COLI: E. coli ER1447 (a recA.sup.+
strain) was transformed with pJALC and, after heat shock, the cells
were incubated overnight in 5 ml of LB containing ampicillin (100
.mu.g/ml). Every 10-12 h, a 100 .mu.l sample was transferred to a
tube containing new medium (up to four rounds of growth). Samples
of 1 ml were taken from each tube and plasmid DNA was isolated,
digested with BglII and dephosporylated with CIP (Boehringer
Mannheim, Germany). These DNA preparations were used to transform
E. coli DH5.alpha..
[0151] RECOMBINATION IN S. LIVIDANS CELLS: Protoplats of S.
lividans 1326 were transformed with pJA680 and plated on R2YE
containing thiostrepton (50 .mu.g/ml). Transformants were incubated
at 30.degree. C. for 5-7 days to allow sporulation. Spore
suspension was prepared according to Hopwood et al. (Hopwood et
al., Genetic Manipulation of Streptomyces: a Laboratory Manual,
Norwich: John Innes Foundation, 1985) and aliquots were stored in
20% glycerol at -80.degree. C. Dilutions from this suspension were
then spread on CG medium supplemented with: 50 .mu.g/ml
thiostrepton, 0.3 g/l L-tyrosine, 0.1 g/l L-methionine, 0.5 g/l
L-leucine, and 5 mg/l CuSO.sub.4. Plates were incubated at
30.degree. C. for 7 days.
[0152] PREPARATION OF CELL-FREE EXTRACTS FROM S. LIVIDANS CELLS:
For each recombinant, a 250 ml baffled flask containing 50 ml of
MST+thiostrepton (5 .mu.g/ml) was inoculated with 5 ml from a seed
culture previously grown in the same medium for 72 h. Cells were
then incubated at 30.degree. C., 200 rpm for 48 h. To prepare the
extracts, cells were harvested by centrifugation (10,000.times.g,
10 min, 4.degree. C.) and washed twice with 50 mM Tris.cndot.HCl pH
7.4 containing 0.1 mM DTT. The pellet was resuspended in 5 ml of
the same buffer and cells were disrupted by four 25-sec sonication
treatments (setting 5; 50% duty cycle) using a Branson Sonifier
(Branson Sonic Power Co, Danbury, Conn.). Cell debris was removed
by centrifugation (14,000.times.g, 30 min, 4.degree. C.). Protein
concentration was measured using the Bio-Rad protein assay
(Bio-Rad, Hercules, Calif.). Bovine serum albumine was used as
standard.
[0153] RING EXPANSION REACTION: Ring expansion activity was
measured in reactions mixtures containing 4 mM ascorbic acid, 8 mM
MgSO.sub.4, 8 mM KCl, 1.8 mM FeSO.sub.4, 1.28 mM
.alpha.-ketoglutarate, 4 mg protein/ml, and 50 mM Tris
Tris.cndot.HCl pH 7.4 in a final volume of 2.5 ml. Penicillin G was
used as substrate at a final concentration of 5.6 mM. The ring
expansion was conducted in test tubes incubated at 30.degree. C.,
200 rpm for 2 h. Samples of 0.5 ml were taken and reaction was
stopped by adding the same volume of methanol.
[0154] BIOASSAY OF PRODUCT: Cephalosporins were assayed by the agar
plate diffusion method described above. Paper discs saturated with
20 .mu.l from the reaction mixtures were placed on LB agar (0.8%
agar) medium seeded with E. coli ESS. The medium also contained
50,000 IU/ml of penicillinase (Bacto Penase Concentrate, Difco,
Detroit, Mich.) which destroys the substrate penicillin G, but not
cephalosporins. The plates were incubated overnight at 37.degree.
C.
[0155] HPLC ANALYSIS: Methanol-treated samples (20 .mu.l) were
analyzed using a Waters Module I with a 486M1 detector, W600 pump
and .mu.Bondapack C18 column (300.times.3.9 mm) (Waters Associate,
Milford, Mass.). The flow rate was 1 ml/min with detection at 260
nm. The elution was done using 10 mM KH.sub.2PO.sub.4 (adjusted to
pH 3 with concentrated H.sub.3PO.sub.4)-methanol (80:20 v/v) in the
isocratic mode during the first 5 min. followed by a 15 min linear
gradient from 100% of the initial solvent
(KH.sub.2PO.sub.4-methanol) to 100% methanol.
[0156] DNA SEQUENCING: To sequence the hybrid cefE genes, we used
two different oligonucleotides depending on where recombination was
located. For those E. coli clones containing both PstI sites (see
Results) we used a 22 bp primer (5'-CCACCAGACCCCTTGCGCGAAC-3')
located 16 bp upstream the PstI site on the cefE of N. lactamdurans
(FIG. 20). For E. coli clones containing only one PstI side and
those obtained in S. lividans, we used a 22 bp primer
(5'-GAGCGGATAACAATTTCACACA-3') located within the Ptac promoter
(FIG. 20).
[0157] Results
[0158] RECOMBINATION IN E. COLI: We first generated hybrid
expandase genes by homologous recombination in E. coli.
Specifically, we produced a bacterial plasmid, pJALC, containing
the S. clavuligerus and N. lactamdurans expandase genes in tandem.
The plasmid was constructed so that a BglII site was situated
between the two genes and could be used as a diagnostic to
determine whether or not recombination between the genes had
occurred.
[0159] The plasmid was transformed into recA.sup.+ E. coli and the
cells were grown up in order to allow recombination to occur.
Plasmids were isolated back out of the cells and digested with
BglII so that plasmids that had not undergone recombination were
linearized, and the digested population was then transformed into
DH5.alpha. cells. Sixty-three DH5.alpha. colonies were analyzed to
determine whether recombination had occurred, as expected.
Seventeen of those colonies were found to contain an approximately
8 kb plasmid, the expected size for a plasmid that had undergone
recombination between the two expandase genes.
[0160] Plasmids were isolated from the seventeen colonies, and were
subjected to restriction analysis (see Table 6). Five of the
plasmids gave two bands after digestion with PstI, indicating that
recombination had occurred at a site after the PstI site in the N.
lactamdurans expandase gene. The remaining plasmids each gave a
single band when digested with PstI, indicating that one of the two
PstI sites originally present in pJALC had disappeared. Further
digestion and sequencing analysis indicated that the PstI site in
the N. lactamdurans expandase was no longer present in these
plasmids.
6TABLE 6 Restriction analysis of different clones (containing a 8
kb plasmid) obtained after transformation of E. coli DH5.alpha..
plasmid undigested + BglII + BamHI + PstI pJALC 11 11 8, 3* 6, 5
pJAR2-4 8 nd** 8 8 pJAR3-4 8 nd 8 8 pJAR4-9 8 nd 8 8 pJAR3-8 8 nd 8
6.1, 1.9 pJAR4-52 8 nd 8 6.1, 1.9
[0161] The nucleotide sequences at the recombination junctions were
determined for the plasmids isolated from DH5.alpha. (see FIG. 22).
As shown, recombination occurred after stretches of identical
sequence in the range of 2-21 bp long.
[0162] RECOMBINATION IN S. LIVIDANS: We also generated hybrid
expandase genes by recombination in Streptomyces. In this case, we
produced a plasmid, pJA680, containing the S. clavuligerus and N.
lactamdurans expandase genes in tandem, separated by the
Streptomyces glaucescens tyrosinase gene. When tyrosinase is
active, it produces the melanin pigment, which diffuses into the
agar as a black splotch. Recombination between the expandase genes
is expected to delete the tyrosinase gene from the plasmid, so that
colonies containing recombinant plasmids can be identified by the
absence of melanin production.
[0163] The plasmid was introduced into Streptomyces lividans cells
and white colonies were identified. Plasmids were prepared from
both white and black colonies. All black colonies contained
plasmids that were 12.5 kb in size, the size of intact pJA680; none
appeared to contain recombinant plasmids, which were expected to be
approximately 6.3 kb in size. Of 58 white colonies that were
analyzed, 37 (63.7%) contained a 6.3 kb plasmid, 17 (19.3%)
contained a 5.8 kb plasmid, 2 (3.45%) contained an 8 kb plasmid,
and 2 (2.45%) contained a 4 kb plasmid.
[0164] Restriction analysis of plasmids isolated from two strains
(W21 and W64) containing a 6.3 kb plasmid, two strains (W25 and
W76) containing an 8 kb plasmid, and one strain (B18) containing
pJA680, showed that plasmids from W21 and W64 were not cleaved by
PstI or XmnI; plasmids from W25 and W76 were cut by XmnI, BamHI,
and ScaI, but not by PstI. From these data, we concluded that W21
and W64 were both missing at least part of the N. lactamdurans
expandase gene; W25 and W76 were also missing part of the N.
lactamdurans gene but appeared to have an intact S. clavuligerus
gene.
[0165] We tested the putative recombinant plasmids that we had
isolated by assaying the ring expansion activity of the enzymes
they encoded (see Table 7). We found that none of the strains
containing plasmids 4 kb or 5.8 kb in size produced zones of growth
inhibition in our bioassay. One strain, W21, containing a 6.3 kb
plasmid produced a clear zone, as did both of the strains (W25 and
W76) containing 8 kb plasmids.
7TABLE 7 Ring Expansion Activity of Strains Containing Hybrid
Plasmids plasmid size (kb) strain clear zone 4 W3 - " W53 - " 5.8
W6 - " W20 - " W23 - " W39 - " W45 - " 6.3 W4 - " W5 - " W21 + "
W26 - " W32 - " W33 - " W34 - " W36 - " W41 - " W48 - " W49 - " W55
- " W61 - " W62 - " W64 - " W69 - " W72 - " W77 - " 8 W25 + " W76 +
12.5 B11 + " B18 + "
[0166] To confirm that the clear zones we were observing in our
growth inhibition assay resulted from the production of DAOG, we
analyzed all positive samples by HPLC. As shown in FIG. 22, the 0 h
samples gave a profile showing several peaks corresponding to
.alpha.-ketoglutarate (3.1 min), FeSO.sub.4 (3.6 min), and
penicillin G (17.2 min). After 2 h of incubation, the peak
corresponding to DAOG (15.5 min) was clearly present on the
chromatograms, as was another unidentified peak eluting at 3.35
min.
[0167] FIG. 21 gives the sequences of the crossover junctions
present in hybrid expandase genes isolated from E. coli. Numbers at
both sides of the sequences indicate the position (bp) within the
genes. Underlined letters indicate identical sequences. Clones
shown in Panel (A) contain two PstI sites. pJAR4-8 and pJAR4-52
showed the crossover junctions at the same position as pJAR3-8,
whereas pJAR4-6 was similar to pJAR4-5. Clones shown in Panel (B)
contain only one PstI site. pJAR3-7 and pJAR3-9 showed crossover
junctions at the same location as pJAR4-9.
Example 7
Expansion of Penicillin G and Ampicillin by Recombinant Strains of
S. lividans
[0168] Materials and Methods
[0169] MICROORGANISM, MEDIUM AND CULTURE CONDITIONS: S. lividans
1326 was used in all experiments. Cultures were grown in 250 ml
baffled flasks containing 40 ml of MST medium. 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.
[0170] TRANSFORMATION: Protoplasts preparation and transformation
were done following the protocols described previously (Hopwood et
al., Meth. Enzymol. 153:116-167, 1987).
[0171] PLASMIDS: The plasmid pUL702-202a was a gift from J. F.
Martin (University of Len, Spain). This vector harboured a 4.3 Kb
BglII fragment containing the epimerase and expandase genes from
Nocardia lactamdurans inserted at the BglII site on pIJ702. The
plasmid pHJ11 was constructed by cloning a 3 Kb BglII fragment
containing the expandase gene from S. clavuligerus 3585 into pIJ702
at the BglII site.
[0172] RING EXPANSION REACTION: The ring expansion mixture
contained 1.8 mM FeSO.sub.4, 1.28 mM .alpha.-ketoglutarate, 6 mg/ml
protein (cell-free extract), 8 mM KCl, 8 mM MgSO.sub.4, 14 mM DTT
and 50 mM TrisHCl (pH 7.4) in a final volume of 2.5 ml. Substrate
concentration was 5.6 mM. Additions were made following the order
established by Shen et al. (Enzyme Microb. Technol. 6:402-404,
1984). Reaction mixtures were incubated in test tubes at 220 rpm,
30.degree. C. At different times, 0.5 ml samples were taken and the
reaction was stopped by adding 0.5 ml of methanol. Samples were
centrifuged and supernatants were transferred to new tubes.
Biotransformation activity was detected by paper disc-agar
diffusion bioassay.
[0173] BIOASSAY OF PRODUCT: Paper discs were saturated with 200
.mu.l of supernatant. Two discs were superimposed and 4.times.50
.mu.l of sample were applied. After each application, the discs
were allowed to dry for 30 min under a laminar hood and, finally,
they were placed on LB 0.8% agar medium seeded with E. coli ESS,
and the plates were incubated overnight at 37.degree. C.
[0174] Results
[0175] When the reaction was carried out using cell-free extracts
of S. lividans containing the plasmid pUL702-202a (expandase from
N. lactamdurans), expansion was only observed when penicillin G was
used as substrate. After 2 h of reaction, production of
cephalosporins was approximately 8 .mu.g/ml. On the other hand, the
strain pHJ11 (a transformant containing the expandase from S.
clavuligerus) was able to expand both penicillin G and ampicillin.
After 4 h of reaction and using penicillin G as substrate,
production of cephalosporins was about 7 .mu.g/ml; cephalosporin
production was about 1.5 .mu.g/ml when ampicillin was used as a
substrate.
Other Embodiments
[0176] Those of ordinary skill in the art will recognize that the
foregoing has been a description only of certain preferred
embodiments of the present invention, which description is not
intended to limit the invention's scope. It will be apparent that
various alterations and substitutions can readily be made without
departing from the spirit or scope of the invention, as that is
defined in the following claims. To give but one example, those of
ordinary skill in the art will appreciate that the system described
herein can also be used for the production of cephamycins so long
as cephalosporin precursors produced by ring expansion from
penicillins can be modified by methoxylating enzymes.
* * * * *