Production of cephalosporin C

Production of undesired desacetylcephalosporin C during fermentation of cephalosporin C-producing microorganisms is substantially reduced by addition of certain phosphorous compounds to the culture medium.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
This invention relates to an improved process for the production of 
cephalosporin C. More particularly, the invention relates to the addition 
of certain organic and inorganic phosphorous compounds to the culture 
medium during fermentation of a cephalosporin C-producing microorganism 
which also produces undesired desacetylcephalosporin C. Addition of the 
phosphorous compounds greatly inhibits formation of the 
desacetylcephalosporin C impurity, thus facilitating recovery of the 
cephalosporin C from the fermentation broth and its subsequent conversion 
to 7-aminocephalosporin acid (7-ACA). 
2. Description of the Prior Art 
Cephalosporin C 
[3-acetoxymethyl-7.beta.-(D-5-amino-5-carboxypentanamido)ceph-3-em-4-carbo 
xylic acid] is a compound which, while having some antibiotic activity per 
se, is of primary importance as a starting material for preparation of 
semi-synthetic cephalosporin antibiotics. Thus, cephalosporin C may be 
converted by known methods to 
3-acetoxymethyl-7.beta.-aminoceph-3-em-4-carboxylic acid (7-ACA) which is 
then used as a key intermediate for preparation of a wide variety of 
commercial cephalosporin antibiotics. 
It is known that cephalosporin C may be obtained by fermentation of various 
microorganisms including especially fungi of the genera 
Emericellopsis-Cephalosporium. Illustrative of cephalosporin C-producing 
microorganisms are the original Brotzu strain of Cephalosporium, i.e. 
Cephalosporium sp. I.M.I. 49137 (ATCC 11550), and mutants thereof such as 
mutant strain 8650 (ATCC 14553), described in U.K. Pat. No. 1,109,362, 
Cephalosporium sp. I.B.I. 1131 described in U.K. Pat. No. 1,503,851 and 
Cephalosporium sp. strain F.12 (ATCC 20339) described in U.K. Pat. No. 
1,400,433. Other examples of cephalosporin C-producing organisms reported 
in the literature include Cephalosporium polyaleurum Y-505 (FERM-P No. 
1160) described in U.K. Pat. No. 1,488,822, Cephalosporium acremonium 
K-121 (ATCC 20427) and Cephalosporium acremonium N-75 (ATCC 20428) 
described in U.K. Pat. No. 1,488,821 and Cephalosporium polyaleurum 199 
(ATCC 20359) and a mutant thereof identified as Y-505 (ATCC 20360) 
described in U.K. Pat. No. 1,389,714. Cephalosporin C is generally 
produced on an industrial scale by use of a high-producing mutant strain 
of Cephalosporium acremonium (also known as Acremonium chrysogenum). 
Examples of such mutants and methods for their preparation have been 
extensively described in the literature. 
Despite exhaustive research over the years, fermentation of cephalosporin C 
on a commercial scale is still not entirely satisfactory. Most 
cephalosporin C-producing microorganisms, especially those high producing 
strains used in commercial production, result in co-production of a 
significant proportion of desacetylcephalosporin C, an impurity which is 
extremely difficult to separate from the desired cephalosporin C product 
because of its similar chemical and physical characteristics. Presence of 
the desacetylcephalosporin C, typically in amounts of about 15% of the 
total cephalosporin nucleus produced during fermentation, results in 
recovery of cephalosporin C (or more commonly, a solvent-extractable 
derivative thereof) contaminated with desacetylcephalosporin C (or 
derivative thereof). Moreover, since on an industrial scale the 
cephalosporin C (or derivative thereof) is usually not purified prior to 
subsequent conversion to 7-ACA, product quality of the 7-ACA is also 
adversely affected by the concomitant production of desacetylcephalosporin 
C in the initial fermentation broth. 
The prior art dealing with cephalosporin C production is primarily 
concerned with finding new microorganisms of higher cephalosporin C 
productivity and providing fermentation additives which increase 
cephalosporin C production. Thus, for example, mutant strains of 
Cephalosporium acremonium have been developed which produce substantially 
higher yields of cephalosporin C. It has been suggested to add various 
additives to the nutrient medium during fermentation of a cephalosporin 
C-producing organism so as to increase the cephalosporin C yield. Thus, 
the use of sulfur compounds such as sodium sulfite, sodium metabisulphite, 
sodium thiosulfate, sodium hydrosulphite, sodium thiosulphate and sodium 
sulphate are disclosed in U.K. Pat. No. 820,422, use of methionine, 
calcium chloride, magnesium chloride, ammonium sulfate and certain 
carbohydrates, oils and fatty acids is disclosed in U.K. Pat. No. 938,755, 
use of norvaline and norleucine is disclosed in U.K. Pat. No. 975,393, use 
of phenylacetic acid is disclosed in U.K. Pat. No. 975,394 and use of 
.epsilon.-caprolactam, 2-butanone, secondary butyl alcohol and 
1,3-butanediol is disclosed in U.K. Pat. No. 1,503,851. The problem of 
co-production of desacetylcephalosporin C during cephalosporin C 
fermentation has been addressed only in terms of providing microorganisms 
which produce higher proportions of cephalosporin C nucleus as 
cephalosporin C or in terms of extraction/isolation procedures (e.g. U.S. 
Pat. No. 4,059,573). 
Desacetylcephalosporin C was first detected in culture filtrates of 
Cephalosporium acremonium. Abraham et al. proposed that formation of this 
substance was due to the enzymatic deacetylation of cephalosporin C 
(Biochem. J. 81: 591-596, 1961). Subsequently, esterase enzymes capable of 
deacetylating cephalosporin C have been isolated from a variety of 
sources, for example, citrus fruits, bacteria, actinomycetes, wheat germ, 
mammalian liver and kidney and Rhodotorula. Pisano et al. in Develop. Ind. 
Microbiol. 8: 417-423, 1967, report that esterase activity is widespread 
in the genus Cephalosporium. The majority of these acetylesterase enzymes 
appear to have broad substrate tolerances, i.e. .beta.-naphthyl acetate 
and triacetin are active substrates, and their activity toward 
cephalosporin C does not appear unique. 
Nuesch et al. in Second International Symp. on Genetics of Industrial 
Microorganisms, Proc., 1975, ed. MacDonald, K. D., New York, Academic 
Press, pg. 451-472 and Fujisawa et al. in Agr. Biol. Chem. 39(6): 
1303-1309 (1975) independently partially purified cephalosporin C esterase 
activity from extracellular broth supernatant of Cephalosporium acremonium 
and concluded that the presence of the enzyme activity was partially 
responsible for the occurrence of desacetylcephalosporin C in C. 
acremonium fermentations. Similar esterase activity has been detected in 
the cephalosporin C producing Streptomycetes Streptomyces clavuligerus 
(Antimicrob. Agents Chemother. 1: 237-241, 1972). Huber in Appl. 
Microbiol. 16(7): 1011-1014 (1968), however, has presented evidence that 
the formation of desacetylcephalosporin C during the fermentation process 
is due to the non-enzymatic hydrolysis of cephalosporin C. It is the 
opinion of the present inventors that desacetylcephalosporin C formation 
is due to both enzymatic and non-enzymatic hydrolysis, with enzymatic 
acetylesterase activity playing a significant role. 
Reports by Liersch et al. in Second International Symp. on Genetics of 
Industrial Microorganisms, Proc., 1976, ed. MacDonald, K. D., New York, 
Academic Press, pg. 179-195 and Felix et al. in FEMS Microbiol. Lett. 8: 
55-58, 1980 have indicated that desacetylcephalosporin C is also an 
intracellular intermediate in the biosynthesis of cephalosporin C from 
desacetoxycephalosporin C. 
The enzyme activity of the partially purified acetylesterase from 
Cephalosporium acremonium was reported to be inhibited by 
diisopropylfluorophosphate, a recognized inhibitor of esterases (Agr. 
Biol. Chem. 39(6): 1303-1309, 1975). The extreme toxicity and high cost of 
this phosphorous acetylesterase inhibitor, however, prevents its use in 
the commercial production of cephalosporin C. 
Cephalosporin C, because of its amphoteric nature, is normally converted 
into a derivative so that it can be more easily recovered from the 
fermentation broth by solvent extraction procedures. Examples of such 
derivatives are given in U.K. patent application No. 2,021,640A. One 
particularly preferred process is disclosed in U.S. Pat. No. 3,573,296. 
The cephalosporin C derivative obtained by such preferred process may be 
recovered as a crystalline bis-dicyclohexylamine salt as disclosed in U.S. 
Pat. No. 3,830,809. The cephalosporin C or derivative thereof recovered 
from the fermentation broth is then cleaved by a conventional procedure, 
e.g. the process of U.S. Pat. No. 3,932,392, to provide 7-ACA. 
As noted above, the desacetylcephalosporin C impurity typically obtained 
during fermentation in amounts of about 15% of the total cephalosporin 
nucleus (cephalosporin C and desacetylcephalosporin C) has chemical and 
physical characteristics quite similar to those of the desired 
cephalosporin C product. Thus, when the cephalosporin C is converted to a 
solvent-extractable derivative, the desacetylcephalosporin C is also 
converted to a similar derivative and the cephalosporin C derivative then 
isolated is contaminated with the desacetylcephalosporin C derivative. It 
can be seen, therefore, that reducing the proportion of cephalosporin 
nucleus obtained as desacetylcephalosporin C will result in a purer 
cephalosporin C derivative product. Moreover, since this derivative is not 
normally purified prior to conversion to 7-ACA, reduced amounts of 
desacetylcephalosporin C in the fermentation broth will also result in a 
better quality 7-ACA product. 
The present invention is directed toward provision of certain phosphorous 
compounds which act as inhibitors of desacetylcephalosporin C production 
during fermentation of cephalosporin C. The resulting fermentation broth 
contains a significantly higher proportion of cephalosporin C to 
desacetylcephalosporin C, thus improving the quality of the recovered 
cephalosporin C product and, in turn, the quality of the ultimate 7-ACA 
intermediate prepared from such cephalosporin C product. 
SUMMARY OF THE INVENTION 
The present invention relates to an improvement in the production of 
cephalosporin C by submerged aerobic culture of cephalosporin C-producing 
microorganisms. More particularly, the present invention relates to a 
method of inhibiting formation of desacetylcephalosporin C during 
fermentation of a cephalosporin C-producing microorganism, said 
microorganism being one which also produces desacetylcephalosporin C, by 
addition of certain organic and inorganic phosphorous compounds to the 
culture medium. 
The phosphorous compound inhibitors provided by the present invention have 
the general formulae 
##STR1## 
wherein R.sup.1, R.sup.2 and R.sup.3 are each independently optionally 
substituted alkyl, aryl or aralkyl, R.sup.4 is optionally substituted 
alkyl or --OR.sup.10 in which R.sup.10 is hydrogen or optionally 
substituted alkyl, aryl or aralkyl, R.sup.5 is hydrogen or optionally 
substituted alkyl, aryl or aralkyl, R.sup.6 is hydrogen, hydroxy, alkenyl, 
alkanoyl or optionally substituted alkyl and R.sup.8 and R.sup.9 are 
either both hydrogen or both chloro. Such compounds effectively decrease 
formation of desacetylcephalosporin C during fermentative production of 
cephalosporin C. Moreover, these compounds are substantially less nontoxic 
than diisopropylfluorophosphate and, in general, relatively inexpensive, 
thus enabling their practical use in large scale cephalosporin C 
production. 
DETAILED DESCRIPTION 
The process of the present invention is applicable to any conventional 
fermentative procedure for preparation of cephalosporin C, providing that 
such procedure utilizes a cephalosporin C-producing microorganism which 
also produces desacetylcephalosporin C in the fermentation broth. Many 
examples of such microorganisms are described in the literature, e.g. U.K. 
patent application No. 2,060,610A. Other cephalosporin C-producing 
microorganisms may be easily tested for desacetylcephalosporin C 
production by conventional assays well known to those skilled in the art. 
The most preferred cephalosporin C-producing microorganism for use in the 
present invention is a strain of Cephalosporium acremonium (also known as 
Acremonium chrysogenum), which produces both cephalosporin C and 
desacetylcephalosporin C. Typical production strains of Cephalosporium 
acremonium result in formation of approximately 15% of the total 
cephalosporin C nucleus (cephalosporin C and desacetylcephalosporin C) as 
desacetylcephalosporin C. 
The process of the invention will desirably be carried out by culturing a 
cephalosporin C-producing microorganism (one capable of producing both 
cephalosporin C and desacetylcephalosporin C) under aerobic conditions, 
preferably in submerged culture, in a conventional cephalosporin C 
nutrient medium according to conventional cephalosporin C fermentation 
procedures. The invention is in the discovery that addition of certain 
phosphorous compounds to the nutrient medium will substantially reduce 
production of desacetylcephalosporin C during fermentation and result in a 
final broth containing a substantially higher proportion of the desired 
cephalosporin C to undesired desacetylcephalosporin C. 
The nutrient medium employed should contain assimilable sources of carbon 
and nitrogen and, if desired, growth promoting substances as well as 
inorganic salts. 
Suitable carbon sources include, for example, glucose, sucrose, starch, 
soluble starch, vegetable and animal oils, dextrin and maltose. 
Suitable nitrogen souces include, for example, natural nitrogen-containing 
substances or materials prepared from them such as meat extracts, peptone, 
casein, cornsteep liquor, yeast extracts, soy bean flour, tryptone, 
cottonseed meal and wheat bran. Nitrogen containing organic or inorganic 
compounds may also be used, for example, urea, nitrates and ammonium salts 
such as ammonium acetate, ammonium chloride or ammonium sulfate. 
Inorganic salts which may be used in the fermentation medium include 
sulfates, nitrates, chlorides, carbonates, etc., which have been employed 
in cephalosporin C production. 
Growth-promoting substances which may be used include, for example, 
cysteine, cystine, thiosulfate, methyl oleate and, in particular, 
methionine and also trace elements such as iron, zinc, copper and 
manganese. 
Culturing conditions such as temperature, pH and fermentation time are 
selected such that the microorganism employed may accumulate a maximum 
amount of the desired cephalosporin C. The temperature is normally kept at 
about 15.degree.-45.degree., preferably at about 25.degree. C., and 
fermentation is carried out for a period of from about 1-20 days, 
preferably 4-10 days and most preferably about six days. 
It has now been found that certain organic and inorganic phosphorous 
compounds when added to the culture medium during cultivation of a 
cephalosporin C-producing microorganism will result in substantially 
reduced production of desacetylcephalosporin C in the fermentation broth. 
It is believed that this reduction in desacetylcephalosporin C production 
results from inhibition of the acetylesterase enzyme typically produced 
during cultivation of cephalosporin C-producing microorganisms. 
The phosphorus compounds which may be used in the process of the present 
invention may be represented by the formulae 
##STR2## 
wherein R.sup.1, R.sup.2 and R.sup.3 are each independently optionally 
substituted alkyl, aryl or aralkyl, R.sup.4 is optionally substituted 
alkyl or --OR.sup.10 in which R.sup.10 is hydrogen or optionally 
substituted alkyl, aryl or aralkyl, R.sup.5 is hydrogen or optionally 
substituted alkyl, aryl or aralkyl, R.sup.6 is hydrogen, hydroxy, alkenyl, 
alkanoyl or optionally substituted alkyl and R.sup.8 and R.sup.9 are 
either both hydrogen or both chloro. 
Preferred phosphorous compounds are those compounds of formulae I, II, III 
and IV wherein R.sup.1, R.sup.2 and R.sup.3 each independently represent 
straight or branched chain C.sub.1 -C.sub.10 alkyl, phenyl or phenyl 
(C.sub.1 -C.sub.4)alkyl, said alkyl group or the alkyl portion of 
phenylalkyl being optionally substituted by one or more, preferably 1-3, 
substituents such as halo (chloro, bromo, fluoro, iodo) or carboxy and 
said phenyl group or the phenyl portion of phenylalkyl being optionally 
substituted by one or more, preferably 1-3, substituents independently 
selected from such groups as C.sub.1 -C.sub.6 alkyl, C.sub.1 -C.sub.6 
alkoxy and halo, R.sup.4 is C.sub.1 -C.sub.6 alkyl optionally substituted 
with one or more, preferably 1-3, halo groups or --OR.sup.10 in which 
R.sup.10 is hydrogen, C.sub.1 -C.sub.10 alkyl, phenyl or phenyl (C.sub.1 
-C.sub.4)alkyl, said alkyl, phenyl and phenylalkyl radicals being 
optionally substituted as defined above for R.sup.1, R.sup.5 is hydrogen, 
C.sub.1 - C.sub.10 alkyl, phenyl or phenyl (C.sub.1 -C.sub.4)alkyl, said 
alkyl, phenyl and phenylalkyl radicals being optionally substituted as 
defined above for R.sup.1, R.sup.6 is hydrogen, hydroxy, C.sub.2 -C.sub.6 
alkenyl, C.sub.2 -C.sub.6 alkanoyl, or C.sub.1 -C.sub.6 alkyl, said alkyl 
group being optionally substituted by one or more, preferably 1-3, 
substituents such as cyano, C.sub.2 -C.sub.6 alkanoyl or carbo (C.sub.1 
-C.sub.6)alkoxy and R.sup.8 and R.sup.9 are either both hydrogen or both 
chloro. 
Phosphite compounds of formula I may be exemplified by trimethyl phosphite, 
triethyl phosphite, triisopropyl phosphite, tributyl phosphite, triphenyl 
phosphite and tris(2-chloroethyl)phosphite. Mixed function phosphites such 
as benzyl diethyl phosphite and diphenyl isodecyl phosphite may also be 
used. 
Phosphorous compounds of formula II may be exemplified by phosphorous acid, 
dibenzyl phosphite, dibutyl phosphite, diethyl phosphite, diisopropyl 
phosphite, dimethyl phosphite, diphenyl phosphite, triethyl 
phosphonoacetate, 2-chloroethyl phosphonic acid, diethyl cyanomethyl 
phosphonate, dimethyl methyl phosphonate, dimethyl phosphate, trimethyl 
phosphonoacetate, diethyl ethyl phosphonate, diethyl carbomethoxymethyl 
phosphonate, diethyl acetyl phosphonate, dimethyl acetylmethyl 
phosphonate, dimethyl cyanomethyl phosphonate, diethyl allyl phosphonate 
and 2-carboxyethyl phosphonic acid. 
Compounds of general formula III may be illustrated by hypophosphorous 
acid, monomethylphosphonate, monoethylphosphonate and 2,2,2-trichloroethyl 
phosphorodichloridite. 
Pyrophosphite compounds of formula IV may be illustrated by 
tetramethylpyrophosphite and tetraethylpyrophosphite. 
Preferred phosphorous compound inhibitors include phosphorous acid, 
hypophosphorous acid, diisopropyl phosphite, triisopropyl phosphite, 
dibenzyl phosphite, dimethyl phosphite, tributyl phosphite, triethyl 
phosphonoacetate, 2-chloroethyl phosphonic acid, tetraethylpyrophosphite, 
diethyl cyanomethyl phosphonate, dimethyl methyl phosphonate, 
2,2,2-trichloroethyl phosphorodichloridite, dimethyl phosphate, diphenyl 
phosphite, triphenyl phosphite, trimethyl phosphite, dibutyl phosphite, 
tris(2-chloroethyl)phosphite, trimethyl phosphonoacetate, diethyl ethyl 
phosphonate, diethyl carbomethoxymethyl phosphonate, diethyl acetyl 
phosphonate, dimethyl acetylmethyl phosphonate, dimethyl cyanomethyl 
phosphonate and diethyl allyl phosphonate. 
Particularly preferred compounds include phosphorous acid, hypophosphorous 
acid, diisopropyl phosphite, triisopropyl phosphite, dibenzyl phosphite, 
dimethyl phosphite and tributyl phosphite. 
The most preferred phosphorous compound inhibitor is phosphorous acid. 
The phosphorous compounds are preferably employed so as to give final broth 
concentrations of from about 100 to 3000 parts per million (based on 
weight) and most preferably about 200 to 1000 parts per million. Inhibitor 
compound may be added all at once or at periodic intervals during the 
course of fermentation. 
Most advantageously the organic phosphorous compounds are added to ongoing 
fermentations between about 70 and 140 hours as single or multiple shots. 
The inorganic phosphorous compounds may advantageously be added to ongoing 
fermentations immediately after inoculation to about 140 hours after 
inoculation. Alternatively, they may be batched into the fermentation 
medium before sterilization. 
Use of the above-mentioned phosphorous compounds according to the process 
of the present invention is found to substantially lower the percentage of 
desacetylcephalosporin C (based on total cephalosporin nucleus which is 
cephalosporin C and desacetylcephalosporin C) in the fermentation broth. 
When used in typical cephalosporin C fermentations, levels of 
desacetylcephalosporin C have been reduced to about 4% of the total 
cephalosporin nucleus compared to about 15% in untreated fermentations. 
In treated shake flask fermentations the total amount of cephalosporin 
nucleus produced appears to remain unchanged and thus the cephalosporin C 
titers are generally increased by an appropriate amount. In larger scale 
fermentations it has not been established that use of the phosphorous 
inhibitor compounds results in any increased levels of cephalosporin C. 
Even if cephalosporin C levels remain constant, however, the reduced 
quantity of desacetylcephalosporin C in treated fermentations greatly 
facilitates recovery of the desired cephalosporin C product in a higher 
state of purity. 
The phosphorous compounds of the present invention were shown to have their 
inhibitory activity at the enzyme level. Thus, cephalosporin C esterase 
activity was partially purified from Cephalosporium acremonium broth 
supernatant by DEAE Sephadex A50 column chromatography and the hydrolytic 
activity of this preparation (as measured by HPLC by following the 
conversion of cephalosporin C to desacetylcephalosporin C) was found to be 
inhibited by phosphorous compounds of formulae I, II, III and IV. 
After fermentation is complete the desired cephalosporin C product is 
preferably converted by known methods such as those described in U.S. Pat. 
No. 3,573,296 to a derivative which can be more easily recovered from the 
broth by solvent extraction procedures. The cephalosporin C or derivative 
thereof obtained from fermentation may then be converted by known methods 
to 7-ACA, a key intermediate in the preparation of many semi-synthetic 
cephalosporins. By employing the phosphorous inhibitor compounds according 
to the present invention, the cephalosporin C or derivative thereof and 
the ultimate 7-ACA intermediate are obtained more efficiently and in a 
greater degree of purity when compared with the prior untreated broth 
procedure. 
The following examples are intended to illustrate the present invention 
without in any way limiting the scope of the invention to the embodiments 
specifically described. The term "ppm" used in the examples refers to a 
weight/weight basis.