Defining an optimal carbon source/methionine feed strategy for growth and cephalosporin C formation by Cephalosporium acremonium.

The effect of the method of methionine addition, growth-limiting carbon source (glucose vs sucrose), and culture growth rate on cephalosporin C production was investigated in a Cephalosporium acremonium defined medium fed batch fermentation. Batch addition of methionine, at a concentration of 3 g/L, prior to the start of a fed sucrose fermentation was found to interfere with the ability of the culture to utilize this sugar, thus limiting growth and decreasing cephalosporin C production. Batch methionine addition had no effect on glucose-limited cultures. Concurrent exponential feeding of methionine with sucrose improved both culture growth and productivity. Under the control of identical carbon source limiting feed profiles, sucrose was observed to support greater cephalosporin C production than glucose. Optimal cephalosporin C production in a C. acremonium defined medium fed batch fermentation was obtained through controlling culture growth during the rapid growth phase at a relatively low level with respect to mumax (mu approximately 0.036 h-1) until achieving a desired cell mass with a concurrent sucrose and methionine feed, followed by maintaining relatively vigorous growth (mu approximately 0.01 h-1) with sucrose for the duration of the fermentation.     

Expression of a cephalosporin C esterase gene in <Emphasis Type="Italic">Acremonium chrysogenum</Emphasis> for the direct production of deacetylcephalosporin C

A recombinant fungal microorganism capable of producing deacetylcephalosporin C was constructed by transforming a cephalosporin C esterase gene from Rhodosporidium toruloides into Acremonium chrysogenum. The cephalosporin C esterase gene can be expressed from its endogenous R. toruloides promoter or from the Aspergillus nidulans trpC promoter under standard Acremonium chrysogenum fermentation conditions. The expression of an active cephalosporin C esterase enzyme in A. chrysogenum results in the conversion of cephalosporin C to deacetylcephalosporin C in vivo, a novel fermentation process for the production of deacetylcephalosporin C. The stability of deacetylcephalosporin C in the fermentation broth results in a 40% increase in the cephalosporin nucleus.     

Development of the cephalosporin C fermentation taking into account the instability of cephalosporin C

Summary Cephalosporin C undergoes chemical hydrolysis during fermentation at 25°C at a rate of 0.48% per hour to give increasing amounts of 2-(D-4-amino-4-carboxybutyl)thiazole-4-carboxylic acid (compound X). Although the cephalosporin C titer in a fermentation levels off at 160–170h, the total of C+X rises almost linearly up to 200h. Cells, therefore, biosynthesize cephalosporin C at an almost constant rate up to 200h. This instability greatly influences attempts to improve the cephalosporin strain by genetic manipulation, or the process by alterations in technology.     

Role of Acetyl CoA: Deacetylcephalosporin C Acetyltransferase in Cephalosporin C Biosynthesis by Cephalosporium acremonium

Existence of an acetyltransferase, which catalizes acetylation of deacetylcephalosporin C to cephalosporin C, was demonstrated for the first time in cell-free extracts of Cephalosporium acremonium. The pH optimum of the enzyme appeared to be 7.0 to 7.5 and the enzyme required essentially Mg²⁺ as a cofactor for its reaction. The activity of this enzyme was not observed in the cell-free extracts of deacetylcephalosporin C-producing mutants Nos. 20, 29, 36 and 40, but was recovered in a revertant obtained from the mutant No. 40. These results indicate that deacetylcephalosporin C accumulation by these mutants was due to the lack of the acetyltransferase and made it reasonable that the terminal reaction of cephalosporin C biosynthesis in Cephalosporium acremonium proceeded by the catalytic action of acetyltransferase.     

Methionine control of cephalosporin C formation

DL -Norleucine, a nonsulfur analogue of methionine was found to markedly stimulate synthesis of cephalosporin C by Cephalosporium acremonium strain CW19 in three different chemically defined media. Methionine, but not norleucine, stimulated cephalosporin C biosynthesis in a crude medium. The lack of stimulation by norleucine in complex medium was shown to be due to lack of uptake of this amino acid by mycelia growing in such a medium. In defined media containing a suboptimal methionine concentration, norleucine stimulated antibiotic production up to the level reached by optimal methionine. At an optimal dose of methionine, norleucine elicited no further increase in cephalosporin C production, indicating that these two amino acids act by the same mechanism. The data strongly indicate that stimulation by methionine is not a function of its ability to donate sulfur for antibiotic formation. Methionine was found to neither repress nor inhibit cysteine metabolism.     

Biosynthesis of Cephalosporin C: I. Factors Affecting the Fermentation

A series of complex and synthetic media have been developed that are suitable for the production of cephalosporin C and cephalosporin N by a mutant strain of Cephalosporium (C.M.I. 49,137). dl-Methionine increased the yield of both antibiotics, with more effect on cephalosporin N. l-Cystine had a greater enhancing effect on formation of cephalosporin C than on formation of cephalosporin N in synthetic media; serine increased yields of cephalosporin C under certain conditions. Disaccharides or polysaccharides appeared to be the best source for carbohydrates. No evidence was found for precursor action such as is found in penicillin fermentations. The ability of resting cells to produce antibiotic was demonstrated.     

Factors affecting cephalosporin C biosynthesis

Summary Caprolactam, butan-2-one, butan-2-ol, and butan-1, 3-diol were found to have a stimulating effect on the biosynthesis of cephalosporin C by a mutant strain ofCephalosporium. Caprolactam was shown to have a synergistic effect, when added to the fermentation medium together with each of the other three compounds.     

Genetic engineering approach to reduce undesirable by-products in cephalosporin C fermentation

Deacetoxycephalosporin C (DAOC) is produced by Acremonium chrysogenum as an intermediate compound in the cephalosporin C biosynthetic pathway, and is present in small quantities in cephalosporin C fermentation broth. This compound forms an undesirable impurity, 7-aminodeacetoxycephalosporanic acid (7-ADCA), when the cephalosporin C is converted chemically or enzymatically to 7-aminocephalosporanic acid (7-ACA). In the cephalosporin C biosynthetic pathway of A. chrysogenum, the bifunctional expandase/hydroxylase enzyme catalyzes the conversion of penicillin N to DAOC and subsequently deacetylcephalosporin C (DAC). By genetically engineering strains for increased copy number of the expandase/hydroxylase gene, we were able to reduce the level of DAOC present in the fermentation broth to 50% of the control. CHEF gel electrophoresis and Southern analysis of DNA from two of the transformants revealed that one copy of the transforming plasmid had integrated into chromosome VIII (ie a heterologous site from the host expandase/hydroxylase gene situated on chromosome II). Northern analysis indicated that the amount of transcribed expandase/hydroxylase mRNA in one of the transformants is increased approximately two-fold over that in the untransformed host. Received 5 January 1998/ Accepted in revised form 29 May 1998     

Extraction of cephalosporin C from whole broth and separation of desacetyl cephalosporin C by aqueous two-phase partition

Cephalosporin C was extracted from diluted or whole broth by PEG/salt aqueous two-phase systems. Parameters such as PEG molecular weight, salt type, pH, and salt concentration were investigated for finding a suitable extraction system. In PEG 600/ammonium sulfate or phosphate systems, K(c) (partition coefficienct of cephalosporin C) was observed to be larger than 1, with K(d) (partition coefficient of desacetyl cephalosporin C) being smaller than 1. The particular values of these coefficients would imply that the difficult separation of cephalosporin C and desacetyl cephalosporin C could possibly be achieved via the aqueous two-phase extraction. The addition of surfactants, water-miscible solvents, and neutral salts for enhancement of the separation efficiency was also investigated. The addition of surfactants to the system did not affect the separation efficiency substantially. K(c) would increase whereas K(d) decreased as a result of the addition of acetone, MeOH, EtOH, IPA, and n-BuOH. Meanwhile both K(c) and K(d) would decrease whenever neutral salts, NaCl, KCl, Kl, or KSCN, were added. The partitioning behavior of cephalosporin C and desacetyl cephalosporin C in filtered, whole, and different batches of broth was notably quite similar to that of diluted broth. The recovery yield of cephalosporin C in whole broth extraction was observed to be a function of centrifugal force used in phase separation. (c) 1994 John Wiley & Sons, Inc.     

Changes in Cellular Fatty Acid Composition of Cephalosporium acremonium during Cephalosporin C Production

Cephalosporium acremonium was cultivated in fermentation medium containing sucrose or methyl oleate as a carbon source for cephalosporin C production. The level of antibiotic production was 48 g of cephalosporin C per liter under optimum conditions when methyl oleate was used. The C_18:1 (oleic acid) methyl ester appeared to be utilized faster than the C_18:2 (linoleic acid) methyl ester in fermentation broth. Physiological characteristics of C. acremonium were investigated by determining the fatty acid composition of the total cellular free lipid. Significant changes in cellular fatty acid composition occurred during inoculum cultivation and fermentation. The percentage of C_18:1 increased from 19.1 to 38.5%, but the percentage of C_18:2 decreased from 56.7 to 36.1%, and there was an increase in pH during inoculum cultivation. The cellular fatty acid composition of C. acremonium grown in fermentation medium containing methyl oleate (methyl oleate medium) was significantly different from that in fermentation medium containing sucrose (sucrose medium). The major fatty acids detected were C_16:0 (palmitic acid), C_18:1, and C_18:2. In methyl oleate medium, the ratio of C_18:1 to C_18:2 increased from 0.34 to 1.37, while the cell morphology changed from hyphae to arthrospores and conidia. In contrast, in sucrose medium, the ratio of C_18:1 to C_18:2 decreased from 0.70 to 0.43, and most of the cells remained hyphal at the end of fermentation. We observed that hyphae contained a higher proportion of C_18:2 but arthrospores and conidia contained a higher proportion of C_18:1.     

Production of cephalosporin C in a fluidized-bed bioreactor

Production of cephalosporin C was investigated in a fluidized-bed bioreactor using bioparticles of Cephalosporium acremonium. Bioparticles were developed by forming a biofilm of growing hyphae around celite particles which contained spores of the microorganism. Production of the antibiotic was significantly improved by using bioparticles over the free mycelial culture, possibly due to the enhanced mass transfer capacity of the bioreactor system and successive generation of highly productive morphological forms of the microorganism. The maximum attainable titer of cephalosporin C from the bioreactor system was almost double that from a jar fermentor operation with a free mycelial culture of the same strain. The biofilm of the bioparticles became unstable as the fermentation proceeded. Morphological differentiation of the microorganism caused a gradual loss of biofilm and an increase of free cells in the culture broth. Additional feeding of a limited amount of methionine to the fermentation broth was not as effective as expected for improving the bioparticle stability. However, repeated use of the bioparticles revealed a strong possibility to improved the overall reactor performance since it allowed an enhanced production of the antibiotic with fewer free cells.     

Cephalosporin C acylase and penicillin V acylase formation by Aeromonas sp. ACY 95

Aeromonas sp. ACY 95 produces constitutively and intracellularly a penicillin V acylase at an early stage of fermentation (12 h) and a cephalosporin C acylase at a later stage (36 h). Some penicillins, cephalosporin C and their side chain moieties/analogues, phenoxyacetic acid, penicillin V and penicillin G, enhanced penicillin V acylase production while none of the test compounds affected cephalosporin C acylase production. Supplementation of the medium with some sugars and sugar derivatives repressed enzyme production to varying degrees. The studies on enzyme formation, induction and repression, and substrate profile suggest that the cephalosporin C acylase and penicillin V acylase are two distinct enzymes. Substrate specificity studies indicate that the Aeromonas sp. ACY 95 produces a true cephalosporin C acylase which unlike the enzymes reported hitherto hydrolyses cephalosporin C specifically.The authors are with Research and Development, Hindustan Antibiotics Limited, Pimpri. Pune 411 018, India     

Growth from spores of nonproteolytic Clostridium botulinum in heat-treated vegetable juice

Unheated spores of nonproteolytic Clostridium botulinum were able to lead to growth in sterile deoxygenated turnip, spring green, helda bean, broccoli, or potato juice, although the probability of growth was low and the time to growth was longer than the time to growth in culture media. With all five vegetable juices tested, the probability of growth increased when spores were inoculated into the juice and then heated for 2 min in a water bath at 80 degrees C. The probability of growth was greater in bean or broccoli juice than in culture media following 10 min of heat treatment in these media. Growth was prevented by heat treatment of spores in vegetable juices or culture media at 80 degrees C for 100 min. We show for the first time that adding heat-treated vegetable juice to culture media can increase the number of heat-damaged spores of C. botulinum that can lead to colony formation.     

Degradation of fructans by epiphytic and inoculated lactic acid bacteria and by plant enzymes during ensilage of normal and sterile hybrid ryegrass

Degradation of grass fructans by epiphytic or inoculated lactic acid bacteria during ensilage was examined using both normal and sterile hybrid ryegrass. It was clear that even in the absence of bacteria fructan degradation occurred, but at a significantly slower rate than in normal grass which had not been inoculated with lactic acid bacteria. Fructan degradation in sterile herbage suggests that plant fructan hydrolases were partially responsible for this process in all herbages, irrespective of treatment. Inoculation of sterile herbage with a strain of Lactobacillus plantarum known to lack the ability to degrade grass fructans resulted in a slower rate of fructan breakdown than when inoculated with Lactobacillus casei subsp. paracasei , a confirmed fructan degrader. In the later stages of the fermentation of uninoculated normal herbage when water-soluble carbohydrate appeared to be limiting, lactic acid was fermented to acetic acid. However, this fermentation pathway was not observed in either of the inoculated silages. The results suggest that silage inoculant bacteria possessing fructan hydrolase activity may have potential for improving silage fermentation, particularly when late cut, low sugar grass containing a high proportion of fructans is ensiled.     

Production of cephalosporin C,and its intermediates,by raised-titre strains of Acremonium chrysogenum

Three different strains of Acremonium chrysogenum have been grown under identical fermentation conditions and their profiles with respect to cephalosporin C and its intermediates were compared. Clear differences were found between the strains; one notably accumulated a large pool of penicillin N, showing a reduced ability to convert this antibiotic to the later intermediates in the pathway, deacetoxycephalosporin C, deacetylcephalosporin C and cephalosporin C.     

The role of valine in the biosynthesis of penicillin N and cephalosporin C by a Cephalosporium sp

1. The production of penicillin N, but not that of cephalosporin C, was inhibited by the addition of d-valine to suspensions in water of washed mycelium of Cephalosporium sp. 8650. The production of cephalosporin C was selectively inhibited by gamma-hydroxyvaline. 2. l-[(14)C]Valine was taken up rapidly and virtually completely by suspensions of washed mycelium but d-[(14)C]valine and alpha-oxo[(14)C]-isovalerate were taken up relatively slowly. 3. Part of the l-valine was rapidly degraded in the mycelium and part was incorporated into protein. Turnover of the valine in the amino acid pool was estimated to occur in 10-17min. 4. No detectable amount of l-[(14)C]valine was converted into the d-isomer in the mycelium. alpha-Oxo[(14)C]isovalerate was rapidly converted into l-[(14)C]valine in mycelium and mycelial extracts. 5. d-[(14)C]Valine was partially converted into the l-isomer in the mycelium and (14)C from d-valine was incorporated into protein. 6. The labelling of penicillin N and cephalosporin C by (14)C from l-[(14)C]valine was consistent with the view that l-valine is a direct precursor of C(5) fragments of both antibiotics and that any intermediates involved are present in relatively small pools in rapid turnover. 7. Labelling of the antibiotics with (14)C from d-[1-(14)C]valine appeared to occur after the latter had been converted into the l-isomer. Unlabelled d-valine did not decrease the efficiency of incorporation of (14)C from l-[1-(14)C]valine. 8. Intracellular peptide material which contained, among others, residues of alpha-aminoadipic acid, cysteine and valine, was rapidly labelled by (14)C from l-[1-(14)C]valine in a manner consistent with it being an intermediate in the biosynthesis of one or both of the antibiotics. 9. Labelling of penicillin N from l-[1-(14)C]valine occurred more rapidly than that of cephalosporin C. However, the effects of d-valine and gamma-hydroxyvaline on antibiotic production and the course of labelling of the antibiotics from l-[(14)C]valine could not readily be explained on the assumption that penicillin N was a precursor of cephalosporin C.     

Effect of Tenuazonic Acid on In Vitro Amino Acid Incorporation by Rice Embryo Ribosomes

Deacetylcephalosporin C negative mutants, lacking a certain step in the pathway of deacetylcephalosporin C biosynthesis, were obtained from the deacetylcephalosporin C producing mutant No. 40 of Cephalosporium acremonium by treatment with N-methyl-N′-nitro-N-nitrosoguanidine. Among these mutants, the strain No. 40-20 was found to mainly accumulate a cephalosporin compound other than deacetylcephalosporin C and cephalosporin C. The cephalosporin was isolated as crystals from the culture broth of the mutant No. 40-20, and identified as deacetoxycephalosporin C, possessing a D-a-aminoadipyl side chain at C-7, by physical, chemical and biological methods. The profile of deacetoxycephalosporin C fermentation and the examination of the biochemical reduction of deacetylcephalosporin C led us to the conclusion that deacetoxycephalosporin C would be produced through de novo synthesis by this mutant.     

Separation of cephalosporin C and desacetyl cephalosporin C by high speed counter-current chromatography in aqueous two-phase systems

Summary In the recovery of cephalosporin C (CPC) from fermentation broth, the separation of desacetyl cephalosporin C (DAC) is a major concern. Multistage extraction in aqueous two-phase systems, mainly PEG/ammonium sulfate systems, proved to be promising. In preparative scale operation, high speed counter-current chromatography (HSCCC) with eccentric columns was used with aqueous two-phase systems to obtain baseline resolution of CPC and DAC. Solvents (e.g. 5% acetone) or neutral salts (e.g. 1.45% KSCN) added into aqueous two-phase systems enhanced the separation efficiency. Operation parameters of HSCCC such as rotational speed and mobile phase flow rate can affect the retention of the stationary phase and HETP.     

Coordinate increase of isopenicillin N synthetase, isopenicillin N epimerase and deacetoxycephalosporin C synthetase in a high cephalosporin-producing mutant of Acremonium chrysogenum and simultaneous loss of the three enzymes in a non-producing mutant

Abstract An early blocked mutant in cephalosporin biosynthesis ( Acremonium chrysogenum N₂) had simultaneously lost 3 enzymes of the cephalosporin biosynthetic pathway (isopenicillin N synthetase, isopenicillin N epimerase and deacetoxycephalosporin C synthetase) and accumulated the tripeptide α-aminoadipyl-cysteinyl-valine. An overproducing mutant ( A. chrysogenum C-10) showed a 2-fold increase in the same 3 enzymes throughout fermentation, with respect to the low-producing strain A. chrysogenum CW-19. These results suggest that expression of the genes coding for cephalosporin biosynthetic enzymes is altered in a coordinate form in these mutants.     

Fermentation of Aqueous Plant Seed Extracts by Lactic Acid Bacteria

The effects of lactic acid bacterial fermentation on chemical and physical changes in aqueous extracts of cowpea (Vigna unguiculata), peanut (Arachis hypogea), soybean (Glycine max), and sorghum (Sorghum vulgare) were studied. The bacteria investigated were Lactobacillus helveticus, L. delbrueckii, L. casei, L. bulgaricus, L. acidophilus, and Streptococcus thermophilus. Organisms were inoculated individually into all of the seed extracts; L. bulgaricus and S. thermophilus were also evaluated together as inocula for fermenting the legume extracts. During fermentation, bacterial population and changes in titratable acidity, pH, viscosity, and color were measured over a 72-h period at 37°C. Maximum bacterial populations, titratable acidity, pH, and viscosity varied depending upon the type of extract and bacterial strain. The maximum population of each organism was influenced by fermentable carbohydrates, which, in turn, influenced acid production and change in pH. Change in viscosity was correlated with the amount of protein and titratable acidity of products. Color was affected by pasteurization treatment and fermentation as well as the source of extract. In the extracts inoculated simultaneously with L. bulgaricus and S. thermophilus, a synergistic effect resulted in increased bacterial populations, titratable acidity, and viscosity, and decreased pH in all the legume extracts when compared to the extracts fermented with either of these organisms individually. Fermented extracts offer potential as substitutes for cultured dairy products.