Ammonium repression of cephalosporin production by Streptomyces clavuligerus

Production of beta-lactam antibiotics took place during growth of Streptomyces clavulgerus in chemically defined medium. The specific activities of isopenicillin N synthetase ("cyclase"), isopenicillin N epimerase, and deacetoxycephalosporin C synthetase ("expandase") increased during the exponential phase of growth. Specific cephalosporin productivity during fermentation followed a similar pattern, reaching a maximum near the end of the growth phase and decaying rapidly in the stationary phase. Ammonium chloride depressed cephalosporin production, presumably as a result of repression of cyclase and expandase formation, but not of epimerase. No inhibitory effects on enzyme activity by ammonium were found. Addition of tribasic magnesium phosphate [Mg3(PO4)2 X 8H2O] prevented the repression of cyclase and markedly stimulated cephalosporin production. Cephamycin C and, in smaller amounts, O-carbamoyldeacetylcephalosporin C were the only cephalosporins detected. Growth with ammonium resulted in lower titers of both compounds, and did not change the relative proportion of each. The correlation found between cephalosporin productivity and cyclase specific activity in different media suggests that formation of this enzyme may be the rate-limiting step in the pathway.     

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.     

Formation of Desacetylcephalosporin C in Cephalosporin C Fermentation

The origin of desacetylcephalosporin C in cephalosporin C fermentation broths was investigated. Esterase activity was detected in cell-free extracts of Cephalosporium acremonium, but these extracts failed to deesterify cephalosporin C. When cephalosporin C was added to sterile and inoculated fermentation media, the antibiotic decayed at nearly identical rates. The formation of desacetylcephalosporin C during the fermentation was measured by quantitative chromatography and by the incorporation of valine-1-(14)C into the molecule. The rate constants obtained from the results of these experiments were equivalent to those for the decay of cephalosporin C in sterile and inoculated media. The data demonstrate that desacetylcephalosporin C is produced by nonenzymatic hydrolysis of cephalosporin C.     

CefR modulates transporters of beta-lactam intermediates preventing the loss of penicillins to the broth and increases cephalosporin production in Acremonium chrysogenum

The Acremonium chrysogenum cephalosporin biosynthetic genes are divided in two different clusters. The central step of the biosynthetic pathway (epimerization of isopenicillin N to penicillin N) occurs in peroxisomes. We found in the “early” cephalosporin cluster a new ORF encoding a regulatory protein (CefR), containing a nuclear targeting signal and a “Fungal_trans” domain. Targeted inactivation of cefR delays expression of the cefEF gene, increases penicillin N secretion and decreases cephalosporin production. Overexpression of the cefR gene decreased (up to 60%) penicillin N secretion, saving precursors and resulting in increased cephalosporin C production. Northern blot analysis revealed that the CefR protein acts as a repressor of the exporter cefT and exerts a small stimulatory effect over the expression level of cefEF that explains the increased cephalosporin yields observed in transformants overexpressing cefR. In summary, we describe for the first time a modulator of beta-lactam intermediate transporters in A. chrysogenum.     

A simulation model for the effect of predation on bacteria in continuous culture

A simulation model was developed for the carbon (C), nitrogen (N), and phosphorus (P) content of bacteria and their medium in a chemostat. Cell components distinguished included the structural component, synthetic machinery, building blocks and intermediates, C reserves, ammonium (NH₄), orthophosphate (PO₄), and polyphosphate. Growth, incorporation of substrates, and production of waste products were related to physiological status, as indicated by the amounts of various cell components. The model was fitted to data from chemostats on the chemical composition of bacteria growing in C-, N-, and P-limiting media and was used to explore the consequences of predation on bacterial populations. In C-limiting media predation (without the return of nutrients to the medium by the predator) increased NH₄ uptake in spite of a decrease in bacterial biomass. In N-limiting media predation decreased both biomass and the rate of N uptake. These results were accounted for by the effect of growth rate on bacterial N demand. In C-limiting media the return of NH₄ and PO₄ by the predator did not change the effect of predation on bacteria. But in N-limiting media the return of nutrients decreased the effect of predation on biomass, and stimulated respiration and NH₄ uptake by the bacteria. The effect of growth rate on the chemical composition of bacteria was proposed as a possible explanation of the stimulatory effect of predators on bacteria.     

Biosynthesis of penicillin N and cephalosporin C: Antibiotic production and other features of the metabolism of a Cephalosporium sp

1. The production of penicillin N and cephalosporin C by two mutants of a Cephalosporium sp. has been studied with cultures grown in a chemically defined medium and with suspensions of washed mycelium in water or a buffered salt solution. 2. Antibiotic synthesis began at an early stage of growth and its rate per unit weight of mycelium appeared to pass its maximum as morphological changes were occurring in young hyphae. This rate subsequently declined, but rapid production could continue after net growth had ceased. 3. In a series of shake-flask fermentations in the growth medium, increases in the yield of penicillin N above the mean were correlated with much smaller increases in the yield of cephalosporin C and vice versa. 4. In suspensions of washed mycelium, moderate decreases in the efficiency of aeration increased the yield of penicillin N and decreased that of cephalosporin C. A similar result normally followed the addition of methionine to the suspension fluid, and in both cases there was usually an increase in the yield of the two antibiotics combined. 5. The apparent intracellular concentrations of the antibiotics were much lower than those attained extracellularly and also much lower than those of most of the amino acids in the intracellular pool. No detectable amount of [(14)C]penicillin N added to the extracellular fluid was found to enter the mycelium. 6. Very small amounts of peptide material whose behaviour was similar to that of the sulphonic acid of delta-(alpha-amino-adipoyl)cysteinylvaline on paper electrophoresis at pH1.8 were found in extracts of the mycelium that had been oxidized with performic acid. 6-Aminopenicillanic acid and 7-aminocephalosporanic acid were not detected. 7. Ultrasonic treatment of the mycelium resulted in rapid fragmentation of mycelial chains, rupture of many individual cells, and the liberation of amino acids and other substances into the medium. 8. Ultrasonically treated preparations synthesized penicillin N and cephalosporin C rapidly after a lag of 12hr. Antibiotic synthesis was accompanied by the growth of hyphae from swollen mycelial fragments and by the re-establishment of permeability barriers resulting in the uptake of amino acids from the medium.     

The transporter CefM involved in translocation of biosynthetic intermediates is essential for cephalosporin production

The cluster of early cephalosporin biosynthesis genes (pcbAB, pcbC, cefD1, cefD2 and cefT of Acremonium chrysogenum) contains all of the genes required for the biosynthesis of the cephalosporin biosynthetic pathway intermediate penicillin N. Downstream of the cefD1 gene, there is an unassigned open reading frame named cefM encoding a protein of the MFS (major facilitator superfamily) with 12 transmembrane domains, different from the previously reported cefT. Targeted inactivation of cefM by gene replacement showed that it is essential for cephalosporin biosynthesis. The disrupted mutant accumulates a significant amount of penicillin N, is unable to synthesize deacetoxy-, deacetyl-cephalosporin C and cephalosporin C and shows impaired differentiation into arthrospores. Complementation of the disrupted mutant with the cefM gene restored the intracellular penicillin N concentration to normal levels and allowed synthesis and secretion of the cephalosporin intermediates and cephalosporin C. A fused cefM-gfp gene complemented the cefM-disrupted mutant, and the CefM-GFP (green fluorescent protein) fusion was targeted to intracellular microbodies that were abundant after 72 h of culture in the differentiating hyphae and in the arthrospore chains, coinciding with the phase of intense cephalosporin biosynthesis. Since the dual-component enzyme system CefD1-CefD2 that converts isopenicillin N into penicillin N contains peroxisomal targeting sequences, it is probable that the epimerization step takes place in the peroxisome matrix. The CefM protein seems to be involved in the translocation of penicillin N from the peroxisome (or peroxisome-like microbodies) lumen to the cytosol, where it is converted into cephalosporin C.     

Comparative characterization of new glutaryl 7-ACA and cephalosporin C acylases

We performed a comparative characterization of three new cephalosporin acylases which were prepared from E. coli recombinant strains and found originally from Pseudomonas sp. A14, Bacillus laterosporus J1 and Pseudomonas diminuta N176. Both A14 and N176 acylases consisted of two non-identical subunits (α, β) whose molecular weights were 28,000 (α), 61,000 (β) and 26,000 (α), 58,000 (β), respectively, whereas J1 acylase consisted of a single peptide with molecular weight of 70,000. The maximum specific activities of A14, J1 and N176 acylases for glutaryl 7-ACA were 7.1, 5.3 and 100 units/mg, respectively, and that of N176 acylase for cephalosporin C was 3.1 units/mg. The K_m values of glutaryl 7-ACA for A14, J1 and N176 acylases were 2.1, 3.2 and 2.6 mM, respectively, and that of cephalosporin C for N176 acylase was 4.8 mM. A14, J1 and N176 acylases exhibited differential activities for cephalosporins having an aliphatic dicarboxylic acid in the acyl side chain and only N176 acylase showed an activity for cephalosporin C. N176 acylase as well as A14 acylase also showed a weak activity for a cephalosporin derivative having a heterocyclic carboxylic acid in the side chain. A14, J1 and N176 acylases catalyzed the reverse reaction to synthesize glutaryl 7-ACA from 7-ACA and glutaric acid, although the rate of the synthesis was 10 to 10⁵ fold slower than that of hydrolysis. The activities of the cephalosporin acylases were considerably inhibited by the reaction products, 7-ACA and glutaric acid. The types of the inhibition by 7-ACA and glutaric acid were both competitive. A14, J1 and N176 acylases were thermostable, their residual activities exceeding more than 90% after treatment at 50°C for 1 h at their optimal pHs.     

Carbon/nitrogen relationships of fungus culture media

Summary and Conclusions Four basic nutrient types are described and discussed in special reference to the ratios between carbon source materials and nitrogen source materials. These include habitat, natural nutrients, semisynthetic nutrients and synthetic nutrients. Special emphasis is placed on the use of such nutrients in agar media for the primary isolation of fungi from various types of habitat materials such as soils and sewage.It is suggested that a basic C:N ratio for such nutrient agars lies at about 9 or 12 to 1, that increasing this ratio tends to overenrich the culture thus encouraging the formation of intracellular or extracellular carbon-carrying compounds, and that decreasing the ratio tends to encourage the formation of nitrogen-carrying storage products. Both conditions tend to produce staling substances more rapidly, tend to decrease discreteness of colonies, tend to produce more diffuse, less easily counted growth, and tend to develop less readily identifiable colonies.It is implied that if a C:N ratio more or less approximating that found in an agar containing plant parts, or limited nutrients plus plant parts, were used in a medium designed for primary isolation of fungi from natural materials, such a medium would have many advantages over currently popular media used for this purpose.     

Influence of medium composition on the cephalosporin C production with a highly productive strain Cephalosporium acremonium.

Cephalosporin production by a highly productive Cephalosporium acremonium strain was carried out and optimized by fed-batch operation in a 40 l stirred tank reactor using a complex medium containing 30-120 g l-1 peanut flour. The concentrations of cephalosporin C (CPC) and its precursors: penicillin N (PEN N), deacetoxy cephalosporin C (DAOC), and deacetyl cephalosporin C (DAC) were monitored with an on-line HPLC. The concentrations of amino acids valine (VAL), cysteine (CYS), alpha-amino adipic acid (alpha-AAA), the dipeptide alpha-amino-adipyl-cysteine (AC), and the tripeptide alpha-amino-adipyl-cysteinyl-valine (ACV), were determined off-line by HPLC. The RNA content and dry weight of the sediment as well as the oxygen transfer rate (OTR) and the CO2 production rate (CPR) were used to calculate the cell mass concentration (X). The influences of peanut flour (PF) and the on-line monitored and controlled medium components: glucose (GLU), phosphate, methionine (MET) as well as the dissolved oxygen (DOC) on the cell growth, the product formation, and the pathway of cephalosporin C biosynthesis were investigated and evaluated. When the glucose fed-batch cycle was optimized and oxygen transfer limitation was avoided (DOC greater than 20% of the saturation value), high process performance (103.5 g l-1 X, 11.84 g l-1 CPC, a maximum CPC productivity of 118 mg l-1 h-1, and the whole concentration of the beta-lactam antibiotics CPC, DAC, DAOC, PEN N 17.34 g l-1) was achieved by using 100 g l-1 PF in the medium with the optimum concentration of phosphate (260-270 mg l-1) and a low glucose concentration (less than 0.5 g l-1). The cultivations with different medium concentrations demonstrated that the product formation was directly proportional to the cell mass concentration. On the average, the cell mass-based yield coefficient of CPC: YCPC/X amounted to 0.115 g CPC per g cell mass.     

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.     

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.     

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     

Collagen biosynthesis; tissue culture experiments to ascertain the role of ascorbic acid in collagen formation

Quantitative studies of collagen formation by chick embryonic lung tissue grown in media deficient in, or completely lacking, ascorbic acid have been carried out. Cell growth and collagen formation in such cultures can proceed almost normally in media lacking ascorbic acid. Ascorbic acid in combination with whole embryo extract, dialyzed media, or synthetic mixture number 703 was found to have no appreciable effect on cell growth or total collagen formation. This is in marked contrast to the almost total failure of collagen formation in scorbutic animals and suggests that for slow collagen biosynthesis as distinct from more prolific collagen-producing systems, ascorbic acid plays an indirect role.     

Effect of norleucine on mycelial fragmentation in Cephalosporium acremonium.

DL-Norleucine, which is known to replace methionine for stimulation of cephalosporin C formation, also mimics methionine's effect on arthrospore formation. Thus, hyphal fragmentation, like antibiotic biosynthesis, is divorced from a sulfur donation role.     

Correlation of cephalosporin C synthesis and proteolytic enzymes in a differentiating culture of Acremonium chrysogenum (Cephalosporum acremonium) mutants

The effect of the growth conditions and composition of the nutrient medium on the synthesis of cephalosporin C, alkaline exoproteases and cell differentiation was studied in various strains of Acremonium chrysogenum. It was observed that the changes in the above processes occurred simultaneously and depended on the aeration rate, temperature, illumination level and concentrations of methionine and soybean meal. Close correlation between the synthesis of alkaline exoproteases, cephalosporin C and formation of secondary structures in the strains of A. chrysogenum was shown.     

COLLAGEN BIOSYNTHESIS : TISSUE CULTURE EXPERIMENTS TO ASCERTAIN THE ROLE OF ASCORBIC ACID IN COLLAGEN FORMATION

Quantitative studies of collagen formation by chick embryonic lung tissue grown in media deficient in, or completely lacking, ascorbic acid have been carried out. Cell growth and collagen formation in such cultures can proceed almost normally in media lacking ascorbic acid. Ascorbic acid in combination with whole embryo extract, dialyzed media, or synthetic mixture number 703 was found to have no appreciable effect on cell growth or total collagen formation. This is in marked contrast to the almost total failure of collagen formation in scorbutic animals and suggests that for slow collagen biosynthesis as distinct from more prolific collagen-producing systems, ascorbic acid plays an indirect role.     

Evolution of an acylase active on cephalosporin C

Semisynthetic cephalosporins are synthesized from 7-amino cephalosporanic acid, which is produced by chemical deacylation or by a two-step enzymatic process of the natural antibiotic cephalosporin C. The known acylases take glutaryl-7-amino cephalosporanic acid as a primary substrate, and their specificity and activity are too low for cephalosporin C. Starting from a known glutaryl-7-amino cephalosporanic acid acylase as the protein scaffold, an acylase gene optimized for expression in Escherichia coli and for molecular biology manipulations was designed. Subsequently we used error-prone PCR mutagenesis, a molecular modeling approach combined with site-saturation mutagenesis, and site-directed mutagenesis to produce enzymes with a cephalosporin C/glutaryl-7-amino cephalosporanic acid catalytic efficiency that was increased up to 100-fold, and with a significant and higher maximal activity on cephalosporin C as compared to glutaryl-7-amino cephalosporanic acid (e.g., 3.8 vs. 2.7 U/mg protein, respectively, for the A215Y-H296S-H309S mutant). Our data in a bioreactor indicate an ~90% conversion of cephalosporin C to 7-amino-cephalosporanic acid in a single deacylation step. The evolved acylase variants we produced are enzymes with a new substrate specificity, not found in nature, and represent a hallmark for industrial production of 7-amino cephalosporanic acid.