A role for alanine in the ammonium regulation of cephalosporin biosynthesis inStreptomyces clavuligerus

Summary It is known that excess ammonium supply decreases cephalosporin production and represses cephalosporin synthases. We wondered whether an additional important effect could be inhibition of synthase action by alanine. We had previously shown that ammonium addition induced alanine dehydrogenase and increased intracellular alanine and that alanine could inhibit resting cell synthesis of cephalosporins. In the present work we confirm the alanine inhibition of antibiotic production by resting cells. We foundl-alanine inhibited three of the four synthases tested: ACV synthetase, cyclase and expandase; the epimerase was not inhibited. These data suggest that interference in cephalosporin production by growth in ammonium salts involves synthase inhibition by intracellular alanine, in addition to the known role of ammonium in synthase repression.     

Prevention of phosphate inhibition of cephalosporin synthetases by ferrous ion

Abstract Phosphate interference in the production of cephalosporins by Streptomyces clavuligerus had been associated with repression of expandase (desacetoxycephalosporin C synthetase) and inhibition of both expandase and cyclase (isopenicillin N synthetase). The present work shows that inhibition of enzyme action could be prevented by increasing the Fe²⁺ added to the cell-free reactions or to resting cells incubated with chloramphenicol. Since excess Fe²⁺ could not reverse phosphate interference of antibiotic synthesis in complete fermentations, it is clear that the major cause of the phosphate effect in fermentations is phosphate repression, rather than phosphate inhibition caused by Fe²⁺ deprivation.     

Relationship between nitrogen assimilation and cephalosporin synthesis inStreptomyces clavuligerus

The levels of three enzymes of the -lactam antibiotic pathway and overall cephalosporin production were subject to nitrogen source repression inStreptomyces clavuligerus. The specific activities of isopenicillin N synthetase (cyclase) and deacetoxycephalosporin C synthetase (expandase) measured during the exponential phase depended on the nitrogen source employed, following a pattern that roughly correlated with the corresponding antibiotic production. The effects on isopenicillin N epimerase (epimerase) activities were less marked than those on the cyclase and expandase.Production of cephalosporins and enzymatic activities were not related to the growth rate of the cultures. Glutamate, glutamine and alanine inhibited production when added to resting cell systems, while lysine and -aminoadipate were stimulatory. No clear relationship could be drawn between cephalosporin production or -lactam synthetase activities and the activities of enzymes of ammonium assimilation (glutamine synthetase, glutamate synthase and alanine dehydrogenase).The intracellular pools of free glutamine, alanine and ammonium were the only ones markedly affected by the nitrogen source in the wild type and mutants, but these amino acids did not seem to play an obvious role as intracellular mediators of nitrogen control.Abbreviations DCW dry cell weight - GS glutamine synthetase - GOGAT glutamate synthase - ADH alanine dehydrogenase - HPLC high performance liquid chromatography     

Phosphate regulation of ACV synthetase and cephalosporin biosynthesis in Streptomyces clavuligerus

Cephalosporin production by Streptomyces clavuligerus was reduced sharply by 60 mM phosphate added to a chemically-defined medium. All the four synthetases in the pathway examined, i.e., ACV synthetase, cyclase, epimerase and expandase, were repressed by phosphate, with ACV synthetase being the main repression target and expandase the next. ACV synthetase activity was inhibited by phosphate to a lesser extent than expandase and cyclase, and this inhibition could be reversed by adding Fe2+. Fe2+ itself was inhibitory to ACV synthetase action.     

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.     

Identification of rate-limiting steps in cephalosporin C biosynthesis in Cephalosporium acremonium: a theoretical analysis

Summary A kinetic model describing the biosynthesis of celphalosporin C in Cephalosporium acremonium has been developed to identify the rate-limiting step(s). Using this model and in-vitro kinetic data of the biosynthetic enzymes, the production kinetics of cephalosporin C were examined theoretically. The predicted time profile of the specific production rate during batch culture is in good agreement with that of experimental results published previously. Sensitivity analysis indicates that -(l--aminoadipyl)-l-cysteinyl-d-valine (ACV) synthetase is the rate-limiting enzyme. Our analysis also predicts that increasing ACV synthetase enhances the production rate initially until expandase/hydroxylase becomes rate-limiting. Furthermore, increasing expandase/hydroxylase reduces the accumulation of penicillin N, and thus, enhances the production of cephalosporin C. Based on our analysis, amplifying both ACV synthetase and expandase/hydroxylase concurrently should enhance the production rate to a great extent.Correspondence to: W. S. Hu     

Enzymatic synthesis of the penicillin and cephalosporin nuclei from an acyclic peptide containing carboxymethylcysteine

The tripeptide delta-(L- carboxymethylcysteinyl )-L-cysteinyl-D-valine (L-CMC-CV) is converted sequentially into the CMC analog of isopenicillin N, the CMC analog of penicillin N, and the CMC analog of desacetoxycephalosporin C by, respectively, isopenicillin N synthetase, isopenicillin N epimerase, and desacetoxycephalosporin C synthetase, all isolated from the beta-lactam producing prokaryote Streptomyces clavuligerus.     

Improvement in the bioconversion of penicillin G to deacetoxycephalosporin G by elimination of agitation and addition of decane

The bioconversion of penicillin G to deacetoxycephalosporin G (DAOG) using resting cells of Streptomyces clavuligerus could be a very valuable step in the economical production of semisynthetic cephalosporin antibiotics. The extent of the reaction, however, is very low due to inactivation of the ring expansion enzyme deacetoxycephalosporin C synthetase ("expandase") by reaction components. We show that elimination of agitation during the reaction lowers the rate but increases the amount of DAOG produced, presumably because the inactivation requires high levels of oxygen. Many additives to the medium were examined for their effect on the reaction. Clearly, the most effective compound was the organic solvent, decane.     

Expression of<Emphasis Type="Italic"> cefD2</Emphasis> and the conversion of isopenicillin N into penicillin N by the two-component epimerase system are rate-limiting steps in cephalosporin biosynthesis

The conversion of isopenicillin N into penicillin N in Acremonium chrysogenum is catalyzed by an epimerization system that involves an isopenicillin N-CoA synthethase and isopenicillin N-CoA epimerase, encoded by the genes cefD1 and cefD2. Several transformants containing two to seven additional copies of both genes were obtained. Four of these transformants (TMCD26, TMCD53, TMCD242 and TMCD474) showed two-fold higher IPN epimerase activity than the untransformed A. chrysogenum C10, and produced 80 to 100% more cephalosporin C and deacetylcephalosporin C than the parental strain. A second class of transformants, including TMCD2, TMCD32 and TMCD39, in contrast, showed a drastic reduction in cephalosporin biosynthesis relative to the untransformed control. These transformants had no detectable IPN epimerase activity and did not produce cephalosporin C or deacetylcephalosporin C. They also expressed both endogenous and exogenous cefD2 genes only after long periods (72–96 h) of incubation, as shown by Northern analysis, and were impaired in mycelial branching in liquid cultures. The negative effect of amplification of the cefD1 - cefD2 gene cluster in this second class of transformants is not correlated with high gene dosage, but appears to be due to exogenous DNA integration into a specific locus, which results in a pleiotropic effect on growth and cefD2 expression. Communicated by P. J. Punt     

Carbon catabolite regulation of cephamycin C and expandase biosynthesis in Streptomyces clavuligerus

Summary Streptomyces clavuligerus produces cephamycin C while growing on chemically defined basal medium. Cephamycin C production takes place during the exponential growth phase and is accompanied by vigorous activity of the cephamycin C synthetase system and of expandase. An excessive amount of glycerol decreases cephamycin C production. Its negative effect appears to be greatest when it is added in the first phase of fermentation either alone or in the presence of starch. Starch excess also reduces cephamycin C production, but its effect is slight compared with glycerol. Glycerol hinders cephamycin C production by the repression of the cephamycin C synthetase system and particularly expandase biosynthesis. Starch and glycerol inhibit neither cephamycin C synthetase nor expandase activities. However, the phosphorylated intermediates of the glycolytic pathway, glucose 6-phosphate and fructose 1,6-phosphate, strongly inhibit expandase activity.     

Regulation of isopenicillin N synthetase and deacetoxycephalosporin C synthetase by carbon source during the fermentation of Cephalosporium acremonium

Summary In a chemically defined medium with glucose and sucrose as major carbon sources (standard medium), Cephalosporium acremonium excretes the intermediate of the -lactam biosynthetic pathway, penicillin N, into the medium during growth; production of cephalosporins is delayed until glucose is completely utilized. Deacetoxycephalosporin C synthetase, the ring-expansion enzyme (expandase), does not appear as long as glucose is present. Afterwards, initiation of its formation is accompanied by the production of cephalosporins. Feeding additional glucose during the fermentation turns off expandase synthesis without affecting formation of isopenicillin N synthetase, the ring-cyclization enzyme (cyclase). The above results point to a strong glucose catabolite repression of the expandase as one of the main regulatory mechanisms in -lactam biosynthesis by Cephalosporium acremonium and the reason for accumulation of penicillin N during the fermentation. Cyclase shows a biphasic pattern in activity, the first very high peak not being correlated with the excretion of any -lactam antibiotic into the medium.     

Glucose regulation of cephamycin biosynthesis in Streptomyces lactamdurans is exerted on the formation of alpha-aminoadipyl-cysteinyl-valine and deacetoxycephalosporin C synthase

Glucose exerted a concentration-dependent negative regulation on the biosynthesis of cephamycin C by Streptomyces lactamdurans. Formation of the cephamycin precursor delta(alpha-aminoadipyl)-cysteinyl-valine was greatly decreased by excess glucose. The ring-expanding enzyme deacetoxycephalosporin C synthase was strongly repressed by glucose in vivo. Isopenicillin N synthase (cyclase) and isopenicillin N epimerase were not repressed by glucose. However, the activity of isopenicillin N synthase was inhibited in vitro by glucose 6-phosphate, and the activity of deacetoxycephalosporin C synthase was inhibited by inorganic phosphate, glucose 6-phosphate, fructose 2,6-diphosphate and fructose 1,6-diphosphate. The intracellular cAMP content decreased as growth proceeded and remained lower in glucose-supplemented cells than in control cultures. cAMP did not seem to be involved in glucose control of cephamycin biosynthesis.     

Further studies on carbon catabolite regulation of β-lactam antibiotic synthesis inCephalosporium acremonium

A high glucose concentration (6%) interfered with production of -lactam antibiotics byCephalosporium acremonium. Production rate of the pathway intermediate, penicillin N, by resting cells harvested from a high glucose fermentation, peaked and declined early in the fermentation. When cells were grown in the standard medium (2.7% glucose + 3.6% sucrose), penicillin N productivity was prolonged, showing two peaks, the first during trophophase and the second afterwards. The decline in productivity was not prevented by addition of the amino acid precursors of -lactam antibiotics. The addition of glucose to resting cells drastically decreased formation of the end product, cephalosporin C, but had only a moderate effect on penicillin N production. Glucose markedly repressed the ring-expansion enzyme (deacetoxy-cephalosporin C synthetase) but had a lesser effect on the tripeptide cyclization enzyme (isopenicillin N synthetase). We conclude that the major effect of a low (2%) or a high (6%) concentration of a rapidly used carbon source (e.g., glucose, glycerol, maltose) onC. acremonium fermentations is repression of the metabolically unstable ring-expansion enzyme and hence of formation of cephalosporins. On the other hand, the lesser degree of repression of the cyclization enzyme and itsin vivo stability allow penicillin N to accumulate normally or even at increased rates except at high carbon source concentrations.     

The effect of thiols and the Ca2+ ionophore A23817 on growth and antibiotic production in Cephalosporium acremonium

Abstract Yeast-like blastosporic growth, lowered levels of isopenicillin N synthetase and deacetoxycephalosporin C synthetase/hydroxylase, and lowered antibiotic production were induced in Cephalosporium acremonium strain C.O. 728 by inclusion of cysteine or glutathione in the growth medium. These effects were similar to those observed under normal conditions with strain M 8650/4, which had higher levels of glutathione reductase and protein disulphide reductase than strain C.O. 728. The addition of cytochalasins A and B slightly increased yeast-like growth in C.O. 728, although levels of biosynthetic enzyme activity and antibiotic secretion were not significantly affected. The Ca²⁺ ionophore A23187 inhibited C.O. 728 growth, slowed penicillin secretion into the medium, and totally inhibited cephalosporin secretion. These results show that maintenance of Ca²⁺ levels and the absence of thiols are important for antibiotic production in Cephalosporium acremonium .     

Isolation of deacetoxycephalosporin C from fermentation broths of Penicillium chrysogenum transformants: construction of a new fungal biosynthetic pathway.

Deacetoxycephalosporin C (DAOC), a precursor of cephalosporins excreted by Cephalosporium and Streptomyces species, has been produced in Penicillium chrysogenum transformed with DNA containing a hybrid penicillin N expandase gene (cefEh) and a hybrid isopenicillin N epimerase gene (cefDh). DAOC from a P. chrysogenum transformant was identified by ultraviolet light (UV), high performance liquid chromatography (HPLC), nuclear magnetic resonance (NMR) and mass spectrum analyses. P. chrysogenum transformed with DNA containing cefEh without cefDh did not produce DAOC. Untransformed P. chrysogenum produced penicillin V (phenoxymethylpenicillin) but not DAOC. Transformants also produced penicillin V but, in general, less than untransformed P. chrysogenum. The cefEh and cefDh genes were constructed by replacing the open reading frame (ORF) of cloned P. chrysogenum pcbC and penDE genes with the ORF of the Streptomyces clavuligerus expandase gene, cefE, and the ORF of the Streptomyces lipmanii epimerase gene, cefD, respectively. Analyses of representative transformants suggested that production of DAOC occurred via cefEh and cefDh genes stably integrated in the P. chrysogenum genome. DNA from untransformed P. chrysogenum did not hybridize to cefE or cefD gene probes.     

Purification and properties of isopenicillin N epimerase from Streptomyces clavuligerus

Isopenicillin N epimerase, which catalyzes conversion of isopenicillin N to penicillin N, has been purified to electrophoretic homogeneity from the cell-free extract of Streptomyces clavuligerus by a procedure involving ammonium sulfate fractionation and chromatographies with DE-52, DEAE Affi-gel blue, Sephadex G-200, calcium phosphate-cellulose, and Mono Q. The purified epimerase is monomeric with a molecular weight of 47,000 or 50,000 as estimated by SDS-polyacrylamide gel electrophoresis or gel filtration, respectively. The enzyme contains 1 mol of pyridoxal 5'-phosphate per mol of protein, and shows absorption maxima at 280 and 420 nm. The epimerase catalyzes the complete 'racemization' on both the L-alpha-aminoadipyl side-chain of isopenicillin N and the D-alpha-aminoadipyl side-chain of penicillin N, so that an approximately equimolar mixture of the two penicillins is produced. The mixture is not truly racemic, since these penicillins are diastereomers rather than optical isomers. The chemical modification of primary amino groups of the epimerase by fluorescamine results in a great loss of the enzyme activity. The activity of purified enzyme is partially stimulated by the addition of sulfhydryl compounds. The activity is strongly inhibited by sulfhydryl group modifiers such as p-chloromercuribenzoate and N-ethylmaleimide.