Metabolic flux distributions in Penicillium chrysogenum during fed-batch cultivations

Based on a review of the Penicillium chrysogenum biochemistry a stoichiometric model has been set up. The model considers 61 internal fluxes and there are 49 intracellular metabolites which are assumed to be in pseudo-steady state. In addition to the intracellular fluxes the model considers the uptake of 21 amino acids. From the stoichiometric model the maximum theoretical yield of penicillin V is calculated to 0.43 mol/mol glucose. If biosynthesis of cysteine is by direct sulfhydrylation rather than by transsulfuration, the maximum theoretical yield is about 20% higher, i.e., 0.50 mol/mol glucose. The theoretical yield decreases substantially if alpha-aminoadipate is converted to 6-oxo-piperidine-2-carboxylic acid (OPC). If only 40% of the alpha-aminoadipate is recycled, the maximum theoretical yield is 0.31 mol/mol glucose. The uptake rates of glucose, lactate, gamma-aminobutyrate, and 21 amino acids were measured during fed-batch cultivations. The rates of formation of penicillin V, delta-(L-alpha)-aminoadipyl-L-cysteinyl-D-valine (ACV), OPC, and the pool of isopenicillin N, 6-APA, and 8-HPA were also measured. Finally the synthesis rates of the biomass constituents RNA/DNA, protein, lipid, carbohydrate, and amino carbohydrate were measured. From these measured rates and the stoichiometric model the metabolic fluxes through the different intracellular pathways are calculated. The calculations show that penicillin formation is accompanied by a large flux through the pentose phosphate (PP) pathway due to a large requirement for nicotinamide-adenine dinucleotide phosphate (NADPH) used in the biosynthesis of cysteine. If cysteine is added to the medium, the flux through the PP pathway decreases. From the stoichiometric model Y(xATP) is calculated to 87 mmol adenosine triphosphate (ATP)/g dry weight (DW), and from the flux calculations m(ATP) is found to 3 mmol ATP/g DW/h. (c) 1995 John Wiley & Sons, Inc.     

Cybernetic modeling based on pathway analysis for Penicillium chrysogenum fed-batch fermentation

A macrokinetic model employing cybernetic methodology is proposed to describe mycelium growth and penicillin production. Based on the primordial and complete metabolic network of Penicillium chrysogenum found in the literature, the modeling procedure is guided by metabolic flux analysis and cybernetic modeling framework. The abstracted cybernetic model describes the transients of the consumption rates of the substrates, the assimilation rates of intermediates, the biomass growth rate, as well as the penicillin formation rate. Combined with the bioreactor model, these reaction rates are linked with the most important state variables, i.e., mycelium, substrate and product concentrations. Simplex method is used to estimate the sensitive parameters of the model. Finally, validation of the model is carried out with 20 batches of industrial-scale penicillin cultivation.     

Analysis of penicillin V biosynthesis during fed-batch cultivations with a high-yielding strain of Penicillium chrysogenum

Metabolites (both intra- and extracellular) involved in penicillin biosynthesis were measured during fed-batch cultivations with a high-yielding strain of Penicillium chrysogenum. The fed-batch cultivations were carried out on a complex medium containing corn steep liqour. Three distinct phases were observed: (a) a rapid growth phase where free amino acids present in the medium are metabolized, (b) a linear growth phase, and (c) a stationary phase. The specific penicillin production (r _p) is initially high and, during the rapid growth phase, it increases slightly. During the linear growth phase r _p is approximately constant [4–6 mg penicillin V (g dry weight)^–1 h^–1 depending on the operating conditions], whereas it decreases during the stationary phase. During the cultivations the tripeptide Aad-Cys-Val (the first metabolite in penicillin biosynthesis) and 8-hydroxypenillic acid (formed by carboxylation of 6-aminopenicillanic acid, 6-APA) were found to accumulate in the medium, whereas the concentrations of isopenicillin N and 6-APA were found to be approximately constant and low. About 3% of the Aad-Cys-Val formed in the first step of the penicillin biosynthetic pathway is lost to the medium and 4% of the isopenicillin N formed in the second step of the pathway is lost as extracellular isopenicillin N, 6-APA or 8-hydroxypenillic acid. Also the cyclic form of -aminoadipic acid, 6-oxopiperidine-2-carboxylic acid, was found to accumulate in the medium and it was found to be formed in an approximately constant ratio to penicillin V of 6 mol/100 mol.     

Effect of glutamine on penicillin formation in Penicillium chrysogenum

Summary Resting-cell studies in Penicillium chrysogenum have indicated that penicillin formation is inhibited by glutamine concentrations higher than 1 mM. Total inhibition was obtained with 10 mM glutamine. This action was neither reverted by the amino acid precursors of the antibiotic moiety nor glutamin affected the in vitro activity of the first enzyme of the penicillin formation pathway. The inhibition was prevented by 1 mM glutathione by mechanisms not related to limitation in the glutamine incorporation nor connected with degradation of the tripeptide.     

Glutathione formation in Penicillium chrysogenum: stimulatory effect of ammonium

Penicillium chrysogenum produced glutathione after growth in a defined medium containing 10 mM-NH4Cl as the sole source of nitrogen. The use of higher ammonium concentrations (100 mM) resulted in stimulation of growth and glutathione formation. In addition, increases in the intracellular pools of glutamate, alanine and glutamine, proportional to the amount of ammonium present in the medium were observed. Resting cell systems, prepared from cells previously grown with ammonium, were able to produce glutathione when incubated with ammonium or the amino acids glutamate, alanine and glutamine. A mutant lacking NADP-dependent glutamate dehydrogenase activity (which has a leaky phenotype on ammonium as sole nitrogen source) required glutamate to synthesize glutathione. Resting cell systems of this mutant, prepared from cells previously grown with ammonium, did not produce glutathione even when incubated with glutamate or glutamine. On the other hand, resting cell systems of this mutant produced glutathione if prepared from cells previously grown with glutamate. The addition of glutamate to resting cell systems of the wild-type strain stimulated the synthesis of gamma-glutamylcysteine synthetase, the first enzyme of glutathione biosynthesis.     

Stability of recombinant protein production by Penicillium chrysogenum in prolonged chemostat culture

Abstract A strain (WKW2) of Penicillium chrysogenum transformed with heterologous fungal acetamidase ( amd S) and bacterial β-galactosidase ( lac Z) was grown at a dilution rate of 0.17 h⁻¹ (doubling time of approx. 4.1 h) for 1600 h in a glucose-limited culture. By the end of the experiment the original strain had been almost completely replaced by spontaneous, morphological mutants, but the acetamidase and β-galactosidase activities of the culture were essentially unaltered. Furthermore, when WKW2 and the non-transformed parental strain (NRRL1951) were grown together in glucose- or NH₄⁺-limited chemostat cultures, neither strain had a selective advantage over the other. Thus, heterologous gene expression does not result in NRRL1951 having a selective advantage over WKW2. These results suggest that continuous flow culture systems could be used for efficient (and cost effective) production of recombinant proteins.     

Yeast-like cell formation and glutathione metabolism in autolysing cultures of Penicillium chrysogenum

The bulk formation of yeast-like (arthrospore-like) cells were typical in carbon-depleted submerged cultures of the high beta-lactam producer Penicillium chrysogenum NCAIM 00237 strain independently of the nitrogen-content of the culture medium. This morphogenetic switch was still quite common in carbon-starving cultures of the low-penicillin-producer strain P. chrysogenum ATCC 28089 (Wis 54-1255) when the nitrogen-content of the medium was low but was a very rare event in wild-type P. chrysogenum cultures. The mycelium-->yeast-like cell transition correlated well with a relatively high glutathione concentration and a reductive glutathione/glutathione disulfite (GSH/GSSG) redox balance in autolysing cultures, which was a consequence of industrial strain development. Paradoxically, the development of high beta-lactam productivity resulted in a high intracellular GSH level and, concomitantly, in an increased y-glutamyltranspeptidase (i.e. GSH-decomposing) activity in the autolytic phase of growth of P. chrysogenum NCAIM 00237. The hypothesized causal connection between GSH metabolism and cell morphology, if verified, may help us in future metabolic engineering of industrially important filamentous fungi.     

Nitrate regulation of alpha-aminoadipate reductase formation and lysine inhibition of its activity in Penicillium chrysogenum and Acremonium chrysogenum

alpha-Aminoadipate reductase (alpha-AAR) is a key enzyme in the branched pathway for lysine and beta-lactam biosynthesis of filamentous fungi since it competes with alpha-aminoadipyl-cysteinyl-valine synthetase for their common substrate L-alpha-aminoadipic acid. The alpha-AAR activity in two penicillin-producing Penicillium chrysogenum strains and two cephalosporin-producing Acremonium chrysogenum strains has been studied. The alpha-AAR activity peaked during the growth-phase preceding the onset of antibiotic production, which coincides with a decrease in alpha-AAR activity, and was lower in high penicillin- or cephalosporin-producing strains. The alpha-AAR required NADPH for enzyme activity and could not use NADH as electron donor for reduction of the alpha-aminoadipate substrate. The alpha-AAR protein of P. chrysogenum was detected by Western blotting using anti-alpha-AAR antibodies. The mechanism of lysine feedback regulation in these two filamentous fungi involves inhibition of the alpha-AAR activity but not repression of its synthesis by lysine. This is different from the situation in yeasts where lysine feedback inhibits and represses alpha-AAR. Nitrate has a strong negative effect on alpha-AAR formation as shown by immunoblotting studies of alpha-AAR. The nitrate effect was reversed by lysine.     

Characterization of a phenylacetate-CoA ligase from Penicillium chrysogenum

Enzymatic activation of PAA (phenylacetic acid) to phenylacetyl-CoA is an important step in the biosynthesis of the beta-lactam antibiotic penicillin G by the fungus Penicillium chrysogenum. CoA esters of PAA and POA (phenoxyacetic acid) act as acyl donors in the exchange of the aminoadipyl side chain of isopenicillin N to produce penicillin G or penicillin V. The phl gene, encoding a PCL (phenylacetate-CoA ligase), was cloned in Escherichia coli as a maltose-binding protein fusion and the biochemical properties of the enzyme were characterized. The recombinant fusion protein converted PAA into phenylacetyl-CoA in an ATP- and magnesium-dependent reaction. PCL could also activate POA, but the catalytic efficiency of the enzyme was rather low with k(cat)/K(m) values of 0.23+/-0.06 and 7.8+/-1.2 mM(-1).s(-1) for PAA and POA respectively. Surprisingly, PCL was very efficient in catalysing the conversion of trans-cinnamic acids to the corresponding CoA thioesters [k(cat)/K(m)=(3.1+/-0.4)x10(2) mM(-1).s(-1) for trans-cinnamic acid]. Of all the substrates screened, medium-chain fatty acids, which also occur as the side chains of the natural penicillins F, DF, H and K, were the best substrates for PCL. The high preference for fatty acids could be explained by a homology model of PCL that was constructed on the basis of sequence similarity with the Japanese firefly luciferase. The results suggest that PCL has evolved from a fatty-acid-activating ancestral enzyme that may have been involved in the beta-oxidation of fatty acids.     

Malachite green treatment of industrial Penicillium chrysogenum protoplasts results in increased penicillin-V formation

We attempted protoplast fusion in order to generate gene transfer between an industrial strain of Penicillium chrysogenum and a fission yeast, Schizosaccharomyces pombe. The Penicillium strain was treated with malachite green. The S. pombe strain was auxotrophic for lysine. The regenerated colonies showed Penicillium morphology. The number of Penicillium colonies was significantly higher when the inactivated Penicillium protoplasts were fused to S. pombe protoplasts than in the self-fusion control experiments. We randomly isolated colonies from the regeneration plates and measured beta-lactam formation in cultures from shaken flasks. Antibiotic production was increased in colonies originated from the malachite green-treated protoplasts. Received 2 June 1998/ Accepted in revised form 30 November 1998     

Penicillin formation by biochemical mutants of Penicillium chrysogenum and their spontaneous revertants

The capacity for the antibiotic production in the auxotrophs of Penicillium chrysogenum with various deficiency and their revertants was studied. It was found that the capacity for penicillin synthesis was impaired to various degrees in the majority of the auxotrophs. Variants with the penicillin production levels by 13--20 per cent higher than those in the initial prototrophic strain were isolated for the first time in selection of the eukaryotes with the method of obtaining highly active revertants from auxotrophs according to the scheme "prototroph-auxotroph-prototroph".