Utilization of side-chain precursors for penicillin biosynthesis in a high-producing strain of Penicillium chrysogenum

Utilization of the side-chain precursors phenoxyacetic acid (POA) and phenylacetic acid (PA) for penicillin biosynthesis by Penicillium chrysogenum was studied in shake flasks. Precursor uptake and penicillin production were followed by HPLC analysis of precursors and products in the medium and in the cells. P. chrysogenum used both POA and PA as precursors, producing phenoxymethylpenicillin (penicillin V) and benzylpenicillin (penicillin G), respectively. If both precursors were present simultaneously, the formation of penicillin V was blocked and only penicillin G was produced. When PA was added at different times to cells that were induced initially for POA utilization and were producing penicillin V, the POA utilization and penicillin V formation were blocked, whereas the cells started utilizing PA and produced penicillin G. The blocking of the POA turnover lasted for as long as PA was present in the medium. If POA was added to cultures induced initially for PA utilization and producing penicillin G, this continued irrespective of the presence of POA. Utilization of POA increased concomitant with depletion of PA from the medium. Analysis of cellular pools from a growing cell system with POA as precursor to which PA was added after 48 h showed that the cellular concentration of POA was kept high without production of penicillin V and at a concentration comparable to the concentration in the medium. The cellular concentration of POA was higher than the concentration of PA that was utilized for penicillin G production. Correspondence to: S. Havn Eriksen     

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.     

alpha-Aminoadipate pool concentration and penicillin biosynthesis in strains of Penicillium chrysogenum

Intracellular amino acid pools in four Penicillium chrysogenum strains, which differed in their ability to produce penicillin, were determined under conditions supporting growth without penicillin production and under conditions supporting penicillin production. A significant correlation between the rate of penicillin production and the intracellular concentration of alpha-aminoadipate was observed, which was not shown with any other amino acid in the pool. In replacement cultivation, penicillin production was stimulated by alpha-aminoadipate, but not by valine or cysteine. Exogenously added alpha-aminoadipate (2 or 3 mM) maximally stimulated penicillin synthesis in two strains of different productivity. Under these conditions intracellular concentrations of alpha-aminoadipate were comparable in the two strains in spite of the higher rate of penicillin production in the more productive strain. Results suggest that the lower penicillin titre of strain Q 176 is due to at least two factors: (i) the intracellular concentration of alpha-aminoadipate is insufficient to allow saturation of any enzyme which is rate limiting in the conversion of alpha-aminoadipate to penicillin and (ii) the level of an enzyme, which is rate limiting in the conversion of alpha-aminoadipate to penicillin, is lower in Q 176 (relative to strain D6/1014/A). Results suggest that the intracellular concentration of alpha-aminoadipate in strain D6/1014/A is sufficiently high to allow saturation of the rate-limiting penicillin biosynthetic enzyme in that strain. The basis of further correlation of intracellular alpha-aminoadipate concentration and penicillin titre among strains D6/1014/A, P2, and 389/3, the three highest penicillin producers studied here, remains to be established.(ABSTRACT TRUNCATED AT 250 WORDS)     

Energetics of growth and penicillin production in a high-producing strain of Penicillium chrysogenum

The results of a large number of carbon-limited chemostat cultures of Penicillium chrysogenum carried out on glucose, ethanol, and acetate as the growth limiting substrate have been used to obtain an estimation of the adenosine triphosphate (ATP) costs for mycelium growth, penicillin production, and maintenance and the overall stoichiometry of oxidative phosphorylation of the fungus. It was found that penicillin production was accompanied by a significant additional energy drain (73 mol of ATP per mole of penicillin-G) from primary metabolism. This finding has been confirmed in independent experiments and has been shown to result in a significantly lower estimate for the maximum theoretical yield of penicillin-G on the carbon source.     

Localization of the pathway of the penicillin biosynthesis in Penicillium chrysogenum.

The localization of the enzymes involved in penicillin biosynthesis in Penicillium chrysogenum hyphae has been studied by immunological detection methods in combination with electron microscopy and cell fractionation. The results suggest a complicated pathway involving different intracellular locations. The enzyme delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase was found to be associated with membranes or small organelles. The next enzyme isopenicillin N-synthetase appeared to be a cytosolic enzyme. The enzyme which is involved in the last step of penicillin biosynthesis, acyltransferase, was located in organelles with a diameter of 200-800 nm. These organelles, most probably, are microbodies. A positive correlation was found between the capacity for penicillin production and the number of organelles per cell when comparing different P. chrysogenum strains.     

Reduced utilization of side-chain precursor for penicillin biosynthesis by mutants of Penicillium chrysogenum

Summary A high yielding strain of Penicillium chrysogenum was mutated with EMS and investigated for selective effects of semi-continuous fermentations. Preferential growth of a class of mutants with different colony type and having reduced ability to utilize side-chain precursor led to reduced Pen V synthesis by the heterogeneous mycelial population.     

Penicillium chrysogenum mycelial productivity in the 1st phase of penicillin biosynthesis]

The course of the mycelium low productivity during the first phase of the usual two-stage process of penicillin biosynthesis was studied. It was found that the low productivity of the mycelium at the beginning of the fermentation process was probably associated with catabolic regression of the penicillin-producing system. The high specific growth rate registered in the experiments (0.06-0.08 hours-1) had no negative effect on the mycelium productivity. It was not possible to connect the productivity level with the mycelium age, because young and mature mycelium had the same producitivity levels at any developmental stage under the same conditions.     

The thioredoxin system of Penicillium chrysogenum and its possible role in penicillin biosynthesis.

Penicillium chrysogenum is an important producer of penicillin antibiotics. A key step in their biosynthesis is the oxidative cyclization of delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine (ACV) to isopenicillin N by the enzyme isopenicillin N synthase (IPNS). bis-ACV, the oxidized disulfide form of ACV is, however, not a substrate for IPNS. We report here the characterization of a broad-range disulfide reductase from P. chrysogenum that efficiently reduces bis-ACV to the thiol monomer. When coupled in vitro with IPNS, it converts bis-ACV to isopenicillin N and may therefore play a role in penicillin biosynthesis. The disulfide reductase consists of two protein components, a 72-kDa NADPH-dependent reductase, containing two identical subunits, and a 12-kDa general disulfide reductant. The latter reduces disulfide bonds in low-molecular-weight compounds and in proteins. The genes coding for the reductase system were cloned and sequenced. Both possess introns. A comparative analysis of their predicted amino acid sequences showed that the 12-kDa protein shares 26 to 60% sequence identity with thioredoxins and that the 36-kDa protein subunit shares 44 to 49% sequence identity with the two known bacterial thioredoxin reductases. In addition, the P. chrysogenum NADPH-dependent reductase is able to accept thioredoxin as a substrate. These results establish that the P. chrysogenum broad-range disulfide reductase is a member of the thioredoxin family of oxidoreductases. This is the first example of the cloning of a eucaryotic thioredoxin reductase gene.     

Quantitative physiology of Penicillium chrysogenum in penicillin fermentation

Using continuous and fed-batch penicillin fermentation systems some important metabolic parameters have been determined for the purpose of achieving process improvement and better process control. The specific uptake rates determined under the optimal conditions are: 0.33 mmol hexose/g cell/hr, 1.6 mmol oxygen/g cell/hr, 2mg NH₃-nitrogen/g cell/hr, 0.6 mg PO₄-phosphorus/g cell/hr, 2.8 mg SO₄-sulfur/g cell/hr, 1.8 mg phenyl acetic acid/g cell/hr. It was also found that during the production phase, or idiophase, the specific growth rate should be maintained at about 0.015 hr^?1 in order to support the maximum penicillin productivity of the given strain. Based on the results of this study a significant process improvement has been achieved through proper control of the supply and demand of the important nutrients and oxygen.     

Analysis of a commercially improved Penicillium chrysogenum strain series: involvement of recombinogenic regions in amplification and deletion of the penicillin biosynthesis gene cluster

Several commercially improved strains of Penicillium chrysogenum have been shown to carry amplifications of the entire penicillin biosynthesis gene cluster. Analysis previously carried out using the strain BW 1890 has here been extended to the characterisation of other members of the SmithKline Beecham strain improvement series. We have determined the length of the amplicon to be 57.4 kb and shown a general increase in copy number and penicillin titre through the series. Sequence analyses of the promoter regions of the acvA, ipnA and aat genes in the high titre strain BW 1901, and comparisons with wild-type sequences have not identified any potentially titre-enhancing mutations. In addition, cDNA screening has failed to identify any further transcribed elements within the co-amplified region. The homogeneity of hybridisation patterns and the identification and analysis of a single copy revertant has shown that the amplification is of a direct tandem nature and we propose a model of chromatid misalignment and recombination as its mode of generation. Hybridisation analysis of penicillin non-producing mutants has indicated the loss, in all those investigated, of the entire penicillin biosynthesis gene cluster, similarities between the deletion junctions in these strains and comparison with previously published data indicating the presence of recombinogenic regions flanking the penicillin biosynthesis gene cluster. Received 05 November 1996/ Accepted in revised form 25 April 1997     

Optimization of a pellicular biocatalyst for penicillin G production by Penicillium chrysogenum

A previously developed immobilization technique involving latex coatings on solid particulate supports was investigated further for penicillin G production by Penicillium chrysogenum. Several modifications were found to decrease the germination lag time, including a higher spore concentration, a thinner latex layer, an increased latex porosity, and a decreased drying time. This approach enabled the development of immobilized mycelial pellets within 2-3 days from the onset of biocatalyst preparation and incubation.A continuous immobilized-cell airlift bioreactor produced penicillin G in a series of runs in which the production phase lasted up to 30 days. The productivity of this system was 3-6 times greater than the productivity of the corresponding free-cell shake flask fermentation.     

Metabolic characterization of high- and low-yielding strains of Penicillium chrysogenum

A recently developed method for analyzing metabolic networks using ¹³C-labels was employed for investigating the metabolism of a high- and a low-yielding strain of Penicillium chrysogenum. Under penicillin-producing conditions, the flux through the pentose phosphate (PP) pathway in the high- and the low-yielding strains was estimated to 70 and 66, respectively. When the high-yielding strain was cultivated in a medium without the penicillin side chain precursor, phenoxyacetic acid, the PP pathway flux was estimated as 71. Thus, in all three experiments, the flux through the PP pathway was almost constant with an average value of 69 ± 3, and the method therefore allows for a very reproducible estimation of the PP pathway flux. Phenoxyacetic acid was found to be a source of cytosolic acetyl-CoA and thereby a source of precursors for the biosynthesis of 2-aminoadipic acid, which is a central amino acid in penicillin biosynthesis. However, the labeling patterns also indicated the presence of an unrecognized pathway to cytosolic acetyl-CoA. Received: 20 December 1999 / Received revision: 7 March 2000 / Accepted: 10 March 2000     

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.