Differences in diploids synthesized between the same parental strains ofPenicillium chrysogenum

Previous work showed that differences in penicillin yield could be demonstrated between diploids of independent origin from the same pair of parental strains ofPenicillium chrysogenum. It was suggested that segregation processes or spontaneous mutation, either of which could occur prior to testing the diploids, might be responsible for such differences. The present work gave results more strongly supporting a mutation hypothesis.

     

Formation of bulges associated with penicillin production in high-producing strains ofPenicillium chrysogenum

Bulges were formed in the hyphae of a high penicillin-producing strain ofPenicillium chrysogenum AS-P-78 when penicillin was accumulated in the broth. The mycelium of cultures grown in the presence of 50 mM lysine, which specifically inhibits penicillin formation, showed a greatly reduced number of bulges. The size and number of bulges increased during the fermentation in parallel with penicillin accumulation. A smaller number of bulges was formed in the mycelium of the low-producing strain Wis 54-1255 than in the high-producing mutant. Three mutants blocked in penicillin biosynthesis did not form these globose structures. Bulges were not osmotically sensitive, although some of them burst out. They may be formed by weakening of the cell wall of hyphae following accumulation of high concentrations of penicillin.     

Production of penicillin by immobilized films ofPenicillium chrysogenum

Summary Various materials were tested and polycarbonate was chosen as the support material for attachment and film growth ofP. chrysogenum in rotating disc fermenters. During batch runs using a penicillin production medium attached biomass increased at a linear rate and penicillin G was produced by the immobilized mycelium over a six day period.     

Contribution to the morphology of the productive strains ofPenicillium chrysogenum thom from the Wisconsin family

The morphology of four productive strains ofPenicillium chrysogenum Thom from the Wisconsin family was studied. The strains Q-176, 47–1564, 49–133, 51–20Z, which were naturally or artificially obtained mutants of thePenicillium chrysogenum NRRL 1951 strain were very variable as to the colony structure and the character of conidiophores. The present study is concerned with the evaluation of their taxonomic position. The macrohabitus of the colonies was not remarkably changed. All different types of colonies (U, D, C, B, rarely A) described by Backus and Stauffer, were found on Czapek agar; they were not recognized on malt agar. Deviations from the normal asymmetric conidiophore were found with every type of colonies, most often with the more floccose or lanose ones showing a higher and a sparser overgrowth. These deviations or changes in the microstructure were divided into three degrees according to their quality and occurrence: (1) A strongly divaricate conidiophore where only metulae and phialides were developed; (2) monoverticilate conidiophore or single phialides on the conidiophore filament; (3) degeneration of phialides or metulae to sterile globose cells or an ultimate reduction of conidiophore to dichotomically branched stump-like hypha. The investigated strains can be involved in the taxonPenicillium chrysogenum Thom; it is necessary, however, to include some additional traits into the characteristics of the taxon: Colonies of the naturally or artificially obtained mutants often have lanose overgrowths sporulating sparsely. Formation of the yellow pigment and the exudate was not obligatory. conidiophores of these strains had a tendency to be more simple. They were scarcer, divaricately open, characterized sometimes by the formation of monoverticilate penicilli. A degeneration was frequently found of the ends of conidiophores (phialides and metulae) to globose enlarged sterile cells as well as the formation of giant cells in the mycelium or reduction of conidiophore to dichotomically branched hypha with stump-like ends.     

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)     

Semicontinuous penicillin production by two Penicillium chrysogenum strains immobilized in calcium alginate beads

Summary The growth rates of immobilized Penicillium chrysogenum strains are important in their application to semicontinuous penicillin production. Immobilized P. chrysogenum strains produced about 10–15% less biomass but about 1–2 times more penicillin than free suspended mycelia.In a chemically defined medium an industrial P. chrysogenum strain, S₁, produced about 10–12 times more penicillin than strain ATCC 12690. In a complex medium the immobilized P. chrysogenum S₁ produced about 12% penicillin more than in shaken cultures. In bubble column fermentations, penicillin production was 163% higher in the complex medium than in the chemically defined medium.     

Lysine regulation of penicillin biosynthesis in low-producing and industrial strains of Penicillium chrysogenum.

The inhibitory effect of L-lysine on penicillin biosynthesis by Penicillium chrysogenum has been compared in a low-producing strain (Wis. 54-1255) and a high-producing strain (ASP-78). Lysine inhibited total penicillin synthesis to a similar extent in both strains. However, in the high-producing strain the onset of penicillin synthesis occurred even at a high lysine concentration, whereas in the low-producing strain lysine had to be depleted before penicillin production commenced.     

Development of high penicillin producing strains for solid state fermentation

Penicillin production with an industrial strain and 4 strains of P. chrysogenum, in solid state fermentation (SSF) and liquid submerged fermentation (LSF), was determined. Their ability to produce the antibiotic in SSF in relation to their capacity to do so in LSF was evaluated. this was done by calculating the ratio PS/PL (production in SSF/production in LSF), which was called relative production. Clones were isolated from each strain and evaluated in a similar way. The strains presented different relative productions (from 1.4 to 2.5). Within the clones, a much wider range of relative productions was observed (0.6 to 16.7). On the other hand, the highest-producing strains in LSF were also the highest producers in SSF. This indicates that the production potential of a strain is an important factor in its production level in SSF. Moreover, the highest penicillin producing ciones (9,500 to 10,500 microg of penicillin/g were generated from high-yielding strains (P2 and ASP-78). However, the higher-producing strains (in LSF) showed lower relative performance, suggesting that higher producing strains tend to express less efficiently their potential in SSF. In this study, several overproducing clones, particularly suited for SSF, were obtained by the procedures followed. Production increases of 500 to 600 %, in this culture system, were achieved.     

Morphology of Penicillium chrysogenum strains immobilized in calcium alginate beads and used in penicillin fermentation

Summary The morphology of two strains of Penicillium chrysogenum immobilized in calcium alginate and used in penicillin fermentation was examined. The degree and distribution of mycelial growth inside and on the surface of the beads depended on the strain, the cultivation media and the fermentation time. P. chrysogenum ATCC 12690 developed as a mycelial network inside the beads. The growth tendency of P. chrysogenum S₁ in micropellets was directed to the outer surface of the beads. At the end of the production phase only a trace of mycelia and no micropellets in the center of alginate beads were observed, while the outer surface and the subsurface were completely covered with mycelia.     

Scale-down of penicillin production in Penicillium chrysogenum

In large-scale production reactors the combination of high broth viscosity and large broth volume leads to insufficient liquid-phase mixing, resulting in gradients in, for example, the concentrations of substrate and oxygen. This often leads to differences in productivity of the full-scale process compared with laboratory scale. In this scale-down study of penicillin production, the influence of substrate gradients on process performance and cell physiology was investigated by imposing an intermittent feeding regime on a laboratory-scale culture of a high yielding strain of Penicillium chrysogenum. It was found that penicillin production was reduced by a factor of two in the intermittently fed cultures relative to constant feed cultivations fed with the same amount of glucose per hour, while the biomass yield was the same. Measurement of the levels of the intermediates of the penicillin biosynthesis pathway, along with the enzyme levels, suggested that the reduction of the flux through the penicillin pathway is mainly the result of a lower influx into the pathway, possibly due to inhibitory levels of adenosine monophosphate and pyrophosphate and lower activating levels of adenosine triphosphate during the zero-substrate phase of each cycle of intermittent feeding.     

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.     

Formate as an auxiliary substrate for glucose-limited cultivation of Penicillium chrysogenum: impact on penicillin G production and biomass yield

Production of beta-lactams by the filamentous fungus Penicillium chrysogenum requires a substantial input of ATP. During glucose-limited growth, this ATP is derived from glucose dissimilation, which reduces the product yield on glucose. The present study has investigated whether penicillin G yields on glucose can be enhanced by cofeeding of an auxiliary substrate that acts as an energy source but not as a carbon substrate. As a model system, a high-producing industrial strain of P. chrysogenum was grown in chemostat cultures on mixed substrates containing different molar ratios of formate and glucose. Up to a formate-to-glucose ratio of 4.5 mol.mol(-1), an increasing rate of formate oxidation via a cytosolic NAD(+)-dependent formate dehydrogenase increasingly replaced the dissimilatory flow of glucose. This resulted in increased biomass yields on glucose. Since at these formate-to-glucose ratios the specific penicillin G production rate remained constant, the volumetric productivity increased. Metabolic modeling studies indicated that formate transport in P. chrysogenum does not require an input of free energy. At formate-to-glucose ratios above 4.5 mol.mol(-1), the residual formate concentrations in the cultures increased, probably due to kinetic constraints in the formate-oxidizing system. The accumulation of formate coincided with a loss of the coupling between formate oxidation and the production of biomass and penicillin G. These results demonstrate that, in principle, mixed-substrate feeding can be used to increase the yield on a carbon source of assimilatory products such as beta-lactams.     

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.