Penicillin production by glucose-derepressed mutants ofPenicillium chrysogenum

Summary Wild-type strains ofPenicillium chrysogenum produce lower penicillin V titers in media containing excess glucose. Two mutant strains were isolated and shown to produce normal penicillin V titers in the presence of excess glucose. These strains, designated as glucose-repression insensitive (GRI) mutants, produced higher penicillin V titers than the wild-type strain in media containing lactose as the main carbohydrate source. In lactose-based media, the production of penicillin V was depressed to a much lesser extent by in-cycle additions of glucose with the GRI mutants when compared to the wild-type strain. In short-term biosynthesis experiments using washed cells in a medium containing glucose as the sole carbon source, the GRI mutants produced penicillin V at a faster rate than the wild-type strain. In fed-batch fermentations in 14-liter fermentors, where glucose was fed continuously and pH controlled, both GRI mutants produced more than 10% higher penicillin V titers than the wild-type strain. These results suggest that isolation of GRI mutants is an effective way to select for higher producing strains and that the synthesis of penicillin synthesizing enzymes in GRI mutants may be less repressed by glucose than in wild-type strains.     

Penicillin production by mutants ofPenicillium chrysogenum resistant to polyene macrolide antibiotics

Summary Polyene-resistant mutants ofPenicillium chrysogenum Wis. 54–1255 have been obtained by stepwise selection in increasing concentrations of polyene antibiotics. From the parent strain, sensitive to 10 g/ml of polyene antibiotics, mutants resistant to fungimycin (1.3 mg/ml), amphotericin B (0. 5 mg/ml), or nystatin (167 g//ml) were obtained. Their penicillin production is different from that of the parent strain and in particular some of the fungimycin-resistant mutants produce higher levels 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.     

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.

     

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.     

Biochemical and ultrastructural studies ofPenicillium chrysogenum,Talaromyces leycettanus andT. thermophilus

A comparative study of the lipids, proteins, amino acids and of the ultrastructure of lipid bodies of Penicillium chrysogenum (mesophilic), Talaromyces leycettanus (thermotolerant) and T. thermophilus (thermophilic) was done. The highest lipid content was found in T. thermophilus and highest protein content in P. chrysogenum whilst a total of 17 amino acids were found in P. chrysogenum and T. thermophilus and only sixteen were detected in T. leycettanus. Ultrastructural features of lipid bodies are reported and compared.     

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.     

Properties of heterozygous diploids between strains ofPenicillium chrysogenum selected for high penicillin yield

Three strains ofPenicillium chrysogenum selected for high penicillin yield and of independent lineage were marked with suitable genetical characters prior to the synthesis of several heterozygous diploids. These parental strains had domestic codes, C, D and Y. Two diploids, between differently labelled mutants of strain C and Y, produced similar amounts of penicillin to strain C, which was less than that produced by strain Y. Previous work had indicated that genes responsible for increased penicillin yield were recessive and the present results suggested that such genes in strains C and Y were allelic, apart from the presence of one or more additional recessive mutations leading to greater penicillin production in the higher yielding parent. Three diploids made between mutants of strains D and Y were lower in penicillin yield than either original parent and only in the case of one diploid compared with one of the parental strains was this difference not significant. In strains D and Y, therefore, there may have been some recessive genes concerned with increasing penicillin yield which were non-allelic. However, no first order segregants arising spontaneously or subsequent to X-ray treatment produced higher levels of penicillin than the better yielding original parent in any cross.     

Response of severed Penicillium chrysogenum hyphae following rapid Woronin body plugging of septal pores

Colonies of Penicillium chrysogenum (Thom) Wisconsin strain Q176 were fixed at varying time intervals after having been severed with a razor blade and were examined by transmission electron microscopy. Within such colonies Woronin bodies were found to have plugged septal pores on either side of the cut, both towards and away from the hyphal apices. A very close association of Woronin bodies with septa was retained in damaged compartments emptied of virtually all other contents. In colonies fixed 3 h after cutting, deposition of material over the plugged pores occurred on the side of the septum away from the cut. The newly deposited material was similar in appearance to hyphal wall and septal plate constituents. This consolidation of the seal was apparently completed within 3 h because no further change was observed in colonies fixed 17 h after cutting.     

Fatty acids reduce the tensile strength of fungal hyphae during cephalosporin C production in Acremonium chrysogenum

Fragmentation rate constants, which can be used to estimate the tensile strength of fungal hyphae, were used to elucidate relationships between morphological changes and addition of fatty acids during cephalosporin C production in Acremonium chrysogenum M35. The number of arthrospores increased gradually during fermentation, and, in particular, was higher in the presence of rice oil, oleic acid or linoleic acid than in their absence. Because supplementation of rice oil or fatty acids increased cephalosporin C, we concluded that differentiation to arthrospores is related to cephalosporin C production. To estimate the relative tensile strengths of fungal hyphae, fragmentation rate constants (k _frag) were measured. When rice oil, oleic acid, or linoleic acid were added into medium, fragmentation rate constants were higher than for the control, and hyphal tensile strengths reduced. The relative tensile strength of fungal hyphae, however was not constant presumably due to differences in physiological state.     

Dependence of penicillium chrysogenum growth, morphology, vacuolation, and productivity in fed-batch fermentations on impeller type and agitation intensity

The influence of the agitation conditions on the growth, morphology, vacuolation, and productivity of Penicillium chrysogenum has been examined in 6 L fed-batch fermentations. A standard Rushton turbine, a four-bladed paddle, and a six-bladed pitched blade impeller were compared. Power inputs per unit volume of liquid, P/VL, ranged from 0.35 to 7.4 kW/m3. The same fermentation protocol was used in each fermentation, including holding the dissolved oxygen concentration above 40% air saturation by gas blending. The mean projected area (for all dispersed types, including clumps) and the clump roughness were used to characterize the morphology. Consideration of clumps was vital as these were the predominant morphological form. For a given impeller, the batch-phase specific growth rates and the overall biomass concentrations increased with agitation intensity. Higher fragmentation at higher speeds was assumed to have promoted growth through increased formation of new growing tips. The mean projected area increased during the rapid growth phase followed by a sharp decrease to a relatively constant value dependent on the agitation conditions. The higher the speed, the lower the projected area for a given impeller type. The proportion by volume of hyphal vacuoles and empty regions decreased with speed, possibly due to fragmentation in the vacuolated regions. The specific penicillin production rate was generally higher with lower impeller speed for a given impeller type. The highest value of penicillin production as well as its rate was obtained using the Rushton turbine impeller at the lowest speed. At given P/VL, changes in morphology, specific growth rate, and specific penicillin production rate depended on impeller geometry. The morphological data could be correlated with either tip speed or the "energy dissipation/circulation function," but a reasonable correlation of the specific growth rate and specific production rate was only possible with the latter. Copyright 1998 John Wiley & Sons, Inc.     

Lowered resistance to lytic agents in sucrose vacuolation.

Vacuolation in fibroblasts cultivated in the presence of sucrose is associated with progressive accumulation of the undigestible sugar. In radioisotope experiments the process lasted several days, and when the cells were subcultured back into a medium devoid of sucrose the label was also lost after several days. This type of vacuolated cells is more fragile when it is challenged with lytic agents. 51Cr-labelled LS fibroblasts released more radioactivity when they had been growing in the presence of sucrose, whether they were suspended in media of decreasing osmolarity, in dilutions of various surfactants, exposed to high temperatures, or subjected to mechanical stress. It is concluded that these cells exhibit a lower resistance when exposed to unfavourable environments, but retain their viability in growth media despite some morphological and biochemical alterations.     

Comparing strategies to preserve evolutionary diversity

The likely future extinction of various species will result in a decline of two quantities: species richness and phylogenetic diversity (PD, or ‘evolutionary history’). Under a simple stochastic model of extinction, we can estimate the expected loss of these quantities under two conservation strategies: An ‘egalitarian’ approach, which reduces the extinction risk of all species, and a ‘targeted’ approach that concentrates conservation effort on the most endangered taxa. For two such strategies that are constrained to experience the same expected loss of species richness, we ask which strategy results in a greater expected loss of PD. Using mathematical analysis and simulation, we describe how the strategy (egalitarian versus targeted) that minimizes the expected loss of PD depends on the distribution of endangered status across the tips of the tree, and the interaction of this status with the branch lengths. For a particular data set consisting of a phylogenetic tree of 62 lemur species, with extinction risks estimated from the IUCN ‘Red List’, we show that both strategies are virtually equivalent, though randomizing these extinction risks across the tip taxa can cause either strategy to outperform the other. In the second part of the paper, we describe an algorithm to determine how extreme the loss of PD for a given decline in species richness can be. We illustrate the use of this algorithm on the lemur tree.