Cytosolic NADPH metabolism in penicillin-G producing and non-producing chemostat cultures of Penicillium chrysogenum

This study addresses the relation between NADPH supply and penicillin synthesis, by comparing the flux through the oxidative branch of the pentose phosphate pathway (PPP; the main source of cytosolic NADPH) in penicillin-G producing and non-producing chemostat cultures of Penicillium chrysogenum. The fluxes through the oxidative part of the PPP were determined using the recently introduced gluconate-tracer method. Significantly higher oxidative PPP fluxes were observed in penicillin-G producing chemostat cultures, indicating that penicillin production puts a major burden on the supply of cytosolic NADPH. To our knowledge this is the first time direct experimental proof is presented for the causal relationship between penicillin production and NADPH supply. Additional insight in the metabolism of P. chrysogenum was obtained by comparing the PPP fluxes from the gluconate-tracer experiment to oxidative PPP fluxes derived via metabolic flux analysis, using different assumptions for the stoichiometry of NADPH consumption and production.     

Role of proteases in autolysis of Penicillium chrysogenum chemostat cultures in response to nutrient depletion

An industrial strain of Penicillium chrysogenum was subjected to carbon or nitrogen limitation in a chemostat and the response monitored in terms of the “classical” indicators of autolysis (biomass decline and ammonia release), culture degradation (as measured by image analysis) and by obtaining profiles for three classes of proteases implicated in autolysis. Under both sets of conditions (carbon or nitrogen limitation), once started, autolysis involved a succession of different protease activities. The first stages of the process of autolysis in starved chemostat cultures was associated with peaks in the activities of both serine and aspartyl proteases, coinciding with the mobilisation of endogenous energy reserves. Conversely, a peak in the activity of metalloproteases was associated with the later stages of autolysis, perhaps occurring in response to depletion of endogenous energy reserves; the activity of these enzymes led to gross culture degradation, disintegration of ordered mycelial structures and signalled the end of metabolic activity (respiration) within the culture. These findings indicate that strategies intended to control/regulate autolysis in large-scale industrial fungal cultures might profitably be focused on regulation of the activity of key classes of proteases involved in the series of events leading to culture degradation. Received: 14 June 1999 / Received revision: 16 September 1999 / Accepted: 17 September 1999     

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.     

Hyphal growth and fragmentation of Penicillium chrysogenum in submerged cultures

A previously derived population model describing the average properties of hyphal elements in submerged cultures of filamentous fungi was revised, and a term for the influence fo spore germination on the average total hyphal length was added. The model was derived from a general balance for the distribution function for the hyphal elements. Based on experimental data and the derived model, simple kinetic expressions for spore germination, tip extension, branching, and hyphal break-up were set up. It is concluded that spore germination can be quantified by three parameters: (1) the time at which spore germination is initiated, (2) the time at which spore germination terminates, and (3) the fraction of viable spores in a spore suspension. The frequency of spore germination can be described with the B-distribution. For growth kinetics it is concluded that the branching frequency is closely correlated with the total hyphal length and that the average tip-extension rate can be described with saturation kinetics with respect to the hyphal length. Finally, the rate of fragmentation is linearly related to the energy input to the bioreactor, and related to the effective hyphal length. (c) 1995 John Wiley & Sons, Inc.     

Characterization of Penicillium chrysogenum physiology in submerged cultures by color and monochrome image analysis

Although filamentous microorganisms are widely used in industrial fermentation processes, their growth and differentation are not yet fully understood, because their biomass is structured, and therefore difficult to descrbie and to quantify. This lack of appropriate tools can hinder the optimization and control of the fermentation. A quantitative image analysis method was therefore developed for characterizing the physiology of the penicillin-producing mold Penicillium chrysogenum. This method is based on a differntial staining procedure showing six physiological states: growing material, three differentiated states characterzied by an increasing granulation, a highly vacuolized state, and dead segments having lost their cytoplasm. The image analysis software, with versions written for monochrome and color images, consisted of a semiautomatic binary mask computation step and a fully automatic segmentation step based on a fuzzy classification. (c) 1995 John Wiley & Sons, Inc.     

Measurement of autolysis in submerged batch cultures of Penicillium chrysogenum

The process of cellular autolysis was studied in an industrial strain of Penicillium chrysogenum by a range of methods, including assessment of biomass decline, NH+4 release, changes in culture apparent viscosity, and by means of a quantitative assessment of changes in micromorphology using a computerized image analysis system. The pattern of total intracellular proteolytic and beta-1, 3-glucanolytic activity in the culture was also examined. The overall aim was to identify a suitable method, or methods, for examining the extent of autolysis in fungal cultures. Autolysis was studied in submerged batch processes, where DOT was maintained above 40% saturation (non-O2-limited), and, under O2-limited conditions. Both N and O2 limitation promoted extensive culture autolysis. Image analysis techniques were perhaps the most sensitive method of assessing the progress of autolysis in the culture. Autolytic regions within some hyphae were apparent even during growth phase, but became much more widespread as the process proceeded. The early stages of autolysis involved continued energy source consumption, increased carbon dioxide evolution rate, degradation of penicillin, and decreased broth filterability. Later stages involved widespread mycelial fragmentation, with some regrowth (cryptic growth) occurring in non-O2-limited cultures. Intracellular proteolytic activity showed two peaks, one during the growth phase, and the other during autolysis. Autolysis was also associated with a distinct peak in beta-1,3-glucanolytic activity, indicating that degradation of cell wall matrix polymers may be occurring during autolysis in this strain of P. chrysogenum.     

Measurement of intracellular metabolites of primary metabolism and adenine nucleotides in chemostat cultivated Penicillium chrysogenum

An experimental platform has been developed for rapid sampling and quenching of chemostat cultivated Penicillium chrysogenum broth for metabolome analysis in highly dynamic experiments, aimed at the elucidation of the in vivo kinetic properties of metabolism. The sampling and quenching protocol available from Saccharomyces cerevisiae had to be modified for Penicillium chrysogenum mainly because of its filamentous character. Intracellular metabolites of glycolysis, TCA cycle, and adenine nucleotides were measured with isotope dilution mass spectrometry (IDMS) using a U-(13)C-labeled metabolite mix produced from yeast cells as internal standard. By addition of the U-(13)C internal standard mix prior to the metabolite extraction procedure, partial degradation of metabolites as well as non-linearity and drift of the LC-MS/MS could be successfully compensated for. It was found that there is a serious matrix effect on metabolite extraction between different organisms, which is however completely corrected for by the IDMS approach. Intracellular metabolites could be analyzed with standard deviations of around 5%. A comparison of the metabolite levels between Saccharomyces cerevisiae and Penicillium chrysogenum showed both significant similarities and large differences, which seem to be related to the presence of the penicillin pathway.     

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.     

Lipids of initial and mutant cultures of Penicillium chrysogenum, producers of penicillin

Comparative investigation of lipogenesis in 2 initial and 4 mutant strains of Penicillium chrysogenum showed that there were no noticeable differences in the composition of the lipid fatty acids in these strains. Certain shifts in the ratio of definite lipid fatty acids in the mutant strain deficient by synthesis of lysine and isoleucine (increased contents of oleic acid) were revealed. A marked influence of the physiological conditions on lipogenesis in the mutant with multiple deficiency by amino acids, vitamins and nucleotides was observed.     

Alternate pathways of linolenic acid biosynthesis in growing cultures of Penicillium chrysogenum

Evidence was obtained that Penicillium chrysogenum can produce linolenate by two biosynthetic pathways, i.e., by elongation of a shorter trienoic acid as well as direct desaturation of 18-C acids. In oxygen deficient cultures, exogenous hexadecatrienoate stimulated [1-¹⁴C]acetate incorporation into labeled octadecatrienoate and [U-¹⁴C]hexadecatrienoate with nonlabeled acetate yielded linolenate that had relatively little label in the 1-C position. With [1-¹⁴C]acetate as the only added substrate, oxygen deficiency inhibited incorporation of label into monoenoic and dienoic acids but not into trienoic acids. Incorporation of the [U-¹⁴C]linoleate into linolenate also was inhibited.In aerated cultures, 1-¹⁴C-label from laurate, palmitate, stearate, oleate, linoleate, and hexadecatrienoate was readily incorporated into linolenate. Decarboxylation and oxidation studies indicated that the longer acids were incorporated largely intact. [U-¹⁴C]Linoleate was incorporated into linolenate in which the fraction of label in 1-C was similar to that of the substrate. These data suggest that this mold has broader synthetic capabilities than do some chloroplast systems for the biosynthesis of linolenate.     

Elicitor effects on reactive oxygen species in liquid cultures of Penicillium chrysogenum

Activity of reactive oxygen species (ROS) was investigated in liquid cultures of Penicillium chrysogenum P2 supplemented with carbohydrates. Oligosaccharides lowered the ROS activity in all samples. The greatest effect occurred when oligosaccharides were added to samples 48 h after inoculation. The ROS decrease in the presence of oligoguluronate, oligomannuronate and mannan oligosaccharides was 18%, 36% and 54%, respectively (ROS levels varied notably with culture age and type of elicitor). The polysaccharides from which the oligosaccharides were derived showed little (5-10%) overall decrease of ROS.     

Influence of n-hexadecane on penicillin fermentation with Penicillium chrysogenum: The morphological effect

Summary In addition to acting as an oxygen transfer enhancer, n-hexadecane plays an important role in dominating the mycelial morphology of Penicillium chrysogenum in a submerged penicillin fermentation. The cell morphology is related to the timing of adding n-hexadecane to the culture. n-Hexadecane also has a function of suppressing foaming and thus can be employed as an antifoam agent in the fermentation.     

Enhancement of chrysogenin production in cultures of Penicillium chrysogenum by uronic acid oligosaccharides

Additions of 50 to 100 g of acid-hydrolysed alginate oligosaccharides ml^–1 and enzyme-hydrolysed pectin oligosaccharides to 24- to 48-h cultures of Penicillium chrysogenum, ATCC 9480, led to enhanced production of chrysogenin by over 30 to 40% in shaken flasks and bioreactors. Some of the oligosaccharides also promoted biomass formation but were not used as a carbon source.     

Quantification of autolysis in Penicillium chrysogenum by semiautomated image analysis

An image analysis method is described for the characterization of empty (autolyzed and inactive) regions within the mycelia of filamentous fungi. It extends a previous method that characterized only regions filled with cytoplasm or vacuoles (i.e., the active biomass). The method is semiautomatic, requiring some manual editing before automated measurements. When the method was used for samples from a batch fermentation of an industrial strain of Penicillium chrysogenum, the empty regions were observed to constitute up to 15% (by projected area) of the biomass during the growth phase. After nutrient exhaustion, however, the proportion of empty regions rose rapidly, eventually representing more than 50% of the biomass by the end of fermentation. The increase in the percentage of empty regions coincided with a decrease in biomass (as measured by dry cell weight) and a fall in penicillin titre. Further morphological analysis revealed that fragmentation of mycelia, particularly clumps, coincided with increases in the levels of empty regions. This new image analysis method gave additional information on hyphal differentiation and a measure of autolysis. It was also a useful indicator of the processes leading to autolysis.