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.     

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.     

Alginate oligosaccharides as enhancers of penicillin production in cultures of penicillium chrysogenum

Oligosaccharide fragments were prepared by partial acid hydrolysis of sodium alginate and consisted of oligomannuronate (OM) and oligoguluronate (OG) blocks. Effects of the OM and OG blocks on penicillin G production by P. chrysogenum were investigated. The oligosaccharides were found to cause significant increases in penicillin G yields. OM blocks at concentrations 10 to 100 mug/mL were used to further evaluate the effects of the oligosaccharides, and were found to enhance the production of penicillin G in shaken flask cultures of P. chrysogenum P2 (high penicillin producer) and NRRL 1951 (low penicillin producer) at the test concentrations. There was an approximately 50% maximum increase in penicillin G yield from biomass in P. chrysogenum P2 cultures and 150% in P. chrysogenum NRRL 1951 cultures, when compared to control cultures without the oligosaccharides. (c) 1997 John Wiley & Sons, Inc.     

The effect of agitation on the morphology and penicillin production of Penicillium chrysogenum

Penicillium chrysogenum strain P1 was grown on complex media in 10 and 100 L agitated fermenters at various aeration rates and stirrer speeds. Samples were removed at intervals for measurements of the culture morphology. At high stirrer speeds (1000 and 1200 rpm) in 10-L fermentations the rate of decrease in the mean effective hyphal length was faster and the rate of penicillin production was lower than fermentations done at 800 rpm. At similar power inputs per unit volume in 100-L fermentations, the change in mean effective hyphal length was less and higher penicillin production rates were observed. This work comparing the results at two scales has shown that neither of the concepts of impeller tip speed or the dissipation rate of turbulence have general validity as a measure of hyphal damage. Our results are reasonaby well correlated by groups similar to circulation rate (ND(i) (3)/V) with lower circulation rates being beneficial. An adaptation of the van Suijdam and Metz relationship, expressed as P/D(i) (3)t(c), was most successful. Our data are insufficient to demonstrate the generality of the relationship but do support the concept of a dispersion zone around the impellers in which mycelia may be damaged. The greater the frequency of circulation of mycelia through the zone the greater the damage and the lower the rate of penicillin synthesis by the culture.     

Autolysis and aging of Penicillium chrysogenum cultures under carbon starvation: Chitinase production and antifungal effect of allosamidin

In carbon-depleted cultures of Penicillium chrysogenum, age-related chitinases were shown to play a crucial role in both autolysis and fragmentation as indicated by in vivo enzyme inhibition experiments using allosamidin. This pseudotrisaccharide even hindered significantly the outgrowth of new hyphal tips from the surviving yeastlike fragments after glucose supplementation. The antifungal effect of allosamidin on autolyzing P. chrysogenum mycelia was fungistatic rather than fungicidal. In growing hyphae, membrane-bound microsomal chitinase zymogen(s) were detected, which may be indicative of some compartmentalization of these hydrolases. Later, during autolysis, no zymogenic chitinase was detected in any enzyme fraction studied, including microsomes. These observations may explain the different sensitivity of growing and autolyzing mycelia to allosamidin. Chitinases taking part in the age-related fragmentation of hyphae and the outgrowth of surviving hyphal fragments seem to be potent targets for future antifungal drug research.     

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.     

Why did the Fleming strain fail in penicillin industry?

Penicillin, discovered 75 years ago by Sir Alexander Fleming in Penicillium notatum, laid the foundations of modern antibiotic chemotherapy. Early work was carried out on the original Fleming strain, but it was later replaced by overproducing strains of Penicillium chrysogenum, which became the industrial penicillin producers. We show how a C(1357)-->T (A394V) change in the gene encoding PahA in P. chrysogenum may help to explain the drawback of P. notatum. PahA is a cytochrome P450 enzyme involved in the catabolism of phenylacetic acid (PA; a precursor of penicillin G). We expressed the pahA gene from P. notatum in P. chrysogenum obtaining transformants able to metabolize PA (P. chrysogenum does not), and observing penicillin production levels about fivefold lower than that of the parental strain. Our data thus show that a loss of function in P. chrysogenum PahA is directly related to penicillin overproduction, and support the historic choice of P. chrysogenum as the industrial producer of penicillin.     

Continuous cultivations of a Penicillium chrysogenum strain expressing the expandase gene from Streptomyces clavuligerus: Growth yields and morphological characterization

The growth stoichiometry of a Penicillium chrysogenum strain expressing the expandase gene from Streptomyces clavuligerus was determined in glucose-limited chemostat cultivations using a chemically defined medium. This strain produces adipoyl-7-aminodeacetoxycephalosporanic acid (ad-7-ADCA) when it is fed with adipic acid. The biomass yield and maintenance coefficients for the strain were similar to those found for penicillin-producing strains of Penicillium chrysogenum. The maximum specific growth rate in the chemostat was found to be 0.11 h(-1). Metabolic degradation of adipate was found to take place in significant amounts only at dilution rates below 0.03 h(-1). After three to five residence times, adipate degradation and ad-7-ADCA production disappeared, and this allowed determination of the biomass yield coefficient on adipate. The morphology was measured at different dilution rates and the mean total hyphal length and mean number of tips both increased with an increase in dilution rate from 0.015 to 0.065 h(-1). Both variables decreased when the dilution rate was increased above 0.065 h(-1). A correlation between mean total hyphal length and productivity of ad-7-ADCA was found.     

Asexual sporulation signalling regulates autolysis of Aspergillus nidulans via modulating the chitinase ChiB production

Aims:  Elucidation of the regulation of ChiB production in Aspergillus nidulans .
Methods and Results:  Mutational inactivation of the A. nidulans chiB gene resulted in a nonautolytic phenotype. To better understand the mechanisms controlling both developmental progression and fungal autolysis, we examined a range of autolysis-associated parameters in A. nidulans developmental and/or autolytic mutants. Investigation of disorganization of mycelial pellets, loss of biomass, extra-/intracellular chitinase activities, ChiB production and chiB mRNA levels in various cultures revealed that, in submerged cultures, initialization of autolysis and stationary phase-induced ChiB production are intimately coupled, and that both processes are controlled by the FluG-BrlA asexual sporulation regulatory pathway. ChiB production does not affect the progression of apoptotic cell death in the aging A. nidulans cultures.
Conclusions:  The endochitinase ChiB plays an important role in autolysis of A. nidulans , and its production is initiated by FluG-BrlA signalling. Despite the fact that apoptosis is an inseparable part of fungal autolysis, its regulation is independent to FluG-initiated sporulation signalling.
Significance and Impact of the Study:  Deletion of chiB and fluG homologues in industrial filamentous fungal strains may stabilize the hyphal structures in the autolytic phase of growth and limit the release of autolytic hydrolases into the culture medium.     

A metabolome study of the steady-state relation between central metabolism, amino acid biosynthesis and penicillin production in Penicillium chrysogenum

The relation between central metabolism and the penicillin biosynthesis pathway in Penicillium chrysogenum was studied by manipulating the steady-state flux in both pathways. A high producing industrial strain was cultivated at a growth rate mu=0.05 h(-1) in glucose-limited chemostat cultures, both under penicillin-G producing and non-producing conditions. Non-producing conditions were accomplished in two ways: (1) by cultivation without addition of the side chain precursor phenylacetic acid and (2) by cultivation of a mutant strain which lost all copies of the gene cluster coding for the penicillin biosynthesis pathway. Manipulation of the fluxes through central metabolism was obtained by cultivation on either glucose or ethanol as sole carbon source. A positive relation was observed between metabolite concentrations and carbon flux in central metabolism. Furthermore, in many cases a positive relation was found between the concentrations of free amino acids and their direct precursors in central metabolism. This corresponds with control of the biosynthesis of these amino acids via feed back inhibition by the end product. With respect to the penicillin production pathway, the flux seems not influenced by two of the three precursor amino acids, namely alphaAAA and valine but is only influenced by cysteine, which requires a large NADPH supply, and the ATP level. An interesting observation is that the absence of penicillin production seems to stimulate storage metabolism (trehalose metabolism). This leads to the final conclusion that the penicillin production flux appears to be mostly influenced by the availability of energy and redox cofactors, where ATP is supposed to exert its influence at ACV-synthetase and NADPH at the cysteine level.     

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     

产黄青霉谷胱甘肽S-转移酶基因PcgstA的克隆与鉴定

从青霉素工业生产菌产黄青霉(Penicilliumchrysogenum)中首次克隆了一个谷胱甘肽S-转移酶(GST)基因,定名为PcgstA.该基因的开放阅读框长840bp,含有两个内含子,编码一个238氨基酸残基的蛋白质.其推断的氨基酸序列与一些已经鉴定的丝状真菌GST具有50%左右的序列一致性.PcgstA的完整编码区经RT-PCR扩增、验证,插入原核表达载体pET11a,转化大肠杆菌BL21(DE3)-RP菌株,表达得到重组PcGSTA蛋白.酶活测定证实,重组PcGSTA具有GST活性,其对底物CDNB(1-chloro-2,4-dinitrobenzene)的比活为(0.159±0.031)μmol/(min·mg).利用TaqMan探针法,对PcgstA的表达情况进行了比较.结果表明,在添加了侧链前体苯乙酸的青霉素生产培养基中,PcgstA的表达水平和在不含苯乙酸培养基中的表达相比明显下调,显示了该基因与苯乙酸代谢的关系.     

Effect of oxygen enrichment on morphology, growth, and heterologous protein production in chemostat cultures of Aspergillus niger B1-D.

The response of steady state chemostat cultures of a recombinant Aspergillus niger (B1-D), secreting both a heterologous enzyme (Hen Egg White Lysozyme [HEWL]) and a native enzyme (Glucoamylase), to varying levels of O2 enrichment of the process gas was evaluated. Formation of both the native and the foreign enzyme increased with increasing O2 supply. Conversely, biomass levels and total extracellular protein levels were generally not increased under O2 enriched conditions. Two distinct micromorphologies were apparent in these cultures, one, typically seen under O2 limiting conditions (i. e. at 0 and 10% enrichment levels), tended to be represented by long, sparsely branched hyphal elements, with low percentages of "active" length (i. e. how much of the hypha is cytoplasm filled); whilst, a second micromorphology, typical of O2 enriched cultures at 30 and 50% O2 enrichment, was represented by shorter hyphal elements, with more branching and a higher % "active" length. At these higher O2 levels, formation of a yellow pigment occurred, and signs of culture autolysis were noted. At 50% enrichment, a "stranded" aggregate morphology was apparent, possibly as a response to a hyperoxidant state. Production of both the native enzyme and HEWL correlated well with a simple morphological measure (tip number) or, with % "active" length. It is proposed the morphological changes noted in the cultures were associated with the increased production of both HEWL and glucoamylase.     

Uptake of phenylacetic acid by two strains of Penicillium chrysogenum

Uptake of phenylacetic acid, the side-chain precursor of benzylpenicillin, was studied in Penicillium chrysogenum Wisconsin 54-1255 and in a strain yielding high levels of penicillin. In penicillin fermentations with the high-yielding strain, 100% recovery of phenylacetic acid in benzylpenicillin was found, whereas in the Wisconsin strain only 17% of the supplied phenylacetic acid was incorporated into benzylpenicillin while the rest was metabolized. Accumulation of total phenylacetic acid-derived carbon in the cells was nonsaturable in both strains at high external concentrations of phenylacetic acid (250-3500 microM), and in the high-yielding strain at low phenylacetic acid concentrations (2. 8-100 microM), indicating that phenylacetic acid enters the cells by simple diffusion, as concluded earlier for P. chrysogenum by other authors. However, at low external concentrations of phenylacetic acid saturable accumulation appeared in the Wisconsin strain. HPLC-analyses of cell extracts from the Wisconsin strain showed that phenylacetic acid was metabolized immediately after entry into the cells and different [14C]-labeled metabolites were detected in the cells. Up to approximately 50% of the accumulated phenylacetic acid was metabolized during the transport-assay period, the conversion having an impact on the uptake experiments. Nevertheless, accumulation of free unchanged phenylacetic acid in the cells showed saturation kinetics, suggesting the possible involvement of a high-affinity carrier in uptake of phenylacetic acid in P. chrysogenum Wisconsin 54-1255. At high concentrations of phenylacetic acid, contribution to uptake by this carrier is minor in comparison to simple diffusion and therefore, of no importance in the industrial production of penicillin.     

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.