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J. Basic Microbiol. 41 (2001) 6, 351–362
WILEY-VCH Verlag Berlin GmbH, 13086 Berlin, 2001 0233-111X/01/0612-0351 $ 17.50+.50/0
(1Department of Microbiology and Biotechnology, Faculty of Science, University of Debrecen, 4010 Debrecen, Hungary; 2Department of Human Genetics, Faculty of Medicine, University of Debrecen, 4012 Debrecen, Hungary)
Carbon source regulation of β-galactosidase biosynthesis in Penicillium chrysogenum
ZOLTÁN NAGY 1, ZSOLT KERESZTESSY
1, ATTILA SZENTIRMAI1 and SÁNDOR BIRÓ1, 2
(Received 04 September 2001/Accepted 17 September 2001)
Growth and β-galactosidase activity of the penicillin producer industrial Penicillium chrysogenum NCAIM 00237 strain were examined using different carbon sources. Good growth was observed using glucose, sucrose, glycerol and galactose, while growth on lactose was substantially slower. β-Galactosidase activity was high on lactose and very low on all the other carbon sources tested. In glucose grown cultures after exhaustion of glucose as repressing carbon source a derepressed low level of the enzyme was observed. cAMP concentration in lactose grown cultures was relatively high, in glucose grown cultures was low. Caffeine substantially decreased glucose consumption and growth but did not increase β-galactosidase activity and did not prevent glucose repression which rules out the involvement of cAMP in the regulation of β-galactosidase biosynthesis in Penicillium chrysogenum.
Penicillium chrysogenum is a widely used penicillin producer industrial fungus. Penicillin biosynthesis has been the subject of many studies, which showed that carbon source regula-tion plays important role acting at several points of the biosynthesis (BRAKHAGE 1998). Glucose, fructose, galactose and other easily utilisable carbon sources, but not lactose sub-stantially reduces penicillin titer (REVILLA et al. 1984, SOLTERO and JOHNSON 1952, MARTIN et al. 1999). Industrial production of penicillin by P. chrysogenum is carried out with lactose as the carbon source and by feeding sub-repressing doses of glucose since the production of penicillin appears to be favoured by sub-optimal growth conditions (BRAKHAGE 1998). Lactose utilisation in fungi takes place by two ways. Lactose is either hydrolysed extracellularly before or in connection with uptake and the product glucose and galactose are taken up (MORTBERG and NEUJAHR 1986, CARVALHO-SILVA and SPENCER-MARTINS 1990), or lactose is transported into the cell and hydrolysed intracellularly (CAR-VALHO-SILVA and SPENCER-MARTINS 1990, BOZE et al. 1987, DICKSON and BARR 1983). Previously we have purified and characterised an intracellular enzyme with β-galactosidase activity (NAGY et al. 2001) which implies that in our strain lactose is probably utilised by the consecutive action of lactose permease and β-galactosidase. Here we present our recent results on the production of β-galactosidase and the influence of different carbon sources on the biosynthesis of the enzyme.
Materials and methods
Culture conditions: P. chrysogenum NCAIM 00237 was grown in a complex medium containing 0.4% peptone, 0.4% yeast extract, 0.2% KH2PO4, 0.8% Na2HPO4 × 12 H2O, 0.025% MgSO4 × 6 H2O and depending on the experiment 2% lactose, glucose, galactose, glycerol or sucrose (sterilised sepa-rately) as carbon source. These media were inoculated with 10 × 106 spores per 100 ml. The fungus was grown in 500 ml ERLENMEYER flasks (each containing 200 ml of the medium) in New Brunswick incubator shaker at 25 °C and 200 rpm.
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In the case of washed cell cultures, the fungus was grown up in complex medium adding 2% glu-cose as carbon source for 35 hours. Growth conditions were the same as before. Following that, in- oculum cultures were filtered and washed using a glass filter and the mycelia were transferred imme-diately into a minimal medium containing 0.2% KH2PO4, 0.8% Na2HPO4 × 12 H2O, 0.025% MgSO4 × 6 H2O, 1% (NH4)2SO4 and 2% carbon source. 200 ml medium was inoculated with inoculum mycelia obtained from 100 ml inoculum culture. Dry weight determination: For determination of the dry weight of fungal mycelium, 5 ml culture broth was filtered through pre-weighted filter papers; the mycelium retained by the filter papers was dried to a constant weight (PÓCSI et al. 1993). Enzyme assay: For enzyme assays, 10 ml aliquots of the culture fluids were filtered and washed using a glass filter. The washed mycelium was suspended in 5 ml 0.1 M phosphate buffer, pH 7.0, and was frozen and kept at –20 °C until further processing. Cells were broken up by X-press (Type X25, AB BIOX, Göteborg, Sweden) according to EMRI et al. (1997) and the cell-free crude extracts were spinned down in EPPENDORF centrifuge at 4 °C for 15 min. The enzyme activities in the supernatants were determined. The activity of β-galactosidase was assayed at 30 °C after 30 min incubation of the enzyme samples with o-nitrophenyl-β-D-galactopyranoside (ONPG) as chromogenic substrate. The 1 ml reaction mix-ture contained 500 µl sample and ONPG at 3 mM final concentration in 0.1 M phosphate buffer, pH 7.0. The reactions were started by the addition of ONPG and terminated on ice. The liberated o-nitrophenol was detected spectrophotometrically at 410 nm and was expressed as specific activity (U mg–1 protein): 1 U is equivalent to 1 nmol o-nitrophenol produced min–1. Protein was determined by the modified LOWRY method (PETERSON 1983) with BSA as standard. Glucose level determination: Determination of the glucose level of glucose grown cultures was carried out by rate assay (LEARY et al. 1992, NÓGRÁDI et al. 1995, PUSZTAHELYI et al. 1997a and b). The reaction mixture contained 0.1 M phosphate buffer (pH 6.6), 4 kUL–1 glucose oxidase, 1 kUL–1 peroxidase, 0.76 mM 4-aminoantipyrine, 11 mM phenol and 1% (v/v) sample. The reaction was followed spectrophotometrically at 500 nm for 3 min. Lactose level determination: Lactose concentration was determined from the filtrates of lactose grown cultures by HPLC analysis (SÁNDOR et al. 1999) on a BIO-RAD Aminex HPX-H+, hydrogen-phase ion-exchange column using isocratic elution with 10 mM H2SO4. The flow rate throughout the chromatographic runs was 0.5 ml min–1 and the temperature was constantly kept at 55 °C. Ammonia level determination: The ammonia concentration was measured in the filtrates of lactose and glucose grown cultures by an ammonia sensitive electrode set (OP-NH3-7113-S, RADELKIS Budapest, Hungary) according to PUSZTAHELYI et al. (1997a). Determination of cAMP concentration: Samples from lactose, glucose and glycerol grown cultures were taken at 24 h or when lactose grown cultures were supplemented by repressing carbon sources at 48 h. For determination of cAMP concentration, 5 ml aliquots of the culture fluids were filtered and washed with water using a glass filter. Cells were extracted with 1 ml 10% ice-cold perchloric acid for 30 min at 4 °C. Mycelia were centrifuged in EPPENDORF tubes at 4 °C, 10.000 rpm for 15 min. The supernatants were neutralised with ice-cold 2 M NaOH. After centrifugation the supernatants were filtered through an 0.45 µm HV-MILLIPORE filter and then 200 µl was loaded onto the column for HPLC analysis (GEBELEIN et al. 1992). Chromatographic conditions and analysis were according to the modified method of DI PIERRO et al. (1995). A Supelcosil LC-18, 150 × 4.6 mm, 3 µm particle size column (SUPELCO), provided of its own guard column, was equilibrated with a mobile phase (buffer A) containing 10 mM tetrabutyl-ammonium hydroxide as the pairing reagent, 10 mM KH2PO4, 0.25% methanol. The pH of buffer A was adjusted by HCl to 7.00. A step gradient was obtained with a second buffer (buffer B) containing 2.8 mM tetrabutyl-ammonium hydroxide, 100 mM KH2PO4, 30% methanol. The pH of buffer B was adjusted by HCl to 5.50. Both buffers were freshly prepared. Gra-dient was formed as follows: 2.5 min 0% buffer B; 2.5 min at up to 30% buffer B; 9 min at up to 60% buffer B; 8 min at up to 100% buffer B; 4 min 100% buffer B. The flow rate throughout the chroma-tographic runs was 1.4 ml min–1. All other chemicals were analytical grade and obtained from SIGMA-ALDRICH Kft. Hungary.
Carbon source regulation of β-galactosidase 353
Results
Growth and β-galactosidase production on different carbon sources
Penicillium chrysogenum NCAIM 00237 was grown in liquid complex culture medium on different carbon sources and the mycelial dry weight and intracellular β-galactosidase activ-ity were monitored (Fig. 1). The growth on different carbon sources varied. Glucose, su-crose and surprisingly galactose were the best, easily metabolised carbon sources giving relatively fast growth. Glycerol was a fairly good carbon source but metabolised slower. Lactose was a rather poor carbon source, giving much less biomass (Fig. 1A). Carbon sources influenced the β-galactosidase production. On lactose grown cultures substantial amount of β-galactosidase was formed, as defined by the ability to hydrolyse o-nitrophenyl-β-D-galactopyranoside (ONPG). When glucose, sucrose, glycerol or galactose were used as carbon source only a very low basal β-galactosidase activity was observed (Fig. 1B). On lactose grown culture we also found high ammonia production increasing until 48 hours of the growth when β-galactosidase activity reached a relatively high level. After that the fungi started to utilise much lactose and ammonia production decreased (Fig. 2A). On the other hand on glucose grown culture the ammonia production was low and started to increase after 20 hours of glucose exhaustion (Fig. 2B). In washed cell cultures the growth on lactose started after 10 hours and was also fairly poor. Glucose metabolised easily giving fast growth and much biomass (Fig. 3A). After washing the mycelium from glucose to lactose as carbon source the β-galactosidase activity A
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Fig. 1 Growth (1A) and β-galactosidase activity (1B) of P. chrysogenum cultures grown on different carbon sources. (�) 2% lactose, (�) 2% glucose, (�) 2% galactose, (×) 2% sucrose or (∆) 2% glycerol
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Fig. 2 Changes of growth (�), β-galactosidase activity (�) and lactose (+), glucose (�) and ammonia (�) level in complex cultures on 2% lactose (2A) and 2% glucose (2B) carbon source
was increased substantially and relatively quickly. On glucose grown minimal cultures only infinitesimal amount of β-galactosidase was formed (Fig. 3C). Consumption of lactose was slow and poor in cultures either on complex or minimal media (Figs. 2A and 3B).
The influence of different carbon sources on β-galactosidase production
To investigate the effect of different carbon sources on β-galactosidase synthesis, glucose, galactose, sucrose or glycerol were added to lactose grown complex cultures at 40 hours of the growth and the enzyme activities were monitored. Adding these carbon sources the growth rate increased rapidly (Fig. 4A) while each carbon source substantially decreased β-galactosidase production. Enzyme activities were less than 20% at 60 h when compared to lactose grown cultures (Fig. 4C). If 2% lactose was added to lactose grown culture at 40 hours it had no effect on growth, enzyme activity and lactose consumption rate (Fig. 4).
Carbon source regulation of β-galactosidase 355
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Synthesis of β-galactosidase after depletion of glucose
We also studied the enzyme activity in cultures grown on glucose as the sole carbon source. The changes of glucose levels and the growth were also monitored. As Fig. 2B shows at about the time of consuming of the high excess of glucose growth has ceased and β-galactosidase activity increased. This probably means that after depletion of glucose the synthesis of the enzyme started without lactose.
Fig. 3 Changes of growth (3A), lactose and glucose level (3B) and β-galactosi-dase activity (3C) in washed cell cultures of P. chrysogenum. (�) 2% lactose, (�) 2% glucose, (�) 2% lactose and 0,15% caffeine, (�) 2% lactose and 1% 2-deoxy-D-glucose or (�) 2% glucose and 0.15% caffeine
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Changes in enzyme activities were low but significant. Values of activities (U mg–1) were 1.17 ± 0.42 (P < 5%) at 48 hours; 1.89 ± 0.26 (P < 0.1%) at 63 hours; 2.30 ± 0.59 (P < 1%) at 72 hours and 1.63 ± 0.82 (P < 5%) at 88 hours in comparison to values of 0.34 ± 0.19 at 24 hours. Mean values and standard deviation were calculated from three independent experiments. P values were calculated using the STUDENT’s t-test.
Fig. 4 Effect of different carbon sources on growth (4A), lactose utilisation (4B) and β-galactosidase activity (4C) of 2% lactose grown cultures of P. chrysogenum (�). The carbon sources were added at 40 h. (�) 2% glucose, (�) 2% galactose, (×) 2% sucrose, (�) 2% glycerol or (+) 2% lactose
Carbon source regulation of β-galactosidase 357
cAMP level in glucose and lactose grown culture
We studied the correlation between the β-galactosidase activity and the cAMP level in P. chrysogenum grown on different carbon sources. cAMP was detectable only in lactose grown cultures. Its amount was 7.67 pmol mg–1 dry weight. cAMP was not detectable in cultures grown on glucose or glycerol or when lactose grown cultures were supplemented at 40 h by glucose or glycerol.
The effect of caffeine on β-galactosidase synthesis
The likely role of cAMP in the regulation of β-galactosidase synthesis in P. chrysogenum was studied by further experiments. Investigating the effect of caffeine on β-galactosidase synthesis we added glucose or caffeine with glucose to lactose grown cultures at 40 hours in complex media (Fig. 5). Interestingly, we observed a pronounced effect of caffeine on growth and glucose consumption. Addition of caffeine significantly reduced growth and glucose consumption but only slightly influenced the decreased enzyme synthesis exerted by glucose (Fig. 5C). After washing glucose grown culture to medium using glucose as carbon source with caffeine we found that caffeine also reduced growth (Fig. 3A) and glucose consumption (Fig. 3B) but did not influence the enzyme synthesis compared to glucose grown culture (Fig. 3C). In lactose grown washed cell culture the caffeine decreased markedly the growth (Fig. 3A) and β-galactosidase activity (Fig. 3C) but later enzyme synthesis started to increase. Adding deoxy-D-glucose with lactose instead of caffeine to minimal medium deoxy-D-glucose had a similar effect on growth and enzyme activity like caffeine (Fig. 3A and 3C).
Discussion
Carbon source regulation plays important role in penicillin biosynthesis. Glucose (and other easily metabolised carbon sources) represses the expression of penicillin biosynthetic genes while no such repression was observed when lactose was used as carbon source (BRAKHAGE 1998). Despite of the critical role of lactose utilisation in penicillin fermentations relatively few studies have been published on the regulation of β-galactosidase synthesis in filamen-tous fungi. In Aspergillus oryzae (GARGOVA et al. 1995), A. niger (WIDMER and LEUBA 1979), Rhyzomucor sp. (SHAIKH et al. 1999) and Penicillium notatum (ROGALSKI and LOBARZEWSKI 1995) the enzyme is extracellular, in Aspergillus nidulans (DIAZ et al. 1996), and Thermomyces lanuginosus (FISCHER et al. 1995) is intracellular and in Phycomyces blakesleeanus both extra- and intracellular forms are present (MONTERO et al. 1989). Neu-rospora crassa has two types of lactases, one inducible by lactose and the other inducible by galactose (BATES et al. 1967). In our P. chrysogenum strain the enzyme is intracellular (NAGY et al. 2001). Detailed study of the enzyme production was performed only in A. nidulans (FANTES and ROBERTS 1973). The enzyme was induced by lactose and galac-tose, and interestingly galactose was the better inducer. Enzyme synthesis was repressed on glucose, sucrose, glycerol and some other carbon sources. In our strain from the carbon sources tested the production of β-galactosidase was high on lactose only (Figs. 1B and 3C). This is in contrast to that of A. nidulans where galactose (FANTES and ROBERTS 1973) and Phycomyces blakesleeanus where galactose and fructose are also effective inducers (MONTERO et al. 1989). Galactose is also an inducer of β-galactosidase in Candida pseu-dotropicalis (PEDRIQUE and CASTILLO 1982), Sacharomyces fragilis (DAVIES 1956) and Neurospora crassa (BATES et al. 1967). Galactose is as poor carbon source as lactose in A. nidulans (FANTES and ROBERTS 1973), while in our strain galactose was as good as glucose and had a similar repressive effect (Fig. 4C).
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In glucose, sucrose, galactose and glycerol grown cultures practically no enzyme was detected during exponential growth (Fig. 1B). When glucose, galactose, sucrose or glycerol were added to lactose grown cultures the enzyme level declined until the exhaustion of these carbon sources (Fig. 4C). The use of excess repressing carbon sources such as glucose or sucrose in the fermentation medium reduced the amount of penicillin produced (BRAKHAGE 1998).
Fig. 5 Effect of caffeine on growth (5A), glucose utilisation (5B) and β-galac-tosidase activity (5C) of lactose grown P. chrysogenum cultures in complex media (�). To the control culture 2% glucose (�) or 2% glucose and 0.15% caffeine (�) were added at 40 h of growth
Carbon source regulation of β-galactosidase 359
Lactose was a hardly metabolised carbon source in Penicillium chrysogenum which was demonstrated by poor growth and slow lactose consumption in lactose grown cultures (Figs. 2A and 3). High ammonia production at the beginning of the growth on lactose grown culture shows that the fungi preferred peptone rather than lactose as an energy source and it needed time to reach a relatively high β-galactosidase activity to utilise lactose, in contrast to glucose grown cultures where glucose was an easily and well utilisable carbon and energy source (Fig. 2) as was previously also shown by PUSZTAHELYI et al. (1997a). Adding more lactose to lactose grown culture no changes were found in growth (Fig. 4A), lactose consumption rate (Fig. 4B) and β-galactosidase activity (Fig. 4C) which shows a limited utilisation of lactose. The phenomenon that growth started after 10 hours in washed cell culture on lactose when the β-galactosidase activity increased markedly suppose an adaptation mechanism to lactose as a carbon source (Fig. 3A and C). In glucose grown complex cultures the correlation between the cessation of growth, depletion of glucose and the appearance of the low level of β-galactosidase suggests that in P. chrysogenum there was an uninduced, derepressed synthesis of the enzyme (Fig. 2B). Derepression was weak but significant. Derepressed synthesis of different enzymes are known in yeasts (ZIMMERMANN 1977) and A. nidulans. To assume the possible role of cAMP in carbon source regulation we determined the cAMP levels of cultures grown on different carbon sources. The role of cAMP in carbon repression in fungi is entirely different from that of bacteria (RONNE 1995) and is controver-sial (RUIJTER and VISSER 1997). Certain studies showed that in A. nidulans the level of cAMP is higher when glucose is present at high concentration (ZONNEVELD 1976), while others showed no or little correlation in Aspergillus (ZONNEVELD 1976) Mucor (PAVETO et al. 1975) and Neurospora species (PALL 1977). The situation is very similar to the yeast Saccharomyces cerevisiae and Schizosaccharomyces pombe. Our results which showed relatively high cAMP in lactose and low in glucose grown cells are in good agreement with that of EMRI et al. (1994) and KOZMA et al. (1992). The endogenous cAMP level can be influenced by drugs and exogenous cAMP or its analogs (PALL 1981). Methylxanthines such as theophylline, caffeine and 1-methyl- 3-isobutylxanthine act as phosphodiesterase inhibitors and are used as drugs to raise endogenous cAMP levels (PALL 1981, SCOTT and SOLOMON 1973, EMRI et al. 1998). In P. chrysogenum, caffeine is an effective inhibitor of phosphodiesterase activity and increases the concentration of cAMP (EMRI et al. 1994). In complex medium caffeine substantially reduced glucose consumption and growth but had little effect on β-galactosidase level (Fig. 5). In washed cell cultures on glucose and caffeine although caffeine decreased the growth but the increased level of cAMP did not increase the β-galactosidase level. However on lactose grown culture caffeine decreased growth mark-edly and reduced enzyme activity. These results suggest that cAMP did not affect the syn-thesis of β-galactosidase directly. Deoxy-D-glucose affect glycolytic flux by its phosphory-lated form (HEREDIA et al. 1963). In our strain deoxy-D-glucose decreased growth and β-galactosidase, therefore we suppose that a glycolytic intermedier can influence the en-zyme activity. In washed cell cultures caffeine also reduced glucose consumption as well as in complex media. Assuming that the main effect of caffeine is to increase cAMP level, cAMP had no role in carbon repression of β-galactosidase synthesis but caffeine strongly antagonised glucose consumption. This is also reflected by the reduced level of glucose-6-phosphate dehydro-genase in cultures grown on glucose and caffeine (EMRI et al. 1994). In pancreatic islets it was shown that caffeine inhibited D-glucose transport in a dose dependent manner while dBcAMP had no effect on glucose transport which means that caffeine inhibition on glucose uptake is independent of its ability to alter intracellular cAMP level (MCDANIEL et al. 1977). It is quite possible that in our case the reduced glucose consumption was also due to the inhibition of glucose uptake.
360 Z. NAGY et al.
From the above discussed data it is very likely that the β-galactosidase activity in P. chry-sogenum was induced by lactose and repressed by glucose, sucrose, glycerol and in contrast to other fungi and yeasts by galactose. An uninduced, derepressed synthesis of the β-galactosidase could be also observed in our Penicillium chrysogenum strain. Although there was a correlation between cAMP level and the presence of repressing carbon source a role for cAMP in carbon repression can not be established and therefore is unlikely.
Acknowledgements
This work was supported by the Ministry of Education (OM-FKFP 0102/1999). Z.N. was the recipient of a short term TEMPUS PHARE JEP student mobility grant and a Ph.D. studentship from the University of Debrecen. We thank ERZSÉBET FEKETE at the Department of Microbiology and Biotechnology University of Debrecen for measuring of lactose level in cultures of Penicillium chrysogenum.
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Mailing address: Dr. SÁNDOR BIRÓ, University of Debrecen, Faculty of Medicine, Department of Human Genetics, 4012 Debrecen, P.O. Box 1, Hungary Tel/FAX: +36 52 416531 E-mail: [email protected]
