Regulation of Ligninase Production in White-Rot Fungi - American


In Phanerochaete chrysosporium and other white-rot fungi, lignin biodégradation ... autocatabolism accompanied by a rapid loss of cell dry weight (8)...
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Chapter 16

Regulation of Ligninase Production in White-Rot Fungi 1

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Pascal Bonnarme , Juana Perez , and Thomas W. Jeffries

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Institute for Microbial and Biochemical Technology, U.S. Department of Agriculture Forest Products Laboratory, Madison, WI 53705

Carbon, nitrogen and manganese are critical nutritional variables in the production of ligninases including lignin peroxidase (LiP) and manganese peroxidase (MnP) by Phanerochaete chrysosporium and other white rot fungi. Excess carbon and nitrogen repress lignin biodegradation. Mn(II) is a specific effector that induces MnP and represses LiP. LiP and MnP also have different sensitivities to carbon and nitrogen supply. Mn(II) regulation is superimposed on carbon and nitrogen regulation and is only apparent when cultures are derepressed for these macronutrients. Supplementing nitrogen during cultivation represses MnP activity but can stimulate LiP production. These findings suggest that the regulatory mechanisms for LiP and MnP isoenzymes differ in several ways.

Lignin peroxidases (LiPs) and manganese peroxidases (MnPs) have been implicated in the biological degradation of lignin by white-rot fungi (7,2,3,4). Their regulation is of interest because such knowledge would help us to better understand the process of lignin biodégradation in nature and because these enzymes might be useful in biological pulping (5) or biological bleaching (6). Nutritional regulation has been studied most extensively. In Phanerochaete chrysosporium and other white-rot fungi, lignin biodégradation and ligninase production occur in response to nitrogen or carbon limitation (7, 8, 9, 10). They are interactive variables with different effects. Carbon limitation causes the rapid onset of lignin mineralization, but it is short-lived as the cells undergo autocatabolism accompanied by a rapid loss of cell dry weight (8). Nitrogen limitation results in a delayed onset of lignin mineralization, occurring only after nitrogen turnover in the cell. (11). Nitrogen limitation is convenient to use in studying lignin 1

Current address: I.N.R.A Laboratoire de Genie des Procédés Biotechnologiques Agro-Alimentaires, 78850 Thiverval-Grignon, France 2

Current address: Departamento de Microbiologia, Facultad de Farmacia, Universidad de Granada, Granada, Spain This chapter not subject to U.S. copyright Published 1991 American Chemical Society

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biodégradation, but in optimizing the production of ligninolytic enzymes, limiting nitrogen also limits the ability of the organism to produce extracellular proteins. Ligninases and other secreted enzymes are degraded by extracellular proteinases, and total enzyme production is limited by the amount of available nitrogen. With balanced carbon and nitrogen supplies one might attain maximal protein production while avoiding carbon catabolite repression. Recently we have discovered that Μη(Π) regulates LiP and MnP in white-rot fungi (72). LiP is formed almost exclusively when Mn(II) is low (1.6 to 0.3 ppm); MnP is formed almost exclusively when Μη(Π) is high (40 to 199 ppm). At interme­ diate levels - and especially the 11 ppm (0.2 mM) level often used for production of lignin-degrading enzymes - both sets of isoenzymes are formed. All isoenzymes of the LiP and MnP families are regulated together, suggesting that common mechanisms are involved. Manganese affects LiP and MnP isoenzymes in a much more specific manner than do carbon and nitrogen. With Μη(Π) regulation, nitrogen and carbon uptake rates are not dramatically altered over the range of Mn(II) levels effective for LiP and MnP regulation; cell dry weight, and oxygen uptake rates are similar at high and low Mn(II) levels (72). The effects of Mn(II) on isoenzyme production are clear-cut; isoenzyme profiles are distinct. These factors make Mn(II) a very useful tool for studying the different effects of LiP and MnP in lignin biodégradation. Several different white-rot fungi are affected by Μη(Π) in a manner similar to that observed with P. chrysosporium, indicating that Mn(II) regulation of P. chrysosporium is not idiosyncratic (72). In fact, organisms such as Lentinula edodes, which was not previously shown to produce LiP, from it in good titer when Mn(II) is present at low concentrations. Mn(II) also affects the rate of lignin mineralization. The rate that P. chrysosporium produces CC>2 from uniformlyring-labeledsynthetic lignin is six times greater at low Mn(II) levels than at high Mn(II) (77). Phlebia brevispora also mineralizes synthetic lignin at a significantly higher rate at low Mn(II), and in this organism, laccase, like MnP is more abundant at higher Μη(Π) levels (73), suggesting that LiP rather than MnP or laccase is principally involved in lignin biodégradation. By regulating the level of Mn(II), it should be possible to maximize production of either enzyme. In the present study, we examined the interactive effects of carbon, nitrogen and manganese in regulation of LiP and MnP. 14

Experimental Microorganism. All experiments reported here were performed with Phanerochaete chrysosporium BKM-F-1767 (ATCC 24725). Cultures were obtainedfromthe Center for Forest Mycology at the Forest Products Laboratory, Madison, WI, and were maintained as previously described (72). Cultural conditions. Unless otherwise stated, the initial medium contained per liter: 10 g glucose, 0.2 g diammonium tartrate, 4.6 g sodium D-tartrate dihydrate (adjusted to pH 4.5), 2.0 g KH2PO4, 0.5 g M g S 0 7 H 0 , 0.1 g CaCl -2H 0, 1 mg thiamine HC1, 0.5 g Tween 20, and 70 ml of a trace elements solution without MnSC>4 added. The trace elements solution contained per liter: 1.5 g nitriloacetic acid, 3.0 g M g S 0 7 H 0 , 1.0 g NaCl, 0.1 g FeS0 -7H 0, 0.1 g C0SO4, 0.1 g CaCl -2H 0, 0.1 g ZnS0 -7H 0,0.01 g C u S 0 5 H 0 , 0.01 g A1K(S0 ) 42H 0,0.01 g H3BO3, 0.01 g Na Mo0 -2H 0. Shake flask studies were carried out at 39°C in 125-ml Erlenmeyer flasks contain­ ing 40 ml of medium (180 rpm, 2.5 cm dia cycle). The carbomnitrogen ratio was varied by increasing or decreasing the diammonium tartrate supply, and Mn(II) added as specified. A spore inoculum was used (0.5 to 1.0 10 spores/ml final concentration), 4

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and veratryl alcohol (2.5 mM) was added at the time of inoculation. After 48 h of growth, Μη(Π) was added to the desired concentration and cultures were flushed daily with pure oxygen for 1 min (flow = 6.5 1/min, 760 mm Hg, 21°C). All other conditions were as previously published (72). Multiple Variant Analysis. Three variables were changed simultaneously: the initial carbon:nitrogen ratio, the initial carbon concentration, and the initial Mn(II) con­ centration. To find an optimum to produce LiP activity, a low range of Mn(II) concen­ trations were used (0 to 0.8 ppm), whereas for the production of MnP activity, a high range of Mn(II) concentrations (0 to 80 ppm) were used. Cultures attained maximal production on different days, so activity was expressed as a percentage of the maximal activity attained under the best conditions.

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Enzyme Assays. All enzyme assays were previously described (72). Results Carbon and Nitrogen Interactions. We found that MnP activity was repressed more at high nitrogen than was LiP. If the carbomnitrogen ratio were kept nearly con­ stant, good LiP and MnP production could be maintained over a range of concentra­ tions. In the experiment shown in Fig. 1, initial carbon and nitrogen concentrations were varied while holding Mn(II) at 11.2 ppm. If carbon were increased without in­ creasing nitrogen, and (especially) if nitrogen were increased without increasing car­ bon, enzyme production decreased. Production of both enzymes increased with in­ creasing carbon and nitrogen up to an apparent optimum range of 3.2 to 4.0 g/1 carbon and 0.11 to 0.14 g/1 nitrogen. The conations routinely used in our experiments corre­ sponded to carbon and nitrogen supplies of 4.0 and 0.030, respectively. An optimum carbomnitrogen ratio of about 30 to 40 is indicated by the data shown, but this will be affected by other nutritional factors.

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Figure 1. Effect of the initial carbon:nitrogen ratio on the production of peroxidases P. chrysosporium. Values in parentheses indicate MnP activity (100% = 900 nmol/min»ml); bold values indicate LiP activity (100% =187 nmol/mimml). Each value corresponds to the activity at its maximum. The concentration of Μη(Π) em­ ployed was 11.15 ppm. All other conditions were as previously published (72). Carbon and Nitrogen Starvation. Carbon and nitrogen starvation affect LiP and MnP production in different manners. As is shown in Fig. 2, carbon starvation leads

Leatham and Himmel; Enzymes in Biomass Conversion ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

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to a rapid appearance of LiP activity, but the effect on MnP activity is much less pro­ nounced. Nitrogen-limited cultures produce both LiP and MnP. Carbon-limited cultures produce mostly LiP. for this reason, the full regulatory effects of Mn(II) can be demonstrated best under nitrogen limitation. Except for the experiments shown in Fig. 4 below, nitrogen was limiting in all experiments to test the effects of Mn(II). In separate experiments, we attempted to examine the effect of Mn(II) under carbon limitation, but no significant MnP or LiP production was obtained (data not shown).

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Figure 2. Effects of carbon and nitrogen limitation on the production of LiP and MnP activities. Carbon limited cultures (•); Nitrogen limited cultures (•). Carbon limited cultures contained per liter 0.66 g diammonium sulfate and 2 g glucose. Nitrogen limited cultures were the same as the controls. They contained 0.2 g diammonium tartrate and 10 g glucose. All cultures received 11.2 ppm Μη(Π). Repression by Supplemental Carbon and Nitrogen. Both LiP and MnP ap­ pear to be under nitrogen-regulation, but the effects on LiP are more stringent than the effects on MnP. Ammonium tartrate is known to repress lignin degrading enzymes and lignin mineralization when it is added during cultivation. As is shown in Fig. 3, when diammonium tartrate was added on day 4 to cultures actively producing lignin-degrading enzymes, a transient repression of LiP activity was observed followed by increased production. In the case of MnP, however, no significant stimulation was observed. Interaction of Mn(II) with Carbon and Nitrogen. We attempted to determine an optimal carbon concentration and carbonmitrogen ratio for LiP and MnP over low and high Mn(II) ranges, respectively. Earlier studies had shown that the optimum Μη(Π) was less than 1 ppm for LiP and greater than 25 ppm for MnP (72). We wanted

Leatham and Himmel; Enzymes in Biomass Conversion ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

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to know if varying Μη(Π) could overcome the repressive effects of glucose and nitrogen. The regulatory effect of Μη(Π) appeared to be separatefromthe effects of carbon and nitrogen. As is shown in Fig. 4, both LiP and MnP activities were completely repressed when the carbon concentration was high (6 g/1 carbon = 15 g/1 glucose) and the carbonmitrogen ratio was low. Both LiP and MnP activities increased with increasing carbonmitrogen ratio. The effect of Mn(II) was seen at intermediate carbon and nitrogen levels. With Lip, enzyme activity increased as Mn(II) decreased; with MnP, the opposite effect was observed. The optimum Μη(Π) concentration for LiP or MnP production appeared to vary with the carbonmitrogen ratio.

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Figure 3. Effects of diammonium tartrate supplements on LiP and MnP production by P. chrysosporium . Additional nitrogen was added to triplicate cultures on day 4. Control ( · ) ; 2.2 mM (O); 4.4 mM (•); 8.8 mM (Δ). Glucose and Mn(II) concentrations were 10 g/1 and 11.2 ppm, respectively. No LiP activity was observed at high carbon and low carbonmitrogen ratio (high nitrogen). Repression of LiP was markedly relieved at each carbon level as the car­ bonmitrogen ratio increased (i.e., as nitrogen became limiting), and repression was also relieved when the initial carbon level was reduced while keeping carbonmitrogen con­ stant. The interactive effects of Mn(II) were ambiguous. At moderate carbonmitrogen ratios, an optimum was obtained at very low Μη(Π) levels (point Β on Fig. 4a), but at higher carbonmitrogen ratios (i.e. under nitrogen limitation) and at higher carbon levels (where nitrogen-limitation would be more effective), moderately higher Μη(Π) levels were necessary (point A on Fig. 4a).

Leatham and Himmel; Enzymes in Biomass Conversion ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

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A similar pattern of carbon and nitrogen catabolite repression of MnP production was observed. Carbon repression of MnP could be overcome somewhat at moderate carbon and nitrogen levels by increasing Mn(II) (point A, Fig. 4b), but at low carbon and high nitrogen levels, decreasing Μη(Π) from 64 to 16 ppm resulted in an almost two-fold increase in MnP activity. Decreasing the carbon supply at a constant carbonmitrogen ratio did not increase MnP activity as much as the same shift increased LiP (Fig. 4a). Three optima were observed for MnP. The two best (points Β and C on Fig. 4b) were both at high carbonmitrogen ratios, relatively high Mn(II) and low carbon. A third was at a moderate carbonmitrogen ratio and carbon concentration, but at the highest Μη(Π) level employed (point A on Fig. 4b).

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Figure 4. Effects of Mn(II), carbon and nitrogen on the production of peroxidases by P. chrysosporium. a) LiP activities over a low range of Μη(Π); b) MnP activities over a high range of Mn(II). Activities are given in percent of maximum. LiP 100% = 454 nmoVmimml; MnP 100% = 874 nmoVmimml). Discussion The experiments depicted in Figs. 1 and 4 did not determine true optima. In the study of carbon and nitrogen interactions, the optima appeared to lie in the range of 2.4 to 4.0 g/1 for carbon and 0.084 to 0.14 g/1 nitrogen. One point fell in this range, and it was the maximum for this series of experiments but it is not necessarily an optimum. Likewise, in the partial factorial design depicted in Fig. 4, all of the maxima occurred at star points rather than within the matrix, so it is apparent that the optimum or optima lie somewhere outside the limits of the experimental design. Despite these limitations, several useful inferences can be drawnfromthese data.

Leatham and Himmel; Enzymes in Biomass Conversion ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

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First, LiPs appear to be regulated by carbon catabolite repression while MnPs are not derepressed by the carbon limitation used here. Since we know also that DHP mineralization shows a sudden onset in response to carbon starvation in this organism (8), one might conclude that this enzyme system is in some way related to respiratory pathways or the acquisition of metabolic energy. However, no direct evidence to date has shown that P. chrysosporium will grow solely at the expense of lignin oxidation. Indeed, cometabolism of carbohydrates or some other carbon source appears to be a necessary requisite. Additional regulatory studies on this enzyme and its relationship to energy-yielding metabolism should be conducted using cellulose or holocellulose as a carbon source. Even though such conditions would result in lower overall enzyme titers, the conditions would be more representative of what occurs in nature. Second, both LiP and MnP are formed in response to nitrogen starvation, but the onset of enzyme production occurs more slowly than with carbon limitation. Unlike carbon with is virtually always present in excess in wood, albeit in a slowly-utilizable form, nitrogen is often a limiting nutrient (at least in laboratory culture), so both LiP and MnP would tend to be derepressed during growth under such conditions. The slightly greater repressive effects of nitrogen on MnP might not be significant, but they suggest that during the phase of initial invasion, when the fungus is growing at the expense of readily-available sugar and when residual cellular nitrogen should be available, neither LiP nor MnP are formed. It is not clear that nitrogen would be a limiting nutrient during extensive degradation in nature where wood is likely to receive nitrogen from soils and through bacterial nitrogen fixation, but it is also unlikely that nitrogen would be present in great excess under such conditions. Third, the effects of Μη(Π) are only conspicuous when nitrogen is limiting. At low carbon levels, LiP is derepressed, but the very low cell yields make even very small Mn(II) concentrations effective in repressing LiP activity. The genetic regulatory elements are not yet known for these enzymes, but recent studies have shown that Mn regulates expression of MnP by P. chrysosporium (14). Literature Cited 1 2 3 4 5 6 7 8 9 10 11 12 13 14

Tien, M. ; Kirk, T. K. Science 1983, 221:661-663. Glenn, J. K.; Morgan, M . B.; Mayfield, M.; Kuwahara, M.; Gold, M . H. Biochem. Biophys. Res. Comm. 1983, 114, 1077-1083. Hunh, V.-B.; Crawford, R. L. FEMS Microbiol. Lett. 1985, 28, 119-123. Glenn, J. K.; Gold, M. H. Arch. Biochem. Biophys. 1985, 242, 329-341. Reid, I.D. Enzyme Microb. Technol. 1989. 11, 786-803. Eriksson, Κ. E.; Kirk, T. K. In The Principles of Biotechnology: Engineering Considerations; Cooney, C., L.; Humphrey, A. E., Eds.; Pergamon:New York, NY, 1985; pp 271-294. Keyser, P. J.; Kirk, T. K.; Zeikus, J. G. J. Bacteriol. 1978, 135, 790-797. Jeffries, T. W.; Choi, S.; Kirk, T. K. Appl. Environ. Microbiol. 1981, 42, 290296. Leatham, G. F.; Kirk, T. K. FEMS Microbiol. Lett. 1983, 16, 65-67. Kirk, T. K.; Croan, S.; Tien, M.; Murtagh, Κ. E.; Farrel, R. L. Enzyme Microb. Technol. 1985, 8, 27-32. Fenn, P.; Kirk, T.K. Arch. Microbiol. 1981, 130, 59-65. Bonnarme, P.; Jeffries, T. W. Appl. Environ. Microbiol. 1990, 56, 210-217. Perez, J.; Jeffries, T. W. Appl. Environ. Microbiol. 1990, 56 (6), 1806-1812. Brown, J. Α.; Glenn, J. K.; Gold, M. H. J. Bacteriol. 1990, 172, 3125-3130.

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