Journal of Biotechnology, 30 (1993) 91-107 91 © 1993 Elsevier Science Publishers B.V. All rights reserved 0168-1656/93/$06.00

BIOTEC 00900

An overview of the recent advances on the physiology and molecular biology of lignin

peroxidases of Phanerochaete chrysosporium

C.A. Reddy

Department of Microbiology and Public Health and the NSF Center for Microbial Ecology, Michigan State University, East Lansing, MI, USA

(Received April 1991; revision accepted 6 February 1992)

Summary

The lignin-degrading white-rot fungus Phanerochaete chrysosporium produces two families of extracellular peroxidases designated lignin peroxidases (LIPs) and manganese-dependent peroxidases (MNPs) which are components of the lignin degradation system of this organism. The number and types of LIP and MNP isozymes produced vary dramatically in response to changes in culture conditions. Protease-mediated degradation of LIPs was shown to be the major cause for the decay of LIP activity in idiophasic cultures of P. chrysosporiurn. Use of biochemical mutants has not only yielded information on the relative importance of LIPs and MNPs in lignin degradation but has given us insights into the regulation of production of LIPs and MNPs. The genes encoding the major LIPs have been cloned and sequenced and were shown to have a high degree of homology to each other. Karyotyping studies indicated that heterokaryotic strains contain ten chro- mosomes and that the L I P genes are distributed on at least two chromosomes.

Lignin peroxidase; Mn-dependent peroxidase; Karyotyping; Fungal mutants; Phanerochaete chrysosporium; Protease; Transformation; White-rot fungus; Sec- ondary metabolism

Correspondence to: C.A. Reddy, Dep. Microbiology, Michigan State University, S-124 Plant Biology Building, East Lansing, MI 48824-1101, USA.

92

Introduction

The lignin peroxidases of Phanerochaete chrysosporium have been the focus of intensive study ever since their discovery in 1983 because of their potential for applications in biopulping and biobleaching, in the conversion of lignocellulosic materials to chemicals and fuels, and in the detoxification of a broad range of toxic xenobiotics including certain dibenzodioxins, benzo(a)pyrenes, and polychlorinated biphenyls (see Boominathan and Reddy, 1992; Buswell and Odier, 1987; Kirk and Farrell, 1987). The lignin peroxidases (LIPs) together with manganese-dependent peroxidases (MNPs) constitute two families of extracellular, glycosylated heme proteins that are produced by P. chrysosporiurn only during secondary metabolism in response to environmental triggers such as nitrogen and carbon starvation. Excess nitrogen or carbon in the medium on the other hand results in complete suppression of LIP and MNP production. LIPs and MNPs are believed to be important in lignin degradation, but their exact role in this function is not well understood. Extensive studies to date have shown that LIPs catalyze H202-depen- dent one electron oxidation of a variety of lignin-related aromatic compounds resulting in the formation of aryl cation radicals. These radicals undergo various non-enzymatic reactions yielding a multiplicity of end products. MNPs, the second family of extracellular peroxidases, catalyze the H202-dependent oxidation of Mn(II) to Mn(III) which in turn oxidizes various phenolic substrates (Gold et al., 1989). The H20 2 required for the activity of LIPs and MNPs is reported to be supplied by several enzymes including glucose oxidase and glyoxalate oxidase (see Boominathan and Reddy, 1992; Kirk and Farrell, 1987).

Recent use of selected biochemical mutants and appropriate manipulation of culture conditions have advanced our understanding of the contribution of LIPs to various biodegradative activities catalyzed by P. chrysosporium (Boominathan et al., 1990a, b; Michel et al., 1991). Current evidence indicates that the multiple isozymes of LIP are encoded by separate genes. Several LIP-encoding cDNAs and genes have been cloned and sequenced and two independent groups have de- scribed transformation systems for this organism (see below). An overview of these recent advances on the physiology and molecular biology of the LIPs, primarily of P. chrysosporium, is presented here.

Physiology

An intensive search for H202-dependent extracellular ligninolytic enzymes followed our earlier studies which demonstrated that a temporal relationship exists between ligninolytic activity and H20 2 production, that the latter compound plays an integral role in lignin degradation, and that environmental parameters such as nitrogen or carbon concentrations in the medium, which profoundly affect lignin degradation, have a parallel effect on H202 production (Boominathan and Reddy, 1992; Forney et al., 1982; Kelley and Reddy, 1986). This search led to the discovery of LIPs by two independent groups and these enzymes have since been studied

93

extensively by numerous investigators around the world (reviewed in Kirk and Farrell, 1987; Boominathan and Reddy, 1992; Buswell and Odier, 1987).

Multiple isozymes The LIP family contains multiple isozymes with a molecular weight range of

38 000 to 43 000 and isoelectric points ranging from 3.3 to 4.7 (Dass and Reddy, 1990; Farrel et al., 1989; Leisola et al., 1987). LIPs are oligomannose-type glyco- proteins with one or more N-glycosylation sites and a number of possible O-glyco- sylation sites. Fast protein liquid chromatography (FPLC) or high pressure liquid chromatography (HPLC) techniques are frequently employed for purification of the LIPs from the concentrated extracellular culture fluid of P. chrysosporium (Dass and Reddy, 1990; Farrell et al., 1989; Tien and Kirk, 1988). The heine proteins separated in this manner from P. chrysosporium strain BKMF (ATCC 24725) cultures were arbitrarily designated H1, H2, H3 . . . . , H10 (Kirk and Farrell, 1987). Of these H1, H2, H6, H7, H8 and H10 were shown to have LIP activity while the rest were shown to have MNP activity (Kirk and Farrell, 1987). The number and the relative abundance of various LIP isozymes identified in P. chrysosporium cultures vary considerably depending on the culture conditions, purification procedures, and the strains employed in different investigations. For example, the number of LIP isozymes was shown to vary from as low as two to as high as 15 under different conditions. FPLC profiles of LIPs in acetate-buffered static cultures of P. chrysosporium strain BKM-F showed major amounts of LIP isozymes H2 and H6 and moderate amounts of H8 and H10 (Dass and Reddy, 1990; Fig. 1). In a similar medium buffered with dimethyl succinate (DMS), instead of acetate, major amounts of H8, moderate amounts of H2, and minor amounts of H6 and H10 were produced (Tien and Kirk, 1988; Dass and Reddy, 1990). Furthermore, a new LIP protein, designated HA, was seen in acetate-buffered cultures that was not seen in DMS-buffered cultures. Also, remarkably large amounts of MNPs were produced in acetate-buffered cultures whereas in DMS- buffered cultures very minor amounts of MNPs were produced (Dass and Reddy, 1990; Tien and Kirk, 1988). In acetate-buffered shaken cultures, MNP protein H4 was the single most dominant peak and the LIP isozymes H2 and H6 were produced in much lower amounts as compared to the static acetate-buffered cultures described above (Fig. 1). Other studies showed that P. chrysosporium produces major amounts of H2 and H8 isozymes in nitrogen-limited, DMS-buffered medium while in an identical but carbon-limited medium, major amounts of H2 were produced (Holzbaur and Tien, 1988). These results strongly suggested differ- ences in the regulation of expression of different LIP genes in response to various environmental triggers. These studies also indicated that, under a broad range of environmental conditions studied, H2 is produced in substantial amounts while the levels of the other LIP isozymes vary a great deal depending on the cultivation regimens employed. The concentration of different LIP isozymes in the extracellu- lar culture fluid also varies with culture age. For example, H6, a major isozyme in 6-day-old acetate-buffered cultures decreases in quantity relatively rapidly after day 8 (Dosoretz et al., 1990). A comparison of the LIP isozymes produced by P.

94

1 6

O1

n 8

.>

re 4

A

O

H2 H4

H3

I H5 ! HA

1'6 2'4 o

H4 B

- 1'6 2 4 O

Elution Volume (ml)

C

H8

H10

H2

1'6 2'4

Fig. 1. Fast protein liquid chromatography profiles of extracellular heme proteins from 6-day-old acetate or Dimethylsuccinate (DMS)-buffered cultures of P. chrysosporium BKM-F (ATCC 24725). H1, H2, H6, H7, H8, and H10 are lignin peroxidases(LIPs); H3, H4, H5, and H9 are manganese-dependent peroxidases(MNPs). The HA peak represents a new LIP protein seen in acetate-buffered cultures but not in DMS-buffered cultures. Relative peak height represents absorbance at 405 nm. Panel A: stationary acetate-buffered cultures grown in nitrogen-limited medium. Panel B: cultures were identical to those described in panel A except that these were agitated at 125 rpm. Panel C: cultures were identical to those described in panel B except that these cultures were buffered with DMS rather than

with acetate. (From Dass and Reddy, 1990.)

chrysosporium strains ME 446 (ATCC 34541) and BKM-F, the two most widely studied strains, showed that ME 446 predominantly produces three LIP and two MNP isozymes while strain BKM-F produces a more complex array of isozymes as described above.

Two important questions stemmed from the discovery that P. chrysosporium produces a multiplicity of LIP isozymes: (1) Why are so many LIP isozymes produced by a single organism and what is the specific role(s) if any of the individual isozymes in lignin degradation and other degradative activities catalyzed by this organism? (2) Are the different LIP isozymes encoded by different genes or do the different isozymes represent different posttranslational modifications of the product of a single gene (or at the most a small number of genes)? The first question largely remains unanswered even today while rapid strides have been made in answering the second question. A comparison of the experimentally determined N-terminal sequences of electrophoretically pure LIP proteins H2, H6, H8, and H10 showed that each of these proteins has its own unique N-termi- nal sequence indicating that each is encoded by a different structural gene (Dass and Reddy, 1990). Furthermore, our data also showed that the N-terminal se- quence of H6 has about 90% homology to that of H8, 70% to that of H10, and only 20% to that of H2 indicating that the LIP isozyme H2 is much less related to LIPs H6, H8, and H10. While the latter three are closely related to each other, H6 and H8 appear to be much more closely related to each other than they are to H10.

95

These results are consistent with the previously reported peptide mapping data which showed that H1 and H2 are more closely related to each other and form one subfamily while H6, H7, and H8 constitute the second subfamily (Farrell et al., 1989). The isozyme H10, while more closely related to the isozymes of the second subfamily was reported to be different enough to form a third subfamily. These results are also consistent with the most recent LIP cDNA/gene sequence data (see below).

Our preliminary studies on the catalytic activities of different LIP isozymes showed that there are considerable differences among the isozymes in degrading the dyes Poly R, Poly B, and crystal violet which have previously been shown to be degraded by the lignin-degrading enzyme system of P. chrysosporium (Dass and Reddy, 1990). These results are consistent with the recent results of Farrell et al. (1989) who also reported wide differences in the catalytic activities of various LIP isozymes against lignin-model aromatic compounds. Furthermore, the LIP isozymes exhibit differing specific activities against veratryl alcohol, the common substrate used for assaying LIP activity (Boominathan et al., 1990b; Farrell et al., 1989). For example, the H2 isozyme often shows 4 to 7-fold greater specific activity as compared to H8 or H10 isozymes. Collectively, these differences in catalytic activities of various LIP isozymes with different substrates suggest differences in affinities and/or active site accessibilities.

Mutants LIPs and MNPs of P. chrysosporium have been implicated in this organism's

ability to degrade lignin (Kirk and Farrell, 1987; Gold et al., 1989); however, the relative contribution of LIPs vs. MNPs to lignin degradation by this organism has never been established. We recently isolated a lip mutant of P. chrysosporium that differs from the wild type in lacking the ability to produce the LIP isozymes, but is similar to the wild type in producing a full complement of MNPs as well as the key H202-producing secondary metabolic enzyme, glucose oxidase (Boominathan et al., 1990a). A comparison of the lignin-degrading ability of the lip mutant with the wild type showed that it exhibits only about 12-15% of the wild type activity. These data not only indicated that the LIPs play a predominant role in lignin degradation but also documented for the first time that a strain of P. chrysospo- rium displaying no LIP activity can still degrade lignin albeit to a minor extent. Independent corroboration of these results came from other studies in which elevation of the manganese concentration in the medium to 100 ppm allowed MNP production but blocked LIP production; under such conditions, the extent of lignin degradation observed was 10-11% of that observed in a positive control medium containing low levels of manganese which allowed production of both LIPs and MNPs (Bonnarme and Jeffries, 1990; Perez and Jeffries, 1990).

The lip mutant appears to be a regulatory mutant in that a regulatory gene which is specifically involved in controlling the expression of the LIP genes (but not the MNPgenes) has apparently been affected in this mutant. These results further indicate that LIP production in P. chrysosporium is regulated differently, at least in some aspects, from that of MNP production. However, it is also equally

9 6

apparent from published data that LIP and MNP production share some common regulatory signals as well. For example, starvation for carbon or nitrogen triggers LIP as well as MNP production whereas excess nitrogen or carbon in the medium result in complete suppression of both LIP and MNP production (Boominathan and Reddy, 1992; Kirk and Farrell, 1987). Furthermore, our most recent data show that there is a sharp rise in intracellular cyclic AMP just prior to the onset of LIP and MNP production and that inhibiting intracellular cyclic AMP levels results in simultaneous blockage of production of both LIPs and MNPs (Boominathan and Reddy, unpublished data).

Availability of the lip mutant also allowed us to determine the relative contribu- tion of LIPs versus MNPs to the decolorization of Kraft bleach plant effluents (BPE). Billions of gallons of intensely colored and toxic BPE are discharged annually into the environment by the pulp and paper industry constituting a serious environmental pollution problem. BPE largely contains high molecular weight chlorinated lignins and its degradation products. Conventional waste water treatment processes employing bacteria are relatively ineffective at removing these pollutants, but white rot fungi such as P. chrysosporium and Trametes versicolor (synonyms: Coriolus uersicolor, Polyporous versicolor) were shown to efficiently dechlorinate and decolorize BPE (see Michel et al., 1991). Our studies showed that negligible decolorization of BPE occurs when a peroxidase mutant of P. chrysospo- rium, which lacks the ability to produce both LIPs and MNPs, was employed (Michel et al., 1991; Fig. 2). Also, little decolorization of was observed when P.

Residual Color (%)

1 0 0

80

60

40

20

A

r

i ~'~ PER-mutant

; LIP- mutant

+ ME-446 ~ ]

t t BKM-F-1767

0 . . . . I . . . . I . . . . I . . . . I . . . . I

0 1 2 3 4 5

Days

Fig. 2. Decolorization of Kraft bleach plant effluent (BPE) by P. chrysosporium wild type strains BKM-F and ME 446 and a lip mutant and a peroxidase mutant (per) of ME 446. The per mutant lacks the ability to produce LIPs and MNPs while the lip mutant lacks the ability to produce LIPs but not MNPs. BPE (3000 color units) was added to the cultures on day four of incubation and the

decolorization was monitored each day for the next five days. (From Michel et al., 1991.)

97

chrysosporium wild type was grown in BPE medium supplemented with high levels of nitrogen under which conditions the production of LIPs and MNPs is blocked. These results showed that LIPs and MNPs are important for BPE decolorization. Inoculation of the lip mutant into the BPE medium resulted in about 50% of the decolorization exhibited by the wild type (Fig. 2). Furthermore, when P. chrysospo- rium wild type was inoculated into nitrogen-limited BPE medium containing 100 ppm Mn(II), high levels of MNPs but no LIPs were produced and this culture also exhibited a high rate of BPE decolorization. On the other hand, when wild type P. chrysosporium was grown in a BPE medium with 0 ppm Mn(II), high levels of LIPs but negligible levels of MNPs are produced and the rate of decolorization was quite low in these cultures indicating that MNPs play an important role in BPE decolorization while the LIPs appear to be relatively less important in this application.

It has been well established that nitrogen starvation favors the production of the lignin degrading enzyme system including the LIPs and the MNPs while nitrogen excess in the medium represses LIP and MNP synthesis and inhibits lignin degradation activity (reviewed in Boominathan and Reddy, 1992; Kirk and Farrell, 1987). We investigated the possibility of obtaining nitrogen-deregulated mutants (der mutants) of P. chrysosporium that produce LIPs and MNPs and degrade lignin both under conditions of nitrogen excess and of nitrogen limitation. Such mutants will be useful in obtaining a better understanding of the nitrogen regula- tion of ligninolytic activity as well as in understanding the regulation of expression of the LIP and MNP genes both at the biochemical and molecular genetic levels. Additionally, the ability of such der mutants to produce high levels of LIPs and MNPs even under conditions of nitrogen excess would make these mutants more suitable for use in various biotechnological applications.

A der mutant of P. chrysosporium strain ME446, designated der8-5, was shown to degrade lignin both under conditions of nitrogen limitation and nitrogen excess (Boominathan et al., 1990b). Furthermore, in nitrogen-limited medium, der8-5 produced several fold greater levels of LIP activity than that produced by the wild type. In nitrogen-rich medium, der8-5 produced high levels of LIP activity whereas the wild type produced no LIP activity under these conditions. FPLC analyses of the extracellular culture fluid of nitrogen-limited cultures of both the wild type and der8-5 showed the presence of three prominent LIP isozymes designated L1, L2, and L3 whereas in nitrogen-rich medium the wild type produced no LIP protein peaks while der8-5 produced L1, L2, and a new LIP protein peak LN, but did not produce L3. These results indicated that the production of L3 protein is subject to nitrogen repression both in the wild type as well as in the der8-5 mutant. Our results also showed that der8-5, besides being nitrogen-deregulated for LIP pro- duction, was deregulated for MNP and glucose oxidase production as well. Our most recent results show that LIP and MNP production by der-8-5, while deregu- lated for nitrogen, is still subject to carbon and Mn(II) regulation indicating that LIP and MNP production by P. chrysosporium is independently regulated by nitrogen, carbon, and manganese (M.W. van Der Woude, Boominathan and Reddy, unpublished data).

98

Protease-mediated degradation of LIPs In nitrogen-limited liquid cultures of P. chrysosporium, the LIP activity peaks

around day 6 of incubation and sharply declines there after regardless of the carbon source, culture conditions or the strains used. Our recent studies showed that this sharp decline in LIP activity is closely correlated with the appearance of idiophasic extracellular protease activity (Dosoretz et al., 1990), the appearance of which was triggered by glucose depletion in the medium. The contribution of the protease to the decline in the LIP activity was further supported by the fact that daily addition of glucose to the culture starting on day 6 resulted in greatly decreased protease levels and this in turn stabilized the LIP activity. Addition of cycloheximide to 6-day-old cultures resulted in negligible levels of protease activity and virtually no drop in LIP activity. These results indicate that the idiophasic protease is synthesized de novo. Incubation of FPLC-purified LIP isozymes with the protease-rich extracellular culture fluid of l 1-day-old cultures resulted in marked time-dependent loss of LIP activity concomitant with the degradation of the LIP isozymes. In contrast to this, incubation of the LIP isozymes with boiled extracellular culture fluid of the day eleven cultures resulted in no loss of LIP activity. These results indicated that protease-mediated degradation of the LIP proteins is a major cause for the decay in LIP activity in idiophasic cultures of P. chrysosporium and that suppression of this protease activity may be important for stabilizing the LIP levels in such cultures.

Molecular biology

The discovery of LIPs rapidly moved lignin biodegradation research into the realm of molecular biology. Soon after the LIPs were discovered, our laboratory as well as several other laboratories started on the trail of cloning the cDNAs/genes encoding the various LIP enzymes because this is an important first step in understanding the structure and regulation of expression of the LIP genes as well as in the use of these clones for the overproduction of the LIPs in appropriate expression systems for the eventual biotechnological exploitation of these enzymes. Rapid advances have been made in recent years in cloning the cDNAs/genes encoding several of the LIP isozymes of P. chrysosporium and the LIP isozymes of two other species of white-rot fungi. A brief overview of these recent developments is presented here.

LIP cDNAs Zhang et al. (1986) first reported the isolation of two distinct types of LIP-en-

coding cDNAs. This was soon followed by the complete sequencing of these two cDNAs, designated CLG4 and CLG5, which encode the LIP isozymes H2 and H10, respectively (de Boer et al., 1987). Tien and Tu (1987) reported the isolation and sequencing of a third species of LIP-encoding cDNA (ML1), which encodes the LIP isozyme H8. The results of these two studies showed that the mature H2, H8, and H10 proteins each contained 344 aa. Consistent with the extracellular

99

nature of these proteins, each is preceded by a leader peptide (27 to 28 aa long) that ends in the Lys-Arg proteolytic cleavage site. The H2 and H8 proteins each contained one putative N-glycosylation site with the general sequence Asn-X-Thr- /Ser while the H10 isozyme contained two such putative N-glycosylation sites. Each of the three proteins contains a number of possible O-glycosylation sites. These data are consistent with the reports that purified LIPs exhibit both N-linked and O-linked glycosylation (Farrell et al., 1989). It is of interest that each of the LIP proteins contains only 344 aa eventhough the experimentally determined molecular weights of purified LIPs range from 38 000 to 43 000 (Dass and Reddy, 1990; Farrell et al., 1989). Thus, the observed differences in molecular weights of different LIPs may be due to differences in glycosylation levels of these proteins. The proximal and distal histidine residues and one arginine residue known to be required for the catalytic activity of all the peroxidase proteins examined to date are found to be conserved in the H2, H8, and H10 protein sequences deduced from the nucleotide sequences of CLG4, ML1, and CLG5, respectively.

The nucleotide(nt) and aa homology between the coding regions of CLG4 and ML1 were 74% and 69%, respectively, while these homologies were 71.5% and 68.5%, respectively, between the coding regions of CLG4 and CLG5 (de Boer et al., 1987). The coding regions of CLG5 and ML1 showed nt and aa homologies of 74% and 81%, respectively. These results indicated a high degree of relatedness among the cDNAs encoding the LIP proteins H2, H8, and H10 in agreement with previous data which showed a high degree of immunological relatedness among the LIP proteins of P. chrysosporium. Northern blot data, using the three cDNAs as probes showed that LIP transcripts are not produced in cultures grown in nitrogen rich medium while high levels of transcripts are seen in cultures grown in nitrogen-limited medium. Furthermore, there was a good correlation between the time of appearance and levels of LIP transcripts and the LIP enzymatic activity. These results indicated that LIP production is controlled at the level of mRNA production.

The cDNA, LGP3, that encodes one of the LIP isozymes of another white rot fungus, Phlebia radiata, has recently been cloned and sequenced (Saloheimo et al., 1989). The coding region of this cDNA encodes a mature LIP protein that contains 337 aa and is preceded by a 24 aa signal peptide that ends in the putative proteolytic cleavage site Arg-Arg, rather than the Lys-Arg proteolytic cleavage site reported above for the signal peptides of P. chrysosporium LIPs. The LIP protein encoded by LGP3 contained one putative N-glycosylation site, similar to that seen in the LIPs of P. chrysosporium. The LGP3 protein showed about 60% aa homology to the LIP isozymes of P. chrysosporium.

LIP genes The isolation and characterization of LIP-encoding cDNAs soon led to the

isolation of a number of the LIP genes of P. chrysosporium. Probing the genomic library of this organism with labeled LIP cDNAs CLG4 and CLG5, six distinct LIP-encoding genomic fragments designated GLG1, GLG2, GLG3 . . . . . and GLG6 were isolated by Zhang et al. (1991; Fig. 3). Of these, GLG1 gave intense

100

kb

23.%

9.4-

6.6-

4 .4-

2,3- 2.0-

A I

1 2 3 4 5 6 1 2 3 4 5 6

i b

B

1 GLG 1

II

B XS[

GLG 2

S~

E X SlI S[ P S~ S]IP

l k b

ST Xb X PXb X S[ B

SI So I ~.~ GLG3

8 HS~KP,~[

GLG4

B XSp X

GLG 5

B E H

GLG6

J.

SLS~ Sli

KSP ~ K

S~XSp p B

Sm S~(b X HX SmS~ B

Sm S[ Xb 1 1 ;

~ I k b

TABLE 1

General features of the LIP genes of P. chrysosporium

101

Characteristic GLG1 GLG2 GLG3 GLG6

LIP protein encoded H2 H10 H8 9 Coding region (aa) 344 344 344 344 Leader peptide (aa) 28 27 28 28 N-Glycosylation sites one two one one TATA box and CAAT box + + + + Number of introns 8 9 8 9 Length of introns (bp) 50-63 50-62 50-60 50-57

hybridization with CLG4, while GLG2 through GLG6 showed homology to CLG5 (Fig. 3); however, of the latter five clones, GLG2 gave the most intense hybridiza- tion with CLG5 as compared to the rest suggesting that GLG2 contains the gene corresponding to the CLG5 cDNA. Sequencing of GLG2 confirmed that it contains the intact LIP gene which encodes the LIP isozyme H10 (Zhang et al., 1991). Subsequently, we sequenced the LIP-encoding regions of GLG1 and GLG3 which were shown to encode, respectively, the LIP proteins H2 and H8 (Naidu and Reddy, 1989 and 1990). Sequencing of the LIP-encoding region of GLG6 showed that it encodes a LIP protein which is different from H2, H8, and H10 and the LIP protein encoded by GLG6 is yet to be identified (Naidu et al., 1990). A number of investigators have reported the sequences of several allelic and nonallelic variants of the H8-encoding gene (Asada et al., 1988; Brown et al., 1988; Schalch et al., 1989; Walther et al., 1988). The general features of the LIP genes based on the cumulative data obtained to date are summarized in Table 1. These data, in agreement with the cDNA data, show that each LIP gene encodes a mature protein that contains 344 aa and is preceded by a signal peptide that contains 27 to 28 aa. All the LIP proteins contain one potential N-glycosylation site except the H10 protein which contains two such sites. Each LIP protein contains a number of O-glycosyslation sites. The C-terminal end of all the LIP proteins contains a characteristic proline-rich region ending with the sequence Pro-Gly-Ala. Archety- pal TATA-box-like and CAAT-box-like eukaryotic promoter elements are readily seen in the 5'-non-coding region of each gene. The coding region of each LIP

Fig. 3. Identification and restriction maps of six LIP-encoding genomic fragments of P. chrysosporium strain BKM-F. (I) Hybridization of LIP genomic clones with LIP cDNAs CLG4 and CLG5, which, respectively, encode LIP isozymes H2 and H10. In each of the three panels shown, lanes 1 to 6, respectively, contained the genomic inserts GLG1 to GLG6 in pUC19. Panel A: agarose gel fractiona- tion. Panels B and C show Southern hybridization of genomic fragments GLG1 to GLG6 with labeled CLG4 and CLG5, respectively. Note hybridization of CLG4 with only GLG1 (panel B) while CLG5 hybridizes with the other five clones GLG2 to GLG6 and only very faintly with GLG1 (panel C). (II) Restriction maps of LIP-encoding genomic fragments GLG1 to GLG6. The limits of the LIP gene in each fragment is indicated by a thick line. The transcriptional direction of the LIP gene in each fragment is denoted by an arrow under each gene. Abbreviations: B, BamHl; E, EcoRI; H, HindlII; K, KpnI; P, PstI; Sm, Smal; Sp, Sphl; SI, SstI; SII, SstlI; X, XhoI; Xb, Xbal. (From Zhang et al., 1991.)

102

gene is interrupted by eight or nine introns which range in size from 50 to 63 bp. A comparison of the positions of introns among the LIP genes showed that the last two introns are highly conserved in all the four genes. GLG2 and GLG6 are almost identical in the position of their introns except for a minor difference in the position of intron 7 (Fig. 4). The position of introns in GLG3 are almost identical to the corresponding introns in GLG2 and GLG6 except that one of the introns is missing in GLG3. The results also show that GLG1, which encodes the LIP protein H2, is much different from the other three genes in the position of its introns.

A comparison of the nucleotide homologies of the various LIP genes as well as the aa homologies of the LIP proteins encoded by these genes showed that GLG1 is very different from the other LIP genes in having a relatively low nucleotide homology of 71-73% and aa homology of 66-72% (see Naidu et al., 1990). In contrast to this, GLG2, GLG3, and GLG6 share relatively high nucleotide and aa homologies (80-82%). Furthermore, a comparison of the N-terminal sequences of the LIP proteins of P. chrysosporium, two each of T. versicolor and Bjerkandera adusta, and one of P. radiata showed that the N-terminal sequence of the H2 isozyme is remarkably more similar to the N-terminal sequences of the LIP proteins of the latter three organisms than it is to the N-terminal sequences of H8, H10, and GLG6 proteins of P. chrysosporium strain BKM-F or to those of two LIP proteins from P. chrysosporium strain ME 446 (Table 2). These results indicate that the H2-encoding gene constitutes a distinct subclass with in the LIP gene family and that genes related to H2 are more widely distributed in various genera of white-rot fungi. These results are also consistent with the other results pre- sented above which showed that the H10 isozyme forms a distinct subclass within the LIP family of proteins of P. chrysosporium.

G L G 1 ~ 2 3 4 5 6 7 8 ~ e m e

G t G ~ j 2 3 4 ~ 6 7 e 5 ...... ~ | ' = " - -

5 , G L G 2 i 2 3 4 5 6 ? e 9 _- _ . - - . , .

G L G 6 i z 3 4 ~ 6 7 8 9 5 . . . . -"

Fig. 4. A comparison of the positions of introns in four LIP genes of P. chrysosporium strain B~M-F. The genes GLGI, GLG3, and GLG2, respectively, encode the LIP isozymes H2, H8, and H10. The LIP

isozyme encoded by GLG6 has not yet been identified. (Modified from Naidu et al., 1990.)

103

TABLE 2

A comparison of the amino-terminal sequences of the lignin peroxidase proteins from different species of white-rot fungi

Lignin peroxidase a Amino-terminal sequence

P. chrysosporium H2 P. chrysosporium H8 P. chrysosporium H10 P. chrysosporium GLG6 P. chrysosporium LIG1 T. versicolor LIP A T. versicolor LIP B B. adusta LIP2 B. adusta LIP4 P. radiata LGP3

Val-Ala-Cys-Pro-Asp-Gly-Val-His-Thr-Ala-Ser-Asn-Ala Ala-Thr-Cys-Ser-Asn-Gly-Lys-Thr-Val-Gly-Asp-Ala-Ser Ala-Thr-Cys-Ser-Asn-Gly-Lys-Val-Val-Pro-Ala-Ala-Ser Ala-Thr-Cys-Ser-Asn-Gly-Ala-Thr-Val-Gly-Asp-Ala-Ser Ala-Thr-Cys-Ser-Asn -Gly-Ala-Thr-Val-Gly-Asp-Ala-Ser Val-Thr-?-Pro-Asp-Gly-Lys-Asn-Thr-Ala-Thr-Asn-Ala Val-Thr-?-Pro-Asp-Gly-Val-Asn-Thr-Ala-Thr-Asn-Ala Val-Ala-Cys-Pro-Asp-Gly-His-Asn-Thr-Ala-Cys-Asn Val-Ala-Cys-Pro-Asp-Gly-Val-Asn-Thr-Ala-Cys-Asn Ala-Thr-Cys-Pro-Asp-Gly-Thr-Gln-Leu-Met-Asn-Ala

a Lignin peroxidases H2, H8, H10, and GLG6 are encoded, respectively, by GLG1, GLG3, GLG2, and GLG6 of P. chrysosporium strain BKMF (Naidu and Reddy, 1989 and 1990; Naidu et al., 1990; Zhang et al., 1991) while LIG1 is encoded by the LIG1 gene of P. chrysosporium strain ME446 (Brown et al., 1988).

Six putative LIP genes were recently isolated from the white-rot fungus, Trametes versicolor (Black and Reddy, 1991). Sequence analysis of one of the genes (VLG1) has been completed and these data showed that it encodes mature LIP protein that contains 341 aa (M r = 36714) that is preceded by a 25 aa signal peptide sequence. This protein has a homology of 55-60% to the LIP proteins of P. chrysosporium. This is comparable to the homology found between the P. radiata and P. chrysosporium LIP proteins.

Transformation A dependable DNA transformation system is critical for the detailed molecular

genetic analysis of the lignin degradation system of P. chrysosporium. To this end, we described a protoplast transformation system for P. chrysosporium using a novel extrachromosomally maintained transformation plasmid, pG12-1 (Randall et al., 1991). This plasmid contains a kan r selection marker, pBR322 ori, and a 2.2 kb fragment (designated ME-l) derived from an endogenous plasmid (pME) of P. chrysosporium.

Plasmid pG12-1 transforms P. chrysosporium to G418 resistance and is main- tained in the fungal transformants in an extrachromosomal circular form. Southern blot analyses showed that pG12-1 is maintained at a low copy number in the fungal transformants as it is demonstrable in the total DNA of the individual G418-re- sistant basidiospore progeny of the transformants only after amplification of the pG12-1 sequences using a polymerase chain reaction. Dam methylation analyses and Exonuclease III digestion data, respectively, confirmed that pG12-1 does replicate in P. chrysosporium transformants and that it is maintained in an extrachromosomal circular form. Plasmid pG12-1 is maintained in the transfor-

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mants even after long term non-selective growth. We were able to show that pG12-1 does not integrate into the chromosome of the transformants and that it can be readily rescued from the total DNA of the transformants via E. coli transformation (Randall et al., 1991).

The pG12-1 transformation system described here is of considerable interest because such low copy extrachromosomal maintenance of the transforming plas- raids is quite rare in filamentous fungi. In fact, a careful examination of most of the previous reports on circular, extrachromosomal vectors in filamentous fungi showed that these vectors were either transiently extrachromosomal prior to integration into the chromosome or that these result from excision of the intact (or altered) form of the vector from the chromosome. It is also of interest that pG12-1, even at a low copy number, confers adequate G418 resistance to P. chrysosporium transformants. It is possible that the coenocytic nature of P. chrysosporium mycelia allows for sufficient cross feeding of the kan r gene product to allow survival in the presence of G418. The pG12-1 based transformation system should be of value not only for the molecular genetic analysis of the lignin degradation system of P. chrysosporium, but also as a useful model for studying the nature of maintenance of low copy extrachromosomal vectors in filamentous fungi. Further studies to improve the copy number of this plasmid and to understand the molecular mechanisms of this novel transformation system are in progress.

Alic et al. (1989) independently reported successful transformation of protoplas- ted basidiospores of a P. chrysosporium adenine auxoti'oph to prototrophy using a plasmid containing the gene for an adenine biosynthetic enzyme from another basidiomycete, Schizophyllum commune. The transformants were reported to be mitotically and meiotically stable. Southern blot analyses of the total DNA from the transformants showed that the transforming DNA was integrated into the chromosomal DNA in multiple tandem copies. Alic et al. (1989) further reported that the transforming DNA integrated into the chromosome at a non-homologous site, i.e. at a site other than the resident adenine biosynthetic gene.

Karyotyping The development of pulsed field gel electrophoresis as exemplified by Contour-

clamped homogeneous electric field electrophoresis (CHEF) and transverse alter- nating field electrophoresis (TAFE) has greatly facilitated resolution of large DNA molecules such as fungal chromosomes. Using both the CHEF and TAFE proce- dures, we have identified 10 chromosome bands in the total genomic DNA preparations of P. chrysosporium BKM-F strain which is known to be a het- erokaryote.

As discussed above, GLG1, which hybridizes to the CLG4 cDNA constitutes one subfamily of LIP genes while GLG2 to GLG6 which hybridize to cDNA CLG5 constitute a second subfamily. Probing of P. chrysosporium chromosome blots with labeled GLG1 and GLG2 showed that the LIP genes are distributed on at least two (perhaps three) chromosomes. Repeating these studies with P. chrysosporium ME446 gave similar results. Further studies using a wider range of probes including the MNP gene probes are in progress.

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Discussion

Considerable progress has been made in understanding the physiology and molecular biology of lignin peroxidases, but much remains to be done. The specific role(s) of individual LIP and MNP isozymes in lignin degradation and a study of the comparative biology of extracellular peroxidases from other important species of lignin-degrading basidiomycetes should be an important area for further study. Although several HzO2-producing enzymes have previously been identified, the relative contribution of each of these enzymes to the overall lignin degradation process is not known and requires further study. Studies with the lip and der mutants have yielded valuable insights about the physiology of LIPs and MNPs, but a much broader range of mutants will be needed to gain further understanding of the lignin-degrading enzyme system of P. chrysosporium. Further studies on the regulation of expression of LIP genes in response to various environmental stimuli are needed to obtain a better understanding of the role of LIP genes in the lignin degradation system of P. chrysosporium. Although both integrative and non-in- tegrative transformation systems have been described, additional research aimed at developing a transformation system that results in a homologous integration of the transforming DNA will be useful. For example, such a system will facilitate disruption of individual LIP genes and thus allow determination of the function of individual LIP isozymes in degrading lignin and related aromatic compounds. There should be a major emphasis on developing expression systems for the over production of active LIP isozymes for use in various industrial applications. This is not a trivial problem and little success has been achieved to date in obtaining expression of active enzymes in significant quantities. There is very little known about the molecular biology of the lignin degradation systems in fungi other than P. chrysosporium. Considering the fact that there are over 1600 species of wood-de- grading fungi known, it is important to investigate in detail the molecular genetic systems of at least a selected number of other white-rot fungi such as T. versicolor, P. radiata, and B. adusta to obtain a better understanding of the comparative biology of the lignin degradation systems in these organisms.

Acknowledgements

The research from the author's laboratory reported in this paper has been supported in part by the Michigan Agricultural Experiment Station, NSF Center for Microbial Ecology, and grants DE-FG02-85ER 13369 from the U.S. Depart- ment of Energy and 1-RO1-GM39032 from the NIH.

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