FEMS Microbiology Reviews 13 (1994) 137-152 © 1994 Federation of European Microbiological Societies 0168-6445/94/$26.00 Published by Elsevier

137

FEMSRE 00345

Physiology and peroxidases of

molecular biology of the lignin Phanerochaete chrysosporium

C. Adinarayana Reddy * and Trevor M. D'Souza

Department of Microbiology, Michigan State University, East Lansing, MI 48824-1101, USA

Abstract: The white-rot basidiomycete Phanerochaete chrysosporium produces lignin peroxidases (LiPs), a family of extracellular glycosylated heme proteins, as major components of its lignin-degrading system. Upto 15 LiP isozymes, ranging in M r values from 38000 to 43000, are produced depending on culture conditions and strains employed. Manganese-dependent peroxidases (MnPs) are a second family of extracellular heme proteins produced by P. chrysosporium that are also believed to be important in lignin degradation by this organism. LiP and MnP production is seen during secondary metabolism and is completely suppressed under conditions of excess nitrogen and carbon. Excess Mn(II) in the medium, on the other hand, suppresses LiP production but enhances MnP production. Nitrogen regulation of LiP and MnP production is independent of carbon and Mn(lI) regulation. LiP activity is also affected by idiophasic extracetlular proteases. Intracellular cAMP levels appear to be important in regulating the production of LiPs and MnPs, although LiP production is affected more than MnP production. Studies on the sequencing and characterization of lip cDNAs and genes of P. chrysosporium have shown that the major LiP isozymes are each encoded by a separate gene. Each lip gene encodes a mature protein that is 343-344 amino acids long, contains 1 putative N-glycosylation site, a number of putative O-glycosylation sites, and is preceded by a 27-28-amino acid leader peptide ending in a Lys-Arg cleavage site. The coding region of each lip gene is interrupted by 8-9 introns (50-63 bp), and the positions of the last two introns appear to be highly conserved. There are substantial differences in the temporal transcription patterns of the major lip genes. The sequence data suggest the presence of three lip gene subfamilies. The genomic DNA of P. chrysosporium strain BKMF-1767 was resolved into 10 chromosomes (genome size of 29 Mb), and that of strain ME-446 into 11 chromosomes (genome size of 32 Mb). The lip genes have been localized to five chromosomes in BKMF-1767 and to four chromosomes in ME-446. DNA transformation studies have reported both integrative and non-integrative transformation in P. cho'sosporium.

Key words." Lignin peroxidase genes; White-rot fungi; Phanerochaete cho,sosporium; Fungal transformation; Manganese-dependent peroxidase: Karyotyping; Gene regulation

Introduction

Lignin peroxidases (LiPs) are a family of extra- cellular heine proteins produced during sec- ondary metabolism by white-rot basidiomycetes

* Corresponding author.

and are believed to play an integral role in lignin biodegradation by these organisms (reviewed in [1-7]). The discovery of LiPs in the white-rot fungus Phanerochaete chrysosporium greatly ac- celerated the pace of research on lignin biodegra- dation by fungi and hundreds of papers have been published on LiPs in the last several years covering the various facets of research on these enzymes. LiPs catalyze hydrogen peroxide

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(H202)-dependent one-electron oxidation of a variety of lignin-related aromatic compounds, re- suiting in the formation of aryl cation radicals. These radicals undergo various non-enzymatic re- actions yielding a multiplicity of end-products. There is intense research interest in LiPs not only because of their importance in lignin biodegrada- tion but also because of their potential applica- tions in the detoxification of a broad range of environmental pollutants such as PCBs and diox- ins, in biopulping, and in the conversion of ligno- cellulosic materials to fuels and chemicals. A number of reviews have appeared covering the physiology, biochemistry, molecular biology, and applications of LiPs [1-7] and the reader is en- couraged to refer to these reviews for detailed information on the early research on these en- zymes. In this review, we have focused on the more recent developments on the physiology and molecular biology of LiPs, primarily in P. chrysosporium and to a lesser extent in other white-rot fungi.

Manganese-dependent peroxidases (MnPs) are a second family of extracellular peroxidases, pro- duced by P. chrysosporium and other white-rot fungi, which catalyze the H 202-dependent oxida- tion of Mn(II) to Mn(III) and the Mn(III) in turn oxidizes various phenolic substrates [5,6]. Little emphasis is given to the MnPs in this review except in relation to some physiological factors which regulate both LiP and MnP production.

Physiology

Studies in the early eighties in our laboratory demonstrated for the first time that a temporal relationship exists between ligninolytic activity and H20 2 production in P. chrysosporium; that H20 2 plays an integral role in lignin degradation; and that nitrogen and carbon concentrations in the medium regulate lignin degradation as well as H20 2 production in a similar fashion [6,8-11]. The idea that HzO 2 production was integral to lignin degradation was also supported by the fact that catalase, which breaks down H20 2, inhibited lignin degradation [12]. The ensuing search for HeO2-dependent enzymes involved in lignin

degradation by P. chrysosporiurn soon led to the discovery of LiPs in this organism [13,14]. Several reviews have been published recently on the bio- chemistry and physiology of these enzymes [1-7].

LiP isozymes LiPs in P. chrysosporium are a relatively large

family of isozymes with M r values ranging be- tween 38000 and 43000 and pl values ranging from 3.3 to 4.7 [15-25]. The LiP isozymes are generally purified from the concentrated extracel- lular culture fluids of P. ch~sosporium by proce- dures such as fast protein liquid chromatography (FPLC), high pressure liquid chromatography (HPLC), hydrophobic interaction chromatogra- phy, and gel filtration [15-25]. The number and relative abundance of the LiP isozymes identified in P. chrysosporium cultures varies from as low as 2 to as high as 15 depending on the culture conditions, purification procedures, and strains employed [18-25]. The heine proteins separated from extracellular culture fluid of P. chrysospo- rium BKMF-1767 (ATCC 24725) grown in de- fined low N medium containing dimethyl succi- nate (DMS) buffer have been arbitrarily desig- nated as H1, H2. . .H10, the 'H' referring to the fact that these are heine proteins. H1, H2, H6, H7, H8, and H10 were shown to have LiP activity [1], as typically assayed spectrophotometrically by measuring the oxidation of veratryl alcohol to veratraldehyde in the presence of H20 2 [23,24]. H3, H4, H5, and H9 were shown to have MnP activity [1], as measured by the oxidation of Mn(II) to Mn(III) in the presence of H202 [26].

Peptide mapping analysis of various LiP isozymes, using V8 proteolysis, showed the pres- ence of four shared peptide fragments, indicating that the LiP isozymes are related to each other [19]. These peptide mapping studies further showed that isozymes H1 and H2 fall into one class; H6, H7 and H8 belong to a second class; and isozyme H10 belongs to a third class. In agreement with these conclusions, Dass and Reddy [20] showed, based on a comparison of the N-terminal sequences (amino acid residues 1-10), that H2 forms a distinct subclass within the LiP isozyme family and that H6, H8 and H10 are closely related to each other although H10 ap-

139

peared to be less related to H8 than the H6 isozyme.

LiPs m other wood-rotting fungi Several reports have been published on the

LiP enzymes of Trametes cersicolor [27-32]. Jo- hansson and Nyman [31] reported 16 LiP isozymes and five MnP isozymes in T. uersicolor strain PRL 572. The LiP isozymes had p I values rang- ing from 3.1 to 3.7 and molecular masses ranging from 41 to 43 kDa. Six of the LiPs and three of the MnPs were characterized by amino-terminal sequencing, amino acid analyses, carbohydrate analyses, and peptide mapping [32]. Based on a comparison of the sequences of the first 35 amino acids in the N-terminal ends of the LiPs and MnPs of T. uersicolor, the degree of similarity among five LiPs was found to be about 80% and about 70% among three MnPs.

The presence of the LiP enzyme system in a number of other white-rot fungi including Bjer- kandera adusta [27], Phlebia (Ph.) radiata [33], Coriolopsis occidentalis [34], Chrysosporium pru- inosurn [27], Panus tigrinus [35], and Merulius tramellosus [28] has been reported. Three LiPs (H2, H3 and H4) with molecular masses of 43-47 kDa and isoelectric points of 3.4-3.5 were par- tially purified from the white-rot fungus Junghuh- nia separabilima, and these enzymes were re- ported to be closely related in their physical characteristics to the LiPs of P. chrysosporium, T. uersicolor and Ph. radiata [36]. Ferrer et al. [37] reported on the characterization of three major LiP isozymes (LIG-I, LIG-II, and LIG-III) from the ascomyete, Chrysonilia sitophila. The molecu- lar masses of LIG-I, LIG-II, and LIG-III were 68, 50, and 47 kDa, respectively. In contrast to the LiPs from P. chrysosporium, these LiPs did not show increased stability at higher pH values and were more stable at their optimum pH values of 3.0-5.0.

Regulation of LiP production Extracellular peroxidases in P. chrysosporium

are produced during secondary metabolism in response to environmental triggers such as nitro- gen (N) or carbon (C) starvation (reviewed in [1,6,7]). The production of LiPs and MnPs is

completely suppressed under conditions of excess N and C in the medium. In defined low-nitrogen liquid medium, MnP activity generally peaks on day 4, while the LiP activity peaks on day 6 [3,38].

Recent studies showed that Mn(II) levels in the medium exert a dramatic effect on the pro- duction of LiPs and MnPs by P. chrysosporium [39-41]. With 12 ppm Mn(II) in the medium, high levels of both LiPs and MnPs were produced while with 40-100 ppm Mn(II) in the medium little or no LiP production was seen but MnP levels were higher than those seen in the medium with 12 ppm Mn(II) [39,40]. In a medium with 0 ppm Mn(II), no MnPs were produced but high levels of LiP production was observed [39-41]. An independent study showed that Mn(II) was required for the transcription of the mnp genes [41].

The relative abundance of LiP isozymes in P. chrysosporium cultures varies considerably de- pending on the culture conditions. For example, major differences were seen in the FPLC profiles of LiPs in acetate-buffered static cultures grown in defined low N medium versus identical cul- tures grown in DMS-buffered cultures of P. chrysosporium BKMF-1767. H8, and to a lesser extent H2 and H10, are the dominant isozymes in DMS-buffered static cultures whereas major amounts of LiPs H2 and H6, and relatively minor amounts of H8 and H10, were produced in ac- etate-buffered static cultures [38]. Furthermore, a new LiP protein, designated HA, was seen in acetate-buffered cultures that was not seen in DMS-buffered cultures. Moreover, in acetate- buffered shaken cultures, the LiPs H2 and H6 were produced in much lower amounts than in identical cultures grown under static conditions [20]. Major amounts of LiP H2 were produced in carbon-limited, DMS-buffered cultures, while major amounts of LiP H2 and H8 were produced in an identical, but nitrogen-limited, cultures [42]. FPLC profiles of wild-type strain ME-446, grown in DMS-buffered cultures showed three major LiP peaks [43] while those of strain INA-12 (a derivative of ME-446), grown in glycerol-contain- ing medium, revealed up to nine LiP peaks [22]. These results collectively indicated that the pro- duction and abundance of different LiPs vary

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greatly with the strain and culture condition em- ployed and in turn suggested that the expression of different lip genes is regulated in response to different environmental signals. These results fur- ther indicated that the LiP H2 is an important isozyme, in that it is produced in substantial amounts under a broad range of environmental conditions [6].

Previous studies showed that LiP activity in nitrogen-limited cultures of P. chrysosporium reaches a peak around day 6 of incubation and declines sharply thereafter [6]. Our studies showed that this sharp decline in LiP activity is positively correlated with the appearance of an idiophasic extracellular protease activity [44], the appearance of which is triggered by glucose de- pletion in the medium. Addition of glucose to the culture starting on day 6 resulted in a dramatic decrease in protease level and concomitant stabi- lization of LiP activity. Our results further showed that this protease is synthesized de novo since the addition of cycloheximide to day 6 cultures re- sulted in negligible protease activity. More direct evidence for protease degradation of LiPs was obtained by demonstrating the degradation of LiPs when FPLC-purified LiP isozymes were in- cubated in vitro with the extracellular culture fluid showing high levels of idiophasic protease activity, while in an identical reaction mixture using boiled extracellular culture fluid, the LiP isoenzymes were stable. Additional studies on protease activity of P. chrysosporium showed that protease activity produced during primary growth and idiophase are distinct and that each can be resolved into several protease activity bands using native gel electrophoresis procedures (Dass, S.B., Dosoretz, C. and Reddy, C.A., unpublished data).

In order to study the contribution of LiP isozymes to lignin degradation by P. chrysospo- rium, we isolated a lip mutant of this organism that lacked the ability to produce LiP isozymes but produced a full complement of MnPs as well as the key H202-producing enzyme glucose oxi- dase [43]. The fact that a single ultraviolet (UV) light-induced mutation resulted in the loss of ability to produce LiP isozymes but not the MnP isozymes and that the revertants regained the ability to produce all the LiP enzymes suggested

global regulation of LiP production. The lip mu- tant exhibited only 12-15% of the lignin-degrad- ing ability ([~4C]synthetic lignin -~ 14CO~) of the wild-type strain. These data not only indicated for the first time that the LiPs play a dominant role in lignin degradation but also showed that a strain of P. chrysosporium which lacked LiP activ- ity could still degrade lignin, albeit to a minor extent. These conclusions were independently corroborated by later studies of Bonnarme and Jeffries [39] and Perez and Jeffries [45], who observed that LiP production is completely sup- pressed in the presence of high levels of Mn(II) and that only 10-11% of the lignin degradation occurs in such a medium, as compared to an identical medium with low levels of Mn(II) in which both LiPs and MnPs are produced. The above results further indicated that LiP produc- tion in P. chrysosporium is regulated differently, at least in some aspects, from MnP production and that LiPs play a dominant role in mineraliz- ing synthetic [14C]lignin in liquid cultures.

It has been well established that nitrogen star- vation favors production of LiPs and MnPs while nitrogen excess in the medium represses the syn- thesis of LiPs and MnPs and inhibits lignin degra- dation by P. chrysosporium [1,3,6]. We isolated a nitrogen-deregulated (der ) mutant of P. chryso- sporium strain ME-446 that produced LiPs and MnPs and degraded lignin under both N-limiting and N-non-limiting conditions [46]. Also, the der8-5 mutant produced upto four-fold greater LiP activity than the wild-type strain in low N medium. FPLC analyses of the extracellular cul- ture 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 [46]. Furthermore, in a nitrogen-rich medium the wild-type produced no LiP protein peaks while der8-5 produced L1 and L2, and a new LiP protein peak, designated LN [46]. Re- cent results on purification and N-terminal analy- sis of LN showed that it is distinct from L1, L2, and L3 of ME-446 (Boominathan, K. and Reddy, C.A., unpublished data). The der8-5 mutant, be- sides being nitrogen-deregulated for LiP produc- tion, was deregulated for MnP and glucose oxi- dase production as well. The availability of thc

A

WT

der 8-5

I Nitrogen Regulation I

I n d e p e n d e n t o f l ~ a r b o n Regulation]

LIP AND MNP PRODUCTION

Excess N Medium Excess C Medium (N - 72 raM; C- 56 raM) (C - 168 raM; N - 24 raM)

+

ManganeSeis Regulation ]

I n d e p e n d e n t o f N i t r o g e n Regulation

LIP AND MNP PRODUCTION

High N Medium Medium (N - 24~nM; C - 56 mM) (N - 2..4 raM; C - 56 mM)

Manganese (parts/10~ 0 12 100

LIP + +

MNP + +

Fig. 1. A schematic illustration of the interrelationships be- tween N, C, and Mn(lI) regulation of LiP and MnP produc- tion in P. chrysosporium. (A) Nitrogen regulation of LiP and MnP production is independent of carbon regulation. (B) Mn(ll) regulation of LiP and MnP production is independent of nitrogen regulation. See text for details. (From Van der

Woude et al. [47]).

der mutant has enabled us for the first time to investigate the question: Is a mutant that is deregulated for N control of LiP and MnP pro- duction, simultaneously deregulated for C and Mn(II) controls as well or is such a mutant, similar to the wild-type, still subject to C and Mn(II) control of LiP and MnP production? The results (Fig. 1) showed that nitrogen regulation of LiP and MnP production is independent of car- bon regulation as evidenced by the fact that der8-5

141

produces both LiPs and MnPs (as expected) in excess N medium; however, in excess carbon medium the LiP and MnP production by this mutant is completely suppressed. WT produces neither LiPs nor MnPs in these two media be- cause LiP and MnP production by it is known to be repressed in the presence of excess N or C [47]. The results further showed that Mn(II) regu- lation of LiP and MnP production is independent of nitrogen regulation in that LiP and MnP pro- duction by der8-5 is subject to Mn(II) regulation regardless of the fact whether it is grown in low N medium or in high N medium. These results indicated that N regulation of LiP and MnP production in P. chrysosporium is independent of C and Mn(II) regulation.

Kuwahara and Asada [48] isolated and charac- terized a nitrogen-deregulated mutant of P. chrysosporium ME-446 that produced high levels of LiPs and MnPs in both low and high nitrogen media, as compared to the wild-type, in less than 2 days of incubation (i.e. apparently during pri- mary metabolic phase). Orth et al. [49] isolated a mutant (PSBL-1) of P. chrysosporium BKMF- 1767 that produced LiPs and MnPs at 4-10-fold higher levels than those produced by the wild-type under nitrogen non-limiting conditions. Further- more, this mutant has been reported to produce LiPs and MnPs during primary metabolism. Ad- dition of low levels of Mn(II) to the medium increased MnP activity in the mutant, as in the wild-type; however, LiP levels were increased in the mutant but not in the wild-type. FPLC analy- ses showed that larger amounts of LiP isozyme H2 were produced in PSBL-1 than in the wild- type. These results indicate that regulation of LiP production in PSBL-1 is substantially different from that in der8-5.

Earlier studies showed that the transition from primary to secondary metabolism in filamentous fungi is positively correlated with a sharp increase in intracellular cAMP levels [50]. MacDonald and co-workers [51,52] showed an elevation in cAMP in cells grown under ligninolytic conditions (i.e. grown in low N medium) but not under non- ligninolytic conditions (i.e. grown in high N medium). In view of the above reports and the well-documented observation that LiPs and MnPs

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are produced immediately following the transi- tion of P. chrysosporium from primary metabolism to secondary metabolism [1,3,6], we investigated the involvement of cAMP in regulating the pro- duction of LiPs and MnPs in P. ch~sosporium. Our results showed that production of LiPs and MnPs was preceded by a sharp rise in intracellu- lar cAMP concentration [53]. Addition of the cAMP inhibitors atropine, theophylline, or his- tamine, at concentrations that had little effect on the growth of the cultures, resulted in a concen- trat ion-dependent drop in intracellular cAMP concentration and a concomitant inhibition of production of LiPs only or of both LiPs and MnPs. LiP production was generally more sensi- tive to the concentration in intracellular cAMP than was MnP production. At high intracellular cAMP concentrations, both LiPs and MnPs were produced; at intermediate cAMP concentrations, only MnPs were produced; and at low concentra- tions, neither LiPs nor MnPs were produced [53]. Northern blot analyses suggested that the cAMP inhibitors affect the production of LiPs and MnPs at the level of transcription.

Molecular biology

lip cDNAs of P. chrysosporium Zhang et al. [54] first isolated two distinct

types of LiP-encoding cDNAs from P. chrysospo-

t

rium BKMF-1767. This was followed by the com- plete sequencing of two cDNAs, CLG4 and CLG5, which encode, respectively, LiPs H2 and H10 [55]. Another cDNA, ML1, which encodes the LiP H8, was independently isolated and se- quenced by Tien and Tu [56]. These two studies revealed that the LiP isozymes H2, H8, and H10 are each encoded by a separate gene and that each lip gene encodes a mature protein contain- ing 344 amino acids (M r = 36 607); however, the M r values of the LiP isozymes have been re- ported to vary from 38000-43000 [19,20]. These observed differences in M r are attributable to differences in the glycosylation levels of the vari- ous LiP isozymes. Northern blots, using the LiP cDNAs as probes, suggested that LiP production is regulated at the transcriptional level [56,57]. The sequences of several other cDNAs that en- code variants of LiP H8 have also been published [58-6O].

lip genes of P. chrysosporium The isolation and characterization of LiP-en-

coding cDNAs soon led to the isolation of a number of the lip genes of P. chrysosporium strain BKMF-1767 (see [2,5,6]). By probing the BarnH1 genomic library of this organism with labeled lip cDNAs CLG4 and CLG5, six distinct LiP-encoding genomic fragments designated GLG1, GLG2 . . . . . and GLG6 were isolated by Zhang et al. [61]. The LiP-encoding regions of

Table 1

Features of the LiP genes/proteins of P. chrysosporium and other white-rot fungi "

Characteristic GLGI GLG2 GLG3 GLG6 LGI LG2 LIG1 VLG1 VLG2 LPG1 LPO-I LGP3 [57] [57] [62] [71] [63] [69] [64] [74] [76] [77] [79] [80]

LiP protein encoded H2 H10 H8 ? H8 H8 H8 VLG1 VLG2 C LPO-2 Llll Mature protein (aa) 344 344 344 344 344 343 343 341 341 346 349 337 Leader peptide (aa) 28 27 28 28 28 28 28 25 26 26 23 24 Propeptide cleavage site b K-R K-R K-R K-R K-R K-R K-R R-R R-R R-R R-R R-R N-glycosylation sites One Two One One One One One One Two One One One TATA&CAAT boxes + + + + + + + + + + + ? Number of introns 8 9 8 9 8 8 8 6 6 6 4 '~ Length ofintrons (bp) 50-63 50-62 50-60 50-57 50-60 48-58 50-59 49-64 55-68 53-80 55-59 ')

" The lip genes GLG1, GLG2, GLG3, and GLG6 are from P. chrysosporiurn strain BKMF-1767; LGI and LIG1 are from P. chrysosporium strain ME-446; LG2 is from P. chrysosporium strain OGC101 (a derivative of strain ME-446); VLGI, and VLG2 are from 7". cersicolor strain ATCC 12679; LPG1 is from T. cersicolor strain PRL 572; LPO-1 is from B. adusta strain IFO 5307; and cDNA LGP3 is from Ph. radiata Fr. strain 79. The references for the respective lip genes are given in brackets under each gene heading.

b Putative propeptide cleavage site. K, lysine; R, arginine.

GLG2 and GLG3 which, respectively, encode the LiP isozymes H10 and H8 [61,62] were se- quenced. Moreover, genomic sequences of a number of allelic and non-allelic variants of the gene encoding LiP H8 of P. chrysosporium BKMF-1767 and ME-446 have been published by several groups independently [63-69]. GLG1, which encodes the LiP isozyme H2, has also been sequenced (EMBL Accession No. X15599; [70]). Sequencing of GLG6 showed that it encodes a LiP protein which is different from H2, H8, and H10, but its protein product in cultures of P. chrysosporium has not yet been identified [71].

Cumulative data on the general features of the lip genes of P. chrysosporium and selected other white-rot fungi, and the proteins encoded by them are summarized in Table 1. The results show that the lip genes of P. chrysosporium encode mature proteins between 343 and 344 amino acids long. Each mature LiP protein contains one putative N-glycosylation site (except H10 which contains two such sites) and a number of putative O-glyco- sylation sites, and is preceded by a 27-28-amino acid leader peptide that ends in a Lys-Arg cleav- age site. These data are consistent with the ear- lier reports that LiPs are oligomannose-type gly- coproteins with one or more N-glycosylated sites and a number of possible O-glycosylation sites [19].

The number and relative positions of introns in different lip genes are presented in Fig. 2. The coding region of the lip genes of P. chrysosporium BKMF-1767 are interrupted by eight or nine rela- tively small introns ranging in size from 50 to 63 bp. The positions of the last two introns are highly conserved in the lip genes. The signal peptide coding region of each lip gene, except that of GLG1, contains an intron (Fig. 2). The intron pattern indicates the presence of three lip gene families in P. chrysosporium: GLG1 consti- tutes one family, GLG2 and GLG6 another fam- ily, and GLG3 a third family. These results are consistent with the results on peptide mapping and other data discussed above [19,20]. The num- ber of introns (eight) and their positions are well-conserved in the LG1 and LIG1 genes of strain ME-446 [63,64], and the LG2 gene of strain OGC101 [69] and are very similar to those

143

l 2 3 4 6 6 7 I I

c ~ . . . | . . _, _. 2

I 2 3 4 5 6 7 41 9 G/.AJ2 - • • • - • • " ' [

i a i i i l l l

! 2 3 4 $ 6 7 8 ( ; z ~ 3 - . . _. . . . .

I 3 J ,it 6 6 7 41 9 G / ~ m • • • m m m= _= -

m i i m i i

L a l ~ 2 J 4 5 6 r a • E m • • • i i m m m m m in

l 2 J 4 $ 6 7 8 L4J2 - i " " m m . l m i

m • • • m

~ a ,, s 6 v 8 LJGI . . : : : • - • -

I 3 3 ,4 S 6 ~[.,GI • • • • • • m m m • • mm

J 'm !m 6 V L G 2 ~. .2 E - • •

F 2 3 4 5 6 ~ 1 • • • • • •

! 2 3 4

z , ~ J - z I I ; •

Fig. 2. Relative positions of introns in the lip genes of P. chrysosporium and other white-rot fungi. See Table 1 for a description of the various lip genes and their sources. Introns are represented by numbered vertical boxes while exons are represented by thick horizontal lines. The leading sequence coding region is interrupted by the first intron in all the lip

genes except in GLG1 and LPO-1.

in GLG3 of strain BKMF-1767 [62]. This is to be expected since all these genes code for LiP en- zymes which are very similar to H8 and its vari- ants.

The codon usage in the H2, H8 and H10 genes is extremely biased in favor of codons rich in C / G residues [55,56,61,66,69,70]. The order of preference of the third (wobble) base within these genes is C > G > T > A. Codons CGG and G G G are not used in either H2 or H10, but CGG is used in H8. Similar tendency to avoid the usage of codons ending in A and to prefer codons ending in pyrirnidines, particularly C, in highly expressed genes has also been reported in Neu- rospora and Aspergillus [72]. Thus, the overall codon usage in the lip genes of P. chrysosporium is not much different from that of the highly expressed genes of other eukaryotes [72,73].

The existence of a pre-pro-form of lignin per-

144

oxidase was first suggested by de Boer et al. [55] based on the putative signal peptide processing region Val~S-Thr-Gln-Ala21-Ala 22 in the transla- tion product of LiP cDNA CLG4 (which encodes for the H2 isozyme) and the putative propeptide cleavage site Lys-Arg found at amino acid posi- tions 27-28. Similar signal peptide processing region (Ala~8-Ala-Glu-Gly-AlaZ2-Ala 23) and propeptide cleavage site Lys-Arg (amino acid po- sitions 26-27) were found in the translation prod- uct of CLG5 which encodes the H10 isozyme. More recently, Ritch et al. [60] demonstrated experimentally that the LiP2 protein of P. chrysosporium OGC101 was synthesized as a pre- proenzyme. In vitro transcription and translation analysis and processing by signal peptidase re- sulted in cleavage at a site following the Ala 21 giving a mature protein that is preceded by a 7-amino acid propeptide.

N-terminal sequence Earlier results based on the peptide mapping

experiments have shown that H1 and H2 are more closely related to each other, and that the isozyme H10, while less related to H2 than it is to H6 and H8, is different from the latter two isozymes [19]. Dass and Reddy [20] analyzed the first 10 amino acids of the N-terminal sequence of the LiP isozymes H2, H6, H8 and H10, and showed that the H6 N-terminal sequence has about 90% homology to that of H8, 70% to that of H10, and 20% to that of H2, indicating that the LiP isozyme H2 is much less related to LiPs H6, HS, and H10. These results together with the intron data presented above are consistent with the idea that the LiP family of isozymes in P. chrysosporium appear to consist of three subfami- lies: H1 and H2 constitute subfamily 1, H6 and H8 constitute subfamily 2, and H10 constitutes subfamily 3. That H2 is distinct among the LiP isozymes was further illustrated in a more recent paper by Ritch et al. [60]. These authors showed that the sequence Ala-Thr-Cys-Ser-Asn-Gly is ex- tremely conserved in the N-terminal ends of the mature LiP proteins H8 (and its variants ML1, ML4, ML5, 0282, LPOA, LPOB) and H10 of P. chrysosporium BKMF-1767, and H8 (and its vari- ants pLG1, Ligl, Lig2, Lig3, Lig4, L18) of ME-446

and OGC101. However, the N-terminal sequence of mature H2 protein is strikingly different from all the other isozymes mentioned above in having the sequence Val-Ala-Cys-Pro-Asp-Gly.

A comparison of the N-terminal sequences of the LiP isozymes of P. chrysosporium, 7". eersi- color, B. adusta, and Ph. radiata showed that the N-terminal sequence of H2 is somewhat 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 LiP isozymes of P. chrysosporium [7]. These results further indicated that the H2-encoding gene constitutes a distinct subclass within the lip gene family of P. chrysosporium and that genes related to H2 are more widely distributed in various genera of white-rot fungi.

Distribution of lip genes in other wood-rotting fungi Using the H2-encoding cDNA (CLG4) of P.

chrysosporium to probe the genomic library of T. t'ersicolor (strain ATCC 12679), which next to P. chrysosporium is the most widely studied white-rot fungus, Black and Reddy [74] isolated six putative lip genes, designated VLG1, VLG2 . . . . . and VLG6. The lip genes of T. uersicolor appear to be clustered in that VLG2, VLG3, and VLG4 were located on one clone, while VLG5 and VLG6 were located on another clone. The linked lip genes were separated by 1.5-2.0 kb of DNA. Clustering of the lip genes has also been reported for P. chrysosporium [68,75]; however, the tran- scriptional orientation of the linked genes in T. t'ersicolor was found to be unidirectional whereas in P. chrysosporium the transcription of the linked genes lpoA and lpoB (which code for H8 vari- ants) was reported to be in opposite directions [68].

Sequence analysis of the T. L, ersicolor lip gene (VLG1) showed that it encodes a mature LiP protein of 341 amino acids (Mr: 37000) that is preceded by a 25-amino acid leader peptide se- quence that ends in Arg-Arg cleavage site [74]. The VLG1 protein contains one N-glycosylation site with the general sequence Asn-Xaa-Thr/Ser . There are six introns (49-64 bp in length) in the VLG1 gene, as compared to the eight to nine introns observed in the lip genes of P. chrysospo-

I45

rium. A second lip gene, VLG2, from T. z'ersi- color has been sequenced. It is similar to VLG1, except that the leader peptide sequence encoded by it was 25 bp long, its six introns varied in length between 55 and 68 bp, and there were two putative N-glycosylation sites present in the pro- tein encoded by this gene [76]. Sequence analysis of the lip gene (LPG1) from T. cersicolor strain PRL 572 showed that this gene, similar to VLG1 and VLG2, contained six introns (53-80 bp) but encoded a LiP protein of 346 amino acids, rather than the 341 amino acids observed in the VLG1- and VLG2-encoded proteins [77]. The positions of the introns appear to be relatively well-con- served in all the three T. z'ersicolor lip genes characterized to date and the first intron is in the leader peptide coding region similar to that in genes encoding H8 (and its variants), H10 and GLG6 proteins of P. chrysosporium (Fig. 2). The mature proteins VLG1, VLG2, and LPG1 had a homology range of 57-61% to the LiP proteins of P. chrysosporium, which is comparable to the amino acid homology found between the LiP protein of Ph. radiata and P. chry, sosporium, and those of B. adusta and P. cho'sosporium (Table 11.

Multiple lignin peroxidases were shown to be produced by B. adusta in glucose-peptone medium [78]. One of these enzymes, LPO-2, was

purified and characterized (M~=41()00: p l = 4.2). Sequence analysis of eDNA LPO-1 and its corresponding gene LPO-I. which encodes kPO- 2. showed that the mature LPO-2 is 349 amino acids long and is preceded by a leader peptide of 23 amino acids that ends in ,'m Arg-Arg cleavage site [79], as reported above for the lip genes of 7". z'ersicolor. LPO-1 contains four introns (ranging in size from 55-59 bp) unlike the P. chr3"so.g~o- rium and T. z'ersicolor lip genes which contained eight to nine and six introns, respectively (Fig. 21. Sequences of two other cDNA clones. LPO-2 and LPO-3 were similar to those of LPO-1, except that LPO-3 had a leader pcptide ending in the proteolytic cleavage site Lys-Arg, similar to that found in the P. cho'sosporium lip genes (Ta- ble 1).

Saloheimo et al. [80] isolated and sequcnccd, from the white-rot fungus Ph. radiata, a cDNA (lgp3), which encodes a 337-amino acid LiP pro- tein and a leader peptide of 24 amino acids that ends in a putative proteolytic cleavage site: Arg- Arg. The lgp3-encoded LiP protein show.ed 58- 62% amino acid homology to the pub[ishcd hp sequences of P. cho'sosporium and other white-rot fungi (Table 2).

In view of the relative similarities in the N- terminal sequence of LiP H2 and that of the LiP proteins of other white-rot fungi, wc screened

Table 2

Nucleotide and amino acid homology among LiP genes/proteins of P. cho'sosporium and other whitc-rot ftmgi "

Amino acid homology (%)

GLGI GLG2 GLG3 GLG6 LGI LG2 LIGI VLGI VLG2 LPGI I.PO-I L.GP3 [57] [57] [62] [71] [63] [69] [64] [74] [76] [77] [Tt~] [8,0]

G L G I 66 71 72 71 70 7(1 61 62 64 ~~11 58 GLG2 71 82 82 82 78 79 57 58 6(1 57 58 GLG3 73 8(1 88 99 82 9(1 58 61 63 ~,l) (, 1 GLGO 71 79 82 88 81 88 58 60 61 5t~ 6() LG1 73 80 99 82 89 90 58 61 63 611 (12 LG2 74 79 85 78 85 84 57 59 61 58 611 LIGI 71 78 82 81 83 82 55 50 611 5 t) 58 VLGI 69 65 67 65 67 70 65 76 74 57 6() VLG2 67 66 67 67 67 69 66 79 86 58 62 LPGI 70 65 69 68 69 68 68 78 82 6{) 61 LPO-1 69 66 58 65 58 68 66 67 68 67 5t, ' .GP3 64 63 44 65 44 66 64 67 67 67 67

Nucleotide homology (%}

a See Table 1 for description of the various lip genes. The gene designations are italicized while the corresponding protein designations are not italicized. The relevant references are given in brackets under each protein heading.

146

species representing seven genera of white-rot fungi, and three genera of brown-rot fungi for the presence of sequences homologous to P. chrysosporium CLG4, which encodes LiP H2. Southern hybridization analyses using high-strin- gency hybridization conditions showed the pres- ence of CLG4 homology in the total DNA of Bjerkandera adusta, Fomes fomentarius, Gano- derma lucidum, Lentinus tigrinus, Lentinus edo- des, Merulius tramellosus, Phlebia brevispora and Pleurotus ostreatus (D'Souza, T.M. and Reddy, C.A., unpublished data).

Karyotyping More recently, we reported on the elec-

trophoretic karyotyping of P. chrysosporium, BKMF-1767 and ME-446 [81]. Using the trans-

verse alternating field electrophoresis (TAFE) procedure, the genomic DNA of wild-type BKMF-1767 was resolved into ten chromosomes ranging in size from 1.8 Mb to 5.0 Mb, amounting to a total genome size of about 29 Mb. The genomic DNA of wild-type ME-446, on the other hand, was resolved into eleven chromosomes, amounting to a total genome size of about 32 Mb (Fig. 3).

To determine the presence of lip gene homol- ogy on different chromosomes of P. cho,sospo- rium, cDNA probes CLG4 and CLG5 and gene probes GLG3 and GLG6 were used. These stud- ies showed tha( in BKMF-1767, CLG4 hybridized to chromosome I (5.0 Mb), while CLG5 hy- bridized to chromosomes IV (3.1 Mb) and V (2.7 Mb). The GLG3 probe hybridized to chromo-

A B Sc i 2 3 4 Sp 1 2 3 4

mb b

2 2 -

15 - 11 -

C D E 1 2 3 4 I 2 3 4 i 2 3 4

, -Iaumk v--

Fig. 3. Electrophoretic separation of P. chrysosporium chromosomes and Sourthern hybridization analysis using lip cDNA and gene probes. The lanes marked Sc and Sp in (A) contained, respectively, Saccharomyces cerevisiae and Schizosaccharomyces pombe chromosomal markers. In all of the panels, lanes 1 and 3 contained P. chrysosporium BKMF-1767 chromosomal DNA, while lanes 2 and 4 contained P. chr?,'sosporiurn ME-446 chromosomal DNA. The Southern blot of the gel shown in (A) was probed with the lip cDNA CLG4 (B); the lip cDNA CLG5 (C); a probe specific for the lip gene GLG3 (D); and a probe specific for the lip gene

GLGO (E). (From D'Souza et al. [81]).

somes I, IV and V, whereas the GLG6 probe hybridized to chromosomes IX (2.0 Mb) and X (1.8 Mb). In strain ME-446, CLG4 hybridized to chromosome I while the GLG6 probe hybridized to chromosomes IX and X, identical to the re- sults observed with BKMF-1767. However, the CLG5 probe gave a single 2.9-Mb hybridization band, designated chromosome IV(i), which is not present in BKMF-1767 (Fig. 3). Chromosome IV(i) was also seen in the densitometric analysis of the TAFE gel of strain ME-446 but not in that of strain BKMF-1767 [81]. The GLG3 probe hy- bridized to chromosomes I and IV(i) of ME-446. These results indicate that the lip gene homolo- gies are present on five chromosomes in wild-type BKMF-1767, and on four chromosomes in wild- type ME-446.

Gaskell et al. [82] independently reported on resolving seven chromosomes in their strain of P. chrysosporium BKMF-1767. Two of these chro- mosomes were claimed to be doublets, based on the relative intensity of ethidium bromide stain- ing. The lip genes LiPA, LipB, 0282, and V4 (genes that code for variants of H8) and GLG5 (which is claimed to be a variant of a gene coding for H10) were reported to be clustered on one dimorphic chromosome (3.5 Mb and 3.7 Mb); of these genes, GLG5, LipB and LipA were re- ported to be linked within a 30-kb region. A subsequent report by the same laboratory [83] localized lip gene GLG4 (reported to be similar to GLG1) to a second dimorphic chromosome, with sizes 4.4 Mb and 4.8 Mb. Similar results were claimed for strain ME-446 of P. chrysospo- rium, but no data were provided. Thus, these results are different from our findings on the localization of the lip genes to five chromosomes in BKMF-1767 and to four chromosomes in ME- 446.

Temporal expression of the lip genes A number of cultural parameters, including

carbon, nitrogen, and Mn(II), are known to affect the amounts and types of LiP isozymes produced by P. chrysosporium [1,6,19,53], and the regula- tion of expression of LiP activity occurs at the transcriptional level [56,57]. However, there has been no information to date on the temporal

147

patterns of expression of the individual lip genes due to the unavailability of gene-specific probes. We recently constructed probes that are specific to the major lip genes GLG1, GLG3, and GLG2, which encode the LiP isozymes H2, H8, and H10, respectively, and the transcript levels were deter- mined from day 2 through day 7 in cultures grown in low nitrogen medium. Transcripts of H2-encoding GLG1 peaked on days 4 and 5, dropped to a relatively low level on day 6, and peaked again on day 7, whereas the H8-encoding GLG3 transcripts peaked on day 4, fell to ap- proximately one-third the peak level on clay 5, and continued to be maintained at this level through day 7. Transcripts of H10-encoding GLG2, on the other hand, peaked on day 4, fell to very low levels on day 5, and were unde- tectable in 6- and 7-day-old cultures (Boomin- athan, K., Naidu, P.S., D'Souza, T.M. and Reddy, C.A., Abstr. Annu. Mtg. ASM, No. K-145, p. 280, 1992). These results indicated substantial differ- ences in the temporal transcription patterns of the major lip genes of P. ch~sosporium.

Transformation A dependable DNA transformation system is

critical for the detailed molecular genetic analysis of the lignin degradation system of P. chrysospo- rium. We described a transformation system for P. chrysosporium using a novel extrachromosoma- lly maintained transformation plasmid, pG12-1, which contains a kanamycin-resistant (kan r) se- lection marker, pBR322 origin of replication (ori), and a 2.2-kb fragment (designated ME-l) derived from an endogenous plasmid (pME) of P. chrys- osporium [84]. Plasmid pG12-1 transformed P. chrysosporium to G418 resistance and was main- tained in the fungal transformants in an extra- chromosomal circular form at a low copy number. Dam methylation analyses and Exonuclease III digestion data, respectively, confirmed that pG12- 1 does replicate in P. chrysosporium transfor- mants and that it is maintained in an extrachro- mosomat circular form, even after long-term non-selective growth. Also, pG12-1 could be readily rescued from the total DNA of the trans- formants via Escherichia coli transformation [84].

148

The pG12-1 transformation system described [84] is of considerable interest because low-copy extrachromosomal maintenance of the transform- ing plasmids is quite rare in filamentous fungi. In fact, similar studies in other filamentous fungi showed that these types of vectors were either transiently extrachromosomal prior to integration into the chromosome or were the result of exci- sion of the intact (or altered) form of the vector from the chromosome. In contrast to these find- ings, we never observed the integration of pG12-1 into the chromosome of P. chrysosporium.

In addition to the rescue of pG12-1 from the total DNA of G418-resistant pGl2-1 transfor- mants of P. chrysosporium, we could also recover, albeit at a low frequency, a smaller plasmid, designated p12-6. We investigated the following questions: (i) Is the transforming plasmid p12-6 maintained in a true autonomous form in P. chrysosporium or is it maintained as part of a larger extra-chromosomal element? (ii) Does the recA allele of the E. coli strain, which is used to rescue the transforming plasmid p12-6 from the total DNA of fungal transformants, have an ef- fect on the structure of the plasmid recovered? (iii) Is the transforming plasmid p12-6, with a lacZ gene cloned into it, potentially useful as a shuttle vector? Based on the results obtained, the following mechanistic model for the establish- ment of transforming plasmids in P. chrysospo- rium was presented [85]. When p12-6 is trans- formed into P. chtysosporium, it initially recom- bines with an endogenous plasmid pME and the recombinant plasmid p12-6 : pME undergoes replication in P. chtysosporium transformants. During recovery in E. coli, this recombinant plas- mid is resolved into the original pl2-6 and a new plasmid, p511, in recA- E. co# strains, whereas only p12-6 is recovered in recA + E. coli strains. Several at tempts to clone the entire pME in E. coli have been unsuccessful to date, probably because a region of pME serves as a substrate for the recA gene product.

To test the usefulness of p12-6 as a P. chr),sosporium-E, coli shuttle vector, we cloned the lacZ gene of pUC19, which encodes the c~-peptide of /3-galactosidase and complements the appropriate lac deletion strains, such as E.

coli DH5a , into p12-6. The recombinant plasmid, pSV7, was then shown to transform P. chrysospo- rium to G418 resistance and, when the' total DNA from these G418-resistant transformants was used to transform E. coli DH5a , blue colonies con- taining pSV7 were obtained. These results indi- cated that lacZ gene can be transferred intact through P. chrysosporium.

Recently, we disrupted the lip gene GLG1, that encodes the LiP protein H2, by inserting into its coding region the kanr determinant of Tn903 [86]. The resul t ing r e c o m b i n a n t plasmid, pUGLGl-kan was transformed into P. ch~'sospo- rium with the expectation that the disrupted lip gene in the transforming plasmid might integrate into the homologous site in the chromosome. The results, however, showed that pUGLGl-kan se- quences do not integrate into the chromosome. Instead, the plasmid was maintained intact as a circular extrachromosomal element and was res- cued intact in E. coli. Our data also showed that pUGLGl-kan undergoes replication in P. chrys- osporiurn, is maintained as a circular element in a relatively high copy number as compared to the pG12-1 plasmid, and is recoverable from meiotic and mitotic progeny although at a low frequency. These results lead us to the conclusion that the GLG1 component of pUGLGl-kan contains as yet unidentified sequences that allow it to au- tonomously replicate in P. chrysosporium trans- formants. It is of interest that integrative trans- formation is not observed with pUGLGl-kan, as is commonly observed with most other fungal transformation systems [87], in spite of the fact that this plasmid contains 4.1 kb of P. chryso- sporium chromosomal DNA.

Alic et al. [88] reported the successful transfor- mation of a P. chrysosporium adenine-requiring auxotroph, ade2, to prototrophy using a p[asmid (pADE2) containing the gene (ade2) for an ade- nine biosynthetic enzyme from Schizophyllum commune [89]. The transformants were reported to be mitotically and meiotically stable on both selective and nonselective media and the trans- forming DNA was shown, by Southern blot analy- ses, to be integrated into the chromosomal DNA in multiple tandem copies, at a site(s) other than the ade gene. Similar results were obtained in a

second study [90], in which another P. chrysospo- rium adenine-requiring auxotroph, adel, was t ransformed with a plasmid (pADE5) containing the ade5 gene from S. commune [89]. In this latter study, however, plasmid rearrangements and deletions among a few of the t ransformant were observed. In a subsequent study Alic et al. [91] reported homologous transformation of the P. chrysosporium adel auxotroph, using an adel gene which was cloned as a 6.0-kb fragment into pUC-18 (pADE1). The transformants were mitot- ically and meiotically stable, and the plasmid integrated in single or multiple copies. Further- more, these transformants grew faster, suggesting that the homologous adel may be functioning more efficiently than ade5 of S. commune [91].

Concluding remarks

There have been significant advances in the past few years in understanding the physiology and molecular biology of lignin peroxidases. The specific role of individual LiP and MnP isozymes in lignin degradation remains a mystery but with the recent availability of appropriate physiologi- cal and molecular biological tools, this area is likely to progress significantly in the coming years. Also, substantial contributions have been made in identifying and characterizing lignin peroxidase enzyme systems of white-rot fungi other than P. chrysosporium, T. versicolor, B. adusta and Ph. radiata, and this area will probably advance rapidly in the coming years. Great progress has been made in isolating and characterizing g e n e s / cDNAs encoding the LiP isozymes of P. chrysosporium and several other species of white- rot fungi. Studies to date show many broad simi- larities among the structural organization of the lip genes of various white-rot fungi although some differences in detail have been observed. Success- ful karyotyping of P. chrysosporium has allowed the determination of lip gene distribution on the chromosomes. Cumulative data to date indicate lip gene clustering both in P. chrysosporium and T, versicolor. Progress is also being made in un- derstanding the regulation of expression of lip genes but a great deal more research is needed in

149

this area. Regulation of lip gene expression in response to various environmental triggers will be an area of particular interest. Some progress has been made in our understanding of both non-in- tegrative and integrative transformation systems but much remains to be done. Lack of significant progress in developing expression systems for the over-production of LiPs has been disheartening but this area is likely to advance significantly with an increase in our basic knowledge about the structure, organization, and regulation of lip genes and the transformation systems in the com- ing years.

Acknowledgements

The research from the author 's laboratory re- ported here was supported in part by D O E grant DE-FG02-85ER 13369, NIH grant RO1-GM 39032, and Michigan Agricultural Experiment Station.

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