Recent advances on the molecular genetics of ligninolytic ... · Recent advances on the molecular...

ELSEVIER Journal of Biotechnology 53 (1997) 273-289

Recent advances on the molecular genetics of ligninolytic fungi

Daniel Cullen *

Institute for Microbial and Biochemical Technology, Forest Products Laboratory, 1 Gifford Pinchot Dr., Madison, WI 53705, USA

Received 7 March 1996; received in revised form 4 October 1996; accepted 8 October 1996

Abstract

This review highlights significant recent advances in the molecular genetics of white-rot fungi and identifies areaswhere information remains sketchy. The development of critical experimental tools such as genetic mappingtechniques is described. A major portion of the text focuses on the structure, genomic organization and transcrip-tional regulation of the genes encoding peroxidases, laccases and glyoxal oxidase. Finally, recent efforts onheterologous expression of lignin-degrading enzymes are discussed. © 1997 Elsevier Science B.V.

Keywords: Lignin degradation; White-rot fungi; Basdiomycetes; Peroxidases

1. Introduction

1.1. Microbial degradation of lignin

The white-rot basidiomycetes degrade ligninmore extensively and rapidly than any otherknown group of organisms. In contrast to otherfungi and bacteria, white-rot fungi are capable ofcompletely degrading lignin to carbon dioxide andwater. The species are widely distributed. occur-ring in tropical and temperate environments.White-rot fungi are also well adapted for utilizingother plant components and the species vary sub-stantially with regard to their relative cellulolytic

* Corresponding author. Tel.: + 1 608 2319468: fax: + 16082319488: e-mail: [email protected]

versus ligninolytic efficiency. The microbiology oflignin degradation has been reviewed (Kirk andShimada, 1985; Buswell and Odier, 1987; Kirkand Farrell, 1987; Eriksson et al., 1990).

The most extensively characterized white-rotfungus is Phanerochaete chrysosporium, previouslyknown as Chysosporium lignorum and Sporo-trichum pulverulentum (Burdsall and Eslyn, 1974;Raeder and Broda, 1984). Four classes of extra-cellular enzymes have been implicated in lignindegradation: lignin peroxidases (LiPs), manganeseperoxidases (MnPs), laccases and the H2O2-gener-ating enzyme glyoxal oxidase (GLOX).

Lignin peroxidase was first discovered based onthe H2O 2-dependent Cx -Cβ cleavage of ligninmodel compounds and subsequently shown tocatalyze the partial depolymerization of methy-

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274 D. Cullen /Journal of Biotechnology 53 (1997) 273-289

lated lignin in vitro (Glenn et al., 1983; Tien andKirk, 1983, 1984; Gold et al., 1984). Severalisozymic fo rms have been de t ec t ed i n P .chrysosporium cultures and a number of otherwhite-rot fungi, e.g. Trametes versicolor, Bjerkan-dera adusta, Phlebia radiata. The principle criteriafor identifying P. chrysosporium LiP isozymes aretheir pI and their order of elution from a Mono Qanion exchange column (Renganathan et al.,1985; Kirk et al., 1986; Leisola et al., 1987). Tenperoxidases are separated by Mono Q chromatog-raphy and are designated H1 through H10. Six ofthese, H1 (pI 4.7), H2 (PI 4.4), H6 (pI 3.7), H7 (pI3.6), H8 (pI 3.5) and H10 (pI 3.3) have veratrylalcohol oxidation activity characteristic of LiP(Farrell et al., 1989). Growth conditions (e.g. Nvs. C starved), purification methods and storagecan affect relative isozymic levels.

Lignin peroxidase catalyzes a variety of oxida-tions, all of which are dependent on H2O 2. Theseinclude Cx -Cβ cleavage of the propyl side chainsof lignin and lignin models, hydroxylation of ben-zylic methylene groups. oxidation of benzyl alco-hols to the corresponding aldehydes or ketones,phenol oxidation and even aromatic ring cleavageof non-phenolic lignin model compounds (Tienand Kirk, 1984; Leisola et al., 1985; Renganathanet al., 1985; Umezawa et al., 1986; Umezawa andHiguchi, 1987). Relative to other peroxidases,LiPs exhibit low pH optima ( < pH 3) and highredox potential. Comprehensive reviews on thebiochemistry of lignin peroxidase are providedelsewhere (Kirk and Farrell, 1987; Higuchi, 1990;Shoemaker, 1990; Hammel, 1992).

Another heme peroxidase first found in theextracellular fluid of ligninolytic cultures of P .chrysosporium is MnP (Kuwahara et al., 1984;Paszczynski et al., 1985). The enzyme is nowknown to be widely distributed among lignin-de-grading fungi (Orth et al., 1993; Hatakka, 1994).The principle function of MnP is to oxidize Mn2+

to Mn3 + using H2O 2 as oxidant. Activity is stim-ulated by simple organic acids which stabilize theMn 3 +, t h u s p r o d u c i n g diffusible oxidizingchelates (Glenn and Gold, 1985; Glenn et al.,1986). Physiological levels of oxalate in P .chrysosporium cultures have been shown to stimu-late MnP activity (Kuan et al., 1993). As with

LiP, the prosthetic group is iron protoporphrynIX and several isozymes can be detected(Paszczynski et al., 1986; Leisola et al., 1987;Mino et al., 1988; Wariishi et al., 1988). Consis-tent with the nomenclature used for the LiPisozymes (Farrell et al., 1989), specific MnPs iden-tified in cultures are H3 (pI = 4.9), H4 (pI = 4.5)and H5 (pI = 4.2) (Pease and Tien, 1992).

The role of MnP in lignin depolymerization hasbeen questioned because, in contrast to LiP, non-phenolic lignin is inefficiently depolymerized byMnP. However, the biomimetic oxidation oflignin model compounds by Mn3+ suggests that itmay play a role in oxidizing both phenolic andnon-phenolic residues of lignin (Hammel et al.,1989). Partial depolymerization of synthetic ligninby MnP has been demonstrated in vitro (Wariishiet al., 1991). Recently, MnP has been shown topromote peroxidation of unsaturated fatty acids.This lipid peroxidation system oxidatively cleavedphenanthrene (Moen and Hammel, 1994), non-phenolic synthetic lignin and a non-phenoliclignin model compound (Bao et al., 1994).

Both MnP and LiP are common in white-rotfungi. However, several efficient lignin-degradingspecies (e.g. Phanerochaete sordida, Ceriporiopsissubvermispora, Dichomitus squalens) produceMnP but apparently lack LiP activity (Perie andGold, 1991; Ruttiman et al., 1992; Orth et al.,1993; Ruttimann-Johnson et al., 1994). The mech-anism(s) of lignin degradation by these fungi isuncertain. Potentially unidentified enzyme mecha-nism(s) or a MnP-promoted lipid peroxidationsystem may play a role. Alternatively, LiP may beinvolved but concentrations are relatively lowand/or detection is complicated by interferingsubstances, particularly in woody substrates. Sup-porting the latter view, Orth et al. (1993) demon-strated the presence of inhibitors to LiP activity incolonized sawdust substrates. Also consistent witha role for LiP is the presence of LiP-like genes inthese ‘LiP-deficient’ species as shown by Southernblot hybridizations (Ruttiman et al., 1992). In anyevent, the failure to detect enzyme activity shouldbe viewed with caution. This point was recently,and unexpectedly, illustrated by a report of lac-case activity in P. chrysosporium cultures (Srini-vasan et al., 1995).

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Laccases are copper-containing enzymes whichreduce molecular oxygen to water and oxidizephenolic compounds. In the presence of certainmediators, non-phenolic compounds may be oxi-dized and this has generated considerable interestas an approach to the enzymatic bleaching ofkraft pulps (Bourbonnais et al., 1995). The possi-ble role of laccases in lignin degradation has beenrecently reviewed (Youn et al., 1995). Like theperoxidases, laccases are widely distributedamong ligninolytic basidiomycetes (Orth et al.,1993; Hatakka, 1994).

Several oxidases have been proposed as thesource of H2O 2 in peroxidative reactions butGLOX is the only oxidase secreted under stan-dard ligninolytic conditions (Kirk and Farrell,1987). The temporal correlation of glyoxal oxi-dase, peroxidase and oxidase substrates in cul-tures suggests a close physiological connectionbetween these components (Kersten and Kirk,1987; Kersten, 1990). Substrates for GLOX aresimple aldehyde-, x-hydroxycarbonyl-, and x-di-carbonyl compounds. Among these substrates isglucoaldehyde, which is produced by the action ofLiPs on lignin (Hammel et al., 1994).

A physiologically significant property of GLOXis its reversible inactivation in the absence of theperoxidase system (Kersten, 1990; Kurek andKersten, 1995). The enzyme is reactivated, how-ever, by reconstituting the complete peroxidasesystem, including both lignin peroxidase and non-phenolic peroxidase substrates. This suggests thatH2O2 supplied by glyoxal oxidase is responsive tothe demand of the peroxidases, thereby providingan extracellular regulatory mechanism for controlof the coupled enzyme systems. GLOX is pro-duced by several white-rot fungi (Orth et al.,1993), and two isozymic forms have been detectedin P. chrysosporium (Kersten and Kirk, 1987).

2. Molecular genetics

Knowledge concerning the molecular geneticsof white-rot fungi. particularly P. chrysosporium,has been advanced considerably over the pastdecade. Standard procedures have been estab-lished for auxotroph production (Gold et al.,

1982), recombination analysis (Alic and Gold,1985; Raeder et al., 1989a; Krejci and Homolka,1991; Gaskell et al., 1994), rapid DNA and RNApurification (Haylock et al., 1985; Raeder andBroda, 1985) and genetic transformation by aux-otroph complementation (Alic et al., 1989, 1990,1991, 1993) and by drug resistance markers (Ran-dall et al., 1989, 1991; Randall and Reddy, 1992;Gessner and Raeder, 1994). Aspects of the molec-ular biology of P. chrysosporium have been re-cently reviewed (Alic and Gold, 1991; Pease andTien, 1991; Cullen and Kersten, 1992; Gold andAlic, 1993; Reddy and D’Souza, 1994; Broda etal., 1996; Cullen and Kersten, 1996).

2.1. Gene structure

Soon after Tien and Tu (1987) first cloned andsequenced the P. chrysosporium cDNA encodingLiP H8, the cDNAs and genomic clones of severalstructurally-related genes were reported (de Boeret al., 1987; Asada et al., 1988; Brown et al., 1988;Holzbaur and Tien, 1988; Smith et al., 1988;Walther et al., 1988). The existence of allelicvariants complicated the identification of LiPgenes, but analysis of single basidiospore culturesallowed alleles to be differentiated from closely-related genes. Alic et al. (1987) showed that singlebasidiospores of the widely used strain BKM-F-1767 contain two identical haploid nuclei whichare the products of meiosis. The presence of spe-cific alleles in these haploid segregants could bedetermined by restriction polymorphisms (Schalchet al., 1989) or by allele-specific probes (Gaskell etal., 1992). It is now clear that P. chrysosporiumLiPs are encoded by a family of at least tenclosely related genes which have been designatedlipA through lipJ (Gaskell et al., 1994).

Recently, P. chrysosporium strain BKM-F-1767was shown to harbor a 1747 bp insertion withinLiP gene lipI (Gaskell et al., 1995). The element.Pcel, lies immediately adjacent to the fourth in-tron of lipI2 and features several transposon-likefeatures including inverted terminal repeats and adinucleotide (TA) target duplication. Southernblots revealed the presence of Pcel in other, butnot all, P. chrysosporium strains. Pcel is present atvery low copy numbers and lacks homology to

276 D. Cullen / Journal of Biotechnology 53 (1997) 273-289

known transposons or transposases. The elementis inherited in a simple 1:1 Mendelian fashionamong single basidiospore progeny. The distribu-tion of Pcel or other transposon-like elements inlignin degrading species is unknown.

Several LiP genes have been characterized fromother fungal species, including four Trametes ver-sicolor clones LPGI (Jonsson and Nyman, 1992),LPGII (Jonsson and Nyman, 1994), VLGI (Blackand Reddy, 1991), LPGVI (Johansson, 1994)),Bjerkandera adusta clone LPO-1 (Asada et al.,1992) and Phlebia radiata lpg3 (Saloheimo et al.,1989). On the basis of Southern blot hybridizationto the P. chrysosporium genes, LiP-like sequencesalso appear to be present in the genomes of Fomeslignosus (Huoponen et al., 1990), Phlebia brevis-pora and Ceriporiopsis subvermispora (Ruttimanet al., 1992).

Sequence conservation is high among the LiPgenes. Pairwise amino acid sequence comparisonsrange from 53 to 98.9% similarity (Table 1, se-quences labeled 1 – 16). Residues believed essentialto peroxidase activity are conserved, i.e. the prox-imal heme ligand (His204 in lipA) and the distalarginine (Arg71) and histidine (His75). The geneencoding isozyme H8. lipA, also features a puta-tive propeptide (residues 22–28; Schalch et al.,1989). A similar sequence was shown in the LiP2gene of strain OGC101 and the proenzyme wasidentified as an in vitro translation product (Ritchet al., 1991). Many of the P. chrysosporium genesfeature a proline rich carboxy terminus, althoughits significance is unknown. The P. chrysosporiumclones also contain 8–9 short introns and thepositions of six of these are highly conserved(Brown et al., 1988; Schalch et al., 1989; Ritchand Gold, 1992).

The crystal structure of LiP is now known(Edwards et al., 1993; Poulos et al., 1993). Moststriking is the similarity of the overall three-di-mensional structure to that of cytochrome c per-oxidase (CCP). even though sequence identity isonly approximately 20%. In both cases. the proxi-mal heme ligand is a histidine that is hydrogenbonded to a buried aspartic acid residue; theperoxide pocket is also similar with distal his-tidine and arginine. In contrast to CCP which hastryptophans contacting the distal and proximal

heme surfaces, LiP has phenylalanines. Further-more, the hydrogen bonding of the heme propi-onate of LiP to Asp (in contrast to Asn withCCP) may explain the low pH optimum of LiP(Edwards et al., 1993). Other notable features ofLiP are four disulfide bonds (CCP has none) anda C-terminal region without regular extended sec-ondary structure, for which there is no equivalentin CCP.

Relative to LiPs, less is known concerning thenumber and structure of the MnP genes. Fourdis t inct sequences have been reported: P .chrysosporium MP-1 (Pease et al., 1989), MnP-1(Pribnow et al., 1989), MnP-2 (Orth et al., 1994)and Trametes versicolor MPG1 (Johansson, 1994).The total number of MnP genes in any of thesespecies remains to be established. The N-terminalamino acid sequences of P. chry sospor iumisozymes (Datta et al., 1991; Pease and Tien,1992) indicate the existence of at least two moregenes.

Recently, a structurally unique Trametes versi-color peroxidase clone, PGV, was discovered(Jonsson et al., 1994). Overall, the sequence ismost closely related to LiPs, but certain residuesare characteristic of MnPs (Fig. 1). The signifi-cance of the PGV sequence is uncertain. No PGVencoded protein has been identified, nor is it clearwhether the gene is transcribed.

Multiple sequence alignments reveal conservedresidues which help distinguish mnps from lips(Fig. 1). Three residues adjacent to the proximalHis (e.g. Asp193, Trp199, Ala203 based on lipAnumbering) as well as Leu115 and Ile113 areinvariant in all LiP genes, but differ in MnPgenes. Useful residues, but less reliable, are thosepurportedly involved in Mn2+ b inding (Sun-daramoorthy et al., 1994) (e.g. Glu59, Glu63,Asp203 based on MP-1 numbering). Excluding T.versicolor clone MPGI, mnps can be distinguishedfrom lips by a 7-11 amino acid surface loop(Sundaramoorthy et al., 1994) (e.g. nos. 52-58 inMP-1) and an extended carboxy terminus. Thelatter insertion contains the fifth disulfide bond.not found in lips. Although T. versicolor MPGIlacks the insertions found on P. chrysosporiumgenes, the sequence clearly encodes MnP isozymeMP2 (Johansson, 1994).

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Glu63 (numbering based on P. chrysosporiumMP-1, Fig. 1) and a heme propionate (Sun-daramoorthy et al., 1994). Recent experimentalsupport for Asp203 in Mn2+ binding has beenprovided by site-directed mutagenesis of theresidue (Kusters-van Someren et al., 1995).

Like the peroxidases, laccases appear to beencoded by complex families of structurally re-lated genes. At least 17 fungal laccase genes havebeen cloned and sequenced. These include fourfrom Rhizoctonia solani (Wahleithmer et al.,1995), five from Trametes villosa (Yaver andGolightly, 1996; Yaver et al., 1996) two fromAgaricus bisporus (Perry et al., 1993) and singlegenes from Neurospora crassa (Germann et al.,1988), Coriolus hirsutus (Kojima et al., 1990),Cryphonectria parasitica (Choi et al., 1992), As-pergillus nidulans (Aramayo and Timberlake,1990), Pleurotus ostreatus (Giardina et al., 1995)and an unidentified basidiomycete (Coll et al.,1993). Overall sequence identity between certainpairs of fungal laccases may be low, but conserva-tion is high within regions involved in copperbinding (Fig. 2). Also, Yaver and Golightly (1996)noted conservation of the intron position in thelaccase genes of T. villosa and other ba-sidiomycetes. Three R. solani laccase genes eachcontain 11 introns at identical positions(Wahleithmer et al., 1995).

Glyoxal oxidase of P. chrysosporium is encodedby a single gene with two alleles (Kersten andCullen, 1993; Kersten et al., 1995). The deducedamino acid sequence lacks significant homologywith any other known proteins (Kersten and Cul-len, 1993). On the basis of catalytic similaritieswith Dactylium dendroides galactose oxidase, po-tential copper ligands were tentatively identified atTyr377 and His378 (Kersten and Cullen, 1993).The deduced amino acid sequences of allelic vari-ants differ by a single residue (Lys308 ++ Thr308),possibly explaining the two isozyme forms ob-served on isoelectric focusing gels (Kersten andKirk, 1987; Kersten, 1990).

2.2. Genomic organization

Meiotic recombination analysis in P. chrysospo-rium (Alic and Gold, 1985; Krejci and Homolka,

1991) has been useful for rudimentary identifica-tion of several linkage groups. While adequate formapping simple nutritional markers, the mulit-plicity of genes involved in the degradation oflignin and organopullutants are not amenable tosuch ‘classical’ genetic analysis.

Genetic linkage based on restriction fragmentlength polymorphisms (RFLPs) has been usefulfor identifying gene clusters and for creating re-combinant genotypes. Using a set of 53 ho-mokaryotic basidiospores and 38 RFLP markers,at least six linkage groups were recognized(Raeder et al., 1989a,b). Probes included ran-domly chosen clones, a cellobiohydrolase (CBH1)gene, and several clones containing LiP-like se-quences. Two clusters of LiP genes were identifiedand one of these was linked to the CBH1 gene(Raeder et al., 1989b). Lignin mineralization wasnot correlated with the segregation of any individ-ual RFLP marker (Wyatt and Broda, 1995).

In agreement with genetic linkage, analysis ofcosmid and λ libraries has provided detailed phys-ical maps of gene clusters. In P. chrysosporium,lipA and lipB are transcriptionally convergentand their translational stop codons separated by1.3 kb (Huoponen et al., 1990; Gaskell et al.,1991), lipC lies approximately 15 kb upstream oflipB (Gaskell et al., 1991). Clustering of CBHIgenes has also been shown in P. chrysosporium(Covert et al., 1992b). In T. versicolor, a MnP andtwo LiP genes are clustered within a 10 kb region(Johansson, 1994; Johansson and Nyman, 1996).The genes have the same transcriptional orienta-tion and they are separated by approximately 2.4kb. Three laccase genes of R. solani are alsoclustered and share the same transcriptional ori-entation (Wahleithmer et al., 1995).

Pulsed field electrophoresis has provided arapid means of localizing cloned genes to chromo-somes. In the dikaryotic P. chrysosporium strainBKM-F-1767, clamped homogeneous electricalfield (CHEF) gels reveal seven or more chromo-some bands. The ten known LiP genes are dis-tributed on three separate chromosomes. two ofwhich are dimorphic with respect to length(Gaskell et al., 1991, 1994; Stewart et al., 1992).Thus. eight lips, including the lipA, lipB, lipCcluster, have been localized to a dimorphic chro-

D. Cullen / Journal of Biotechnology 53 (1997) 273-289, 281

mosome of 3.5/3.7 mb (Gaskell et al., 1991). TheLiP gene encoding isozyme H2, lipD, was local-ized to another dimorphic pair of 4.4/4.8 mb(Stewart et al., 1992), along with the CBHI cluster(Covert et al., 1992a). In contrast to many of theCBHI and LiP genes, Southern blot analysis ofCHEF gels suggests that MnP and GLOX genesare unlinked to each other and to LiP genes (Orthet al., 1994; Kersten et al., 1995). Recently, CHEFgel blots have shown the five known T. villosalaccase genes to be distributed on three separatechromosome bands (Yaver and Golightly, 1996).

Although convenient, pulsed field gel blots havedisadvantages. The common occurrence of dimor-phic chromosomes necessitates the simultaneousanalysis of parent dikaryon and several ho-mokaryotic progeny. Electrophoresis run timesare often quite long ( > 5 days) and several chro-mosomes co-migrate. Resolution is poor as mostbasidiomycete chromosomes are quite large,greater than 1000 kb.

Genetic linkage relationships in P. chrysospo-rium have been further refined by monitoring thesegregation of specific alleles in single ba-sidiospore progeny (Gaskell et al., 1994). Themethod involves PCR amplification of genomicDNA from single basidiospore cultures and thenprobing with allele-specific oligonucleotides. Us-ing these techniques, the ten LiP genes of P .chrysosporium were mapped to three separatelinkage groups. Entirely consistent with pulsedfield gel blots. no lips were linked to mnps or glx,and lipD was distantly linked to a CBHI cluster.Eight lips, including the lipA, lipB, lipC cluster,were closely linked. Four genes, lipG, lipH, lipIand lipJ, showed 100% cosegregation. This geneclustering was recently confirmed by cosmid map-ping (unpublished data). An integrated physicaland genetic linkage map is shown in Fig. 3.

Preliminary data suggest a relationship betweengenetic linkage of LiP genes and their intron/exonstructure. Gold and coworkers delineated foursubfamilies on the basis of intron conservation(Ritch and Gold, 1992; Gold and Alic, 1993). TheLiP genes unlinked to all others, lipD and lipF,were also assigned to separate subfamilies. Not allgenomic lip clones have been sequenced and untilthe intron structure of all LiP genes is determined

it remains to be seen if the relationship betweengenomic organization and gene structure holds.

2.3. Gene expression

Transcription of P. chrysosporium strain BKM-F-1767 LiP genes is dramatically modulated byculture conditions. Holzbaur and Tien (1988) ex-amined transcript levels of the genes encodingisozymes H8 (lipA) and H2 (lipD) by Northernblot hybridization. Under carbon limitation, lipDtranscripts dominated and lipA transcripts werenot detected. Under nitrogen limitation, lipA wasthe most abundant transcript and lipD expressionwas relatively low. Quantitative RT-PCR and nu-clease protection assays have been developed todetect all known LiP transcripts (Stewart et al.,1992; Reiser et al., 1993). The genes lipA, lipBand lipI were expressed at similar levels in bothC- and N-limited cultures. In contrast, lipC a n dlipJ transcript levels were substantially increasedunder N-limitation. Consistent with Holzbaur andTien (Holzbaur and Tien, 1988), lipD transcriptswere the most abundant under C-limitation. Arecent report suggests that nutrient nitrogen limi-

Fig. 3. Integrated physical and genetic map of P. chrysospo-rium dikaryon BKM-F-1767 (Gaskell et al., 1994). Approxi-mate chromosome size in mb is based on CHEF migration andshown on the vertical bar. Dimorphic chromosome pairs haveidentical shading. Genetic linkage, established by monitoringthe segregation of specific alleles in single basidiospore cul-tures, is presented on horizontal lines to the right. Thus, thecbh1-1, cbh1-2, cbh1-3 cluster shows 25% recombination withlipD, but their relative orientation is unknown. Positions ofmnp1, mnp2, cbh1-4 and cbh1-5 were established solely byCHEF blots. Four LiP genes (lipG, lipH, lipI, lipJ) show100% cosegregation.


tation may regulate LiP expression post-transla-tionally by heme processing (Johnston and Aust,1994), but this view has been contradicted by Li etal, (1994).

Strain variation plays an important role in tran-scriptional regulation of P. chrysosporium LiPgenes. In contrast to the above mentioned studieswith BKM-F-1767, lipD (= LIG5) transcripts ofstrain ME446 appear to dominate in N-limiteddefined media (James et al., 1992) as well as inmore complex substrates such as ball-milled straw(Broda et al., 1995). In strain OGC1O1, l i pE(= LG2) is expressed at highest levels (Ritch andGold, 1992).

Manganese peroxidase product ion in P .chrysosporium is dependent upon Mn concentra-tion (Bonnarme and Jeffries, 1990; Brown et al.,1990). Regulation by Mn is at the transcriptionallevel (Brown et al., 1990; 1991). Potential tran-scriptional control elements have been identified in5'-untranslated regions of MnP genes (Godfrey etal., 1990; Alic and Gold, 1991; Brown et al., 1993)and reporter gene constructions may be useful inconfirming these putative regulating elements(Godfrey et al., 1994). Pease and Tien (1992)showed differential regulation of MnP isozymes inC- and N-limited cultures. Each isozyme alsoresponded differently to Mn concentration (Peaseand Tien, 1992).

GLOX is also transcriptionally regulated in P.chrysosporium (Kersten and Cullen, 1993). Consis-tent with a close physiological relationship betweenGLOX and LiP, glx transcript appearance is coin-cident with those of lips and mnp (Stewart et al.,1992).

The regulation of laccase expression differs sub-stantially among species. Transcripts of P. radiatalaccase are readily detected under N-limited, ligni-nolytic conditions. In T. villosa, lcc1 is stronglyinduced by 2,5-xylidine addition to cultures, whilelcc2 transcript levels remained unchanged. In con-trast, Northern blots failed to detect lcc3, lcc4 andlcc5 transcripts under any conditions. Three R .solani laccases (lcc1, lcc2, lcc3) are transcribed atlow constitutive levels which can be further re-pressed by the addition of p-anisidine to cultures.However. R. solani lcc4 is expressed at muchhigher levels and induced by additions of p-ani-sidine. In R. solani, lcc1. lcc2. lcc3 are clustered

but are separate from lcc4 suggesting a relation-ship between genomic organization and transcrip-tional regulation.

No obvious relationship exists between genomicorganization and transcriptional control in P .chrysosporium. The eight closely linked LiP genesshow dramatic differences in their response tomedia composition. For example lipC resides lessthan 15 kb upstream of lipB, but lipB transcriptlevels remain unchanged in C- vs N-limitedmedium while lipC transcript levels are greatlyincreased in N-limited medium (Gaskell et al.,1991; Stewart et al., 1992). As a further example,glx is unlinked to peroxidase genes (Gaskell et al.,1994; Kersten et al., 1995) regardless of the closephysiological connections (Kersten, 1990; Kurekand Kersten, 1995) and coordinate transcription ofglx and lips (Stewart et al., 1992; Kersten andCullen, 1993). In contrast to the situation in P .chrysosporium, a cluster of two lips and one mnphave been detected in T. versicolor and all threegenes may be coordinately expressed under certainculture conditions (Johansson, 1994; Johanssonand Nyman, 1996).

Elucidating the role and interaction of individualisozymes in lignin degradation and organopollu-tant degradation requires the identification of spe-cific isozymes in complex substrates. Immunogoldlabeling experiments have identified LiPs andMnPs in P. chrysosporium colonized wood(Blanchette et al., 1989; Daniel et al., 1989, 1990)and Datta et al. (1991) partially purified MnP, LiP,and GLOX from colonized Aspen pulp. However,enzyme yields from solid wood samples are too lowfor precise isozyme identification.

Recently, quantitative RT-PCR has been ap-plied to lip expression in P. chrysosporium colo-nized soil during organopollutant degradation(Lamar et al., 1995). Transcript analysis in suchcomplex substrates has been made possible by theuse of magnetic capture techniques for the rapidpurification of poly(A) RNA. Initially designed forthe detection of lipA, lipC, lipD, lipE, cbh1-1 andcbh1-4 transcripts the methodology has been ex-tended to quantitative analysis of glx and allknown lip and mnp transcripts (Bogan et al.,1996a,b). To summarize, MnP genes are coordi-nately expressed and the patterns of LiP genetranscription are unlike those observed in definedmedia. Extreme sensitivity and specificity is pro-


vialed by RT-PCR, but caution is warranted astranscript and enzyme levels are not necessarilydirectly correlated.

Peroxidases are needed for a variety of bio-chemical investigations, but yields of purifiedisozymes from fungal cultures are low and het-erologous expression has been problematic. In E.coli, relatively low levels of aggregated LiP apo-protein are produced in inclusion bodies. At-tempts to recover and reconstitute active LiP fromE. coli have had limited success (reviewed byPease and Tien, 1991), although recently Whit-wam et al. (1995) developed techniques for recov-ery of active P. chrysosporium MnP isozyme H4from inclusion bodies. S. cerevisiae expressionsystems yield no extracellular and little or nointracellular apoprotein (Pease and Tien, 1991).Expression of the P. radiata LiPs in T. reesei hasbeen limited to transcripts even when placed un-der the control of the highly expressed and in-ducible cbh1 promoter (Saloheimo et al., 1989).

Baculovirus systems have been used to produceactive recombinant MnP isozyme H4 (Pease et al.,1991) and LiP isozymes H2 (Johnson et al., 1992)and H8 (Johnson and Li, 1991). Although yieldsare relatively low, baculovirus production may beuseful for experiments requiring limited quantitiesof recombinant protein, e.g. site specific mutagen-esis. In contrast, highly efficient secretion of activeP. chrysosporium MnP isozyme H4 has beendemonstrated in Aspergillus oryzae. Expressionwas under the control of the A. oryzae T A K Aamylase promoter and like the baculovirus sys-tem, addition of herein to the cultures increasedyields substantially (Stewart et al., 1996). Thesecreted MnP is fully active and the physical andkinetic properties of the recombinant protein weresimilar to the native protein.

A ‘homologous expression’ system, in whichmnp transcriptional control is placed under theglyceraldehyde-3-phosphate dehydrogenase pro-moter, temporally separates production of therecombinant protein from other peroxidases(Mayfield et al., 1994). The system has been suc-cessfully used in site-directed mutagenesis experi-ments (Kusters-van Someren et al., 1995).

In contrast to peroxidases, the heterologousexpression of fungal laccases has been straightfor-

ward. The A. oryzae TAKA amylase system hasbeen successfully used for the production of T .villosa and R. solani laccases (Wahleithmer et al.,1995; Yaver et al., 1996). The P. radiata laccasehas been efficiently expressed in Trichoderma ree-sei under the control of the T. reesei cbh1 pro-meter (Saloheimo and Niku-Paavola, 1991). TheC. hirsutus laccase gene was expressed in S. cere-visiae (Kojima et al., 1990).

Glyoxal oxidase is efficiently expressed in A.nidulans under the control of the A. niger glu-coamylase promoter (Kersten et al., 1995). Undermaltose induct ion, ful ly-act ive GLOX wassecreted by A. nidulans at levels 50-fold greaterthan optimized P. chrysosporium cultures. Sitespecific mutagenesis enabled production of recom-binant GLOX isozymes corresponding to the na-tive allelic variants. Several recent biochemicalinvestigations have been aided by A. nidulans-pro-duced GLOX (Hammel et al,, 1994; Kurek andKersten, 1995; Whittaker et al., 1996).

3. Conclusions

Microbial degradation of lignin plays a pivotalrole in carbon cycling. Ligninolytic fungi are alsocritical to emerging biotechnologies such asbiomechanical pulping (Wegner et al., 1991;Akhtar et al., 1992), enzymatic bleaching of pulps(Eriksson and Kirk, 1985; Eriksson et al., 1990;Bourbonnais et al., 1995), and organopollutantdegradation (Hammel, 1992; Lamar, 1992). Pro-gress has been made. but the genetics and physiol-ogy of lignin-degrading fungi is still poorlyunderstood.

A central unanswered question is the role ofindividual genes in the degradation of lignin and/or xenobiotics. Rapidly developing experimentaltechniques such as transcript analyses in complexsubstrates, gene replacements and heterologousexpression will help establish the importance ofspecific genes. In this regard, there continues to bea critical need for an efficient expression systemfor LiPs.

In addition to basic mechanism(s) of ligninoly-sis, P. chrysosporium is a model system for investi-gating eukaryotic genome organization. The


species offers interesting and challenging featuressuch as complex families of structurally relatedgenes, differential transcriptional regulationamong related genes. polymorphic chromosomesand transposon-like elements. A variety of power-ful experimental tools are available includingtechniques for physical/genetic mapping and fortransformation and gene replacement. By study-ing P. chrysosporium. fundamental questions re-lating to the mechanismsvariation can be addressed.

References

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