The Role of Lytic Polysaccharide Monooxygenases in Wood Rotting Basidiomycetes
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GLYCOREVIEW Trends in Glycoscience and Glycotechnology
Vol. 32 No. 188 (July 2020) pp. E135–E143
doi: 10.4052/tigg.2020.7E
Japanese Edition: doi.org/10.4052/tigg.2020.7J
The Role of Lytic Polysaccharide Monooxygenases in
Wood Rotting Basidiomycetes
1 2 2 3
Yuka Kojima ; Anikó Várnai ; Vincent G. H. Eijsink ; and Makoto Yoshida
1
Institute of Global Innovation, Tokyo University of Agriculture and Technology, Fuchu, Tokyo 183–8509, Japan
2
Faculty of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences, 1432 Ås, Norway
3
Department of Environmental and Natural Resource Science, Tokyo University of Agriculture and Technology,
Fuchu, Tokyo 183–8509, Japan
TEL: +81–42–367–5252, E-mail: fu5654@go.tuat.ac.jp
(Received on May 15, 2020, accepted on June 1, 2020)
Key Words: wood decay, brown-rot fungi, chelator-mediated Fenton (CMF) reaction,
lytic polysaccharide monooxygenase, LPMO9
Abstract
The group of filamentous fungi called wood rotting fungi comprises the main decomposers of woody biomass in forest ecosys-
tems. These wood rotting fungi secrete various extracellular enzymes, such as cellulases and lytic polysaccharide monooxygenases
(LPMOs) to degrade cellulose in wood cell walls. Interestingly, one particular group of wood-rotting fungi, called brown-rot fungi,
lacks key cellulases for degrading crystalline cellulose, namely cellobiohydrolases (CBHs), with only a few exceptions. On the other
hand, genes encoding LPMOs are widely conserved among brown-rot fungi, suggesting the importance of these enzymes in the
brown-rot system. In this paper, after reviewing the wood degradation process by wood rotting fungi, we describe the history of the
discovery of LPMOs and then review current knowledge on the characteristics of these enzymes. We then review our research on
LPMOs derived from a brown-rot fungus and discuss possible physiological roles of LPMOs in the brown-rot system. Finally, we ad-
dress the significance of LPMOs in the evolution of brown-rot system.
A. Introduction all wood cell wall components such as lignin, cellulose, and hemi-
Plant-degrading filamentous fungi play an important role as cellulose, although to various extent (3). On the other hand, brown-
decomposers in forest ecosystems. They can degrade plant cell rot fungi preferentially degrade cellulose and hemicellulose with-
wall polysaccharides, such as cellulose and hemicellulose, using out depolymerization of lignin (4). During brown rot, the chemical
various extracellular enzymes, and the degradation products can structure of lignin is partly modified via demethylation, β-O-4
be used as nutrient source. While most filamentous fungi degrade ether cleavage, and side chain oxidation (5–7). These composition-
herbaceous plants well, only a limited number of fungi can degrade al differences affect the physical properties of decaying wood. The
woody plants. Plant biomass-degrading fungi with a high ability strength, especially the bending strength, of brown-rotted wood
to degrade woody plants are referred to as wood rotting (or wood is significantly reduced at an early stage of the decay process (8),
decay) fungi, and the majority of them belong to the class Agari- probably because cellulose and hemicellulose have been rapidly
comycetes in the subphylum Agaricomycotina and phylum Basid- degraded (9, 10). In wood undergoing white-rot decay, on the other
iomycota (1, 2). Wood rotting basidiomycetes are important not hand, the reduction of bending strength is known to be more mod-
only in the global carbon cycle but also because they are a major erate (8).
contributor to the decay of wooden buildings. Furthermore, the en- There are also microscopic differences in the morphologi-
zymes produced by these fungi may be exploited in the industrial cal characteristics of decayed wood. After their hyphae penetrate
conversion (saccharification) of woody biomass. the lumen of wood cell walls, white-rot fungi start degrading the
cell wall near their hyphae, attacking the innermost, so-called S3
B. Wood Rotting Basidiomycetes and Their Mecha- layer of the secondary cell wall. On the other hand, it has been
nism for Wood Degradation suggested that brown-rot fungi initially degrade the intermediate
Wood decay by wood rotting basidiomycetes is usually classi- S2 layer (which is located just under the S3 layer) of the second-
fied into two different decay types, white-rot and brown-rot, based ary cell wall, without any remarkable structural changes of the
on the appearance of the decayed wood, and the causative fungi are S3 layer (11–13) (Fig. 1). It is understood that such differences in
called white-rot fungi and brown-rot fungi, respectively (1, 2). In morphological characteristics are due to the different degradation
addition to appearance, the chemical composition of the decayed mechanisms. Since enzymes are larger than the size of the micro-
wood varies between the two decay types. White-rot fungi degrade pores in the S3 layer (14, 15), it makes sense that plant cell wall is
E135 © 2020 FCCA (Forum: Carbohydrates Coming of Age)
to lignin degradation in conjunction with other enzymes, and these
PODs are often considered as the driving force behind the high
lignin-degrading ability of white-rot fungi (2, 18). White-rot fungi
also possess a plethora of enzymes that degrade plant cell wall
polysaccharides such as cellulose and hemicellulose (18). Cellu-
lolytic enzymes include well-known hydrolytic enzymes, such as
cellobiohydrolases (CBHs), which are considered key enzymes in
the degradation of crystalline cellulose, and endoglucanases (EGs),
which are thought to primarily act on amorphous regions in cel-
lulose, as well as redox enzymes such as Auxiliary Activity (AA)
family 9-type lytic polysaccharide monooxygenases (LPMO9s),
which oxidatively attack cellulose chains (2, 18, 19). Some of
these enzymes have a carbohydrate-binding module belonging to
family 1 (CBM1) as an additional domain that promotes adsorp-
tion on cellulose (20). CBM1 modules have been shown to be in-
dispensable for the efficiency of CBH-type catalytic domains (i.e.,
processive cellulases) in depolymerizing crystalline regions of cel-
lulose (21–23). Importantly, while many of the above-mentioned
Fig. 1. Schematic overview of the woody plant cell wall and the enzymes occur in most plant-degrading filamentous fungi, the
CMF system employed by brown-rot fungi. (A) Schematic dia- majority of brown-rot fungi lack (or have much less) CBHs and
gram of plant cell wall and fungal hyphae. The woody plant cell wall
consists of three layers from the outside inward, namely the middle PODs, and at the same time also CBM1 (18) (Table 1). Neverthe-
lamella, characterized by high (up to 90–95%) lignin content, the pri- less, brown-rot fungi are known for their ability to rapidly depo-
mary cell wall, characterized by being rich in lignin as well as pectin- lymerize cellulose, in contrast to a more gradual degradation by
and xyloglucan-type hemicelluloses and structural glycoproteins, and
the secondary cell wall, which contains ca. 40–50% cellulose and white-rot fungi (24). It is possible that brown rot fungi have partic-
provides the plant with mechanical support. Secondary cell walls can ularly powerful endoglucanases that can rapidly depolymerize cel-
be further divided into three distinct layers based on the orientation of lulose (25, 26), but this remains to be documented. Most often, the
cellulose fibers, namely the S1, S2 and S3 layers. The S1 layer may be
viewed as a transition layer between the primary cell wall and the S2 ability of brown-rot fungi to rapidly depolymerize cellulose in the
layer, the S2 layer is the thickest layer of the cell wall and comprises absence of CBHs has been ascribed to the presence of a comple-
ca. 75% of the cell wall mass, and the S3 layer is the innermost layer mentary non-enzymatic redox system for biomass degradation (1,
of the cell wall, separating the S2 layer from the cell lumen (16). The
small grey circle in the cell lumen represents the fungal hypha pen- 4, 17, 27).
etrating the cell. (B) The principle of the proposed CMF system (4, In 1974, Koenigs proposed that hydroxy radicals generated
17). In the lumen, with low pH, the fungus produces H2O2, reducing by a chemical reaction between iron and hydrogen peroxide (Fen-
compounds and oxalic acid which can penetrate the cellulose-rich
S2 layer. Fe(oxyhydr)oxide complexes in the lumen and iron bound ton reaction: Fe2++H2O2→Fe3++·OH+OH−) could play a role
to wood components are bound to oxalic acid and will co-migrate in brown-rot biomass degradation (28). There were mainly two
3+
into the S2 layer. In the S2 layer, with higher pH, Fe becomes more reasons for this hypothesis. Firstly, it was noted that wood treated
soluble, is reduced and can react with H2O2 in a Fenton reaction.
with an artificial Fenton reaction and brown-rot decayed wood
both showed a rapid decrease in the degree of polymerization of
degraded from near the fungal hyphae if degradation is based on cellulose without weight loss (29). Secondly, it was noted that
using enzymes. Since brown rot fungi seem to initially attack the brown-rot fungi accumulate hydrogen peroxide, which is a key re-
middle layer distant from their hyphae and “behind” the barrier actant in the Fenton reaction, in their medium (28).
posed by the S3 layer, it has been proposed that they use low mo- Many studies have been conducted based on this hypothesis,
lecular weight compounds that can penetrate the cell wall, in what and current ideas about this brown-rot mechanism below are sum-
essentially is a chemical degradation process, as described below marized in Fig. 1 (4, 17). In this system, often referred to as the
in detail. Chelator-Mediated Fenton (CMF) mechanism, oxalic acid pro-
The different decay mechanisms are reflected in different en- duced by the fungus contributes to solubilization of insoluble iron
zymatic repertoires. White-rot fungi have genes encoding class II (oxyhydr)oxide in the lumen and iron bound to plant cell walls by
peroxidases (PODs), which degrade lignin directly or contribute creating acidic conditions around the hyphae, and forms a com-
© 2020 FCCA (Forum: Carbohydrates Coming of Age) E136
Table 1. Occurrence of genes encoding selected wood-degrading enzymes in fungal genomes.
Table 1 was created based on the JGI genome portal (38) and Floudas et al. (37). EG, endoglucanases (GH5, GH9, GH12, GH45); CBH, cello-
biohydrolases (GH6 & GH7); CBM1, cellulose binding domain appended to cellulolytic domain (GH5, GH6, GH7, GH12, GH45, AA9); POD,
class II peroxidases (AA2); CDH, cellobiose dehydrogenases containing both AA3 and cytochrome domains (AA8); LPMOs, lytic polysaccharide
monooxygenases classified in AA9, AA11, AA14 or AA16. *, lacking a Mn(II)-oxidation site; **, three of them lack catalytic residue(s).
plex with iron. This oxalate-Fe complex can diffuse through the regarding the diversity of degradative enzymatic systems in
micropores in the secondary plant cell wall where an increasing brown-rot fungi. As mentioned above, almost all brown-rot fungi
pH will weaken the interaction between Fe3+ and oxalate, allowing lack genes encoding PODs and CBHs (Table 1). As an excep-
subsequent reduction of the iron by reducing agent. As the reduc- tion, Coniophora puteana, Hydnomerulius pinastri and Serpula
ing agent for ferric ion, low molecular weight aromatic compounds lacrymans, all belonging to Boletales, have genes encoding CBH,
produced by brown-rot fungi (30) as well as lignin fragments have however, the putative CBHs from C. puteana lack a cellulose-
been suggested (31), although the true nature of the physiological binding domain, while transcription studies have shown that puta-
2+
reductant is still in discussion. The Fe ions can react with hydro- tive CBH-encoding genes from S. lacrymans are hardly, or not at
gen peroxide, which is readily produced by fungi (32), to produce all, expressed during wood decay (39). Some reports suggest that
hydroxyl radicals. EGs secreted by Fomitopsis palustris and Gloeophyllum trabeum
It has been reported that CMF occurs at a relatively early can processively degrade cellulose chains similar to CBH (25,
stage of the brown-rot process, generating pores in the second- 26), however, their role in degrading crystalline cellulose remains
ary cell wall through which enzymes can diffuse (17, 33, 34). It controversial. Regarding LPMOs, it is now clear that almost
has also been suggested that so-called selective white-rot fungi, all brown-rot fungi contain genes encoding AA9-type LPMOs
which preferentially degrade lignin, use a similar hydroxyl radical- (LPMO9s) (2). However, the enzymatic potential of these enzymes
generating system to degrade plant cell walls (35, 36). However, has hardly been addressed (40) until our group recently reported
in contrast to brown-rot fungi, white-rot fungi have a complete set a detailed functional study of a LPMO9 from G. trabeum, as de-
of enzymes that degrade all wood cell wall components (Table 1) scribed below.
(2, 18, 37). Therefore, CMF may be more important for brown-rot
decay, compared to white-rot decay. C. History and Function of LPMOs
Recent genome-wide analyses have generated new insight LPMOs, which are classified into AA families 9, 10, 11, 13,
E137 © 2020 FCCA (Forum: Carbohydrates Coming of Age)
14, 15, 16 in the Carbohydrate Active enZymes (CAZy) database lic acid are commonly used in experiments as an electron donor
(41), catalyze oxidative depolymerization of polysaccharides. So for LPMO9, AA8 cytochrome domains of cellobiose dehydroge-
far, cellulose, other β-glucans such as xyloglucan, chitin, starch, nase (CDH; domain composition AA8-AA3_1-CBM1) and PQQ-
and xylan have been reported as substrates of LPMOs (42). dependent pyranose dehydrogenase (domain composition AA8-
LPMOs belonging to family AA9 (LPMO9) are among the most AA12-CBM1) can fulfill the same role and may perhaps do so in
studied LPMOs as this type of enzyme is conserved in many cellu- nature (43, 45, 56, 57), although it is still in discussion. In addition,
lolytic filamentous fungi. The first LPMO9s that oxidatively cleave lignin-derived compounds and other phenolic compounds derived
β-1,4-glucosidic bonds in cellulose were reported in 2011 (43–46). from fungi or wood, as well as certain glucose-methanol-choline
Because it had been thought that only hydrolytic enzymes such as oxidoreductases (AA3) have been reported as potential electron
EGs and CBHs were involved in the degradation of cellulose by donors (57–59). Oxidative cleavage of the β-1,4-glucosidic bond
filamentous fungi, the discovery of LPMOs had a great impact on in cellulose by LPMO9s may, depending on the LPMO, lead to
research in the field, both in academia and in industry. Interest- oxidation of the C1 or C4 carbon or may result in a mixture of C1-
ingly, in retrospect, several earlier studies indicated the impor- and C4-oxidized products, and these LPMOs are called type 1,
tance of proteins now known as LPMOs in cellulose degradation. type 2, and type 3, respectively (60). It has been suggested that the
For example, in 1992, Raguz et al. described a cellulose-induced differences in regioselectivity between LPMOs are determined by
protein from the basidiomycete Agaricus bisporus (AbCel1), the aromatic amino acid residues exposed on the surface near the ac-
function of which was unknown at that time (47) and that we now tive site (61, 62).
know is an LPMO9. A Trichoderma protein called EGIV and ho- LPMOs had been thought to utilize oxygen as the electron ac-
mologous to AbCel1 was reported to show weak EG activity, and ceptor, but recently it has been experimentally shown that their cat-
therefore both EGIV and AbCel1 were classified into Glycoside alytic efficiency significantly increases when hydrogen peroxide is
Hydrolase family 61 (GH61) in the CAZy database (48, 49). These used as the electron acceptor instead of molecular oxygen (63, 64).
GH61 proteins remained enigmatic for a while, perhaps even more When oxygen is used as the electron acceptor, catalysis requires
so after determination of the first crystal structure, which showed the timely delivery of two protons and two electrons in at least two
that GH61 did not have an obvious active site architecture typi- steps. On the other hand, when hydrogen peroxide is used as the
cal for known hydrolases (50). In 2010, just before the discovery electron acceptor, the reaction proceeds by first supplying only one
of LPMO activity, Harris et al. confirmed that the EG activity of electron (i.e., reduction of the copper), after which the enzyme can
GH61 is indeed very weak, and showed that, nevertheless, GH61s
can boost the activity of other cellulases (51). Already in 2005,
Vaaje-Kolstad et al. had shown that a (bacterial) CBM33 protein
known to bind to chitin and structurally similar to GH61s (52)
boosts the activity of chitinases (53). In 2010 everything came to-
gether, when these same authors showed that the CBM33 protein
oxidatively decomposes crystalline chitin when supplied with oxy-
gen and an electron donor (54). They predicted that GH61s would
oxidatively cleave cellulose, as was indeed shown in 2011 (43–46).
Following these findings, CBM33s and GH61s were re-named
Fig. 2. Schematic illustration of LPMO catalysis. This sche-
lytic polysaccharide monooxygenase (LPMO), and they were matic and simplified picture is based on the assumption that the O2
classified in Auxiliary Activities families 10 and 9 of the CAZy and H2O2 mechanisms converge in that the O2 mechanism involves
database, respectively (41). Most filamentous fungi possess several formation of H2O2, as recently discussed by Wang et al. (65). For
more details on possible catalytic mechanism, see (32, 63, 66–68).
genes encoding LPMO9s (Table 1). The arrows labeled with * reflect the process where a copper-oxyl
The catalytic center of LPMOs contains a single copper ion abstracts a hydrogen from a carbon in the scissile bond followed
(44), which is retained by two fully conserved histidine ligands. by hydroxylation of the carbon through a rebound mechanism; the
hydroxylated product is unstable, leading breakage of the glucosidic
One of these is an LPMO-characteristic histidine at the N-terminal bond (45). The arrows labeled with ** reflect the process where a
end, of which the imidazole group and the N-terminal amino group substrate-free LPMO produces H2O2, which may then be used by
interact with the copper ion. The other histidine, usually in the another LPMO to degrade the substrate. It should be noted that the si-
multaneous occurrence of Cu(I) and H2O2 in the absence of substrate
residue 70–90 region in LPMO9s, interacts with the copper ion via may lead to side reactions that oxidize the LPMO and may perma-
its imidazole side chain (44, 55). Although ascorbic acid and gal- nently damage the enzyme (63); these side reactions are not shown.
© 2020 FCCA (Forum: Carbohydrates Coming of Age) E138Table 2. Activity of experimentally characterized LPMO9s.
The Table shows the total number of LPMO9s for which activity toward a particular substrate has been detected. For hemicelluloses, values in
brackets show the number of LPMO9s that only show activity on that hemicellulose when cellulose is added to the reaction mixture. Note that not
all LPMO9s described in the literature have been tested on all listed substrates. PASC, phosphoric acid-swollen cellulose; RAC, regenerated amor-
phous cellulose; CMC, carboxymethylcellulose; BMCC, bacterial microcrystalline cellulose; *, one of them show minor activity (77); **, all show
minor activity (78); ***, two of them show minor activity (79, 80).
catalyze multiple H2O2-driven catalytic cycles. In natural environ- ble cello-oligosaccharides, providing insight into how the LPMO
ments, hydrogen peroxide can be produced by various enzymes catalytic site interacts with substrate (72). In addition, the catalytic
such as glucose oxidase, alcohol oxidase, or as a result of the oxi- activity of two LPMO9s from Heterobasidion irregulare has been
dase activity of the LPMO itself (32) (Fig. 2). described, along with the crystal structure of one of these (73, 74).
All experimentally characterized LPMO9s have activity to- In a recent study, Li et al. showed that, like other LPMOs (75), an
ward cellulose. In experiments, pulp, microcrystalline cellulose LPMO9 from Pleurotus ostreatus produces H2O2 in the presence
(Avicel), phosphoric acid swollen cellulose (PASC), and carboxy- of a reductant (AscA) and this production was sufficient to drive a
methyl cellulose (CMC) have been used as cellulosic substrates for versatile peroxidase, which oxidizes lignin using hydrogen perox-
activity measurements (Table 2). Many LPMO9s show activity not ide as an electron acceptor (76).
only on cellulose but also on other β-glucans and cello-oligosac- On the other hand, little research has been conducted on
charides (Table 2). For a few LPMO9s, activity on xylan has been LPMO9s derived from other wood-rot fungi until recently we
demonstrated (69, 70). Among the β-1,4-linked hemicelluloses, have studied two LPMO9s from G. trabeum to clarify their func-
xyloglucan is the most common substrate for LPMO9s. Based tions and physiological roles (77). A search of the genomic se-
on current findings, it is considered that many LPMO9s basically quence of G. trabeum indicated that there are four LPMO9 genes,
recognize β-1,4-D-glucans, whereas some LPMO9s can recognize and we named the encoded LPMOs GtLPMO9A, GtLPMO9B,
β-1,4-linked xylose-xylose bonds in xylan. GtLPMO9C, and GtLPMO9D. Interestingly, we detected two types
of transcripts, having different 3′-terminal ends, for the gene en-
D. LPMO9s from Wood-Rotting Basidiomycetes coding GtLPMO9A, possibly due to alternative splicing. The pro-
Among LPMO9s from wood-rot fungi, PcLPMO9D (previ- tein encoded by the shorter transcript was named GtLPMO9A-1,
ously PcGH61D) from one of the most studied white-rot fungus and the other, encoded by the transcript with a longer sequence
Phanerochaete chrysosporium was the first LPMO to be character- at the 3′-terminal end, was named GtLPMO9A-2. Both have a
ized. It was shown that PcGH61D is active on Avicel, filter paper, common sequence from the 5′-terminal end comprising 706 bp,
PASC, and woody biomass with C1-oxidation (46, 71). The three- which is followed by unique sequences comprising 53 bp for
dimensional structure of PcGH61D was determined as early as GtLPMO9A-1 and 407 bp for GtLPMO9A-2. Homologues of the
in 2013 and simulation studies of adsorption onto cellulose and C-terminal extension in GtLPMO9A-2 occur in multiple fungal
structural comparisons with other LPMO9s provided indications LPMOs but the function of this extra domain, potentially involved
as to which amino acid residues and loop structures are involved in in substrate binding, remains to be uncovered.
interacting with the cellulose substrate (71). Another characterized Recombinant GtLPMO9A-2 (351 residues) was heterologous-
white-rot fungal LPMO is LsAA9A from Lentinus similis, which ly expressed in the yeast Pichia pastoris and activity assays with
has gained considerable attention because it has been possible to the purified recombinant enzyme showed activity not only toward
determine crystal structures of this enzyme in complex with solu- cellulosic substrates, such as CMC and PASC, but also on hemi-
E139 © 2020 FCCA (Forum: Carbohydrates Coming of Age)celluloses, such as xyloglucan and glucomannan. MALDI-TOF/ expression pattern of genes encoding LPMO9s along the hyphae
MS analysis of the reaction products generated from xyloglucan indicates a controlled and time-dependent regulation of LPMO
showed that GtLPMO9A-2 is capable of cleaving the xyloglucan genes during brown rot. Zhang et al. (86) have proposed that CMF
backbone at almost any position, in contrast to multiple known xy- reaction occurs at the hyphal front, while enzymes involved in sac-
loglucan-active LPMOs which can only cleave next to a non-sub- charification are highly expressed consecutively, at an area distant
stituted glucose. Of note, GtLPMO9B, which comprises 232-resi- from the hyphal front. Considering that different LPMO9 genes are
dues sharing 76.6% sequence identity with the catalytic domain of expressed at different areas along the hyphae, some LPMO9s may
GtLPMO9A-2, showed almost the same cleavage capabilities (81). work synergistically with hydrolytic enzymes while others work
There are some examples that multiple LPMOs from the same in parallel with the CMF system. Consequently, although the role
organism have different activities (e.g (82)), while GtLPMO9A-2 of LPMOs has been mainly discussed based on their synergistic
and GtLPMO9B showed similar enzymatic activities, suggesting action with the hydrolytic enzyme system, their interaction with
variation in natural LPMO systems. CMF should also be discussed to clarify the physiological role of
From these initial results, it is clear that brown-rot fungi have LPMOs in the brown-rot system.
“regular” LPMO9s, but additional comparative studies, of multiple Both the LPMO and the CMF reactions involve powerful
LPMO9s from the same organism and of LPMO9s from brown-rot redox chemistry, possibly both involving formation of hydroxyl
fungi other than G. trabeum, are needed to obtain a comprehensive radicals (63). Most importantly, the recent finding that LPMOs,
picture of the role of these enzymes. It is worth noting that many including LPMO9s (90), use H2O2 as a co-substrate (63), and that
brown-rot fungi lack or have few genes encoding GH74, an en- they do this very efficiently (91), shed new light on a potential in-
zyme that is crucial for hydrolyzing xyloglucan. As an example, terplay between the CMF reaction and LPMOs. In a recent review,
G. trabeum has only one GH74-encoding gene (18), while it has at Bissaro et al. (32) discussed the potentially central role of H2O2
least two xyloglucan-active AA9 LPMOs (77, 81) and one GH12 in fungal biomass conversion, but many questions remain unan-
endoglucanase with good activity on xyloglucan (83). Thus, it is swered. Interestingly, although highly speculative, it is conceivable
conceivable that G. trabeum decomposes xyloglucan not only by that the relatively low number of LPMOs in brown-rot fungi (Table
hydrolases but also by oxidative degradation system, and the abil- 1) relates to the fact that these fungi have another useful purpose
ity of the GtLPMO9s to cleave xyloglucan may have evolved to for available H2O2, namely the CMF reaction.
increase the contribution of the oxidative degradation system in
brown-rot mechanism. We are currently investigating this hypoth- E. Evolutionary Considerations on the Acquisition of
esis. Oxidative Degradation Systems in Wood Rotting Basid-
Next to biochemical characterization of individual en- iomycetes
zymes, comprehensive -omics analyses give insight into the Many filamentous fungi produce not only hydrolases, such as
roles of LPMO9s during brown-rot decay. Enabled by extensive cellulases, but also oxidoreductases involved in cellulose degrada-
sequencing of fungal genomes during the last decade, several tion, such as LPMO9 and CDH, while white-rot fungi also produce
transcriptome and secretome studies of brown-rot fungi have oxidoreductases involved in lignin degradation, such as PODs and
been reported. When brown-rot fungi are cultivated in shaking laccases (18). Furthermore, it has been considered that brown-rot
cultures or stationary cultures containing woody material as a fungi may use CMF, a non-enzymatic oxidative degradation sys-
carbon source, their LPMO9 genes generally are not highly ex- tem, in addition to the enzymatic degradation system. Thus, differ-
pressed (84–88), and regulation of LPMO9 genes and secretion ent species of filamentous fungi may have acquired and developed
of LPMO9s show no clear trends over time (39, 84, 89). On the different redox mechanism as well as enzymatic hydrolytic mecha-
other hand, growing brown-rot fungi along a wood wafer enables nisms in the process of evolution, in order to decompose wood
spatial separation of the hypha (e.g. root or tip), which means that efficiently (18). This chapter addresses how filamentous fungi may
the stages of brown-rot decay can be separated (86). The study by have obtained and subsequently lost oxidoreductases, especially
Zhang et al. showed a clear difference between the expression of PODs and LPMOs, and weighs the potential ecological advantages
the two LPMO9s in brown-rot fungus P. placenta: one LPMO9 of the non-enzymatic oxidative degradation system in brown-
(Pospl1 ID 135008/24502/36097) was expressed in invariably rot fungi against those of a large set of lignin- and plant cell wall
low quantities along the hyphae, while the other LPMO9 (Pospl1 polysaccharide-specific oxidoreductases in white-rot fungi.
ID 126811/115618) was expressed in higher amounts and is up- The phyla of basidiomycete and ascomycete fungi belong
regulated preferentially during later decay (86). A difference in the to the fungal sub-kingdom of Dikarya, the common ancestor of
© 2020 FCCA (Forum: Carbohydrates Coming of Age) E140which is thought to have emerged more than 600 million years ago wood, the brown-rot system can thus be advantageous compared
(18). Over a very long period spanning from that time to the pres- to the systems employed by other fungi, including the white-rot
ent, plant-degrading filamentous fungi have evolved, in part by fungi. Nevertheless, genes encoding LPMO9s have been retained
acquiring genes encoding plant cell wall-degrading enzymes, as in almost all brown-rot fungi, clearly indicating that LPMO9 is a
well as by duplication or loss of those genes (2). Recent compara- vital enzyme in the brown-rot system.
tive genomic analysis has suggested that Dikarya acquired vari- Interestingly, Dacryopinax primogenitus, the taxonomically
ous plant cell wall-degrading enzymes, including LPMO9s, at an oldest brown-rot fungus, does not have any LPMO9 genes, but has
early stage of evolution, however, it is believed that, at that time, three LPMO14 genes. Indeed, while some brown-rot and white-
the filamentous fungi did not have sufficient redox mechanisms rot fungi lack LPMO9s, they all contain genes encoding LPMO14s
to catalyze oxidative degradation of wood (18). It is assumed to (Table 1). Therefore, it is possible that the not yet very well studied
be approximately 300 million years ago when filamentous fungi LPMO14s (95) may be important in wood cell wall degradation by
explosively increased their content of genes encoding oxidoreduc- wood rotting basidiomycetes.
tases involved in wood decomposition (e.g. oxidases generating
H2O2) (18, 92). During this period, the number of LPMO9 genes F. Concluding Remarks
increased rapidly. After expansion of the LPMO9 genes, the num- So far, research on the mechanism of wood degradation by
ber of POD genes also started increasing (92, 93). The point where brown-rot fungi has focused on the CMF system, because of its
filamentous fungi acquired the gene encoding manganese peroxi- uniqueness. On the other hand, the role of degrading enzymes
dase is considered the birth of white-rot fungi, which happened from brown-rot fungi has not been assessed in much detail. It is
approximately 290 million years ago, at the end of the Carbonifer- clear that some enzyme types that are today known to be very im-
ous period (18). In this period, the oxygen concentration in the at- portant for biomass conversion, such as the LPMO9s, have been
mosphere is estimated to have been more than 35% and this highly preserved even in brown-rot fungi, and such enzymes likely play a
aerobic environment may have contributed to the rapid increase in crucial role in the brown-rot mechanism. It is also conceivable that
oxidoreductase-encoding genes. LPMOs to some extent compensate for the lack of certain GHs,
After the appearance of the white-rot fungi, further evolution for example the lack of CBHs and xyloglucan-degrading enzymes,
of the plant-degrading fungi included the emergence of brown-rot as discussed above. Recent observations suggesting interaction
fungi (18, 92). Considerable loss of genes encoding PODs, AA3_2 between the CMF process and LPMO reaction and showing a role
oxidoreductases, CBHs and LPMO9s, which play a major role in of H2O2 in both these processes raise the question as to how the
wood cell wall degradation, is considered a key step in this evolu- two (non-enzymatic and enzymatic) degradation mechanisms may
tionary event (2). For example, it is clear that brown-rot fungi lost be connected. It cannot be excluded that the roles of LPMO9s in
all, or almost all, of the POD-encoding genes. Similarly, almost brown-rot fungi differ from, and perhaps are more specific than,
all brown-rot fungi lack CBHs and CBM1s (2, 37, 92) (Table 1). the roles of LPMO9s in other filamentous fungi. In this respect,
It has been claimed that the evolution to brown-rot fungi occurred potential differences between various LPMO families from differ-
several times (18, 94), which may be taken to indicate that these ent lineages of brown-rot fungi and the role of the LPMO14s need
gene-losses are biologically beneficial. Interestingly, the brown-rot further attention.
fungi belonging to Polyporales have a gene encoding POD that is
incomplete, and the brown-rot fungi belonging to Boletales have Acknowledgments
genes encoding CBH and CDH (Table 1). These observations sug- We thank the Japan Society for the Promotion of Science
gest that brown-rot fungi belonging to these different clades may (JSPS) for funding through grants 16J03946 (Yuka Kojima), and
employ slightly different wood degradation mechanisms. Grant-in-Aid for Scientific Research (B) 18H02252 (Makoto Yo-
It is generally accepted that brown-rot fungi have developed shida). We also thank the Research Council of Norway for funding
CMF system for efficient degradation of wood cell walls. This non- through grants 257622, 268002 and 262853.
enzymatic system, which, notably, depends on the generation of
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© 2020 FCCA (Forum: Carbohydrates Coming of Age) E142Information of the Authors
Yuka Kojima received her Ph.D. degree in agriculture from Tokyo University of Agriculture and Tech-
nology in 2019 under the supervision of Professor Makoto Yoshida. After graduation, she has worked as
an assistant professor at the Institute of Global Innovation at the same University. She studied LPMO9
enzymes at the Norwegian University of Life Sciences in 2014. She visited VTT Technical Research
Centre of Finland in 2017, where she studied biomass saccharification. She also visited the laboratory of
Finn L. Aachmann at the Norwegian University of Science and Technology in 2019, where she learned
about the three-dimensional structural analysis of proteins. She received the Japan Wood Research So-
ciety Outstanding Woman Student Award in 2017. She is interested in elucidating the phenomenon of
wood decay caused by wood-rotting fungi from the perspective of wood-degrading enzymes.
Vincent Eijsink received his Ph.D. in 1991 at the University of Groningen, The Netherlands, on engi-
neering of protein thermal stability. He started working at the Norwegian University of Life Sciences
in 1993, where he became a full Professor in 1997. Today, he leads a research group working on the
enzymology of biomass conversion, covering a broad spectrum of fundamental and applied aspects. His
group is well known for work on enzyme processivity and for being the first to describe, in 2005, the
boosting effects of certain proteins, now known as LPMOs, on the efficiency of polysaccharide degrada-
tion by glycoside hydrolases. This was followed, in 2010, by the discovery that these proteins carry out
oxidative cleavage of glycosidic bonds.
Anikó Várnai received her Ph.D. in 2012 at the University of Helsinki, Finland, on optimization of
enzymatic saccharification of plant biomass. Since then, she has been working as a researcher at the
Protein Engineering and Proteomics group led by Prof. Vincent Eijsink at the Norwegian University of
Life Sciences (NMBU). Her major research interest is in biomass valorization, with particular focus on
understanding the role various enzyme components, including cellulases, hemicellulases and LPMOs,
play in plant cell wall degradation and applying that knowledge for process optimization. She is cur-
rently coordinating the ‘Biochemical Conversion’ subproject and leading the Enzyme Technology work
package of the Norwegian Centre for Sustainable Bio-based Fuels and Energy (Bio4Fuels), funded by
the Research Council of Norway in 2016.
Makoto Yoshida received his Ph.D in 2005 at the University of Tokyo (Supervisor, Prof. Masahiro
Samejima). He started working as a post-doc at National Food Research Institute in 2005. He moved to
Tokyo University of Agriculture and Technology as a tenure-track associate professor in 2006, and then
got a tenure position in 2011. He became a professor in 2017. He also worked as a visiting researcher at
Virginia Polytechnic Institute and State University (Virginia Tech) from 2015 to 2016. He was elected
as a member of fellows of International Academy of Wood Science (IAWS) in 2019. He is interested in
the mechanisms of wood deterioration caused by wood rotting fungi for protection of wood architecture,
and also interested in plant biomass conversion using the ability of wood rotting fungi.
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