Applied Microbiology and Biotechnology
https://doi.org/10.1007/s00253-020-11087-9
GENOMICS, TRANSCRIPTOMICS, PROTEOMICS
Comparative transcriptional analyses of Pleurotus ostreatus mutants
on beech wood and rice straw shed light on substrate-biased
gene regulation
Hongli Wu 1 & Takehito Nakazawa 1 & Haibo Xu 1 & Ruiheng Yang 2 & Dapeng Bao 2 & Moriyuki Kawauchi 1 &
Masahiro Sakamoto 1 & Yoichi Honda 1
Received: 7 October 2020 / Revised: 15 December 2020 / Accepted: 28 December 2020
# The Author(s), under exclusive licence to Springer-Verlag GmbH, DE part of Springer Nature 2021
Abstract
Distinct wood degraders occupying their preferred habitats have biased enzyme repertoires that are well fitted to their colonized
substrates. Pleurotus ostreatus, commonly found on wood, has evolved its own enzyme-producing traits. In our previous study,
transcriptional shifts in several P. ostreatus delignification-defective mutants, including Δhir1 and Δgat1 strains, were analyzed,
which revealed the downregulation of ligninolytic genes and the upregulation of cellulolytic and xylanolytic genes when
compared to their parental strain 20b on beech wood sawdust medium (BWS). In this study, rice straw (RS) was used as an
alternative substrate to examine the transcriptional responses of P. ostreatus to distinct substrates. The vp1 gene and a
cupredoxin-encoding gene were significantly upregulated in the 20b strain on RS compared with that on BWS, reflecting their
distinct regulation patterns. The overall expression level of genes encoding glucuronidases was also higher on RS than on BWS,
showing a good correlation with the substrate composition. Transcriptional alterations in the mutants (Δhir1 or Δgat1 versus 20b
strain) on RS were similar to those on BWS, and the extracellular lignocellulose-degrading enzyme activities and lignindegrading ability of the mutants on RS were consistent with the transcriptional alterations of the corresponding enzymeencoding genes. However, transcripts of specific genes encoding enzymes belonging to the same CAZyme family exhibited
distinct alteration patterns in the mutant strains grown on RS compared to those grown on BWS. These findings provide new
insights into the molecular mechanisms underlying the transcriptional regulation of lignocellulolytic genes in P. ostreatus.
Key Points
• P. ostreatus expressed variable enzymatic repertoire-related genes in response to distinct substrates.
• A demand to upregulate the cellulolytic genes seems to be present in ligninolysis-deficient mutants.
• The regulation of some specific genes probably driven by the demand is dependent on the substrate.
Keywords Lignin . Cellulose . Basidiomycete . Transcriptomes . White-rot
Introduction
Lignocellulosic biomass is an abundant source of carbon that
has attracted increased interest due to its applicability as a
* Takehito Nakazawa
nakazawa.takehito.8u@kyoto-u.ac.jp
1
Graduate School of Agriculture, Kyoto University, Oiwakecho,
Kitashirakawa, Sakyo-ku, Kyoto 606-8502, Japan
2
Institute of Edible Fungi, Shanghai Academy of Agricultural
Sciences, Shanghai 201403, China
renewable energy source and in chemical products. There
are three main components of lignocellulosic biomass.
Among these, lignin and hemicellulose provide a matrix in
which cellulose gathers into a highly ordered network structure via various intermolecular and intramolecular forces,
making lignocellulose resistant to enzymatic attacks and recalcitrant to decomposition (Zoghlami and Paës 2019). Whiterot fungi utilize not only cellulose and hemicellulose, but also
uniquely depolymerize the most recalcitrant component of
lignin, and are thus one of the key players in the global carbon
cycle. White-rot fungi utilize lignocellulose by secreting a
number of lignocellulose-degrading enzymes, including cellulolytic, xylanolytic, and pectinolytic enzymes, as well as
Appl Microbiol Biotechnol
oxidative enzymes, which function coordinately to decompose lignocellulose (Floudas et al. 2012; Rytioja et al. 2014).
Most of these enzymes have been biochemically characterized
(Lundell et al. 2010; Manavalan et al. 2015) and classified as
carbohydrate-active enzymes (CAZymes) (Lombard et al.
2013). Six classes have been identified in CAZymes: glycoside hydrolases (GHs) (Henrissat 1991), glycosyltransferases
(GTs) (Campbell et al. 1997), polysaccharide lyases (PLs)
(Lombard et al. 2010), carbohydrate esterases (CEs)
(Lombard et al. 2010), carbohydrate-binding modules
(CBMs) (Boraston et al. 2004), and auxiliary activities
(AAs) (Levasseur et al. 2013). Typically, white-rot fungi are
thought to attack lignin by producing a series of oxidative
enzymes, including manganese peroxidase (MnP, EC
1.11.1.13), versatile peroxidase (VP, EC 1.11.1.16), and lignin peroxidase (Lip, EC 1.11.1.14) (Janusz et al. 2017). In
addition to these enzymes, enzymes such as glyoxal oxidases,
pyranose dehydrogenases, and methanol oxidases may also
play indispensable roles in lignin degradation (Kersten and
Cullen 2014). The transcriptomes of white-rot fungi have been
studied widely, and it has been suggested that their high variability is dependent on the substrates and growth period (Hori
et al. 2014; Alfaro et al. 2016; Fernández-Fueyo et al. 2016;
Rytioja et al. 2017).
Colonizing various lignocellulosic biomasses, white-rot
fungi obtain nutrients from the substrates, while these substrates affect the colonization strategies of these biomasses
in return (Rytioja et al. 2017). Wood and herb biomass differ
in both composition and structures. Generally, wood biomass
contains higher amounts of lignin than herb biomass
(Sjostrom 1993). There are also more guaiacyl and syringyl
units and smaller amounts of p-hydroxyphenyl units in hardwood compared to softwood, in which guaiacyl units play a
predominant role, with very small amounts of p-hydroxyphenyl units. All three types of lignin units are present
in comparable amounts in herb biomass (Billa and Monties
1995; Vanholme et al. 2010; Kotake et al. 2015). In addition
to lignin, hemicellulose, which is mainly composed of xylose
units connected by β-1,4-glucosidic bonds, also differs in the
fraction of hardwood and graminaceous plants. The hemicellulose of birchwood contains 89.3% xylose, 1% arabinose,
8.3% anhydrouronic acid, and 1.4% glucose (Kormelink and
Voragen 1993). The hemicellulose in the bran of rice contains
46% xylose, 44.9% arabinose, and 6.1% galactose (Shibuya
and Iwasaki 1985), whereas rice straw hemicellulose contains
43.2% glucose, 42.2% xylan, 11.2% arabinose, and 3.07%
galactose (Xiao et al. 2001). In addition to xylan backbones,
the side chains in hemicellulose also vary between hardwood
and graminaceous plants. In plant heteroxylans, the
arabinofuranosyl residues are esterified with p-coumaric acids
and ferulic acids (Mueller-Harvey et al. 1986; Sun et al. 2001),
while O-acetyl substitution commonly occurs in hardwood
(Koutaniemi et al. 2013).
Pleurotus ostreatus has been studied widely not only as a
popular edible mushroom but also as one of the model whiterot fungi of the order Agaricales. Most members of Agaricales
grow naturally on various lignocellulosic substrates
(Fernández-Fueyo et al. 2016). Several studies have explored
the effects of distinct substrates on the growth of P. ostreatus
and fruiting body formation (Ashraf et al. 2013; Hoa et al.
2015). Transcriptome or secretome analyses of P. ostreatus
PC9 on several substrates have recently been reported
(Fernández-Fueyo et al. 2016; Alfaro et al. 2016, 2020). A
comparative secretome analysis of poplar, wheat straw, and
glucose medium suggested several specific genes that were
highly expressed on lignocellulosic substrates (FernándezFueyo et al. 2016). Alfaro et al. (2016) also reported the transcriptome and secretome analysis of PC9 grown on sucrosemalt-yeast extract (SMY) medium under different culture conditions (shaken and static cultures). However, these studies are
still not sufficient to fully elucidate the mechanism underlying
lignocellulose degradation.
In addition to transcriptome analysis, considerable efforts
have been devoted to exploring the lignocellulose-degrading
mechanisms using molecular genetics, especially since efficient gene targeting using homologous recombination has
been established in P. ostreatus (Salame et al. 2012;
Nakazawa et al. 2016). The genes and proteins involved in
the transcriptional regulation of the ligninolytic system have
been identified in some white-rot fungi (Álvarez et al. 2009;
Toyokawa et al. 2016). In P. ostreatus, it was reported that the
disruption and overexpression of cre1 enhanced and reduced
the cellulolytic activity, respectively, in a liquid medium containing wheat straw (Yoav et al. 2018). Feldman et al. (2017)
reported that the overexpression of the gene ssp1, which encodes a small secreted protein, elevated the expression of vp1
in glucose-peptone medium. In our previous studies, singlegene mutations in a putative chromatin remodeler gene chd1,
a peroxisome biogenesis gene pex1, a histone chaperone gene
hir1, and two putative Agaricomycetes-specific DNA-binding
transcription factor (TF) genes, wtr1 and gat1, were shown to
reduce the expression levels of specific mnp/vp genes as well
as the wood lignin-degrading abilities of P. ostreatus on beech
wood sawdust medium (BWS) (Nakazawa et al. 2017a, b,
2019; Wu et al. 2021). Additionally, certain cellulolytic genes
were shown to be upregulated in most of the mutants (Wu
et al. 2020). Gene expression in P. ostreatus could be induced
variably across different substrate species (Alfaro et al. 2016).
Thus, transcriptional alterations caused by the mutations could
be different on BWS compared with the other substrates.
Investigating mutants generated from the deletion of genes
encoding TF or functional proteins on different substrates
may cue us in which way or what extent these TF/functional
proteins affects gene expressions.
In this study, rice straw was used as the substrate. The
transcriptional expression of P. ostreatus strain 20b grown
Appl Microbiol Biotechnol
on rice straw medium (RS) for 13 d was examined and compared with those grown on BWS. Two mutants obtained from
our previous study, Δgat1 and Δhir1, were also cultured on
rice straw medium to compare the transcriptional expression
between 20b and the mutants when grown on rice straw medium, with the aim of improving our understanding of the
transcriptional shifts of P. ostreatus in response to lignocellulosic substrates.
Materials and methods
P. ostreatus strains and media
The strains used in this study are all monokaryon and are
listed in Table 1. YMG agar medium [0.4% (w/v) yeast
extract, 1% (w/v) malt extract, 1% (w/v) glucose, and 2%
(w/v) agar] (Rao and Niederpruem 1969) was used for
routine cultures. Rice straw (strain name is Koshihikari,
one of the popular cultivars of Japonica rice in Japan) was
purchased from Honda Nojo, Ishikawa, Japan. Rice straw
medium [RS, 1.9 g size-fractionated rice straw (250−500
μm), 0.1 g wheat bran, and 6 mL H2O] was used as the
substrate for the transcriptional analysis of the
P. ostreatus strains. Beech wood sawdust media [BWS,
1.9 g beech wood sawdust (almost all of the particles
were smaller than 1 mm), 0.1 g wheat bran, and 6 mL
H2O] was used in our previous study of transcriptome
analysis of Δgat1 and Δhir1 mutants (Wu et al. 2020,
2021). Each P. ostreatus strain was precultured on YMG
agar medium and fresh mycelia were inoculated into 6-cm
glass Petri dishes containing RS under solid conditions at
28°C for 13 d. The growth rate of the 20b strain on RS
and BWS was almost the same, since it takes 13 d to fully
cover the plate. The rice straw, beech wood sawdust, and
wheat bran used in this study were subjected to organic
solvent extraction by soaking the samples in toluene/
ethanol (2:1, v/v) for 1 h at 80°C, repeating 4 times.
Wheat bran was added to the cultures to increase the production of ligninolytic enzymes (Tsukihara et al. 2006).
The cultures were maintained under continuous darkness.
Table 1 The P. ostreatus strains
used in this study
RNA-seq and bioinformatic analysis
Each strain was grown on RS for 13 d, after which the total
RNA was isolated from each sample in two biological replicates, using the RNeasy Plant Mini Kit (Qiagen, Venlo,
Netherland). To remove genomic DNA, all RNA samples
were subjected to DNase digestion using the Turbo DNAfree Kit (Ambion, Austin, TX). The kits used to prepare samples for sequencing and the accession numbers of the sequencing raw data are shown in Supplemental Table S1. Paired-end
raw reads were quality trimmed by the CLC Genomics
Workbench tool version 20.0 (Qiagen, Venlo,
The Netherlands). Trimmed reads were mapped to the PC9
strain genome (http://genome.jgi.doe.gov/PleosPC9_1/
PleosPC9_1.home.html) using the RNA-seq analysis package
in CLC with at least 80% sequence identity over at least 80%
of the read lengths. The RPKM (reads per kilobase of transcript per million mapped reads) and CPM (count per million)
values were exported from the CLC workbench for subsequent analysis.
Differentially expressed genes (DEGs) were defined using
edgeR (http://bioconductor.org) with the following
parameters: fold change >4 and FDR (false discovery rate)
<0.05. The “calcNormFactors” from R (Robinson et al.
2010) were used to perform data scaling with the trimmed
mean of M values (TMM) method. CPM (count per million)
was used for DEG assessment as required by the function of
edgeR (Robinson et al. 2010). Heatmap analysis was calculated using Heml 1.0-Heatmap Illustrator (Deng et al. 2014)
and the Euclidean squared distance metric was used. Gene
annotations were based on the available PC9 genome on JGI
(http://genome.jgi.doe.gov/PleosPC9_1/PleosPC9_1.home.
html) and dbCAN (http://csb1.bmb.uga.edu/dbCAN).
Hierarchical clustering analysis was performed using
Euclidean distance. Principal component analysis (PCA) was
performed using the RPKM values of each sample.
Co-expression and putative shared motif analysis
Co-expression network analysis was performed using the
Comparative Co-Expression Network Construction and
Visualization (Cytoscape) tool with the Pearson correlation
Strain
Genotype/description
Source
20b
A2B1 ku80::CbxRa
Salame et al. (2012)
Δhir1
A2B1 ku80::Cbx hir1::hph / a hir1 disruptant derived from 20b
Wu et al. (2021)
Δgat12
A2B1 ku80::CbxR gat1::hph / a gat1 disruptant derived from 20b
Nakazawa et al. (2017a)
1
R
a
CbxR indicates the carboxin resistance gene (Honda et al. 2000)
1
The strains hir1d#1 and hir1d#2 used in this study represent duplicate samples of the strain Δhir1
2
The strains gat1d#1 and gat1d#2 used in this study represent duplicate samples of the strain Δgat1
Appl Microbiol Biotechnol
coefficient (Tzfadia et al. 2016). The RPKM values of DEGs,
mainly CAZyme-encoding genes, were used as the input files.
The gene gat1 (protein ID 83134 in the PC9 genome database)
was used as a bait gene. We used a 1 kb DNA fragment in the
promoter region of co-expressed genes associated with the
gene gat1 to predict the putative shared motifs. The meme
suite webserver was used to identify common binding motifs
shared by co-expressed genes, allowing for a width from 6 to
10 with 1 or 2 mismatches (Bailey and Elkan 1994). The
generated motifs were compared against the JASPAR Core
(fungi) 2018 database using the TOMTOM webserver
(Gupta et al. 2007).
Quantitative reverse transcription-PCR (qRT-PCR)
20b as well as the mutant strains gat1d#1 and hir1d#1 were
cultured on RS for 9, 13 and, 20 days; moreover, the parental
strain 20b was also cultured on BWS for 9, 13, and 20 days.
Afterward, total RNA was isolated from each sample in two
biological replicates. Of note, rice straw with particle size
larger than 500 μm (almost all of the particles were smaller
than 2 mm), and the FastGene RNA Premium Kit (Nippon
Genetics, Tokyo, Japan; this kit includes DNase) were used.
qRT-PCR was performed as described by Nakazawa et al.
(2019).
Assay for extracellular enzyme activities
Results
P. ostreatus strains were cultured on RS for 13 d. The RS
covered with mycelial cells was suspended in 0.1 M Nalactate buffer (pH 4.5) and then centrifuged at 2000×g to remove the rice straw and mycelial cells. The resulting supernatant fluids were used to determine the ligninolytic enzyme
activities. Guaiacol (2-methoxyphenol) oxidation was used
as the substrate to examine H2O2-independent oxidase and
Mn2+-independent/dependent peroxidase activities, as described by Kamitsuji et al. (2004). One unit of guaiacol oxidation activity was defined as the amount of enzyme that
increased the absorbance at 465 nm by 1.0 per min. The
CMCase (carboxymethyl cellulase) and xylanase activities
were assayed as described by König et al. (2002). Before the
examination, the resulting supernatant fluids were subjected to
ultrafiltration with a 10-kDa cutoff to remove molecules affecting the quantification of reducing sugars. A 3,5dinitrosalicylic acid reagent was used to quantify the reducing
sugars. Xylans derived from beech wood (Nacalai Tesque,
Kyoto, Japan) and CMC (carboxymethyl cellulose) were used
as substrates to determine xylanase and CMCase activity, respectively. One unit of xylanase/CMCase was defined as the
amount of enzyme required to liberate 1 μmol of reducing
sugar as xylose/glucose per minute under the assay conditions.
The protein concentration was estimated using the Bradford
assay. Extracellular enzymes boiled at 95°C for 10 min were
used as controls. The absorbance was measured and monitored using a Multiskan GO plate reader (Thermo Fisher
Scientific, MA, USA).
Quantification of Klason lignin content in rice straw
medium
Each P. ostreatus strain was grown on RS for 28 d, followed
by solvent extraction using toluene and ethanol (2:1, v/v). The
residual amount of acid-insoluble Klason lignin contained in
each BWS solution after the cultivation of the P. ostreatus
strains was quantified using the acidolysis method as described by Ritter et al. (1932).
Transcriptional expression of CAZyme-encoding
genes in the P. ostreatus strain 20b grown on RS and
BWS
White-rot fungi harness lignocellulolytic enzymes to forge
sugars from the cell wall. These enzymes (or enzymeencoding genes) are characterized by high variety in protein
families and redundancy in copy numbers. In this study, transcripts of such enzyme-encoding genes were examined to
provide an overview of different lignocellulolytic strategies
in P. ostreatus strain 20b on RS and BWS (Fig. 1a).
Enzymes that had similar targeting polysaccharide-substrates
were also identified (Fig. 1a). A lower transcriptional expression level (overall RPKM values in each CAZy family) of
ligninolytic genes encoding enzymes from AA2 on RS was
observed (Fig. 1b). However, genes encoding enzymes from
AA3_2, AA3_3, and AA5 families, catalyzing the production
of H2O2, which supports peroxidase oxidation processes,
shared comparable expression levels when grown on RS and
BWS. It was also suggested that genes which encode enzymes
(CH6/7, GH10/11, AA9 families) targeting cellulose and xylan exhibited lower expression level on RS. We observed a
more abundant accumulation of transcripts encoding laccases
from AA1 family and β-glucuronidases from the GH79 family when grown on RS.
DEG analysis of the P. ostreatus strain 20b grown on
RS and BWS
We also examined differentially expressed genes in 20b_RS
versus 20b_BWS to identify various specific genes with distinct functions, which could provide more clues for elucidating gene regulation in the adaption of distinct substrates. A
total of 139 and 67 down- and upregulated genes
(Supplemental Table S2) (fold change [FC] > 4, FDR <
0.05) were observed in the fungus grown on RS compared
with those grown on BWS, respectively. It is worth noting
Appl Microbiol Biotechnol
Fig. 1 RPKM values of
CAZyme-encoding genes associated with plant cell wall degradation in P. ostreatus strain 20b
grown on RS and BWS. a
Heatmap analysis of genes belonging to each CAZyme families
and substrates predicted to be associated with each CAZymes are
also listed; heatmap was built
using the result generated from
the formula [Log10(RPKM
values)] of genes belonging to
each CAZy families. b Overall
expression levels shown by each
substrate. RS, rice straw medium;
BWS, beech wood sawdust; PLs,
pectate lyase, pectinase; ABF,
arabinofuranosidase
that the gene vp1 (corresponding to protein ID 116738), which
was expressed lightly in BWS, was strikingly upregulated in
RS (Supplemental Table S2). The expression level of one
cupredoxin-encoding gene (protein ID 87572) was also found
to be extremely high (RPKM value, 5148.3) on RS
(Supplemental Table S2). We also obtained some intriguing
findings among the downregulated genes. It is worth noting
that 15 putative peptidase-encoding genes were downregulated on RS (Supplemental Table S2). Apart from peptidaseencoding genes, many genes encoding sugar transporters
and CAZymes showed lower expression level when parental
strain 20b were grown on RS.
Global transcriptional expression of mutant strains
grown on RS
In our previous study, we observed transcriptional alterations
with respect to lignocellulolytic genes in two single-gene
disruptants, Δgat1 and Δhir1, when grown on BWS (Wu
et al. 2020, 2021). However, it remains unclear whether these
alterations are consistent with other substrates. Thus, the comparative transcriptional analysis of parental strain 20b and mutant
strains Δgat1 and Δhir1 grown on RS was performed.
Hierarchy cluster and PCA analysis were performed using transcriptomics of mutants Δhir1 and Δgat1 and parental strain 20b
(Fig. 2a, b). Two major clusters were observed: parental strain
20b and mutant strains, which revealed some co-upregulated or
downregulated genes caused by mutations. PCA analysis
showed a strong clustering of two biological replicates in all
strains. A clear separation of parental strain 20b and mutants
Δgat1 and Δhir1 was identified on the distribution along PC1.
Along the distribution of PC2, Δgat1 and Δhir1 exhibited some
differences. Furthermore, some strains from the same substrate
were found to cluster together, which suggested that gene expression was more dependent on the type of strains other than the
substrates they were colonized on. Differentially expressed genes
were examined in each mutant strain. There were 427 and 591
upregulated and downregulated genes identified in Δgat1, respectively, while 362 and 326 up- and downregulated genes were
observed in hir1 disruptants.
Transcriptional alterations of CAZyme-encoding
genes in Δgat1 grown on RS
The expression patterns of CAZyme-encoding genes in
Δgat1 versus 20b were determined by carrying out five
Appl Microbiol Biotechnol
Fig. 2 The global transcriptional profiles of P. ostreatus strains grown on
RS for 13 d. a Heatmap analysis showing the clustering of 6 samples by
calculating the Pearson’s correlation values. b Principle component
analysis (PCA) was built based on the normalized transcriptional
expression level of 12 samples. Samples grown on BWS were used for
comparison with those on RS. c Bar plot showing the number of DEGs in
two pairwise comparisons, Δgat1 vs 20b and Δhir1 vs 20b
groups (AA, AA9, GH, PL, and GL-encoding genes) (Fig. 3)
to evaluate the transcript alterations of genes encoding enzymes from these CAZyme families. The RPKM values of
all CAZyme-encoding genes are provided in Supplemental
Table S3. AA9 was excluded from the AA families because
it represents an oxidative mechanism that likely plays a role in
cellulose attack other than lignin degradation. There were 158
CAZyme-encoding genes identified as DEGs in Δgat1.
Among them, AA-encoding genes accounting for 15.7%,
AA9-encoding genes accounting for 39.2%, and GHencoding genes accounting for 43.1% were upregulated, while
the proportion of these identified groups for downregulated
genes was 30.2%, 0%, and 62.8%, respectively (Fig. 3b).
Genes encoding enzymes from AA9 families exhibited high
expression levels in the mutant Δgat1 versus 20b on RS. The
AA-encoding genes, which are mainly involved in the lignin
oxidation process, showed a high proportion among downregulated genes. Among them, the vp2 (protein ID 60432) gene,
which was predominantly accumulated in the 20b strain, was
significantly downregulated in the Δgat1 mutant strain. Other
vp/mnp genes, which were not highly expressed in parental
strain 20b, were also inactivated in the mutant Δgat1. Six
genes (protein IDs 62347, 67424, 77373, 89214, 96655, and
134564) encoding putative glyoxal oxidases from AA5 families were also strikingly downregulated. The RPKM values
and fold changes of the genes encoding putative enzymes
from the AA2 and AA5 families are shown in Table 2.
These transcriptional alterations were consistent with those
observed in BWS (Wu et al. 2020). In addition to downregulated genes, upregulated genes also revealed some intriguing
findings. Some cellulolytic (protein IDs 43698, 49686, and
130231, Table 3) and xylanolytic genes (protein IDs 81650,
89740, 110996, and 125911, Table 4), which encode putative
endo-β-1,4 glucanases and xylanases, respectively, were
found to be strongly upregulated in the mutants Δgat1 when
grown on RS, supporting the changes in the expression pattern
found on BWS. However, the transcriptional alterations of
some genes were not consistent on these two different substrates. For example, one gene (protein ID 83320, GH7,
Table 3) was highly upregulated (121-fold) in Δgat1 when
grown on BWS but not remarkably activated (2-fold) when
grown on RS. In contrast, three genes (protein IDs 47406,
100231, and 129772, GH7, Table 3) that were not activated in
Δgat1 when grown on BWS, were highly upregulated on RS.
We also observed similar transcriptional alterations in the expression of genes encoding lytic polysaccharide monooxygenases
Appl Microbiol Biotechnol
Table 2 Fold changes of ligninolytic genes including vps/mnps and
cros in hir1 and gat1 disruptants versus 20b strain, respectively
CAZy
Gene or protein IDa
FC (RS)b
FC (BWS)c
Table 3 Fold changes of cellulolytic genes encoding endo-β-1,4
glucanases belonging to GH6 and GH7 in hir1 and gat1 disruptants
versus 20b strain, respectively
CAZy
AA2
AA5
Δgat1
Δhir1
Δgat1
Δhir1
vp1
0.01
1.40
1.80
418.32
vp2
vp3
mnp1
mnp2
mnp3
mnp4
mnp5
mnp6
101121
134564
98389
121363
62166
62347
67424
77373
84350
88952
0.00
0.03
0.39
0.03
0.00
0.53
0.05
0.06
0.28
0.20
1.15
0.82
0.67
0.05
0.07
0.04
1.04
1.11
0.00
0.10
0.26
0.27
0.15
0.77
0.04
0.85
0.90
0.97
0.84
0.90
0.70
0.05
0.18
0.04
1.11
1.30
0.00
0.01
0.71
0.01
0.01
1.06
0.13
0.08
0.94
0.03
1.48
1.93
1.47
0.04
0.13
0.09
1.57
1.22
0.01
0.29
1.28
1.70
2.87
1.82
0.18
4.60
0.61
0.59
0.60
1.83
0.66
0.04
0.94
0.15
1.19
1.13
89214
91068
94009
96655
99670
0.01
1.25
15.93
0.01
0.78
0.03
0.81
1.29
0.01
0.38
0.04
2.28
2.58
0.01
0.17
0.17
1.27
0.72
0.05
0.31
a
A genomic fragment containing the genes that corresponds to each
Protein ID were from the genome database of strain PC9 (JGI
Pleurotus ostreatus PC9 v1.0, https://genome.jgi.doe.gov/PleosPC9_
1/PleosPC9_1.home.html)
b
FC, fold change. Ratios were calculated by comparing the RPKM
value of mutants and that of the parental control strain 20b grown on
rice straw
c
FC, fold change. Ratios were calculated by comparing the RPKM value
of mutants and that of the parental control strain 20b grown beechwood
sawdust (Wu et al. 2020, 2021)
(LPMOs) from the AA9 family (Protein IDs 96461, and 97339,
Table 4) on RS compared with that on BWS.
Transcriptional alterations of CAZyme-encoding
genes in Δhir1 grown on RS
A total of 125 CAZyme-encoding genes were identified as
DEGs in mutant strain Δhir1. In the mutant Δhir1, 7.0%,
29.6%, and 57.8% AA, AA9, and GH-encoding genes were
upregulated, while those for downregulated genes were
39.0%, 0%, and 56%, respectively (Fig. 3b). AA9-encoding
genes were mostly found in the upregulated group, and AA-
GH6
GH7
Protein IDa
FC (RS)b
FC (BWS)c
Δgat1
Δhir1
Δgat1
Δhir1
45206
130231
43698
100231
100398
107842
9.41
17.51
202.96
24.73
1.19
21.78
36.39
31.93
243.72
16.76
0.39
5.79
7.65
15.86
858.68
1.06
13.02
26.24
6.38
14.75
93.95
1.85
0.66
1.86
114771
129772
129783
47406
49445
83320
83849
83987
90281
90565
94368
47295
49686
1.94
24.84
14.21
57.14
0.41
1.99
0.86
1.30
0.93
0.52
0.71
0.44
12.45
14.85
2.18
1.95
33.99
11.04
472.33
144.53
0.85
1.61
0.39
0.56
0.51
60.87
1.52
1.71
3.94
0.35
0.05
121.23
7.98
1.22
1.81
1.85
1.59
0.17
10.82
2.62
1.15
0.81
0.55
2.22
270.89
65.82
0.83
2.48
1.11
0.83
0.17
7.75
a
A genomic fragment containing the genes that corresponds to each
Protein ID were from the genome database of strain PC9 (JGI
Pleurotus ostreatus PC9 v1.0, https://genome.jgi.doe.gov/PleosPC9_1/
PleosPC9_1.home.html)
b
FC, fold change. Ratios were calculated by comparing the RPKM value
of mutants and that of the parental control strain 20b grown on rice straw
c
FC, fold change. Ratios were calculated by comparing the RPKM value
of mutants and that of the parental control strain 20b grown beechwood
sawdust Wu et al. 2020, 2021)
encoding genes were mostly identified in the downregulated
group. We observed similar transcriptional alterations with the
mutant Δgat1, in which most cellulolytic (AA9, GH6, and
GH7) and xylanolytic genes (GH10 and GH11), were upregulated, while most ligninolytic genes (AA2 and AA5, Table 2)
were downregulated. However, we also observed some differences in the transcriptional alterations of several genes (AA2)
in Δhir1 grown on RS compared to those grown on BWS.
The mnp3 and mnp6 genes were both upregulated by 2.87and 4.60-fold in BWS (Δhir1 vs 20b) and could be considered
redundantly in the ligninolytic system (Wu et al. 2021), while
these genes were both downregulated on RS (Δhir1 vs 20b).
As mentioned previously, the vp1 gene (protein ID 116738,
Table 2), which was slightly expressed in parental strain 20b
on BWS, was upregulated (81.7-fold) on RS. As for the mutant Δhir1, the vp1 gene was only slightly upregulated (1.4fold) when it was cultured on RS, while it was highly
Appl Microbiol Biotechnol
Table 4 Fold changes of genes encoding LPMOs belonging to AA9
and xylanases belonging to GH10 and GH11 in hir1 and gat1 disruptants
versus 20b strain, respectively
CAZy
AA9
GH10 and GH11
Protein IDa
FC (RS)b
100006 and 122311, AA9) (Tables 3, 4) also exhibited distinct transcriptional alterations in the Δhir1 mutant in response to RS and BWS.
FC (BWS)c
Δgat1
Δhir1
Δgat1
Δhir1
100006
100072
117057
122311
125666
130437
31.79
4.64
1.89
22.19
26.57
2.99
130.93
38.28
0.73
67.20
7.68
2.26
15.58
3.33
1.43
0.61
0.34
191.12
7.59
1.84
1.00
6.78
1.68
2.26
134258
134259
20839
21077
21397
44265
45362
46220
46385
56431
59310
82144
82613
83777
83978
84841
87701
90424
2.09
1.27
0.37
0.50
136.65
88.76
4.14
21.01
2.64
12.26
175.74
2.92
54.22
41.32
12.36
0.82
132.87
109.69
1.15
0.83
0.48
0.24
139.37
273.07
3.64
102.94
2.27
87.82
274.68
25.25
27.73
61.79
11.57
20.23
132.96
309.49
30.41
6.29
34.09
16.53
4.42
61.40
0.30
12.47
75.05
68.41
0.73
522.70
176.82
53.35
3.81
53.70
62.57
92.73
1.34
0.67
0.40
0.14
13.99
34.51
1.55
10.26
1.14
4.60
11.04
3.24
3.20
26.28
3.35
7.90
39.67
98.88
90521
94095
94230
96461
97339
125911
81650
96691
110996
89740
33.23
31.28
25.73
103.56
39.82
52.04
5.81
0.14
77.61
4.41
12.63
15.71
37.32
181.59
69.72
104.96
36.26
0.08
126.27
62.27
15.58
3.33
1.43
0.61
0.34
23.97
2.45
0.10
394.47
4.38
6.90
2.19
13.98
43.34
52.34
19.25
2.12
0.10
45.56
3.62
a
A genomic fragment containing the genes that corresponds to each
Protein ID were from the genome database of strain PC9 (JGI
Pleurotus ostreatus PC9 v1.0, https://genome.jgi.doe.gov/PleosPC9_1/
PleosPC9_1.home.html)
b
FC, fold change. Ratios were calculated by comparing the RPKM value
of mutants and that of the parental control strain 20b grown on rice straw
c
FC, fold change. Ratios were calculated by comparing the RPKM value
of mutants and that of the parental control strain 20b grown beechwood
sawdust Wu et al. 2020, 2021)
upregulated (300 to 400-fold) on BWS (Wu et al. 2021). Other
cellulolytic genes (protein IDs 47406 and 100231, GH7;
Transcriptional analysis of small secreted proteinencoding genes and hydrophobin genes
CAZymes are key players in the lignocellulose degrading process. Some intriguing findings on CAZyme-encoding genes
have been identified in our previous and current studies.
However, in addition to CAZyme-encoding genes, there are
a number of genes that may play important roles in lignocellulose degradation processes that have not been extensively
studied (Nagy et al. 2020). In addition to the enzymatic degradation of substrates by CAZymes, the interaction between
substrates and fungi is facilitated through, for example, small
secreted proteins (SSPs) and hydrophobins (Bayry et al. 2012;
Pellegrin et al. 2015). SSPs and hydrophobins all feature
short-chain amino acids (aa): less than 300 aa with a conserved secretion signal peptide for SSPs (Kim et al. 2016)
and approximately 100 aa in length with eight conserved cysteine residues for hydrophobins (Wösten 2001). In this study,
we compared the transcription of genes that encode SSP and
hydrophobins (Fig. 4) to find more clues about the wooddecay process in mutants and parental strains grown on RS
and BWS.
There are totally six ssp genes predicted in the genome of
P. ostreatus PC9 strain currently (Feldman et al. 2017). We
observed a very low expression level for all ssp genes in the
parental strain 20b grown on both RS and BWS (RPKM value
<30). However, the ssp1 gene (protein ID 65712), which was
slightly expressed in parental strain 20b, was found to be
significantly upregulated in the mutant Δgat1 on both RS
(330.3-fold) and BWS (3518.4-fold) (Fig. 4, Supplemental
Table S4). This upregulation was not observed in the Δhir1
strain.
Twenty-five hydrophobin-encoding genes were predicted
in the genome of P. ostreatus PC9 strain, among which three
genes (protein IDs 74127, 80078, and 114483) were predominantly accumulated in the parental strain 20b on both RS and
BWS (Fig. 4, Supplemental Table S4). Among these three
genes, two genes (protein IDs 74127 and 80078) have been
identified and designated as vmh2 and vmh3, respectively
(Peñas et al. 2002). The vmh3 gene was reported to be the
most highly expressed hydrophobin-encoding genes in
P. ostreatus PC9 grown on SMY liquid medium under both
shaken and static conditions (Alfaro et al. 2016), different
from the parental strain 20b grown on BWS. All three genes
were significantly downregulated in the Δgat1 mutant on both
substrates, especially vmh2, which was downregulated by
17.2- and 31.0-fold in RS and BWS, respectively. In contrast,
we did not observe a significant downregulation of the three
genes in the Δhir1 mutant on both substrates. In addition,
Appl Microbiol Biotechnol
Fig. 3 Heatmap analysis of CAZyme-encoding genes in the parental
strain and mutants grown on rice straw for 13 d. a Heatmap analysis of
two groups: Δgat1 vs 20b and Δhir1 vs 20b. b The proportion of five
groups: AA, AA9, GH, PL, and GL-encoding genes in up- and
Fig. 4 Heatmap analysis of
hydrophobin-encoding genes and
small secreted protein-encoding
genes predicted in the JGI
P. ostreatus PC9 genome (http://
genome.jgi.doe.g.,ov/PleosPC9_
1/PleosPC9_1.home.html) grown
on BWS and RS
downregulated sets, respectively. The percentage was calculated using
the number of genes in each group divided by the number of all up- or
downregulated genes
Appl Microbiol Biotechnol
vmh2, which was strikingly downregulated in the Δgat1 mutant, was found to be slightly upregulated in the Δhir1 mutant.
Time-course expression analysis of some of the DEGs
using qRT-PCR
In this study, transcriptomic analysis of the strains grown for
13 days was performed. However, different growth periods
may result in gene expression bias in some wood-decaying
fungi (Hori et al. 2014; Alfaro et al. 2016; Fernández-Fueyo
et al. 2016). Therefore, to examine the time-course expression
patterns of some of the DEGs identified in this study (17
genes; Supplemental Table S6), qRT-PCR was performed.
As shown in Supplemental Fig. S1, different relative expression levels at different culture periods were observed in the
context of most of the analyzed genes, indicating that different
growth periods can indeed affect gene expression; however,
the transcriptomic alterations regarding the DEGs were maintained. The relative expression levels of up- and downregulated genes identified by RNA-seq analysis were also higher and
lower in the mutant strain(s), respectively, with two exceptions: 59310 was not significantly upregulated in hir1d#1
grown on RS, and 87582 was not highly expressed in 20b
grown on BWS. Overall, these qRT-PCR results mostly validated the RNA-seq data, further supporting the reliability of
the transcriptome analysis performed in this study.
Promoter analysis of genes co-expressed on distinct
substrates
Transcription factor alterations could affect the transcriptional
expression of specific genes (Hobert 2008). As the gene gat1
encodes a putative Agaricomycete-specific DNA-binding
transcription factor, we identified co-expressed genes using
the gene gat1 (protein ID 83134) as the bait gene in the
DEG lists of Δgat1 versus 20b grown on BWS and RS, respectively, to determine whether most transcriptional alterations are associated with the gat1 gene. There were 257 and
72 genes co-expressed on BWS and RS, respectively (Fig. 5,
Supplemental Table S5). These numbers are quite small when
compared to the DEGs identified on each substrate, which
suggested that most genes are regulated indirectly by Gat1.
Among these co-expressed genes, 43 and 13 genes were
CAZyme-encoding genes. Using these co-expressed
CAZymes genes, we searched for putative shared-regulatory
elements using de novo motif discovery in the promoter regions (Fig. 5b, d). However, considering that the number of
co-expressed CAZyme-encoding genes is smaller on RS than
that on BWS, which could make the shared motifs less ubiquitous, we added 13 more DEGs from the co-expressed
datasets of RS samples. As a result, a total of 43 and 26 genes
from the DEG lists identified from Δgat1 versus 20b on RS
and BWS, respectively. One motif in each co-expressed set
covered almost all co-expressed genes and was identified on
BWS and RS. The consensus binding motifs within the 1-kb
promoter regions of co-expressed CAZyme-encoding genes
showed conservation of two critical bases: C and T (Fig. 5).
Measurement of extracellular enzyme activities of
mutants grown on RS
To determine whether lignocellulolytic enzymes were affected by the transcript alterations of lignocellulolytic genes on
RS, we compared the extracellular lignin-modifying enzymes,
cellulase, and xylanase activities of the mutants Δgat1 and
Δhir1 with the parental strain 20b. We cultivated the mutants
Δgat1 and Δhir1, as well as the parental strain 20b, for 13 d
on RS, and subsequently examined the activities of extracellular guaiacol oxidizing activities (H2O2-independent, Mn2+independent/dependent peroxidase activities), and the specific
activities of carboxymethylcellulase (CMCase) and xylanase.
As shown in Fig. 6, extracellular laccase activities were almost
lost in the Δgat1 mutant, but increased in the Δhir1 mutant.
Mn2+-independent and Mn2+-dependent peroxidase activities
were all inactivated in both mutants, which was consistent
with the transcriptional alterations observed in the mutants.
Both specific activities of CMCase and xylanase were higher
in Δgat1 and Δhir1 than in 20b. These results suggest that the
up- and downregulation of lignocellulolytic genes are correlated with the increase and decrease of lignocellulolytic enzymes in both RS and BWS.
Examination of lignin-degrading abilities of mutants
grown on RS
In a previous study, Δgat1 and Δhir1 showed total defects
and decreased lignin-degrading ability on BWS compared to
the parental strain 20b (Nakazawa et al. 2019; Wu et al. 2021).
In this study, we examined lignin-degrading capacity again by
evaluating the Klason lignin loss after culturing the strains on
RS. As shown in Fig. 6c, similar results were obtained as those
on BWS, where Δgat1 lost the ability to degrade lignin and
Δhir1 retained approximately 20% of the lignin-degrading
ability. These results suggest that changing the substrate from
BWS to RS did not change their lignin-degrading capacity,
despite the fact that changes in the transcriptional expression
of several specific genes were observed.
Discussion
In this study, we aimed to examine whether distinct substrates
affect the transcriptional alterations present in the ligninolysisdeficient mutants. Comparative transcriptional analysis of the
parental strain 20b grown on BWS and RS was firstly examined. Genes which encode enzymes from AA2 family
Appl Microbiol Biotechnol
Fig. 5 Co-expressed transcription
factor (Gat1)-related genes of
P. ostreatus strains grown on
beech wood (a) and rice straw (c).
The sequenced logos showing
motifs shared by all co-expressed
CAZyme-encoding genes associated with the gene gat1 on beech
wood (b) and rice straw (c)
Fig. 6 The extracellular enzyme activities and Klason lignin loss in the
indicated strains grown on RS for 13 d (n = 3) and 28 d (n = 3),
respectively. a Extracellular lignin-modifying enzyme activities of indicated strains, one unit of activity for guaiacol oxidation was defined as the
amount of enzyme that increased the absorbance at 465 nm by 1.0 per
min. b Extracellular CMCase and xylanase activities, one unit of
xylanase/CMCase (U) is defined as the amount of enzyme required to
liberate 1 μmol of reducing sugar as xylose/glucose per minute under the
assay conditions. c Klason lignin loss was determined by detecting the
decrease in the amount of Klason lignin in the substrate relative to the nofungus control plate. Error bars represent the standard deviations of three
bioreplicates; *p < 0.05 and **p < 0.01 were determined by t test
Appl Microbiol Biotechnol
exhibited lower expression level on RS (Fig. 1). Considering
previous reports that a lower number and expression level of
ligninolytic genes in wheat bran and cotton seed hulls in the
white-rot fungus Dichomitus squalens were detected (Rytioja
et al. 2017), ligninolytic genes might also be more likely induced by specific components present in wood biomass in
P. ostreatus. However, most genes encoding putative H2O2producing enzymes possibly ascribed to lignin-degrading system exhibited comparable amount on both substrates (Janusz
et al. 2017). Given that lignin-modifying enzymes exhibited
lower expression levels on RS, a possible explanation may be
that these H2O2-producing enzymes may have other functions
exemplified by supporting LPMOs in cellulose oxidative degradation (Kracher et al. 2016). Rytioja et al. (2017) examined
the transcript responses of D. squalens to distinct substrates,
and found a similar cellulose/xylan response by the fungus
with a lower expression level of (hemi)-cellulolytic genes on
wheat bran substrates compared to spruce substrates when
grown for 16 d. This is similar with our findings in
P. ostreatus which showed lower transcript accumulations of
genes targeting cellulose and xylan on RS when compared
with that on BWS. It has been documented that graminaceous
plants and softwood consist mainly of arabino-4-Omethylglucuronoxylans, while hemicellulose of hardwoods
contains O-acetyl-4-O-methylglucuronoxylan, with a lower
amount of methylgulcuronic acid (Sunna and Antranikian
1997). This may explain why P. ostreatus expressed more
β - g l uc ur on i da s e- en co di n g g en es on R S t ha n o n
methylgulcuronic acid-rich BWS. Considering that the total
accumulation of transcripts targeting each substrate on BWS
was higher than that on RS, we assume that P. ostreatus may
be more suited to beech wood substrates, as this fungus is
commonly found on wood in nature. However, many factors
could be involved to affect the growth of P. ostreatus. To
confirm the above-mentioned hypothesis, a more complex
and comprehensive analysis is needed.
We did not observe a large amount of DEGs when
P. ostreatus was grown on RS compared with that on BWS
(Supplemental Table S2). But some specific genes showing
significantly different expression levels on distinct substrates
may cue us that P. ostreatus may switch their transcriptional
preferences in response to various substrates. Two genes
which encode VP1 and cupredoxin were significantly upregulated on RS. The expression of vp1 was reported to be predominant in P. ostreatus PC9 after being cultured on Mn2+deficient GP liquid medium (Knop et al. 2014). However,
when the 20b and PC9 strains were cultured on BWS and
Mn2+ amendment cotton stalk solid substrates, respectively,
the vp2 transcript accumulated most abundantly among the vp
genes (Salame et al. 2014; Nakazawa et al. 2017a). The secretion of VP1 was higher when P. ostreatus PC9 was cultured
on poplar wood and wheat straw for 21 d compared to glucose
medium (Fernández-Fueyo et al. 2016). These reports
suggested that vp1 or vp2 are two predominant expressed
genes in P. ostreatus and which one is selected to play a
predominant role is dependent on the substrate or the culture
stage. Cupredoxin-encoding genes were shown to be highly
expressed when the PC9 strain was cultured on SMY liquid
medium in trophophase (Alfaro et al. 2016), but not by strain
20b on BWS in this study. The altered expression of vp1 and
the cupredoxin-encoding gene in response to different substrates suggests some gene regulations induced by the different components of these substrates. Among downregulated
genes on RS, many peptidase-encoding genes were found
(Supplemental Table S2). Fungi secrete peptidases to breakdown proteins and polypeptides into small molecules to support osmotrophic nutrition of the growing hyphae (Petrini
et al. 1993). It has been reported that the secretion of peptidases could be affected by trophic status and phylogeny
(Semenova et al. 2017). However, our study suggests that
different substrates may also affect the expression of genes
encoding peptidases. A secretomic analysis showed that the
production of several peptidases differs when the P. ostreatus
PC9 strain was grown on lignocellulosic substrates and glucose medium, especially for the protein ID 71759, which was
abundantly secreted on glucose medium, but completely absent from lignocellulosic substrates (Fernández-Fueyo et al.
2016). Notably, in the list of identified DEGs above, there
are still many genes that encode enzymes with unknown functions. These genes could also provide key information in this
study, as described by Nagy et al. (2020). Thus, more attention
should be paid on the study of these genes in the future.
The transcriptional alterations of ligninolysis-deficient mutants (Δgat1/Δhir1 vs 20b) grown on RS were also examined
and compared with that on BWS. The downregulation of
ligninolytic genes in Δgat1 grown on RS was consistent with
that on BWS, suggesting that the transcriptional alterations of
vp/mnp genes in gat1 disruptants on BWS (Wu et al. 2020)
were also found on RS. However, as for the mutant Δhir1, the
vp1 gene was only slightly upregulated when it was cultured
on RS, while it was highly upregulated on BWS (Wu et al.
2021); in addition, some mnp genes upregulated on BWS
were shown to be downregulated on RS. These results suggest
that a redundant ligninolytic system in the mutant strain Δhir1
may not be induced properly when grown on RS. It has been
reported that putative CRE-A regulatory elements were only
found in the promoter region of the vp1 gene among nine vp/
mnp genes (Fernández-Fueyo et al. 2014). The regulation system of vps is different, especially between the genes vp1 and
vp2, which could result in distinct transcriptional expression
patterns. This should be confirmed with promoter analysis in
the future. Regarding the upregulation of the vp1 gene in the
Δhir1 mutant on RS, we assumed that the expression level of
the vp1 gene had a threshold in the Δhir1 mutant strain, such
that when the expression reached a certain level on RS, the
elevation became limited. Cellulolytic and xylanolytic genes
Appl Microbiol Biotechnol
were also upregulated in both mutants (Δgat1 and Δhir1)
grown on RS as they were observed on BWS, suggesting that
upregulation of cellulolytic or xylanolytic genes seems to be a
demand in ligninolysis-deficient mutant strains. However, we
observed some specific cellulolytic genes which are quite differently regulated regarding to distinct substrate.
Environmental stimuli and adaption to different substrates
may be the reason why mutants alter the expression bias of
specific genes (Jaenisch and Bird 2003). Antoniêto et al.
(2014) reported that many DEGs identified in the Δcre1 mutant versus the Trichoderma reesei wild type strain were quite
different on glucose and cellulose (Avicel) medium. Thus,
distinct substrates could induce different regulation patterns.
Regarding to which specific gene is going to be significantly
upregulated in the mutant strains, it seems to be decided depending on the transcriptional regulation systems in response
to different substrates independent from gat1 or hir1.
Furthermore, the results of qRT-PCR (Supplemental
Table S1) suggested that different growth periods are associated with gene expression bias in P. ostreatus in the context of
RS and BWS media; therefore, time-course expression patterns should be investigated in detail in the future.
In addition to CAZyme-encoding genes, we also observed
some genes which encode SSPs and hyrophobins differentially expressed in mutants grown on both substrates. The gene
ssp1 was found highly upregulated in Δgat1 mutant grown on
both substrates. A previous study found that the expression
level of ssp1 was also slightly expressed in P. ostreatus PC9 in
either static or shaken cultures on SMY liquid medium (Alfaro
et al. 2016). Potential functions of SSPs have been suggested,
including an “effector” function in pathogenic fungi (review
in Dodds et al. 2009), degradative capabilities in T. reesei
(Saloheimo et al. 2002), and a symbiosis role in Laccaria
bicolor (Plett et al. 2011). Feldman et al. (2017) proposed that
SSPs may function as partial regulators of the ligninolytic
system in P. ostreatus PC9 after demonstrating that modifications to ssp1 expression affected the transcriptional expression
of genes encoding aryl-alcohol dehydrogenases, aryl alcohol
oxidases, and VPs. In our study, all strains were cultured on
RS and BWS solid medium, and the levels of gene expression
were quite different from those of the strains grown on GP
liquid medium. However, the transcriptional alterations of the
ssp gene in our study strengthened our belief that these genes
play a direct or indirect role in the lignocellulolytic system.
Hydrophobins allow fungi aerial hyphae to escape from
aqueous environments and come into contacting with the air
(Wösten 2001). In wood decay fungi, the role of
hydrophobins in wood colonization has yet to be fully elucidated (Peddireddi et al. 2006) and evidence to support the
necessity of having a hydrophobic surface for lignocellulose
decomposition is still needed. In this study, hydrophobinencoding genes in the Δgat1 mutant was found to be remarkably downregulated on both substrates while that in the Δhir1
mutant was slightly upregulated. In Aspergillus nidulans,
hydrophobins were shown to promote biofilm formation on
sugarcane bagasse and may enhance lignocellulose utilization
by promoting the close-packed structure of enzyme-substratefungi (Brown et al. 2016). Transcriptome analysis of
Aspergillus niger also revealed that the upregulation of
hydrophobins when grown on wheat straw substrate may play
a role in recruiting hydrolases to the surface of wheat straw
(Delmas et al. 2012). Zong et al. (2016) reported that
pretreating cellulases with hydrophobins improved the bioconversion of cellulose in corn stover. Thus, the downregulation of hydrophobin-encoding genes in the mutant Δgat1 may
contribute to its defects in lignocellulose degradation. Our
results support the hypothesis that hydrophobin-encoding
genes in P. ostreatus are involved in the lignin-degrading
process.
The co-expressed genes exhibited differences on distinct
substrates, suggesting that different substrates could affect
the alterations associated with TF. In the brown rot fungi
Wolfiporia cocos, TFs have been shown to play important
roles in regulating biased expression on different substrates
(Wu et al. 2019). Motifs shared by co-expressed genes suggest
common regulatory mechanisms with respect to the TF
Δgat1. The two consensus motifs provided in this study provide targets for further experimental testing to confirm these
hypotheses.
The extracellular enzyme activities identified in this study
on RS were consistent with the alteration of transcriptional
alterations and also showed consistency with that we observed
on BWS (Wu et al. 2020, 2021). This strengthened our belief
that there seems a demand to elevate the cellulolytic and
xylanolytic systems. This should be confirmed in the future.
To conclude, our study found that P. ostreatus expressed
variable enzymatic repertoire-related genes in response to distinct substrates. The overall expression level of most genes
from the same CAZyme families was higher on BWS than
on RS. However, the different levels of expression of some
specific genes, such as the upregulation of β-glucuronidasesencoding genes, indicates that P. ostreatus adjusted its transcriptome in response to different substrates. Changing the
substrate from BWS to RS did not significantly affect the
trend of transcriptional shifts identified in Δgat1 and Δhir1
when grown on BWS. However, we observed that the activation of genes encoding cellulolytic or xylanolytic enzymes
from the same CAZyme families differs on distinct substrates,
suggesting that the upregulation of cellulolytic or xylanolytic
genes seems to be a demand in ligninolysis-deficient mutant
strains. Recently, Alfaro et al. (2020) showed that the wooddependent induction of the secretion of lignocellulolytic enzymes is suppressed in the presence of glucose in P. ostreatus
in the context of liquid media; therefore, comparative
transcriptome using the ligninolysis-deficient mutants
grown on the medium containing lignocellulose and/or
Appl Microbiol Biotechnol
monosaccharides, such as glucose, may provide some mechanistic clues. In regard to the specific gene that will be significantly upregulated in the mutant strains, this seems to depend
on the transcriptional regulation systems in response to different substrates, independent of gat1 or hir1. As such, this study
provides insights into the regulatory mechanisms of
lignocellulolytic genes.
Supplementary Information The online version contains supplementary
material available at https://doi.org/10.1007/s00253-020-11087-9.
Acknowledgments We would like to thank Prof. Yitzhak Hadar (Hebrew
University of Jerusalem, Israel) for providing P. ostreatus strain 20b.
Authors’ contributions TN conceived and designed the study. HLW carried out the experiment and drafted the manuscript. HLW, HBX, RHY,
DPB and MK performed the analyses, TN, MK, MS, and YH provided
editorial suggestions and revisions. All authors read and approved the
final manuscript.
Funding This work was supported in part by the Institute for
Fermentation, Osaka [to T.N.], JSPS KAKENHIs [16K18729 and
19H03017 to T.N.], and the China Scholarship Council [to H.W.].
Data availability All data supporting the claims of this manuscript are
presented and made available in this manuscript.
Compliance with ethical standards
Competing interests The authors declare that they have no competing
interests.
Ethics approval and consent to participate This article does not contain
any studies with human participants or animals performed by any of the
authors.
References
Alfaro M, Castanera R, Lavín JL, Grigoriev IV, Oguiza JA, Ramírez L,
Pisabarro AG (2016) Comparative and transcriptional analysis of
the predicted secretome in the lignocellulose-degrading basidiomycete fungus Pleurotus ostreatus. Environ Microbiol 18:4710–4726.
https://doi.org/10.1111/1462-2920.13360
Alfaro M, Majcherczyk A, Kües U, Ramírez L, Pisabarro AG (2020)
Glucose counteracts wood-dependent induction of lignocellulolytic
enzyme secretion in monokaryon and dikaryon submerged cultures
of the white-rot basidiomycete Pleurotus ostreatus. Sci Rep 10:
12421. https://doi.org/10.1038/s41598-020-68969-1
Álvarez JM, Canessa P, Mancilla RA, Polanco R, Santibáñez PA, Vicuña
R (2009) Expression of genes encoding laccase and manganesedependent peroxidase in the fungus Ceriporiopsis subvermispora
is mediated by an ACE1-like copper-fist transcription factor.
Fungal Genet Biol 46:104–111. https://doi.org/10.1016/j.fgb.2008.
10.002
Antoniêto ACC, dos Santos CL, Silva-Rocha R, Persinoti GF, Silva RN
(2014) Defining the genome-wide role of CRE1 during carbon catabolite repression in Trichoderma reesei using RNA-Seq analysis.
Fungal Genet Biol 73:93–103. https://doi.org/10.1016/j.fgb.2014.
10.009
Ashraf J, Ali MA, Ahmad W, Ayyub CM, Shafi J (2013) Effect of
different substrate supplements on oyster mushroom (Pleurotus
spp) production. Food Sci Technol 1:44–51. https://doi.org/10.
13189/fst.2013.010302
Bailey TL, Elkan C (1994) Fitting a mixture model by expectation maximization to discover motifs in bipolymers. Proc Int Conf Intell Syst
Mol Biol 2:28–36
Bayry J, Aimanianda V, Guijarro JI, Sunde M, Latge JP (2012)
Hydrophobins—unique fungal proteins. PLoS Pathog 8(5):
e1002700. https://doi.org/10.1371/journal.ppat.1002700
Billa E, Monties B (1995) Molecular variability of lignin fractions isolated from wheat straw. Res Chem Intermed 21(3-5):303–311. https://
doi.org/10.1007/BF03052260
Boraston AB, Bolam DN, Gilbert HJ, Davies GJ (2004) Carbohydratebinding modules: fine-tuning polysaccharide recognition. Biochem
J 382:769–781. https://doi.org/10.1042/BJ20040892
Brown NA, Ries LN, Reis TF, Rajendran R, Dos Santos RAC, Ramage
G, Riaño-Pachón DM, Goldman GH (2016) RNA-seq reveals
hydrophobins that are involved in the adaptation of Aspergillus
nidulans to lignocellulose. Biotechnol Biofuels 9:p145. https://doi.
org/10.1186/s13068-016-0558-2
Campbell JA, Davies GJ, Bulone V, Henrissat B (1997) A classification
of nucleotide-diphospho-sugar glycosyltransferases based on amino
acid sequence similarities. Biochem J 326:929–939. https://doi.org/
10.1042/bj3260929u
Delmas S, Pullan ST, Gaddipati S, Kokolski M, Malla S, Blythe MJ,
Ibbett R, Campbell M, Liddell S, Aboobaker A, Tucker GA,
Archer DB (2012) Uncovering the genome-wide transcriptional responses of the filamentous fungus Aspergillus niger to lignocellulose using RNA sequencing. PLoS Genet 8:e1002875. https://doi.
org/10.1371/journal.pgen.1002875
Deng W, Wang Y, Liu Z, Cheng H, Xue Y (2014) HemI: a toolkit for
illustrating heatmaps. PLoS One 9:e111988. https://doi.org/10.
1371/journal.pone.0111988
Dodds PN, Rafiqi M, Gan PH, Hardham AR, Jones DA, Ellis JG (2009)
Effectors of biotrophic fungi and oomycetes: pathogenicity factors
and triggers of host resistance. New Phytol 183:993–1000. https://
doi.org/10.1111/j.1469-8137.2009.02922.x
Feldman D, Kowbel DJ, Glass NL, Yarden O, Hadar Y (2017) A role for
small secreted proteins (SSPs) in a saprophytic fungal lifestyle:
ligninolytic enzyme regulation in Pleurotus ostreatus. Sci Rep 7:
1–13. https://doi.org/10.1038/s41598-017-15112-2
Fernández-Fueyo E, Castanera R, Ruiz-Dueñas FJ, López-Lucendo MF,
Ramírez L, Pisabarro AG, Martínez AT (2014) Ligninolytic peroxidase gene expression by Pleurotus ostreatus: differential regulation
in lignocellulose medium and effect of temperature and pH. Fungal
Genet Biol 72:150–161. https://doi.org/10.1016/j.fgb.2014.02.003
Fernández-Fueyo E, Ruiz-Dueñas FJ, López-Lucendo MF, Pérez-Boada
M, Rencoret J, Gutiérrez A, Pisabarro AG, Ramírez L, Martínez AT
(2016) A secretomic view of woody and nonwoody lignocellulose
degradation by Pleurotus ostreatus. Biotechnol Biofuels 9:49.
https://doi.org/10.1186/s13068-016-0462-9
Floudas D, Binder M, Riley R, Barry K, Blanchette RA, Henrissat B,
Martinez AT, Otillar R, Spatafora JW, Yadav JS, Aerts A, Benoit I,
Boyd A, Carlson A, Copeland A, Coutinho PM, de Vries RP,
Ferreira P, Findley K, Foster B, Gaskell J, Glotzer D, Gorecki P,
Heitman J, Hesse C, Hori C, Igarashi K, Jurgens JA, Kallen N,
Kersten P, Kohler A, Kües U, Kumar TK, Kuo A, LaButti K,
Larrondo LF, Lindquist E, Ling A, Lombard V, Lucas S, Lundell
T, Martin R, McLaughlin DJ, Morgenstern I, Morin E, Murat C,
Nagy LG, Nolan M, Ohm RA, Patyshakuliyeva A, Rokas A, RuizDueñas FJ, Sabat G, Salamov A, Samejima M, Schmutz J, Slot JC,
John FS, Stenlid J, Sun H, Sun S, Syed K, Tsang A, Wiebenga A,
Young D, Pisabarro A, Eastwood DC, Martin F, Cullen D,
Grigoriev IV, Hibbett DS (2012) The Paleozoic origin of enzymatic
Appl Microbiol Biotechnol
lignin decomposition reconstructed from 31 fungal genomes.
Science 336:1715–1719. https://doi.org/10.1126/science.1221748
Gupta S, Stamatoyannopoulos JA, Bailey TL, Noble WS (2007)
Quantifying similarity between motifs. Genome Biol 8:R24.
https://doi.org/10.1186/gb-2007-8-2-r24
Henrissat B (1991) A classification of glycosyl hydrolases based on amino acid sequence similarities. Biochem J 280:309–316. https://doi.
org/10.1042/bj2800309
Hoa HT, Wang CL, Wang CH (2015) The effects of different substrates
on the growth, yield, and nutritional composition of two oyster
mushrooms (Pleurotus ostreatus and Pleurotus cystidiosus).
Mycobiology 43:423–434. https://doi.org/10.5941/MYCO.2015.
43.4.423
Hobert O (2008) Gene regulation by transcription factors and
microRNAs. Science 319:1785–1786. https://doi.org/10.1126/
science.1151651
Honda Y, Matsuyama T, Irie T, Watanabe T, Kuwahara M (2000)
Carboxin resistance transformation of the homobasidiomycete fungus Pleurotus ostreatus. Curr Genet 37:209–212. https://doi.org/10.
1007/s002940050521
Hori C, Gaskell J, Igarashi K, Kersten P, Mozuch M, Samejima M,
Cullen D (2014) Temporal alterations in the secretome of the selective ligninolytic fungus Ceriporiopsis subvermispora during growth
on aspen wood reveal this organism's strategy for degrading lignocellulose. Appl Environ Microbiol 80:2062–2070. https://doi.org/
10.1128/AEM.03652-13
Jaenisch R, Bird A (2003) Epigenetic regulation of gene expression: how
the genome integrates intrinsic and environmental signals. Nat
Genet 33:245–254. https://doi.org/10.1038/ng1089
Janusz G, Pawlik A, Sulej J, Świderska-Burek U, Jarosz-Wilkołazka A,
Paszczyński A (2017) Lignin degradation: microorganisms, enzymes involved, genomes analysis and evolution. FEMS
Microbiol Rev 41:941–962. https://doi.org/10.1093/femsre/fux049
Kamitsuji H, Honda Y, Watanabe T, Kuwahara M (2004) Production and
induction of manganese peroxidase isozymes in a white-rot fungus
Pleurotus ostreatus. Appl Microbiol Biotechnol 65:287–294.
https://doi.org/10.1007/s00253-003-1543-9
Kersten P, Cullen D (2014) Copper radical oxidases and related extracellular oxidoreductases of wood-decay Agaricomycetes. Fungal
Genet Biol 72:124–130. https://doi.org/10.1016/j.fgb.2014.05.011
Kim KT, Jeon J, Choi J, Cheong K, Song H, Choi G, Lee YH (2016)
Kingdom-wide analysis of fungal small secreted proteins (SSPs)
reveals their potential role in host association. Front Plant Sci 7:
186. https://doi.org/10.3389/fpls.2016.00186
Knop D, Ben-Ari J, Salame TM, Levinson D, Yarden O, Hadar Y (2014)
Mn2+-deficiency reveals a key role for the Pleurotus ostreatus versatile peroxidase (VP4) in oxidation of aromatic compounds. Appl
Microbiol Biotechnol 98:6795–6804. https://doi.org/10.1007/
s00253-014-5689-4
König J, Grasser R, Pikor H, Vogel K (2002) Determination of xylanase,
β-glucanase, and cellulase activity. Anal Bioanal Chem 374:80–87.
https://doi.org/10.1007/s00216-002-1379-7
Kormelink FJM, Voragen AGJ (1993) Degradation of different
[(glucurono) arabino] xylans by a combination of purified xylandegrading enzymes. Appl Microbiol Biotechnol 38:688–695.
https://doi.org/10.1007/BF00182811
Kotake T, Kawamoto H, Saka S (2015) Pyrolytic formation of monomers
from hardwood lignin as studied from the reactivities of the primary
products. J Anal Appl Pyrolysis 113:57–64. https://doi.org/10.1016/
j.jaap.2014.09.029
Koutaniemi S, van Gool MP, Juvonen M, Jokela J, Hinz SW, Schols HA,
Tenkanen M (2013) Distinct roles of carbohydrate esterase family
CE16 acetyl esterases and polymer-acting acetyl xylan esterases in
xylan deacetylation. J Biotechnol 168:684–692. https://doi.org/10.
1016/j.jbiotec.2013.10.009
Kracher D, Scheiblbrandner S, Felice AK, Breslmayr E, Preims M,
Ludwicka K, Haltrich D, Eijsink VG, Ludwig R (2016)
Extracellular electron transfer systems fuel cellulose oxidative degradation. Science 352:1098–1101. https://doi.org/10.1126/science.
aaf3165
Levasseur A, Drula E, Lombard V, Coutinho PM, Henrissat B (2013)
Expansion of the enzymatic repertoire of the CAZy database to
integrate auxiliary redox enzymes. Biotechnol Biofuels 6:41.
https://doi.org/10.1186/1754-6834-6-41
Lombard V, Bernard T, Rancurel C, Brumer H, Coutinho PM, Henrissat
B (2010) A hierarchical classification of polysaccharide lyases for
glycogenomics. Biochem J 432:437–444. https://doi.org/10.1042/
BJ20101185
Lombard V, Golaconda Ramulu H, Drula E, Coutinho PM, Henrissat B
(2013) The carbohydrate-active enzymes database (CAZy) in 2013.
Nucleic Acids Res 42:D490–D495. https://doi.org/10.1093/nar/
gkt1178
Lundell TK, Mäkelä MR, Hildén K (2010) Lignin-modifying enzymes in
filamentous basidiomycetes-ecological, functional and phylogenetic
review. J Basic Microbiol 50:5–20. https://doi.org/10.1002/jobm.
200900338
Manavalan T, Manavalan A, Heese K (2015) Characterization of
lignocellulolytic enzymes from white-rot fungi. Curr Microbiol 70:
485–498. https://doi.org/10.1007/s00284-014-0743-0
Mueller-Harvey I, Hartley RD, Harris PJ, Curzon EH (1986) Linkage of
p-coumaroyl and feruloyl groups to cell-wall polysaccharides of
barley straw. Carbohydr Res 148:71–85. https://doi.org/10.1016/
0008-6215(86)80038-6
Nagy LG, Merényi Z, Hegedüs B, Bálint B (2020) Novel phylogenetic
methods are needed for understanding gene function in the era of
mega-scale genome sequencing. Nucleic Acids Res 48:2209–2219.
https://doi.org/10.1093/nar/gkz1241
Nakazawa T, Tsuzuki M, Irie T, Sakamoto M, Honda Y (2016) Marker
recycling via 5-fluoroorotic acid and 5-fluorocytosine counter-selection in the white-rot agaricomycete Pleurotus ostreatus. Fungal Biol
120:1146–1155. https://doi.org/10.1016/j.funbio.2016.06.011
Nakazawa T, Izuno A, Kodera R, Miyazaki Y, Sakamoto M, Isagi Y,
Honda Y (2017a) Identification of two mutations that cause defects
in the ligninolytic system through efficient forward genetics in the
white-rot agaricomycete Pleurotus ostreatus. Environ Microbiol 19:
261–272. https://doi.org/10.1111/1462-2920.13595
Nakazawa T, Izuno A, Horii M, Kodera R, Nishimura H, Hirayama Y,
Tsunematsu Y, Miyazaki Y, Awano T, Muraguchi H, Watanabe K,
Sakamoto M, Takabe K, Watanabe T, Isagi Y, Honda Y (2017b)
Effects of pex1 disruption on wood lignin biodegradation, fruiting
development and the utilization of carbon sources in the white-rot
Agaricomycete Pleurotus ostreatus and non-wood decaying
Coprinopsis cinerea. Fungal Genet Biol 109:7–15. https://doi.org/
10.1016/j.fgb.2017.10.002
Nakazawa T, Morimoto R, Wu H, Kodera R, Sakamoto M, Honda Y
(2019) Dominant effects of gat1 mutations on the ligninolytic activity of the white-rot fungus Pleurotus ostreatus Fungal. Biol 123:
209–217. https://doi.org/10.1016/j.funbio.2018.12.007
Peddireddi S, Velagapudi R, Hoegger PJ, Majcherczyk A, Naumann A,
Polle A, Kües U (2006) Multiple hydrophobin genes in mushrooms
In Pisabarro AG, Ramírez L (eds): VI Meeting on Genetics and
Cellular Biology of Basidiomycetes (GCBB-VI) Pamplona:
Universidad Pública de Navarra/Nafarroako Unibertsitate
Publikoa, pp 151-163
Pellegrin C, Morin E, Martin FM, Veneault-Fourrey C (2015)
Comparative analysis of secretomes from ectomycorrhizal fungi
with an emphasis on small-secreted proteins. Front Microbiol 6:
1278. https://doi.org/10.3389/fmicb.2015.01278
Peñas MM, Rust B, Larraya LM, Ramírez L, Pisabarro AG (2002)
Differentially regulated, vegetative-mycelium-specific
hydrophobins of the edible basidiomycete Pleurotus ostreatus.
Appl Microbiol Biotechnol
Appl Environ Microbiol 68:3891–3898. https://doi.org/10.1128/
AEM.68.8.3891-3898.2002
Petrini O, Sieber TN, Toti L, Viret O (1993) Ecology, metabolite production, and substrate utilization in endophytic fungi. Nat Toxins 1:
185–196. https://doi.org/10.1002/nt.2620010306
Plett JM, Kemppainen M, Kale SD, Kohler A, Legué V, Brun A, Martin
F (2011) A secreted effector protein of Laccaria bicolor is required
for symbiosis development. Curr Biol 21:1197–1203. https://doi.
org/10.1016/j.cub.2011.05.033
Rao PS, Niederpruem DJ (1969) Carbohydrate metabolism during morphogenesis of Coprinus lagopus (sensu Buller). J Bacteriol 100:
1222–1228
Ritter GJ, Seborg RM, Mitchell RL (1932) Factors affecting quantitative
determination of lignin by 72 per cent sulfuric acid method. Ind Eng
Chem Anal Ed 4:202–204
Robinson MD, McCarthy DJ, Smyth GK (2010) edgeR: a Bioconductor
package for differential expression analysis of digital gene expression data. Bioinformatics 26:139–140. https://doi.org/10.1093/
bioinformatics/btp616
Rytioja J, Hildén K, Yuzon J, Hatakka A, de Vries RP, Mäkelä MR
(2014) Plant-polysaccharide-degrading enzymes from basidiomycetes. Microbiol Mol Biol Rev 78:614–649. https://doi.org/10.
1128/MMBR.00035-14
Rytioja J, Hildén K, Di Falco M, Zhou M, Aguilar-Pontes MV, Sietiö
OM, Tsang A, de Vries RP, Mäkelä MR (2017) The molecular
response of the white-rot fungus Dichomitus squalens to wood and
non-woody biomass as examined by transcriptome and
exoproteome analyses. Environ Microbiol 19:1237–1250. https://
doi.org/10.1111/1462-2920.13652
Salame TM, Knop D, Tal D, Levinson D, Yarden O, Hadar Y (2012)
Predominance of a versatile-peroxidase-encoding gene, mnp4, as
demonstrated by gene replacement via a gene targeting system for
Pleurotus ostreatus. Appl Environ Microbiol 78:5341–5352. https://
doi.org/10.1128/AEM.01234-12
Salame TM, Knop D, Levinson D, Mabjeesh SJ, Yarden O, Hadar Y
(2014) Inactivation of a Pleurotus ostreatus versatile peroxidaseencoding gene (mnp2) results in reduced lignin degradation.
Environ Microbiol 16:265–277. https://doi.org/10.1111/14622920.12279
Saloheimo M, Paloheimo M, Hakola S, Pere J, Swanson B, Nyyssönen E,
Penttilä M (2002) Swollenin, a Trichoderma reesei protein with
sequence similarity to the plant expansins, exhibits disruption activity on cellulosic materials. Eur J Biochem 269:4202–4211. https://
doi.org/10.1046/j.1432-1033.2002.03095.x
Semenova TA, Dunaevsky YE, Beljakova GA, Borisov BA,
Shamraichuk IL, Belozersky MA (2017) Extracellular peptidases
as possible markers of fungal ecology. Appl Soil Ecol 113:1–10.
https://doi.org/10.1016/j.apsoil.2017.01.002
Shibuya N, Iwasaki T (1985) Structural features of rice bran hemicellulose. Phytochemistry 24:285–289. https://doi.org/10.1016/S00319422(00)83538-4
Sjostrom E (1993) Wood chemistry: fundamentals and applications Gulf
professional publishing. Academic Press, San Diego. https://doi.org/
10.1016/B978-0-08-092589-9.50005-X
Sun RC, Sun XF, Zhang SH (2001) Quantitative determination of
hydroxycinnamic acids in wheat, rice, rye, and barley straws, maize
stems, oil palm frond fiber, and fast-growing poplar wood. J Agric
Food Chem 49:5122–5129. https://doi.org/10.1021/jf010500r
Sunna A, Antranikian G (1997) Xylanolytic enzymes from fungi and
bacteria. Crit Rev Biotechnol 17:39–67. https://doi.org/10.3109/
07388559709146606
Toyokawa C, Shobu M, Tsukamoto R, Okamura S, Honda Y, Kamitsuji
H, Izumitsu K, Suzuki K, Irie T (2016) Effects of overexpression of
PKAc genes on expressions of lignin-modifying enzymes by
Pleurotus ostreatus. Biosci Biotechnol Biochem 80:1759–1767.
https://doi.org/10.1080/09168451.2016.1158630
Tsukihara T, Honda Y, Sakai R, Watanabe T, Watanabe T (2006)
Exclusive overproduction of recombinant versatile peroxidase
MnP2 by genetically modified white rot fungus, Pleurotus
ostreatus. J Biotechnol 126:431–439. https://doi.org/10.1016/j.
jbiotec.2006.05.013
Tzfadia O, Diels T, De Meyer S, Vandepoele K, Aharoni A, Van de Peer
Y (2016) CoExpNetViz: comparative co-expression networks construction and visualization tool. Front Plant Sci 6:1194. https://doi.
org/10.3389/fpls.2015.01194
Vanholme R, Demedts B, Morreel K, Ralph J, Boerjan W (2010) Lignin
biosynthesis and structure. Plant Physiol 153:895–905. https://doi.
org/10.1104/pp.110.155119
Wösten HA (2001) Hydrophobins: multipurpose proteins. Annu Rev
Microbiol 55:625–646. https://doi.org/10.1146/annurev.micro.55.
1.625
Wu B, Gaskell J, Zhang J, Toapanta C, Ahrendt S, Grigoriev IV,
Blanchette RA, Schilling JS, Master E, Cullen D, Hibbett DS
(2019) Evolution of substrate-specific gene expression and RNA
editing in brown rot wood-decaying fungi. ISME J 13:1391–1403.
https://doi.org/10.1038/s41396-019-0359-2
Wu H, Nakazawa T, Takenaka A, Kodera R, Morimoto R, Sakamoto M,
Honda Y (2020) Transcriptional shifts in delignification-defective
mutants of the white-rot fungus Pleurotus ostreatus. FEBS Lett 594:
3182–3199. https://doi.org/10.1002/1873-3468.13890
Wu H, Nakazawa T, Morimoto R, Shivani, Sakamoto M, Honda Y
(2021) Targeted disruption of hir1 alters the transcriptional expression pattern of putative lignocellulolytic genes in the white-rot fungus Pleurotus ostreatus. Fungal Genet Biol 147:103507. https://doi.
org/10.1016/j.fgb.2020.103507
Xiao B, Sun X, Sun R (2001) Chemical, structural, and thermal characterizations of alkali-soluble lignins and hemicelluloses, and cellulose
from maize stems, rye straw, and rice straw. Polym Degrad Stab 74:
307–319. https://doi.org/10.1016/S0141-3910(01)00163-X
Yoav S, Salame TM, Feldman D, Levinson D, Ioelovich M, Morag E,
Yarden O, Bayer EA, Hadar Y (2018) Effects of cre1 modification
in the white-rot fungus Pleurotus ostreatus PC9: altering substrate
preference during biological pretreatment. Biotechnol Biofuels 11:
212. https://doi.org/10.1186/s13068-018-1209-6
Zoghlami A, Paës G (2019) Lignocellulosic biomass: understanding recalcitrance and predicting hydrolysis. Front Chem 7:874. https://doi.
org/10.3389/fchem.2019.00874
Zong Z, He R, Fu H, Zhao T, Chen S, Shao X, Zhang D, Cai W (2016)
Pretreating cellulases with hydrophobins for improving bioconversion of cellulose: an experimental and computational study. Green
Chem 18:6666–6674. https://doi.org/10.1039/C6GC02694J
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.