polymers

Review
Studies of Cellulose and Starch Utilization and the
Regulatory Mechanisms of Related Enzymes in Fungi
Bao-Teng Wang, Shuang Hu, Xing-Ye Yu, Long Jin , Yun-Jia Zhu and Feng-Jie Jin *
College of Biology and the Environment, Co-Innovation Center for Sustainable Forestry in Southern China,
Nanjing Forestry University, 159 Longpan Road, Nanjing 210037, China
* Correspondence: jinfj@njfu.edu.cn; Tel.: +86-25-8542-7210




Received: 9 January 2020; Accepted: 16 February 2020; Published: 2 March 2020


Abstract: Polysaccharides are biopolymers made up of a large number of monosaccharides joined

together by glycosidic bonds. Polysaccharides are widely distributed in nature: Some, such as
peptidoglycan and cellulose, are the components that make up the cell walls of bacteria and plants,
and some, such as starch and glycogen, are used as carbohydrate storage in plants and animals. Fungi
exist in a variety of natural environments and can exploit a wide range of carbon sources. They play
a crucial role in the global carbon cycle because of their ability to break down plant biomass, which is
composed primarily of cell wall polysaccharides, including cellulose, hemicellulose, and pectin. Fungi
produce a variety of enzymes that in combination degrade cell wall polysaccharides into different
monosaccharides. Starch, the main component of grain, is also a polysaccharide that can be broken
down into monosaccharides by fungi. These monosaccharides can be used for energy or as precursors
for the biosynthesis of biomolecules through a series of enzymatic reactions. Industrial fermentation
by microbes has been widely used to produce traditional foods, beverages, and biofuels from starch
and to a lesser extent plant biomass. This review focuses on the degradation and utilization of plant
homopolysaccharides, cellulose and starch; summarizes the activities of the enzymes involved and
the regulation of the induction of the enzymes in well-studied filamentous fungi.

Keywords: fungi; polysaccharides; enzyme; regulator; cellulase; amylase

1. Introduction
Polysaccharides are relatively complex carbohydrates that are widely distributed in nature.
They are biopolymers made up of a variety of monosaccharides joined together by glycosidic bonds.
Plant polysaccharides are the most abundant carbon source and can be divided into plant cell wall
polysaccharides (such as cellulose, hemicellulose, and pectin) and storage polysaccharides (such as
starch and inulin) [1–3].
All plant cells are surrounded by complex cell walls, and secondary cell walls form the architecture
of plant biomass. Different plant cell wall polysaccharides are interconnected with each other and linked
to the aromatic polymer lignin to provide the mechanical strength and structural integrity of plant
cells. Among them, cellulose fibrils are synthesized at the plasma membrane, while hemicelluloses and
other matrix polysaccharides are produced in the Golgi apparatus [4]. The final step of secondary cell
wall formation is lignification, which is caused by monolignol secretion by the lignifying cell and/or
neighboring cells [5,6]. Lignin polymer deposition in the apoplast provides physical and chemical
recalcitrance to plant tissues through the formation of lignocellulosic complexes [4]. In addition to
polysaccharides and lignins, plant cell walls also contain several types of structural proteins, such as
arabinogalactan proteins, extensins, and lectins [7,8].
Cellulose, as the major component of plant biomass, is the most abundant polysaccharide in the
world. Cellulose is a linear polymer consisting of β-1,4-linked D-glucose residues. These glucose

Polymers 2020, 12, 530; doi:10.3390/polym12030530 www.mdpi.com/journal/polymers

Polymers 2020, 12, 530 2 of 17

chains are tightly bonded by hydrogen bonds to form insoluble fibrous materials. The cellulosic
polymer has been described by a two-phase model, consisting of crystalline and amorphous phases
often interrupted by a series of semicrystalline structures, which makes it difficult to be utilized by
active carbohydrate enzymes [9]. Compared with cellulose, another major component of plant cell wall
polysaccharides, hemicelluloses, are more diverse and complex heterosaccharides, which are derived
from a heterogeneous group of sugars including D-xylose, D-galactose, and D-mannose. Among
them, the most abundant hemicellulose is xylan, whose backbone is a chain of β-1,4-linked D-xylose
residues [10]. Relative to cellulose, hemicellulose has a smaller molecular weight and can be dissolved
in alkaline solutions.
As one of the most abundant natural resources, cellulose has been used in many different ways,
such as in industrial fermentation, fiber material, and papermaking. As a polysaccharide, cellulose
has been regarded as an important cornerstone of developments in bioenergy [11–15]. At present,
using lignocellulose biomass as a carbon source substrate to produce methane, ethanol, and biofuels
by microbial fermentation has become a hotspot in renewable energy research [16–18]. In addition,
the cellulose utilization of microorganisms also has an important effect on the carbon cycling process,
which is one of the largest material flows in the biosphere. Crop straw is a valuable bioenergy resource
in agroecosystems. To date, the crop straw industry treatment is still in a compromised state, with high
energy consumption, high pollution, low output, and low efficiency; therefore, the comprehensive
utilization of crop straw resources will be of great significance to promote resource conservation,
environmental protection, and sustainable agricultural development. Recently, some studies have
shown that crop straw can be used as a substrate for fermentation to produce biogas or methane [19,20].
Bioenergy research on the production of biogas from lignocellulose biomass through anaerobic
fermentation has great potential but has not been widely adopted. Because of its complex structure,
lignocellulose is not easily decomposed and utilized by microorganisms [21]. The slow degradation
rate of lignocellulose seriously affects the reaction time of anaerobic digestion in biogas production and
undermines the economic feasibility. Therefore, in the past few decades, various physical, chemical,
and biological pretreatment technologies have been developed for the better use of lignocellulose
biomass to obtain high-yield biogas [22–24]. Most of the pretreatments for plant biomass are directed to
get rid of lignin, which is the main polymer that hampers cellulose and hemicellulose utilization, and
many of them simultaneously disrupt the other polysaccharides in the cell wall. However, these studies
were mostly based on the pretreatment of the whole corn stalk [21,25], and it is difficult to unify the
pretreatment conditions because the chemical components of different parts of the corn stalk are quite
different. In addition, physical and chemical pretreatment methods can also cause serious energy
consumption and environmental pollution [26]. Therefore, it is an important task for us to further
improve the fermentation capacity of microorganisms by increasing the activities of enzymes related to
lignocellulose biomass decomposition and optimizing the cultivation conditions of microorganisms.
In addition, the gradually increasing global energy crisis requires us to further develop and explore
new bioenergy and other renewable energy sources, such as the use of lignocellulose biomass resources
by microbial anaerobic fermentation [27]. Another application of cellulose is nanocellulose materials,
which are nontoxic, biodegradable, and biocompatible and have no adverse effect on the environment
and human health. Because of their good physical and chemical properties, nanocelluloses are widely
used in thermoreversible hydrogels, food packaging, flexible screens, coating additives, paper, optical
transparent films, and biopharmaceuticals, for example [28–31].
In recent years, as increasing numbers of cars are produced and used in the world, the demand
for vehicle fuel is expanding annually. Some studies have shown that ethanol can be used as an
alternative fuel [32–34]. The production of ethanol from plants has been known since ancient times, but
its substrate is amyloid polysaccharides. The major amyloid polysaccharide is starch, which consists
of multiple glucose units that are linked by α-1,4-glycosidic bonds and branched by α-1,6-glycosidic
bonds. As an important plant storage polysaccharide, the main sources of starch are cereal grains,
which are widely used in traditional food fermentation production, such as liquor and soy sauce, for
Polymers 2020, 12, 530 3 of 17

example [35,36]. Recent studies have shown that starch granules can also be used to prepare nanoscale
starch particles, which have unique physical properties. Because starch is an environmentally friendly
material, starch nanoparticles are considered to be a promising new biomaterial for use in foods,
medicines, cosmetics, and various composite materials [37].
In this review, we discuss recent advances in the utilization of major plant polysaccharides
(cellulose and starch), related enzyme production, and their molecular regulatory mechanisms in
fungi. We aimed to better understand the degradation of plant polysaccharides and the regulatory
mechanisms of related enzymes that can help us to acquire better strains that are more suitable for
industrial fermentation utilization.

2. The Fungi and Their Potential in the Utilization of Plant Polysaccharides

The filamentous fungi of the genus Aspergillus could release large amounts of conidia and are
widely distributed in the environment, such as soil, grain, and organisms. Aspergillus species, as
important environmental microorganisms, play important roles not only in the traditional food
fermentation industries but also in the growth and development of plants and animals because of
their strong capacity for the decomposition of organic matter and pathogenicity. Some Aspergillus
species, such as Aspergillus niger, Aspergillus awamori, and Aspergillus oryzae, are used to produce a
variety of fermented products, which are mostly related to liquor, vinegar, and soy sauce [35,38,39].
Among them, A. oryzae is an industrially important filamentous fungus, which could utilize the cereal
grain to produce traditional fermented foods such as liquor [40,41]. A. oryzae has the ability to produce
and secrete large amounts of amylase, which could break down starch into glucose. Glucose can be
further degraded into low-carbon sugar through the glycolysis pathway, and finally converted into
ethanol by anaerobic fermentation. At the same time, A. oryzae can also secrete large amounts of
proteolytic enzymes essential for soy sauce production [42]. Another filamentous fungus, A. niger,
is widely used in the industrial production of citric acid and gluconic acid because of its ability to
secrete organic acids in large quantities [39,43]. In recent years, using starch as an inducer and a
strong starch-inducible promoter, some Aspergillus species have also been used widely in the efficient
production of heterologous proteins, including various active enzymes, and even some heterologous
proteins derived from higher eukaryotes, such as plants and animals [44–46].
In addition to the Aspergillus species, some yeast strains are also often used for alcohol fermentation
in industrial production. For example, the yeast Saccharomyces cerevisiae can convert polysaccharides
into ethanol and some other vital metabolites, such as acetate, glycerol, pyruvate, succinate, and esters.
Some of the most significant food and beverages known to humans (such as beer, wine, and bread)
are made through the alcoholic fermentation process. These traditional fermentation products are
produced by using yeast strains to spontaneously ferment carbon-rich substrates (e.g., bread/beer from
cereals, wine from grapes) [36,47–49]. Some studies have shown that despite many kinds of microbes
involved in the brewing of wine and other fermentation production, S. cerevisiae has always been the
dominant species [50–52]. The yeast population dynamics of winemaking follow a consistent growth
pattern, in which a large number of non-Saccharomyces initially appear in the early stages but are soon
replaced by S. cerevisiae strains to finish the fermentation production. The advantage of S. cerevisiae over
other yeast species in alcoholic fermentation has traditionally been attributed to its high fermentation
capacity and adaptation to harsh environmental conditions [53]. Although these fungi can use starch
from grains to produce alcohol, this substrate is too expensive to be used to produce alcohol as an
alternative to biofuels and can cause serious food waste. Therefore, it is urgent to use lignocellulosic
biomass as a carbon source to produce alcohol and other bioenergy efficiently.
Plant biomass is the most abundant renewable carbon resource on earth, and the bioconversion of
plant polysaccharides has already attracted extensive attention due to the potential applications as
described above. Plant biomass is degraded and utilized by a variety of microorganisms that play
crucial roles in the carbon cycle of the ecosystem [54]. Among them, the filamentous fungi Trichoderma
reesei is an effective cellulase-producing strain and the most studied cellulose-decomposing fungus.
Polymers 2020, 12, 530 4 of 17

The filamentous fungus T. reesei is an ascomycete that can grow rapidly and is widely distributed in soil
environments. It was originally isolated from the South Pacific [55] and is well known for the ability
to secrete large amounts of cellulase, especially when cellulose is used as the carbon source. To date,
a large number of studies have deeply explored not only the function of glycoside hydrolase but also
the molecular mechanism of regulation of related enzyme–gene expression in T. reesei [56–60]. Due to its
industrial importance and the multiple uses of cellulase in T. reesei, many mutants that increase cellulase
yield have been acquired through conventional mutagenesis techniques. Currently, some mutants
have been reported to secrete high yields of cellulase into the medium for industrial utilization [61–63].
In addition to T. reesei, there are also some other microorganisms that can use cellulose as a carbon
source to produce useful substances. For examples, several wood-rotting basidiomycetes, white rot and
brown rot fungi, some plant pathogens, the basidiomycetous yeast Rhodotorula glutinis, etc. have been
isolated [64–66]. Basidiomycetes are the most potential cellulose degraders since many species grow
on dead wood or litter, in environments rich in cellulose, and they have been studied extensively [67].
Different strains are suitable for different fermentation industries, and researchers are constantly trying
to choose better ones to exploit [68,69]. Recent studies also showed that the saccharification of wheat
straw was importantly enhanced by mixing enzymes from T. reesei and Aspergillus species [70,71].
Cocultivation of multiple fungi may be an excellent system for producing various active enzymes in a
single bioreactor.

3. Cellulolytic Enzymes and Their Regulatory Mechanisms in Fungi

3.1. Classification of Cellulolytic Enzymes

Cellulases are complex enzymes that degrade cellulose into cellobiose or glucose. In generally,
the degradation of cellulose requires three types of enzymes: endoglucanases (EGs: EC 3.2.1.4),
cellobiohydrolases (CBHs: EC 3.2.1.91), and β-glucosidases (BGLs: EC3.2.1.21) [72]. Among them,
endoglucanases could cleave long cellulose into the shorter oligosaccharides; cellobiohydrolases could
further degrade these shorter oligosaccharides into cellobiose; and cellobiose is finally broken down
by β-glucosidases, resulting in the conversion of cellulose into D-glucose. However, some fungi
also produce other enzymes to promote the decomposition of cellulose, such as cellodextrinase (EC
3.2.1.74), which can remove disaccharide (cellobiose) from the cello-oligosaccharide. In addition,
cellodextrin phosphorylase (EC 2.4.1.49), cellobiose phosphorylase (EC 2.4.1.20), and cellobiose
epimerase (EC 5.1.3.11) were also found to relate to the degradation of cellulose [73]. In addition to
these, the lytic polysaccharide monooxygenases (LPMOs) are also considered as effective auxiliary
enzymes for cellulose degradation [74,75]. These enzymes could form a cellulase system that hydrolyzes
cellulose through synergistic action. In addition, the deconstruction of cell wall polymers is in a
certain order. For example, an acetyl xylan esterase from Bjerkandera adusta is inhibited by ferulic acid,
therefore the ferulate esterases gene could be expressed once the acetyl groups have been removed
from hemicellulose [76]. Except for these, a set of non-hydrolytic proteins, fungal swollenins, that are
homologous to canonical plant expansins, can improve the accessibility and efficiency of the enzymes
involved in saccharification of cellulose substrates by loosening macrofibrils [77].

3.2. Mechanism of Cellulase Induction

Cellulose-degrading fungi can be directly used in industrial production, but the optimum
conditions for enzyme production and utilization must be considered simultaneously, because their
environmental requirements are not uniform. For example, some enzymes have maximum activity at
80 ◦ C, while fungi often produce enzymes at the optimum temperature of 30 ◦ C. Therefore, how to
create a suitable working environment for both requires further exploration. Of course, cellulase
production and its industrial applications can be separated, but this also increases the extraction and
purification steps of the enzyme. Both methods have their advantages and disadvantages. At present,
the main method to increase cellulase yield is through molecular biotechnology.
Polymers 2020, 12, 530 5 of 17

The first step is for the fungi to sense an external carbon source. According to available carbon
sources, the production of cellulase and xylanase is regulated at the transcriptional level in fungi,
and only when plant polysaccharides (such as cellulose and xylan) are provided as carbon sources
does the fungus begin to produce these enzymes in large quantities [78–81]. However, when using
easily metabolized carbon sources such as glucose, the production of these enzymes is inhibited [79].
These suggested that several signal transduction pathways responsible for each of these inducers
might control the expression of cellulase and xylanase. For example, the heterotrimeric G-protein
GanB(alpha)-SfaD(beta)-GpgA(gamma) is a carbon source sensor that controls cAMP/PKA signaling
in response to glucose [82]. The GanB may be involved in sensing various carbon sources and
subsequently activating downstream signal transduction. In addition, HxtB, a glucose and xylose
transporter, has been confirmed to localize to the plasma membrane and may play a role in downstream
glucose signaling and metabolism [83]. Furthermore, the protein kinase PskA has an important
function in the control of sugar flux and metabolism [84]. However, carbon source sensors, subsequent
transport, and cellular signaling pathways still remain largely unelucidated. It has been reported
that coregulation of these cellulolytic and xylanolytic enzymes can effectively degrade plant cell wall
polysaccharides. Since these polymers from plants cannot enter fungal cells directly, it has been
suggested that the expression of these cellulase- (or hemicellulase)-encoding genes is induced by
the existence of soluble sugars degraded from cellulose [78,85,86]. The primary product of cellulose
degradation by cellulase is called cellobiose. Studies have shown that cellobiose could induce the
production of cellulase in many fungi, such as T. reesei [86–88]. However, cellobiose can be further
hydrolyzed into glucose by extracellular β-glucosidases, and the presence of glucose inhibits the
uptake of cellobiose, therefore resulting in the inhibition of cellulase expression [79,89]. Some studies
have indicated that reduction in BGL activity can lead to an increasing cellulose production, such as
the deletion of the extracellular BGL encoding gene or addition of the inhibitor of β-glucosidase in
the media [90]. Based on these results, current molecular biology techniques have been applied to
improve cellulase production.

3.3. Molecular Regulation Mechanisms of Cellulase Gene Expression

The efficient degradation of plant cell wall polysaccharides requires not only a wide range
of cellulases to be secreted in large quantities but also a complex regulatory system to control the
expression of these genes. Most of the transcription factors involved in these regulatory mechanisms
belong to the Zn2Cys6 zinc binuclear cluster family, which to date has only been found to be specific
to fungi [91]. Understanding of the molecular mechanism involved in cellulase and xylanase gene
transcription regulation in filamentous fungi has made great progress. Through extensive analysis
of these hydrolase genes and their promoter regions, several transcription regulators involved in the
expression of these enzymes have been identified. The molecular mechanisms of transcription factors
that regulate the expression of cellulase genes are shown in Figure 1.
XlnR, as a fungal-specific Zn2Cys6-type positive regulator, was first identified in A. niger [92],
and then its orthologous gene was further isolated and designated Xyr1 (xylanase regulator 1) in
T. reesei [93]. Some studies have demonstrated that it regulates the expression of not only cellulolytic
genes [94,95] but also xylanolytic genes [92,95]. It has been reported that the deletion of the Xyr1 gene
resulted in importantly decreased expression levels of major xylanolytic genes (xyn1 and xyn2) and
cellulolytic genes (cbh1, cbh2, egl1, and bgl1), even under induction culture conditions [95]. Cellulose
and xylan are the most abundant polysaccharides in nature; therefore, XlnR/Xyr1 is a critical regulator
in the biomass utilization process. In addition to polysaccharide degradation, XlnR/Xyr1 also regulates
the expression of enzymes (D-xylose reductase, XyrA) involved in first step of the pentose catabolic
pathway, which is the main pathway for the conversion of L-arabinose and D-xylose [96]. Studies
in Aspergillus species have shown that the regulation of the pentose catabolic pathway also requires
synergies with another transcription factor, AraR [97,98]. In industrial fermentation production, a large
amount of hemicellulose is produced, and the presence of these hemicelluloses will inhibit the secretion
Polymers 2020, 12, 530 6 of 17

of fungal cellulase. By exploring the regulation of Xyr1 on the xylanase pathway, cellulase production
may be enhanced by an external hemicellulose, such as xylan as carbon source [99]. Of course, Xyr1
is not the only transcription factor that can sense the external carbon source. BglR (β-glucosidase
regulator) is also involved in the sensing of cellobiose, but the specific mechanism of its action has not
been thoroughly studied, and needs further exploration [100]. In addition, the transcription factor BglR
has a low
Polymers 2020,homology the AmyR of A. oryzae, which is a key activator of amylase gene expression.
withREVIEW
12, x FOR PEER 6 of 18

Figure 1. Schematic diagram of a transcriptional regulatory network of genes encoding cellulolytic

Figure 1. Schematic diagram of a transcriptional regulatory network of genes encoding cellulolytic
enzymes in fungi. The solid line with arrowhead represents activated gene expression. The dotted line
enzymes in fungi. The solid line with arrowhead represents activated gene expression. The dotted
with arrowhead represents suppressed gene expression.
line with arrowhead represents suppressed gene expression.
In recent years, a novel Zn2Cys6-type fungal-specific transcription factor, ClbR, was identified
to beXlnR, as a in
involved fungal-specific
the degradation Zn2Cys6-type
of cellulosepositive regulator,
and cellobiose was first identified
in Aspergillus in A. niger
aculeatus [101]. ClbR[92],
was
and then its orthologous gene was further isolated and designated Xyr1 (xylanase
found to induce the expression of several cellulolytic genes, such as the FII-carboxymethyl cellulase regulator 1) in T.
reesei
(cmc2)[93].
andSome studies have
FIII-avicelase demonstrated
(cbhI). Additionally,that it regulates
it also coregulated the expression
the expression of not onlyFIb-xylanase
of the cellulolytic
genes [94,95] but also xylanolytic genes [92,95]. It has been reported that
(xynIb) and FI-carboxymethyl cellulase (cmc1) genes with XlnR [101]. From the above studies, the deletion of the Xyr1 we gene
can
resulted in importantly decreased expression levels of major xylanolytic genes
see that in addition to the genus Trichoderma, the molecular regulation mechanism of cellulase is also (xyn1 and xyn2) and
cellulolytic
fully studied genes (cbh1, cbh2,
in Aspergillus egl1, and bgl1), even under induction culture conditions [95]. Cellulose
species.
and xylan are the most abundant
Clr-1 is another transcription factor polysaccharides in nature;
that is necessary therefore,utilization.
for cellulose XlnR/Xyr1 However,
is a critical its
regulator in the biomass utilization process. In addition to polysaccharide
regulation of cellulase gene expression is strongly influenced by inducers: Clr-1 cannot induce the degradation, XlnR/Xyr1
also regulates
expression of the expression
the cellulase of enzymes
gene without an (D-xylose
inducerreductase,
[102]. In theXyrA) involved
presence in inducer,
of an first stepsuch of the as
pentose catabolic pathway, which is the main pathway for the conversion
cellobiose, a degradation product of cellulose, Clr-1 induces the expression of some genes containing of L-arabinose and D-
xylose [96]. Studies
β-glucosidases and in Aspergillus
Clr-2, species have
a transcription shown[103].
regulator that the Theregulation of the pentose
Clr-2 regulator was found catabolic
to be
pathway also requires synergies with another transcription factor, AraR
responsible for the expression of major cellulolytic genes [104]. The deletion of the two genes may [97,98]. In industrial
fermentation
block cellulose production,
utilizationaas large amount
a carbon of hemicellulose
source by Neurosporais crassa
produced,
[103].and the presence
Interestingly, even of in
these
the
hemicelluloses will inhibit the secretion of fungal cellulase. By exploring the
absence of inducers, Clr-2 expression leads to activation of cellulase gene expression [104]. Through regulation of Xyr1 on
the xylanase pathway, cellulase production may be enhanced by an external
the screening of mutants in the absence of an inducer, a Clr-3 that inhibits the activity of Clr-1 washemicellulose, such as
xylan as carbon
identified sourceIt[99].
in N. crassa. was Of course,
found that Xyr1 is not the
the deletion only
of the transcription
clr-3 gene led to factor thatgene
cellulase canexpression
sense the
external carbon source. BglR (β-glucosidase
of Clr-1–dependent even without an inducer [102,105]. regulator) is also involved in the sensing of cellobiose,
but the specific mechanism of its action has not been thoroughly studied, and needs further
exploration [100]. In addition, the transcription factor BglR has a low homology with the AmyR of A.
oryzae, which is a key activator of amylase gene expression.
In recent years, a novel Zn2Cys6-type fungal-specific transcription factor, ClbR, was identified
to be involved in the degradation of cellulose and cellobiose in Aspergillus aculeatus [101]. ClbR was
found to induce the expression of several cellulolytic genes, such as the FII-carboxymethyl cellulase
Polymers 2020, 12, 530 7 of 17

These transcription factors often recognize and bind to specific sequences in target gene promoters.
Some transcription factors are pathway specific and can be clustered in the same gene cluster with
the target genes, while others have their own substrate preferences, such as activation by specific
inducers or regulation by carbon catabolite repression (CCR) [106]. CCR, as a universal regulatory
mechanism, is mediated by the Cre-transcription factor, which inhibits the expression of many genes
by binding to specific sites in the target gene promoter region. The CreA transcriptional regulator
was first identified in Aspergillus nidulans [107,108]. CreA inhibits the transcription of genes encoding
enzymes that are involved in the polysaccharides’ degradation when in the presence of simple sugars
such as glucose or fructose or other monomeric carbon sources, such as mannose or xylose. [109].
It has been reported that the CreA repressor may specifically bind to the SYGGRG sequence on the
promoters of the target genes and inhibit their expression [109,110]. It is likely that this inhibitory
mechanism requires further posttranslational modification of the CreA protein or interaction between
proteins [111]. In A. niger, CreA has been shown to inhibit gene expression involved in the utilization of
xylan, cellulose [109], and arabinan [112]. The final monomer product of polysaccharide degradation,
such as glucose, is actually a suppressor; therefore, the entire regulatory mechanism is based on a
concentration-dependent balance between transcriptional induction and CreA inhibition. In addition,
T. reesei Cre1 was also identified and isolated as an ortholog of the A. nidulans CreA [113]. Studies
have shown that in the presence of D-glucose, Cre1 is phosphorylated by casein kinase II-like protein,
which is necessary for the DNA binding of Cre1 [114]. It has been demonstrated that the expression
of xyn1 and cbh1 is directly regulated by the glucose repressor Cre1 [115,116]. However, Cre1 is not
directly involved in the expression of xyn2 and cbh2 [117,118]. The CCR of cellulase and hemicellulase
genes was confirmed to be mediated by Cre1 through complementary experiments in the cre1 mutant
T. reesei Rut C-30 by the full-length cre1 gene [115]. In addition to Cre1, another regulator that is able
to control CCR has been discovered in T. reesei, named Cre2. T. reesei Cre2 protein is an ortholog of
A. nidulans CreB, which has been identified as an ubiquitin c-terminal hydrolase associated with the
deubiquitination of Cre1 [119]. The Cre2/CreB protein has been shown to interact with the Cre3/CreC
WD40-repeat protein under both carbon catabolite repressing and derepressing conditions. The
interaction is necessary and may stabilize the Cre2/CreB protein by preventing its proteolysis [120].
Another member of the Cre protein family is CreD, which is involved in an opposing process to the
complex of Cre2/CreB and Cre3/CreC proteins and inhibits the activity of Cre1/CreA [121].
Moreover, two other genes encoding cellulose regulators, AceI and AceII, were identified in
T. reesei [58,122]. Of the two, AceII is a transcriptional activator of all major cellulolytic enzyme genes,
including cbh1, cbh2, egl1, egl2, and xylanolytic gene xyn2, whereas AceI is an inhibitor of cellulase and
xylanase expression [86]. To date, only the ortholog of T. reesei AceI has been isolated in Aspergillus [123];
however, the Ace2 homolog has not yet been found in the other filamentous fungi, suggesting that
Ace2 is a species-specific regulator in T. reesei [101]. In addition, using a specific screening strategy for
candidate regulators of cellulase production, the activator Ace3 was identified [59]. The overexpression
of ace3 led to the increase in cellulase gene expression, while its deletion not only resulted in markedly
reduced activity of cellulase and hemicellulase but also influenced the expression of the regulator Xyr1
gene [60,124]. The growth of the strain and secretion of a large number of proteins in filamentous fungi
is often affected by the ambient pH. The pH signal transduction pathway has been well investigated in
these fungi, such as A. nidulans and T. reesei, and includes PacC/Pac1, a pH-responsive transcription
factor [125,126], and six pal proteins. The PacC/Pac1 regulator activates alkali-expressed genes and
suppresses acid-expressed genes under high pH conditions. Studies have shown that PacC/Pac1
can also promote or inhibit cellulase production in response to changes in the external environment.
The deletion of the pac1 gene leads to an increase in Xyr1 activity at neutral pH. However, the
effect of Pac1 on cellulase production is often masked by other regulatory mechanisms [59,127]. In
addition, based on the transcriptomic profiling during solid-state and submerged fermentation, a novel
transcription factor PoxMbf1 involved in cellulase production was also identified [128]. Cellulase
production is determined by both the external production environment and internal genes. The CCAAT
Polymers 2020, 12, 530 8 of 17

sequences are found on a wide range of fungal promoters, and the protein complex bound to CCAAT
sequences was identified as hap complexes in some important filamentous fungi, such as HapB/C/E in
A. nidulans and Hap2/3/5 in T. reesei [129,130]. These complexes have been confirmed to regulate the
expression of some genes, including polysaccharidase genes, such as A. oryzae taka-amylase (taa) and
T. reesei cellulase and xylanase genes (cbh2 and xyn2) [118,130].

4. Amylolytic Enzymes and Their Regulatory Mechanisms in Fungi

4.1. Amylolytic Enzymes

Starch is a major storage polysaccharide in plants. It consists of multiple glucose units that are
linked by α-1,4-glycosidic bonds and branched by α-1,6-glycosidic bonds. Filamentous fungi secrete a
large number of starch-hydrolytic enzymes, including α-amylase, glucoamylase, and α-glucosidase,
all of which are induced by starch, dextrin, or maltose. These amylolytic enzymes play a crucial role
in traditional food and beverage fermentation production. Among them, α-amylase breaks down
the 1,4-glycosidic linkages in starch into glucose, maltose, and other oligosaccharides. To date, three
α-amylase encoding genes (amyA/B/C) have been first identified in A. oryzae [131–133]. Next, the
glucoamylase gene glaA and α-glucosidase gene agdA were identified, respectively, in A. oryzae [134–136].
In addition, a glucoamylase gene glaB, which was purified from rice Koji [137], is highly induced under
solid-state culture conditions and, therefore, plays a key role in solid-state fermentation. By comparing
the sequences in the promoter region of these amylolytic genes, it has been found that in addition to
the TATA and CCAAT sequences, there were three highly conserved regions I, II, and III potentially
involved in gene expression [138–140]. Further deletion analysis verified that region III was crucial
for the high expression of amylolytic genes. The modified promoter is constructed by introducing
multiple copies of region III into the promoter regions of the glaA and agdA genes. The constructed
promoters induce high-level expression of related genes downstream of the promoters, which could be
used for improving the production of heterologous protein [141].

4.2. Molecular Regulation Mechanism of Amylase Gene Expression

By screening the protein binding to region III in the amyB promoter, a transcription factor gene
amyR was successfully isolated [142]. AmyR is a Zn(II)2Cys6-type regulator that induces the expression
of starch-degradation genes, and it has been well studied in A. nidulans and A. oryzae [142,143].
With starch as the carbon source, the amyR mutants displayed extremely poor growth and produced
significantly fewer amylolytic enzymes, such as approximately 10-fold lower levels of glucoamylase and
100-fold lower levels of α-amylase relative to the control strain, suggesting that AmyR is importantly
involved in the regulation of amylolytic enzyme gene expression in A. oryzae [142]. Genome sequencing
analysis showed that the three genes amyR, amyA, and agdA constituted an amylolytic gene cluster
that successively appeared in adjacent parts of the same chromosome in A. oryzae [41]. In addition
to these two adjacent genes, AmyR has also been found to regulate the expression of α-amylase
encoding gene amyB and glucoamylase gene glaA in A. oryzae [143]. In addition, the expression of the
glucoamylase gene glaB, which is expressed exclusively in solid-state culture, was also regulated by
the positive transcription factor AmyR, since the deletion of the amyR gene led to the loss of glaB gene
expression [144]. After screening and identification of transcription factors of glaB genes from the mutant
library of A. oryzae, a C2 H2 -type transcription factor, FlbC, was found to be involved in regulating glaB
gene expression [145]. The disruption of the flbC gene caused a significant reduction in glucoamylase
activity under solid-state culture conditions relative to the control. It has been reported that FlbC
is also related to the regulation of conidiospore development in Aspergillus species [146,147]. Some
research also showed that the loss of amyR function also indirectly affects the production of cellulolytic
and hemicellulolytic enzymes in A. oryzae [144]. Recent studies have revealed that the basic-region
helix–loop–helix (bHLH) transcription factor DevR is significantly involved in polysaccharide (such
as chitin and starch) metabolism in A. oryzae [148]. The overexpression of AodevR led to a strong
Polymers 2020, 12, 530 9 of 17

inhibition of strain growth and amylase-related gene expression. The yeast one-hybrid assay indicated
that DevR potentially interacts with the amyR promoter, providing a novel insight for further revealing
the regulatory mechanism of amylolytic enzyme production [148].
Interestingly, the disruption of A. nidulans amyR prevented growth on a medium in which starch
or maltose was used as a carbon source, implying that the regulatory mechanism of gene expression
under the control of AmyR is different in A. nidulans and A. oryzae. This could be explained by the
existence of an additional MAL cluster involved in maltose utilization in A. oryzae [149]. The MAL
cluster contains three genes, which encode a maltose-responsive regulator (malR), an intracellular
α-glucosidase (malT), and a maltose permease (malP) [149,150]. The deletion of the malR gene led to
the loss of expression of both malT and malP, suggesting that the transcription factor MalR is necessary
for the expression of the two genes in A. oryzae. The deletion of malR and malP also caused a dramatic
delay in the production of α-amylase [151]. In addition, the addition of glucose generally results in a
significant decrease in amylolytic enzyme gene expression because of carbon catabolite repression
(CCR), which is regulated by the negative regulator CreA [107,152], consistent with the cellulase
regulatory mechanism described above.

5. Conclusions
Some filamentous fungi have the ability to produce and secrete large amounts of enzymes;
therefore, they are widely used in the food, pharmaceutical, detergent, textile, biofuel, and other
industries, especially Trichoderma, Penicillium, and Aspergillus strains [153–155]. In addition, they can
use lignocellulosic waste to reduce environmental pollution. Among them, the molecular regulatory
mechanism of related enzymes (such as cellulase and amylase) of the genera Trichoderma and Aspergillus
has been well studied. Aspergillus species, which can use starch as a substrate for the traditional
fermentation production of foods and beverages, have been utilized for thousands of years. However,
the utilization of plant cell wall polysaccharides and the production of related enzymes (such as
cellulases and hemicellulases) still remain relatively expensive for commercial application. Therefore,
it is important to improve enzyme production and fermentation efficiency by screening for effective
microbial species, constructing genetically engineered strains, and further selecting appropriate culture
processes. Previous studies have shown that in addition to the genus Trichoderma, Aspergillus species
not only secrete a large amount of amylase and protease during fermentation but also produce cellulase
to make use of polysaccharides in plant cell walls. Moreover, the regulatory mechanisms for cellulase
genes in Aspergillus species have been extensively studied. The genera Aspergillus and Trichoderma
have similar enzyme gene regulation mechanisms and the ability to secrete a large number of active
enzymes; therefore, coculture of multiple strains to ferment plant polysaccharides to produce useful
substances is a new research direction. With the recent developments in biotechnology, these fungi
will open up new prospects in the field of microbial industrial utilization.

Author Contributions: The main author of this work, B.-T.W.; original draft preparation and references
investigation, B.-T.W., S.H., X.-Y.Y., Y.-J.Z. and L.J.; writing—review and editing, F.-J.J. All authors have read and
agreed to the published version of the manuscript.
Acknowledgments: This study was supported by the Natural Science Foundation of China (31570107), the
National Key Research and Development Program of China (no. 2016YFD0600204), the Six Talent Peaks Program
of Jiangsu Province of China (TD-XYDXX-006), and the Priority Academic Program Development of Jiangsu
Higher Education Institutions (PAPD).
Conflicts of Interest: The authors declare no conflict of interest.

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