TIBTEC 1405 No.

of Pages 13

Review
The Post-genomic Era of
Trichoderma reesei: What's
Next?
Vijai Kumar Gupta,1,* Andrei Stecca Steindorff,2
Renato Graciano de[15_TD$IF] Paula,3 Rafael Silva-Rocha,4
Astrid R. Mach-Aigner,5 Robert L. Mach,5 and
Roberto N. Silva3,*
The ascomycete Trichoderma reesei is one of the most well studied cellulolytic
Trends
microorganisms. This fungus is widely used in the biotechnology industry, mainly
Synthetic construction of regulatory ele-
in the production of biofuels. Due to its importance, its genome was sequenced in ments such as transcriptional factors
2008, opening new avenues to study this microorganism. In this ‘post-genomic’ and promoters for T. reesei expands
the toolbox for fungal engineering.
era, a transcriptomic and proteomic era has emerged. Here, we present an
overview of new findings in the gene expression regulation network of T. reesei. The identification of novel cis-regula-
We also discuss new rational strategies to obtain mutants that produce hydrolytic tory elements using systems biology
approaches has begun to provide
enzymes with a higher yield, using metabolic engineering. Finally, we present how building blocks for constructing com-
synthetic biology strategies can be used to create engineered promoters to plex circuits in T. reesei.
efficiently synthesize enzymes for biomass degradation to produce bioethanol.
The use of synthetic biology standards,
such as the BioBricks assembly for-
Trichoderma reesei is an Efficient Biomass Degrader mat, for constructing modular systems
Trichoderma reesei encodes a range of cellulases and hemicellulases for biomass degra- for T. reesei has been reported, and its
generalization should have a strong
dation. The biomass degradation system of T. reesei (Box 1) is one of the most studied systems
impact in the field.
within the filamentous fungi, and its enzymes are commonly used as industrial cellulase source,
reaching a production up to 100 g/[18_TD$IF]l of secreted proteins under industrial fermentation conditions While the simultaneous implementation
[1]. This ability to produce a high cellulase content arises from the efficient ability of T. reesei to of multiple expression systems in
T. reesei has been reported, there is
sense and transport nutrients for rapid induction and secretion of cellulases at low energy cost still a pressing need to develop novel
[2]. Furthermore, T. reesei responds rapidly to changes in the nutritional composition of its tools and techniques that could pro-
environment in order to compete with other microorganisms [3]. After the T. reesei genome was vide an efficient strain engineering plat-
sequenced [4], ushering in the ‘post-genomic era,’ large-scale transcriptome and proteome form for this organism.

studies reported the efficient regulation of a complex gene expression network [5–7], leading to
new applications of this fungus in the biotechnology industry, specifically biofuel production [8].
However, a major challenge for the successful implementation of T. reesei in the biofuel industry
is reducing the costs of producing enzymatic cocktails. Better understanding the biology of 1
Molecular Glyco-biotechnology
T. reesei can lead to new approaches in synthetic biology to construct strains that are able to Group, Discipline of Biochemistry,
produce biomass-degrading enzymes more efficiently than they can today. Critically reviewing School of Natural Sciences, National
University of Ireland Galway, Galway,
the available literature is important for both the scientific community and for biotechnology Ireland
companies to produce biofuel from biomass material at decreased costs. 2
Departamento de Biologia Celular,
Universidade de Brasília, Campus
Universitário Darcy Ribeiro, Instituto
The Genome of T. reesei de Ciências Biológicas, Brasília, DF,
Comparative genomics is a very important tool to understand different lifestyles and how different Brazil
3
organisms interact with the environment. Figure 1 compares the T. reesei genome [4] with those Department of Biochemistry and
Immunology, Ribeirão Preto Medical
of four well-studied biomass degraders: Neurospora crassa [9], Aspergillus niger [10], Aspergillus School, University of São Paulo,
nidulans [11], and Penicillium chrysogenum [12], as well as Saccharomyces cerevisiae [13]. The Ribeirão Preto, SP, Brazil

Trends in Biotechnology, Month Year, Vol. xx, No. yy http://dx.doi.org/10.1016/j.tibtech.2016.06.003 1

© 2016 Elsevier Ltd. All rights reserved.
 TIBTEC 1405 No. of Pages 13

4
Box 1. Biomass Degradation System of T. reesei Department of Cell and Molecular
Biology–Ribeirão Preto Medical
Biomass is a complex structure formed by polysaccharides, including cellulose, hemicellulose, and the aromatic polymer,
School, University of São Paulo,
lignin. T. reesei efficiently degrades cellulose and hemicellulose, but not lignin. Therefore, lignin needs to be removed by
Ribeirão Preto, SP, Brazil
thermochemical pretreatment to allow T. reesei cellulases and hemicellulases to degrade biomass [75]. 5
Department for Biotechnology and
Microbiology, Institute of Chemical
Biomass hydrolysis requires a variety of enzymes that act cooperatively to liberate monomeric sugars, along with accessory
Engineering, TU Wien, Wien, Austria
enzymes that cleave side chains of polymers, oligosaccharides, and copper oxidases that can oxidatively cleave cellulose.

The cellulolytic system of T. reesei is composed of exoglucanases, endoglucanases, b-glucosidases, and accessory
proteins such as swollenin and PMOs. Its hemicellulolytic system includes a variety of enzymes including endox- *Correspondence: vijaifzd@gmail.com
ylanases, xylosidases, manosidases, galactosidases, and arabinofuranosidases. (V.K. Gupta) and rsilva@fmrp.usp.br
(R.N. Silva).

sizes of their genomes are similar, except that N. crassa has a larger genome (>40 Mb). T. reesei
has the smallest genome within the Trichoderma genus, probably due to gene losses during its
evolution as a saprophytic fungus, or one that obtains nutrients from dead organic matter [14]. All
of the compared genomes show low-complexity DNA sequences (or repetitive sequences), which
comprise less than 5% of the DNA in all of the strains (Figure 1B). Clustering analysis has shown
that the majority of proteins are shared by at least two species, and approximately 3000 are
shared by all species (Figure 1C). T. reesei has the smallest set of unique proteins, and A. niger
has the largest when compared with other species (excepting S. cerevisiae). The proportion of
transcription factors, transporters and CAZymes seems to be the same among the species
independently of proteome size (Figure 1D), which is noted when comparing T. reesei and
N. crassa, both from class Sordariomycetes (a subdivision of Ascomycota). T. reesei contains a
large proportion of all three categories of proteins despite its smaller genome and proteome. Most
likely, this is one of the most important characteristics of T. reesei that causes it to be more efficient
in substrate recognition, transport, and activation of gene expression (Box 2).

(A) (B) (C) Key: (D) Key:

Shared by all Transcripon factors
Key:
Shared by 2–5 species Transporters
Repeats
Sordariomycetes Unique CAZymes

T. reesei

N. crassa

P. chrysogenum

A. nidulans

A. niger
Euroomycetes
Saccharomycetes
S. cerevisiae

0 10 20 30 40 0 3000 6000 9000 12000 15000 0 1000 2000 3000

Genome size (Mb) Gene models Proteins

Figure 1. Comparative T. reesei Genome Characteristics to Five [4_TD$IF]Well-studied Biomass Degraders and to S. cerevisiae. (A) Phylogenetic tree
representation based on ITS 1 and 2 of 6 fungi representing classes Sordariomycetes (T. reesei AF510497; N. crassa JX981479)[5_TD$IF], Eurotiomycetes (A. niger
FJ011542; A. nidulans KP131592; P. chrysogenum KF417579)[6_TD$IF], and Saccharomycetes (S. cerevisiae KU311155). (B) Genome size and repeat content. (C) Number
of predicted genes/proteins. (D) Protein counts of transcription factors, transporters, and CAZymes. The data in this figure are visual representations of information from
Mycocosm [76] or Ensembl Fungi [77]. Abbreviations: CAZymes, carbohydrate active enzymes.

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TIBTEC 1405 No. of Pages 13

Box 2. [1_TD$IF]Trichoderma reesei CAZyome Conservation and Saprotrophic Specialization Glossary

Filamentous fungi grow on a wide range of substrates and efficiently degrade polymers. Therefore, they are an attractive Activator of cellulase expression
resource for obtaining new enzymes. Figure I illustrates protein conservation among these six fungi, with 114 proteins 1 (ACE1): ranscription factor that
common to all six species. S. cerevisiae has the smallest set of carbohydrate active enzymes (CAZyome) and negatively regulates cellulase
consequently shares only 0–4 proteins with any given set of species. High protein counts were associated with the expression.
Sordariomycetes (26 proteins, box AB) and Eurotiomycetes (44 proteins, box CDE); most proteins are unique to each Activator of cellulase expression
species (boxes A, B, C, D, E, and F). T. reesei has 71 proteins with orthologs (box A). 2 (ACE2): transcription factor that
positively regulates the expression of
Among the proteins unique to T. reesei, 51% of the proteins are glycoside hydrolases (GH), 15% are glycosyl- xylanase genes.
transferases (GTs), 8% are carbohydrate-binding modules (CBMs), 6% are classified as having auxiliary activity Activator of cellulase expression
(AA), 6% are plant-like-expansins (EXPNs), 6% are carbohydrate esterases (CEs), 4% are polysaccharide lyases 3 (ACE3): transcription factor that
(PLs), and 4% are GH + CBM domains that are in the same protein (Figure II). positively regulates the expression of
xylanase genes.
The most abundant family is GH18, the chitinase family, with five proteins. Three of these five belong to chitinase subgroup ATP-binding cassette
C, which consists of large proteins (140–170 kDa) with multiple LysM motifs [78]. T. reesei also contains a unique transporters (ABC transporters):
chitosanase (GH75); these proteins hydrolyze chitosan, a partially deacetylated form of chitin [79]. Another chitin-related members of a transport system
enzyme is NAG2, a GH20. Four proteins are in the GH16 family. This family has 14 different activities, such as endo-1,3(4)-b- consisting of multiple subunits, one
glucanase and glucanosyltransferase. GH2 (b-mannosidases) and GH30 (exo-1,4-xylanases) contain three proteins each, or two of which are transmembrane
followed by two proteins in GH3 (BGL3f and CEL3e), GH11 (XYN1 and XYN5), GH55 (exo-b-1,3-glucanases)[12_TD$IF], and GH89 proteins, and one or two of which are
(/-N-acetylglucosaminidase). [13_TD$IF]Twelve families have only one protein each: GH1 (CEL1b), GH54 + CBM42 (ABF3)[14_TD$IF], and a set membrane-associated ATPases,
of enzymes involved in the degradation of glucan and mannan (GH: 5, 17, 47, 71, 75, 76, 79, 81, 95, 125, and 128). Looking which utilize the energy of adenosine
at these data, a pattern arises: because T. reesei is not a specialized mycoparasite [14], these enzymes might be involved in triphosphate (ATP) binding and
degrading organic material from dead plants (cellulases, xylanases, arabinofuranosidases) or microorganisms [/(b)-1,3(6)- hydrolysis to energize the
glucanases and chitinases], or in a nutrient competition strategy, supporting a predominant lifestyle as a saprophyte. This set translocation of various substrates
of unique proteins of T. reesei can be used as targets for future genetic manipulation and improving biomass degradation. across membranes, either for uptake
or for export of the substrate.
BioBrick: DNA sequences that
Key: conform to a restriction-enzyme
A – T. reesei assembly standard, used to design
B – N. crassa and assemble larger synthetic
C – P. chrysogenum biological circuits.
cAMP: a second messenger
D – A. nidulans
important in many biological
E – A. niger
processes. cAMP is a derivative of
F – S. cerevisiae ATP and used for intracellular signal
transduction.
Carbohydrate-binding modules
(CBMs): amino acid sequence within
a carbohydrate-active enzyme with a
discrete fold having carbohydrate-
binding activity.
Carbohydrate esterases (CEs):
enzymes that catalyze the de-O or
de-N-acylation of substituted
saccharides.
CAZymes: carbohydrate-active
enzymes involved in the synthesis,
metabolism, and transport of
carbohydrates.
Cellulases: a group of enzymes that
act synergistically to decompose
cellulose molecules into
monosaccharides such as glucose,
or shorter polysaccharides and
oligosaccharides.
Carbon catabolite repression 1
(CRE1): transcription factor of
T. reesei involved with carbon
Figure I. Distribution of CAZyome Orthologs from Six Fungi. Each block on this diagram represents one catabolite repression.
intersection of all 63 possibilities when grouping six sets, and the number inside the block is the number of proteins Glycoside hydrolases (GHs):
shared among the species indicated by the letters in the box. The central dark blue block shows 114 proteins shared by enzymes that catalyze the hydrolysis
all fungus represented by the letters A, B, C, D, E[9_TD$IF], and F. The red blocks represent the unique proteins for each species. of the glycosidic linkage of
The intermediate blocks represent proteins shared by 2–5 species (light blue,[10_TD$IF] five species; green, four species; yellow, glycosides.
three species; orange, two species). The data in this figure are visual representations of information from the Carbohy-
drate-Active enZYmes database (http://www.cazy.org/) [80].

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 TIBTEC 1405 No. of Pages 13

PL Glycosyltransferase (GTs):
enzymes that catalyze the formation
AA 4%
EXPN CE of the glycosidic linkage to form a
6% 6% 6% CBM glycoside.
8% GH+CBM Hemicellulases: a collective term for
4% a group of enzymes that break down
hemicellulose.
GT LAE1: a putative S-
adenosylmethionine-dependent (SAM)
15%
methyltransferase protein that
controls cellulase gene expression in
T. reesei.
Mannosidase: an enzyme that
removes mannose residues from
GH glycans.
51% Major facilitator superfamily
(MFS): a class of membrane
transport proteins that facilitate
GH89 GH95 (1) GH1 (1)
GH11 (2)
movement of small solutes across
GH81 (1) (2)
GH125 (1) cell membranes.
GH79 (1)
GH128 (1) Polysaccharide monooxygenases
GH76 (1)
(PMOs): Copper-dependent
GH75 (1)
enzymes formerly known as GH61
GH71 (1)
GH16 (4) proteins, PMOs were recently shown
GH55 (2) to catalyze the O2-dependent
GH17 (1) oxidative cleavage of recalcitrant
GH5 (1) polysaccharides.
GH18 (5)
GH47 (1) GH30 Polysaccharide lyases (PLs): a
(3) group of enzymes that cleave uronic
GH2 (3)
acid-containing polysaccharide chains
GH3 (2)
GH20 (1) via a b-elimination mechanism to
generate an unsaturated hexenuronic
acid residue and a new reducing
end.
Figure II. Unique CAZymes from T. reesei. CAZy classes of the 71 proteins identified as unique to T. reesei: Saprotrophic: living and feeding on
glycoside hydrolases (GH), glycosyltransferases (GT), carbohydrate esterases (CE), polysaccharide lyases (PL), auxiliary dead organic matter.
activities (AA), and carbohydrate-binding modules (CBM). Glycoside hydrolases represent 51% of all of the unique Secretome: a set of secreted
proteins. The diversity of these proteins is shown with their respective counts in parentheses. Abbreviations: EXPN, proteins of an organism.
expansin. Swollenin: a non-hydrolytic
disruptive protein similar to the plant
expansins.
Transactivator: transcription factor
Global Regulation of Biomass Degrading Enzymes and Gene Regulation or molecule that binds to a specific
Networks promoter region of DNA, stimulating
Proteomic approaches exploring the gamut of cellulases and hemicellulases in the filamentous gene expression.
VELVET protein: protein complex
fungus T. reesei have focused on analysis of the secretome during growth on different carbon
essential for cellulase gene
sources. A number of studies have tried to identify the complete protein repertoire of T. reesei expression, which in turn might
involved with lignocellulosic biomass degradation. Besides the classical cellobiohydrolases regulate both LAE1 and XYR1
(CBHI/Cel7A and CBHII/Cel6A), other enzymes have been found to compose the arsenal used expression and activity.
Xylanase regulator 1 (XYR1):
to degrade the biomass, including seven b-glucosidases, one b-xylosidase, eight endogluca-
transcription factor involved with
nases, three xylose-encoding genes, one mannase, and 10 new genes encoding hydrolytic activation of cellulase and
enzymes encompassing b-glucosidases and xylosidases [15]. Furthermore, a comparative hemicellulase gene expression.
secretome profile of two hypersecretory industrial strains, Rut-C30 and CL847, grown on
different carbon sources revealed the presence of new players in biomass degradation, called
accessory enzymes. Their activity is not hydrolytic but appears important to prepare the biomass
for hydrolysis by cellulases. These proteins include cellulose-induced protein 1 (CIP1), CIP2,
swollenin, and GH61 cellulose monooxygenases [16]. A variety of studies have pointed out that
the T. reesei extracellular proteome differs considerably depending on the carbon sources used
[2,17,16]. Compared to other filamentous fungi, T. reesei expresses a wide repertoire of
enzymes in response to the same induction conditions. However, a conserved and an inde-
pendent secretion pattern for glycoside hydrolases can be observed between different fungi

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 TIBTEC 1405 No. of Pages 13

Table 1. Comparative Secretome of CAZymes in Different Filamentous Fungi during Growth in Sugarcane
Bagasse and Cellulose. The table entries indicate the number of identified proteins
CAZymes SUGARCANE BAGASSE CELLULOSE

Trichoderma Trichoderma Trichoderma Aspergillus Trichoderma Neurospora Aspergillus

reesei asperellum harzianum niger reesei crassa fumigatus

Refs [81,82] [82] [83] [81] [2,86] [84] [85]

Glycosyl hydrolase families

GH1 3 2 0 0 2 0 0

GH3 10 7 6 5 7 1 2

GH5 2 5 7 6 7 1 1

GH6 2 1 1 2 1 2 1

GH7 3 2 2 2 7 2 2

GH17 1 1 2 2 1 0 0

GH55 3 1 4 2 3 0 0

AA9 (GH61) 3 0 2 3 3 3 2

GH11 4 4 3 1 0 0 2

GH30 4 0 0 1 3 0 1

GH74 2 1 1 1 0 0 0

GH16 2 2 2 4 2 0 1

GH27 3 2 1 3 0 0 0

GH31 2 2 0 0 1 0 0

GH65 1 0 0 1 0 0 0

GH72 4 3 2 3 9 0 0

GH18 1 3 3 2 0 0 0

GH13 1 0 0 2 1 0 0

GH28 1 2 0 2 0 0 0

GH53 0 0 0 1 1 0 0

GH10 0 1 1 1 0 0 2

GH43 0 1 0 5 0 0 4

GH51 0 0 0 1 0 0 0

GH54 2 2 1 1 2 0 0

GH62 1 2 2 1 0 0 1

GH67 1 1 0 1 0 0 0

GH35 1 1 0 1 0 0 0

GH12 0 1 0 3 0 0 1

GH32 0 0 0 1 0 0 0

GH95 0 1 1 1 0 0 0

GH81 0 0 1 1 0 0 0

GH15 0 2 1 1 1 0 1

GH20 1 1 0 1 0 0 0

GH47 0 1 1 1 1 0 0

GH92 1 3 2 1 1 0 0

GH2 2 2 3 0 0 0 0

GH25 1 1 1 0 0 0 0

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Table 1. (continued)
CAZymes SUGARCANE BAGASSE CELLULOSE

Trichoderma Trichoderma Trichoderma Aspergillus Trichoderma Neurospora Aspergillus

reesei asperellum harzianum niger reesei crassa fumigatus

GH36 1 1 0 0 0 0 0

GH79 0 1 0 0 0 0 0

GH93 0 1 0 0 0 0 0

GH4 0 0 0 0 2 0 0

GH71 0 0 3 0 4 0 0

GH109 0 0 0 0 1 0 0

GH38 0 0 0 0 1 0 0

GH9 0 0 0 0 1 0 0

GH64 0 0 2 0 0 0 0

GH76 0 0 1 0 0 0 0

Carboydrate-module binding

CBM1 1 0 0 0 1 0 0

CBM2 0 0 0 0 1 0 0

CBM13 0 0 0 0 1 0 0

Glycosyl transferase

GT20 2 0 0 0 0 0 0

GT31 1 0 0 0 0 0 0

GT35 1 0 0 0 1 0 0

GT2 1 0 0 0 1 0 0

Carboydrate esterase

CE5 1 0 0 0 0 0 0

CE8 0 0 0 2 0 0 0

CE1 0 0 0 4 0 0 0

CE16 0 0 0 1 0 0 0

Polysaccharide lyase

PL1 0 0 0 1 0 0 0

species (Table 1). These data provide evidence for the ability of Trichoderma to compete for and
colonize a range of habitats (Box 3).

Although the different gene and protein expression in T. reesei is well understood in response to
different inducers, there are still aspects of gene regulation that remain to be elucidated. Many
reports in the literature have been trying to identify the proteins involved in the regulation of
cellulose degradation. Hakkinen and colleagues [18] screened candidate regulators for cellulase
and hemicellulase production in T. reesei cultivated in different lignocellulose-derived materials,
and they demonstrated that deleting the ACE3 transcription factor (until then not described)
significantly reduced xylanase activity and expression of xylan-degrading enzyme genes. The
role of transcription factors in cellulase gene expression is complex. Interestingly, a range of
transcriptome analyses have shown that the main transcription factors (XYR1, CRE1, ACE1,
and ACE2) involved in cellulase expression in T. reesei had no significant changes in their
expression during growth in different carbon sources [2].

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 TIBTEC 1405 No. of Pages 13

Box 3. Quantitative Secretomic and Transcriptomic Analysis as a Biotechnology Target

A quantitative secretome analysis of fungi grown in complex carbon sources can help to choose which strains are most
applicable for the biofuel industry. For example, if biofuel is produced from sawdust, corn stover, wood, or sugar cane
bagasse, a different complex of enzymes will be secreted in different yields [21,87].

In the same way, quantitative transcriptomic studies have shown the importance of different expressions of genes that
encode known and predicted cell-wall degrading and accessory enzymes and proteins. This regulation may be important
to understand how different fungi sense carbon substrates and induce enzyme formation [2,7].

The use of these two approaches together in a platform database can help the biofuel industry to rapidly screen fungi for
hydrolyzing specific biomass.

Recently, two other transcription factors were described that control cellulase synthesis: LAE1
(protein methyltransferase) and VELVET [19–21]. Deletion of vel1 completely blocked the
expression of cellulases, xylanases, and the cellulase regulator XYR1 on lactose as a carbon
source. Likewise, in a lae1 deletion mutant, a complete loss of expression of cellulases, auxiliary
factors for cellulose degradation, b-glucosidases[19_TD$IF], and xylanases was observed. Interestingly,
these studies show the interplay between the LAE1/VELVET complex in the regulation of XYR1
(Figure 2). XYR1 remains the master regulator of the cellulase expression in T. reesei. Silva-
Rocha et al. [22] identified XYR1-binding sites in the promoter regions of the main cellulase
genes (cel7a, xyn1, cel6a, cel7b, cel61a, cel61b, cel3c, cel3d, xyn3[20_TD$IF], and swollenin genes).
Moreover, strong sites for CRE1 binding were found in the xyr1 and cel7b promoters, suggest-
ing a mechanism for signal integration by XYR1 and CRE1 at cellulase promoters. Dos Santos
Castro et al. [5] showed that among 409 CAZy genes differentially expressed in the
Dxyr1 T. reesei strain grown in cellulose, glucose[21_TD$IF], and sophorose, only 16 genes are directly
regulated by XYR1. Antonieto et al. [23] showed that only three and one CAZy genes are directly
regulated by the CRE1 strain during growth on sophorose and glucose, respectively. These
results suggest that there is a robust regulatory network, still unknown, involving other tran-
scription factors. Further, this regulation seems to be indirect, ultimately regulated by a master
regulator (Figure 2).

The genes belonging to the MFS permease and ABC transporter families have their
expression strongly modulated in the presence of cellulose, sophorose[2_TD$IF], and glucose [2,23].
Since extracellular sugar transport into the cell is an important process involved with the
induction and synthesis of cellulases, these transporters may be central players controlling
the expression of cellulases and hemicellulases in response to lignocellulosic biomass. Ivanova
et al. [24] showed that a lactose-induced MFS permease involved in the transport of hemicellu-
lose monomers is essential for cellulase induction. Together, these results suggest that different
transporter families and transcription factors (still not characterized) in T. reesei play important
roles in the signaling pathways involved in cellulase induction. Furthermore, the data reviewed
here summarizes new knowledge for developing industrial strains of T. reesei that can be more
efficient in substrate recognition and transportation, as well as the activation of cellulase and
hemicellulase gene expression in high-yield production.

Strategies for Metabolic Engineering of T. reesei

New strategies for metabolic engineering in T. reesei include manipulating factors that regulate
carbon catabolite repression (CCR), sensing of nutrients, and chromatin remodeling.

CCR is a mechanism used to maintain the metabolic efficiency of an organism by using only the
most favorable (i.e., easy to metabolize) carbohydrates. As a saprotrophic fungus T. reesei is
able to degrade plant cell wall material using sets of plant cell wall degrading enzymes
(PCWDEs). The formation of these enzymes is triggered when certain inducers, which are
commonly biopolymer degradation products (such as cellobiose or sophorose), enter the cell

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 TIBTEC 1405 No. of Pages 13

Cellulose polymer

Sophorose Cellobiose

Crystallineregion

Amorphous region

Cellobiohydrolases Endoglucanases
Glucose transporter

Swollenin β-glucosidases Dissacharide transporter

PMOs (GH61s) CDHs MFS permeases(?) ABC transporters

Cellobiose Cellobiose Cellotriose

Cellobiose
Sophorose ???
BGL1

ACE2
HAP2/3/5 Cellodextrins Glucose
Gl
XYR1 ACE3 Cellulase complexinducon ???
TFs Glucose
Gl
cbh1, cbh2, eg11, eg12, ???
xylansaes, transporters
??? Ou
ACE1
CRE1 tsi
CRE1
XYR1 ACE2 ACE3 HAP2/3/5 Ins de
VELVETs ACE1
Glucose LAE1
ide
XYR1
CAZymes, TFs, Transporters
(MFS permeases, copper, iron)
Ferric reductases
Celluases, xylanases, auxilary factors, Cellulases, hemicellulases, transporters,TFs
β-glucosidases

Figure 2. Overview of Global Regulation of Cellulase Expression in T. reesei. This figure shows the processes that are expected to be involved in regulating
lignocelluosic enzymes in T. reesei. The main enzymes involved with lignocellulosic material mobilization are endoglucanases (EGs), cellobiohydrolases (CBHs)[7_TD$IF], and b-
glucosidases. These enzymes act synergistically to break down biomass. EGs hydrolyze cellulose bonds internally, while CBHs cleave cellobiose units from the ends of
the polysaccharide chains. The released cellobiose units (disaccharide) are finally hydrolyzed by b-glucosidases, releasing glucose. Swollenins rapidly induce the
extension of plant cell walls by weakening the noncovalent interactions that help to maintain their integrity. Polysaccharide monooxygenases (PMOs) are newly thought to
play a role in this process. Cellobiose dehydrogenase (CDH) is a potential electron donor for PMOs. EGs and PMOs cleave internally cellulose chains releasing chain ends
that are targeted by CBHs. The expression of all degrading enzymes is controlled by the main transcription factors (TFs): XYR1, ACE1, ACE2, ACE3, CRE[8_TD$IF]1, and the
HAP2/3/5 complex. Additionally, a putative protein methyltransferase LAE1 and VELVET were shown to be important in cellulase gene expression. Under conditions of
high glucose, T. reesei cannot induce the expression of cellulases, and the TF CRE1 mediates this carbon catabolite repression. New insights to be investigated as
potential areas of research for enhancing enzyme secretion are indicated by dotted arrows. At this point, TFs still not characterized might activate or repress (directly or
indirectly) the master TFs involved with cellulase expression. Moreover, these TFs may regulate the expression of major facilitator superfamily (MFS) and ATP-binding
cassette (ABC) transporters that in turn are responsible for sugar uptake in response to different carbon sources.

[25]. However, the presence of monosaccharides derived from plant biopolymers, such as
[23_TD$IF]D-glucose or [23_TD$IF]D-xylose, reduces the expression of PCWDE-encoding genes by CCR [26].
Generally, the expression of these genes is regulated at the transcriptional level. Important
regulatory proteins are activators like [24_TD$IF]XYR1 [27] and [25_TD$IF]ACE2 [28][3_TD$IF] and repressors like [26_TD$IF]CRE1 [29]
and [25_TD$IF]ACE1 [30]. The transcription factors [26_TD$IF]CRE1 and [26_TD$IF]CRE2 [31] were identified as mediators of
CCR, but [26_TD$IF]CRE2 had much less impact. [26_TD$IF]CRE1 is a zinc finger protein that binds to 50 -SYGGRG-
30 DNA motifs (reviewed in [25]). It represses both the transcription of a number of PCWDE-
encoding genes and the expression of the transactivator-encoding gene xyr1 [32], leading to

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 TIBTEC 1405 No. of Pages 13

an efficient double-lock mechanism for certain genes (e.g., xyn1 [32]). Unfortunately, deleting the
cre1 gene leads to strongly reduced growth and hampers subsequent transformation, so this is
not a useful strategy. Notably, the strains used for industry-scale production of PCWDEs bear a
truncated version of [26_TD$IF]CRE1 ([26_TD$IF]CRE1-96), as does their ancestor strain Rut-C30, resulting in missing
CCR [33,34]. Another mutation in the cre1 gene (T78P) detected in strain PC-3-7 resulted in a
lower binding affinity of [26_TD$IF]CRE1 to DNA and also a partial mitigation of CCR [35]. However, a recent
study revealed that [26_TD$IF]CRE1-96 positively contributes to cellulase expression and is involved in
chromatin opening of the promoters of the regulated genes. These findings suggest that
modifying transcription factors is the best approach to circumvent CCR. An alternative or
additional strategy to avoid CCR might be the directed alteration of promoters of target genes.

The presence of cellobiose and its transglycosylation product, sophorose, leads to the rapid
formation of cellulases in T. reesei. Unexpectedly, lactose also induces the formation of
cellulases, though slower and less pronounced than on cellulose (reviewed in [36]). In both
cases, transporters and b-glucosidases have been identified as essential for cellulase induction
[24,37,38]. In either case, the inducing signals are delivered to the main transactivator [24_TD$IF]XYR1
[27,39]. Upregulation of xyr1 expression directly results in the upregulation of cellulase expres-
sion [33]. [24_TD$IF]XYR1 is also essential for the formation of xylanases, which are best induced by low
extracellular levels of D[23_TD$IF] -xylose [26]. [23_TD$IF]D-[27_TD$IF]Xylose does not trigger upregulation of xyr1 transcription
[40], and the strength of xylanase expression does not depend on the [24_TD$IF]XYR1 concentration [33].
To date, it is still unclear how exactly the induction signals are delivered.

A variety of environmental and physiological factors can affect enzyme production by T. reesei. A
complex signaling cascade and regulatory network is important for efficient hydrolytic enzyme
production. The following signal transduction mechanisms and factors have been reported to
influence the expression of PCWDEs. G-protein signaling has been demonstrated to respond to
different carbon sources and is involved in the regulation of cellulolytic enzymes [41,42], and in
modulating cAMP signaling, which is considered the major output of heterotrimeric G-protein
signaling [43]. The G-protein signaling network includes the phosducin-like protein PhLP1, and
signal transduction is light-dependent with the PAS/LOV domain protein ENVOY as a central
regulator (reviewed in [44]). The influence of cAMP levels and its signaling in cellulase expression
has been investigated in a number of studies [45–47]. Furthermore, the MAP-kinases Tmk3,
Tmk2, and TrIme2 have been reported to be involved in cellulase formation [48–50]. Finally,
abiotic nutrient signals are related to cellulase expression: for example, ambient pH mediated by
the transcription factor Trpac1 [51] or sulfur metabolism seems to be linked to cellulase induction
because expression of the central regulator, LIMPET (Lim1), responds to cellulase-inducing
signals [52]. In summary, many sensing mechanisms influence cellulase expression, although to
different extents. Due to their wide impact on the physiology of T. reesei, any modification might
result in unpredictable pleiotropic effects. Thus, these are not targets that can be recommended
for strain engineering purposes. An obvious modification target would be [24_TD$IF]XYR1, which receives
the inducing signals. In particular, the directed activation of [24_TD$IF]XYR1 in response to an extrinsic
signal should be considered as an engineering strategy, leading to the construction of strains
expressing PCWDEs constitutively.

Chromatin remodeling is an emerging method for metabolic engineering to improve industrial

fungal strains. Initially, the effects of chromatin packing on metabolic processes were investi-
gated with respect to secondary metabolism [53]. In T. reesei, genes encoding for PCWDEs are
frequently found in clusters along with genes involved in the production of secondary metabolites
[4]. The deletion of lae1 (the A. nidulans laeA ortholog), as well as the VELVET protein-encoding
gene, vel1, decreased [24_TD$IF]xyr1 expression and cellulase formation [19,54]. Histone acetylation also
seems to be related to cellulase expression. In the absence of GCN5 (the S. cerevisiae Gcn5
ortholog), cellulase gene expression and acetylation of H3K9 and H3K4 in the cbh1 promoter

Trends in Biotechnology, Month Year, Vol. xx, No. yy 9

 TIBTEC 1405 No. of Pages 13

were strongly reduced. Moreover, recent RNAseq analysis revealed that several genes poten-
tially involved in histone acetylation are differentially expressed on sophorose compared to
glucose [55].

In addition, transcription factors affect chromatin remodeling in T. reesei. In [26_TD$IF]CRE1 mutant strains,
nucleosomes positioned in the coding regions or promoters of cbh1 and cbh2 were reduced
[56,57]. The truncated version of [26_TD$IF]CRE1 ([26_TD$IF]CRE1-96) seems to act on these promoters and triggers
chromatin opening [58]. In addition, [26_TD$IF]CRE1-96 may indirectly contribute to chromatin remodeling
by regulating the expression of htf1, a putative helicase that might participate in an ATP-
dependent chromatin-remodeling complex [58]. Supporting this hypothesis, there is evidence
that hft1 (also referred as snf2) is controlled by [26_TD$IF]CRE1 [59]. The main transactivator [24_TD$IF]XYR1 also
contributes to chromatin remodeling. During PCWDE expression, [24_TD$IF]XYR1 partially coordinates the
sophorose-inducing signal [55]. Interestingly, investigations on the chromatin accessibility of the
xyr1, cbh1, and cbh2 promoters revealed that, during early induction, the chromatin rearrange-
ment primarily affects the xyr1 promoter [60]. Identification and knowledge-based modification
of chromatin remodeling proteins that act on targets with strong effects on PCWDE expression
(such as [24_TD$IF]XYR1) are certainly a promising method for strain development.

Toward Synthetic Biology of T. reesei

While T. reesei has a remarkable potential for high-level protein production, exploiting this potential
would require both a deep understanding of the cellular physiology of this microorganism and the
development of high-performance tools suitable to perform the engineering tasks required for the
intended biotechnological applications. The understanding part is crucial because the intrinsic
regulatory network of the microorganism would impose some limitations for expressing the genes
of interest. Because some applications would require inserting multiple genes into the same strain,
increasing the efficiency of the engineering tools available for this organism would strongly affect
the capability to modify T. reesei for novel biotechnological applications. Several studies have
focused on the implementation or development of novel expression systems for T. reesei using, at
least in part, the concepts of synthetic biology (a comprehensive review of heterologous expres-
sion in T. reesei can be found elsewhere [61]). This approach has involved either generating
modular vectors endowed with different expression systems or engineering synthetic promoters
using well-known expression systems as a starting point.

The use of inducible systems has mainly focused on naturally strong promoters such as Pcbh1
and Pcbh2 [61–65]. Additionally, genomic and transcriptomic approaches have been used to
identify novel inducible systems, such as copper and L[18_TD$IF] -methionine inducible promoters with
strong activity in T. reesei [66,67]; both systems display high performance for constructing stable
phenotypes. On the other hand, several studies have tried to identify and characterize strong
constitutive expression systems for T. reesei, using, for example, promoters from ribosomal
proteins[28_TD$IF], such as Prp2 [68], the pyruvate decarboxylase promoter Ppdc [69], and the enolase
promoter Peno [70]. In a more synthetic biology-driven approach, Wang et al. engineered a
modular synthetic regulator through the fusion of short Gal4, Vivid[29_TD$IF], and VP16 domains [71]. This
regulator, in combination with a synthetic promoter harboring five repetitions of the Gal4 UAS,
provided a novel light-inducible expression cassette for T. reesei [71]. This type of approach has
remarkable potential because newly described transcriptional factors such as Xpp1 [72] could
also be used to construct synthetic, modular expression systems with enhanced performance.
In this way, the implementation and characterization of novel expression system allowed a
significant expansion of the molecular toolbox available for the engineering of novel strains
expressing high levels of proteins of interest.

In a different approach, several studies have focused on the construction of novel strains
expressing enzymatic activities of interest. In these cases, native or engineered promoters

10 Trends in Biotechnology, Month Year, Vol. xx, No. yy

 TIBTEC 1405 No. of Pages 13

were used alone or in combination to express multiple targets at high levels [62,73]. Wang et al. Outstanding Questions
used the BioBrick assembly approach to construct a series of vectors dedicated to the The integration of omics science with
co-expression of target genes in T. reesei [74]. Protein co-expression has also been exploited knowledge of Trichoderma reesei biol-
ogy will lead to obtaining metabolic
by Zhang et al. by using different resistance markers such as hygromycin B and phosphinothricin mutants to improve the enzyme pro-
[62], while Miyauchi et al. introduced multi-copies of the xynB gene under the control of different duction for biomass degradation. What
native promoters (Pxyn2, Pcbh2, and Pegl2) to generate a high xylanase-producing strain [73]. else is necessary to produce these effi-
cient metabolic strains of T. reesei for
We expect that the growing number of available expression systems should promote the use of
biomass degradation?
rational and more complex circuits in T. reesei for novel applications, and such sophisticated
circuits should be pursued in future research. Systems biology knowledge of T. ree-
sei regulatory networks can be inte-
Concluding Remarks grated using transcriptomic and
proteomic data. With this information,
The use of T. reesei as an industrial cellulase and hemicellulase producer for biofuel production is
through functional genomics, we can
already a global reality. However, production costs, principally in enzyme production, are still improve cellulase production. How can
high. The transcriptomic and proteomic era has led to an increased understanding of how to we better regulate gene expression in
improve cellulase and hemicellulase production for the enzymatic hydrolysis of biomass. T. reesei to further improve cellulase
production?
Biotechnological developments, such as metabolic engineering of T. reesei, will allow production
of a variety of enzyme cocktails at lower costs. Furthermore, implementing synthetic biology
How will synthetic biology provide new
tools will achieve cost-effective biomass hydrolysis and sugar fermentation for biofuel and expression systems in T. reesei to
biorefinery technologies in one step. Thus, the future of T. reesei in omics-scale research is achieve a higher level of protein pro-
highly promising. New perspectives for the study of this fungus and their application in duction, including foreign proteins to
improve biomass degradation? Fur-
biotechnology are ready to be implemented (see Outstanding Questions). thermore, how will synthetic biology
lead to using native or engineered pro-
Acknowledgments moters to be used alone or in combi-
This work was supported by The State of São Paulo Research Foundation (FAPESP) (proc. 2014/23653-2) and (2012/ nation to express multiple enzyme
22921-8). Authors are very thankful to Professor David W. Wilson (Department of Molecular Biology and Genetics, Cornell cocktails?

University, Ithaca, NY 14853, USA) for doing necessary English proof reading of the article.
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