Fungal Biology 124 (2020) 723e734

Contents lists available at ScienceDirect

Fungal Biology
journal homepage: www.elsevier.com/locate/funbio

Solid-state fermentation increases secretome complexity in Aspergillus

brasiliensis
Daniel Salgado-Bautista a, Tania Volke-Sepúlveda a, Francisco Figueroa-Martínez c,

pez b, Ernesto Favela-Torres a, *
Ulises Carrasco-Navarro a, Alicia Chagolla-Lo
a
Departamento de Biotecnología, Universidad Auto
noma Metropolitana-Iztapalapa, San Rafael Atlixco 186, Col. Vicentina, Iztapalapa, 09340, Ciudad de
M
exico, Mexico
b
mica- Cinvestav Unidad Irapuato, Km 9.6 Libramiento Norte Carretera Irapuato-Leo
Laboratorio de Proteo
n, Irapuato, 36824, Guanajuato, Mexico
c
CONACyT Research Fellow, Departamento de Biotecnología, Universidad Auto
noma Metropolitana-Iztapalapa, San Rafael Atlixco 186, Col. Vicentina,
Iztapalapa, 09340, Ciudad de M
exico, Mexico

a r t i c l e i n f o a b s t r a c t

Article history: Aspergillus is used for the industrial production of enzymes and organic acids, mainly by submerged
Received 18 September 2019 fermentation (SmF). However, solid-state fermentation (SSF) offers several advantages over SmF.
Received in revised form Although differences related to lower catabolite repression and substrate inhibition, as well as higher
19 April 2020
extracellular enzyme production in SSF compared to SmF have been shown, the mechanisms undelaying
Accepted 25 April 2020
such differences are still unknown. To explain some differences among SSF and SmF, the secretome of
Available online 7 May 2020
Aspergillus brasiliensis obtained from cultures in a homogeneous physiological state with high glucose
concentrations was analyzed. Of the regulated proteins produced by SmF, 74% were downregulated by
Keywords:
Aspergillus niger
increasing the glucose concentration, whereas all those produced by SSF were upregulated. The most
Proteomics abundant and upregulated protein found in SSF was the transaldolase, which could perform a moon-
Kinetic parameters lighting function in fungal adhesion to the solid support. This study evidenced that SSF: (i) improves the
Branching kinetic parameters in relation to SmF, (ii) prevents the catabolite repression, (iii) increases the branching
Protein regulation level of hyphae and oxidative metabolism, as well as the concentration and diversity of secreted proteins,
and (iv) favors the secretion of typically intracellular proteins that could be involved in fungal adhesion.
All these differences can be related to the fact that molds are more specialized to growth in solid ma-
terials because they mimic their natural habitat.
© 2020 British Mycological Society. Published by Elsevier Ltd. All rights reserved.

1. Introduction solid-state fermentation (SSF) processes offer several advantages

Various species of the genus Aspergillus are used for a large scale
production of enzymes such as amylases (Kammoun et al., 2008),
xylanases, galactosidases and pectinases (de Vries and Visser,
2001), and organic acids (Yang et al., 2017). Aspergillus brasiliensis
ATCC9642, previously named Aspergillus niger ATCC9642
(Houseknecht et al., 2008) has been used for enzymes production
(Zeni et al., 2011; Zhao et al., 2019), and hydrocarbon biodegrada-
tion at lab level (Volke-Sepúlveda et al., 2006). Most of these pro-
cesses are performed by submerged fermentation (SmF); however,
* Corresponding author.
E-mail addresses: em.daniel.alf@gmail.com (D. Salgado-Bautista), tvs@xanum.
uam.mx (T. Volke-Sepúlveda), franfigmtz@gmail.com (F. Figueroa-Martínez),
ulises.c.n@gmail.com (U. Carrasco-Navarro), alicia.chagolla@cinvestav.mx

pez), favela@xanum.uam.mx (E. Favela-Torres).
(A. Chagolla-Lo
over SmF, such as the reduction in catabolite repression and sub-
strate inhibition (Aguilar et al., 2001; Solís-Pereira et al., 1993),
greater enzyme yields and volumetric productivities, extended
product stability and low production costs (Viniegra-Gonza
lez
et al., 2003; M et al., 2016). Nonetheless, studies explaining the
metabolic differences of microorganisms when cultured in SSF or
SmF are scarce to date.
The differences between the culture conditions in SSF and SmF
influence fungal phenotypes, such as growth and enzyme produc-
tion, through modifying the expression of many genes (Iwashita,
2002). To understand these differences, proteomic studies are a
valuable alternative, since they can reveal details about post-
transcriptional and post-translational regulation of gene expres-
sion that cannot be detected using only transcriptional analyzes.
Proteomics has proven to be a suitable method for the identifica-
tion of proteins and the study of changes in their expression levels

https://doi.org/10.1016/j.funbio.2020.04.006
1878-6146/© 2020 British Mycological Society. Published by Elsevier Ltd. All rights reserved.
724 D. Salgado-Bautista et al. / Fungal Biology 124 (2020) 723e734
depending on the type of culture and the environmental conditions
(Oda et al., 2006). For instance, the carbon source has a greater
effect on the secretome than on the intracellular proteome in
A. niger (Lu et al., 2010); during amylases production by A. niger in
SSF with starch and glucose, analysis of the secretome demon-
strated both the absence of catabolic repression, and the presence
of several enzyme isoforms (Carrillo-Sancen et al., 2016); addi-
tionally, the increase in glucose concentration favors protein
secretion by non-conventional secretion mechanisms (Volke-
Sepulveda et al., 2016). Secretome analysis also gives a sight into
metabolic changes in filamentous fungi grown in SSF using high
glucose concentrations; in this regard, the culture of A. brasiliensis
under SSF conditions, showed that the increase in glucose con-
centration (up to 180 g/L) favored catabolism and reduced the
expression of stress-related proteins (Volke-Sepulveda et al., 2016).
Thus, a secretomic approach can help understand some of the
differences between SSF and SmF at the biochemical and molecular
level. Therefore, this work aimed to explain some differences be-
tween SSF and SmF through the study of the secretome of
A. brasiliensis obtained from cultures in a homogeneous and well-
defined physiological state e regardless of the type of culture and
glucose concentration in the media e with high glucose concen-
trations as the sole carbon source.
2. Materials and methods
2.1. Aspergillus brasiliensis strain
The study was carried out with the strain ATCC9642 of
A. brasiliensis, formerly A. niger ATCC9642 (Houseknecht et al.,
2008). The strain was conserved as a conidia suspension in glyc-
erol (20% v/v) at
20  C. For inoculum production, the strain was
propagated in slants with 10 mL of potato dextrose agar (PDA,
Bioxon) and incubated at 30  C for 7 days. Conidia were suspended
in 10 mL of sterile Tween 80 (0.05%, v/v), and 1 mL of the suspen-
sion was inoculated in 30 mL of PDA in 125 mL Erlenmeyer flasks.
After incubation (30  C, 7 days), conidia were recovered with sterile
Tween 80 (0.05%), and the obtained suspension was used as inoc-
ulum for SmF and SSF.
2.2. Submerged fermentation
Erlenmeyer flasks (250 mL) with 50 mL of modified Hill and
Kafer medium (Hill and Kafer, 2001) with glucose (J.T. Baker) as the
sole carbon source were used. The initial pH was adjusted to 6.5,
and the sterilized (94  C, 15 min) culture medium was inoculated
with 1  106 conidia/mL. The initial glucose concentrations were
20, 40 and 60 g/L, and salts concentration increased at the same
ratio than glucose. Cultures were incubated (30  C, 200 rpm), and
aerated with sterile air (20 mL/min).
2.3. Solid-state fermentation
For SSF studies, washed and dried (60  C, 24 h) perlite (AGROlita,
Mexico) with a particle size of 0.85e1.70 mm was used as inert solid
support. The modified Hill and Kafer medium (pH 6.5) with initial
glucose concentrations of 60, 120 and 180 g/L, and increasing salts
concentration at the same ratio than glucose was used. Perlite and
the culture medium were separately sterilized for 15 min at 120 and
94  C, respectively. Then, perlite was moistened with inoculated
medium (1  107conidia/mL) at a ratio of 1.8/0.2/1 (v/v/w) culture
medium/inoculum/perlite, leading to moisture contents of 60.6,
57.3 and 52.8% for 60, 120 and 180 g/L of glucose, respectively.
Twenty-two grams of the above SSF medium were packed in pre-
viously sterilized (120  C, 15 min) glass columns (2.15 cm diameter),
obtaining a volume filled with 50.5 cm3 (±0.51 cm3) per column.
Cultures were incubated at 30  C and aerated with sterile air
(20 mL/min).
2.4. CO2 analysis
The CO2 concentration in the exhaust gas was analyzed and used
to estimate kinetic parameters (Volke-Sepulveda et al., 2016). At
least, four replicates were assessed for each condition tested.
2.5. Recovery of extracellular protein
For the secretome analysis, samples were taken 15e30 min after
the maximum CO2 production rate (MCPR) was attained (Volke-
Sepulveda et al., 2016). For SmF, once the MCPR was reached,
20 mL of protease inhibitor cocktail (P2714, SigmaeAldrich) were
added to the culture medium, and flasks were immediately cooled
at 4  C for 30 min. Then, the culture medium was vacuum-filtered
(Whatman 41), and the filtrate was stored at 4  C for further pro-
cessing. For SSF, once the sampling time was reached, 35 mL of
20 mM TriseHCl buffer (pH 7.2) with 20 mL of protease inhibitor
were added to each column; then, the columns were cooled at 4  C
for 30 min. Afterward, the extracellular fraction was recovered by
vacuum extraction from the solid support. To increase the protein
recovery, 25 mL of 20 mM TriseHCl buffer (pH 7.2) were added to
each column and allowed to stand for 10 min at 4  C; then, the
protein extract was recovered by vacuum extraction. This proced-
ure allows obtaining about 85% of the total extracellular protein
(data not shown). Both extracts were combined and stored at 4  C
for further processing.
2.6. Morphometric analysis
For SmF cultures, small pellets were taken directly from the
culture medium (after 53e62 h of culture), placed on a slide and
covered with a coverslip (Colin et al., 2013). For SSF, the samples
were prepared on slides with perlite and culture medium with the
corresponding glucose concentration; these were covered with
coverslips and incubated at 30  C for 38e57 h. Microscopic obser-
vations were performed in a light microscope (BX 50, Olympus);
image acquisitions were done using a 10 objective with a digital
camera (Evolution VF Monochrome Cooled, Media Cybernetics, Inc,
using an exposure time of 250 ms and 64-bites of resolution), and
processed using the software Image Pro-plus (Media Cybernetics,
Inc). Morphometric analysis (i.e. hyphae length and branching
points) was done with the ImageJ software (NIH, Bethesda MD)
previously calibrated at 1.64 pixel/mm. Images were filtered for
Variance 3D with the following preset values: x, y and z radius
equal to 2. Terminal hyphae with a length longer than 400 mm were
considered for morphometric analysis. The branching level was
defined as the number of tips per hypha length.
2.7. Protein concentration
Protein extracts obtained from SmF and SSF were centrifuged
(7000 rpm, 15 min), and the supernatants were concentrated and
diafiltrated (20 mM TriseHCl buffer, pH 7.2) with membranes of
MWCO 10 KDa (Ultra-15, Amicon). Concentrated extracts
(1.5e2 mL) were centrifuged (10,000 rpm, 2 min), and protein
concentration was determined spectrophotometrically using a BCA
Protein Assay Kit (23,225, Thermo-Fisher Scientific). Aliquots con-
taining 50 mg of protein were lyophilized and used for protein
separation by SDS-PAGE.
 D. Salgado-Bautista et al. / Fungal Biology 124 (2020) 723e734
2.8. Protein separation by SDS-PAGE
Lyophilized samples of protein were cleaned with the Ready-
Prep 2-D cleanup kit (1632130, Bio-Rad). The obtained pellets were
dissolved in 30 mL of Laemmli sample buffer (Mini-PROTEAN® Tetra
Cell-Bio-Rad) according to the instruction manual, homogenized
(30 min), and centrifuged (10,000 rpm, 10 min). The solubilized
proteins were heated (95  C, 5 min), centrifuged (10,000 rpm,
5 min) to eliminate the suspended solids, and separated in a 12%
SDS-PAGE precast gel (Mini-PROTEAN TGX Precast Gels, Bio-Rad).
Electrophoresis was done at 300 V (PowerPac Universal power
supply, Bio-Rad) until the migration distance of the dye front
reached 3 cm. Protein bands were visualized by staining with
Coomassie Brilliant Blue G-250 (161e0406, Bio-Rad).
2.9. In-gel tryptic digestion
After separation of proteins by SDS-PAGE, the gel lanes were
manually excised into three segments (1 cm), and each segment
was cut into smaller pieces (~1 mm2) for in-gel digestion
(Shevchenko et al., 2007). Briefly, the gel pieces were washed twice
with 200 mL of dH2O with gentle shaking for 15 min, destained with
100% acetonitrile/100 mM NH4HCO3 (1:1, v/v) for 15 min, dehy-
drated with 100% acetonitrile (15 min), and vacuum dried (Cen-
trivap, Labconco). The pieces were rehydrated with 10 mM
dithiothreitol (DTT) in 100 mM NH4HCO3, and proteins were
reduced at 56  C for 45 min. DTT solution was removed, and gel
pieces were washed with 100% acetonitrile/100 mM NH4HCO3 (1:1,
v/v) at room temperature for 15 min. After removing the washing
solution, proteins were alkylated with 55 mM iodoacetamide in
100 mM NH4HCO3 at room temperature in the dark for 30 min.
Finally, the pieces were washed with 50% acetonitrile in 100 mM
NH4HCO3 for 15 min, dehydrated with 100% acetonitrile, and vac-
uum dried.
For tryptic digestion the gel pieces were rehydrated with a
porcine trypsin (Promega, Sequencing Grade) solution (10 ng/mL in
50 mM NH4HCO3) at 4  C for 1 h, followed by addition of 50 mM
NH4HCO3 to cover gel pieces and incubated overnight at 37  C.
Peptides were recovered after centrifugation at 10,000 rpm for
2 min. To optimize the peptide extraction, gel pieces were washed
with 50% acetonitrile in 5% formic acid, and then with 100%
acetonitrile/5% formic acid (2:1 v/v). The obtained supernatants
were vacuum-dried and stored at
20  C until LC-MS/MS analysis.
For each culture condition, four biological replicates were inde-
pendently run by SDS-PAGE, two of which were selected for tryptic
digestion and LC-MS/MS analysis.
2.10. LC-MS/MS analysis
Peptides were dissolved in 16 mL of 3% acetonitrile/0.1% formic
acid, centrifuged (10,000 rpm, 1 min), and analyzed in a nano-
Acquity liquid chromatography (LC) system (nano-UPLC System,
Waters), coupled to a linear ion trap LTQ Velos mass spectrometer
(Thermo-Fisher Scientific), equipped with a nano electrospray ion
source. Eight microliters of peptides were injected in a Symmetry
C18 (180 mm  20 mm) precolumn (Waters), and desalted with 3%
acetonitrile at 10 mL/min for 1 min. The separation was performed
switching the flow to 10 cm nano-UPLC column (100 mm ID BEH-
C18 1.7 mm particle size). Peptides were separated with a binary
gradient (solution A: 0.1% formic acid, solution B: 100% acetonitrile
in 0.1% formic acid) at 400 nL/min, and 35  C. The gradient program
modified the concentration of solution B from 3 to 40% over 60 min,
followed by 40e85% over 2 min, and leave washing the column for
10 min before starting the equilibration step with 3% solvent B for
26 min. The column eluate was directed into the mass spectrometer
725
through the Nano Electrospray ion source equipped with a stan-
dard coated silica tip (New Objective).
The mass spectrometer was operated in data-dependent
acquisition mode, to automatically alternate between full scan
(400e1600 m/z) and subsequent Top 5 MS/MS scans in the linear
ion trap with dynamic exclusion enabled. The collision-induced
dissociation (CID) using helium as collision gas at a normalized
collision energy of 35% and 10 ms activation time. Data were ac-
quired using Xcalibur 2.0 software (Thermo-Fisher Scientific).
2.11. Database searching and protein identification
Tandem mass spectra were obtained with Proteome Discover
version 1.4 (Thermo-Fisher Scientific); the identification of proteins
with statistical validity in the mass spectra was performed with the
target-decoy-based control at a 1% protein false discovery rate
(FDR). Peptides search was done with Sequest Program against
A. brasiliensis (13,042 entries) from the NCBI database (downloaded
on February 9, 2017). Program’s settings were as follows:
carbamido-methylation of cysteine as a fixed modification, oxida-
tion of methionine as a variable modification, and two trypsin
missed cleavage. The precursor mass tolerance was set to 2 Da, and
the fragment mass tolerance was set to 1 Da. The following bio-
informatic tools were used to characterize the identified se-
quences: homology search was performed using BlastP from the
NCBI database (www.ncbi.nlm.nih.gov) with the search limited to
entries from A. brasiliensis and A. niger. The minimum percent of
identity considered for all proteins was 80%. The function of the
identified proteins was assigned according to UniProt knowledge-
base (www.uniprot.org), and KEGG database (www.genome.jp/
kegg). The protein subcellular localization was predicted with the
WoLF-PSORT server (http://wolfpsort.org). The prediction of pro-
tein secretion by the classical signal peptides-dependent pathway
was done with the SignalP 4.1 server, while to predict non-classical
mechanisms (i.e. not signal peptide triggered secretion), the
SecretomeP 2.0 server was used.
2.12. Classification and regulation of identified proteins
Proteins were classified into three groups: unique, unregulated
abundant, and regulated. Unique proteins are those identified in
just one of the conditions tested. Protein abundance and regulation
are based on weighted spectral counts (WSC) per milliliter of pro-
tein extract for each type of culture, which was calculated as fol-
lows (WSC)i ¼ (SpC/L)i; where, SpC is the MS spectral count for the
protein i per milliliter of medium, and L is the predicted length of
the protein i. The level of regulation for each protein (i) concerning
glucose concentration, was estimated by plotting the normalized
WSC value obtained for each glucose concentration vs. the
normalized values of glucose. The normalization was made
regarding the maximum WSC value, and the maximum glucose
concentration. Data were adjusted with the least-squares regres-
sion model. Proteins with r2 > 0.75 were considered as regulated,
and the slope value (SV) was used to define the up- (positive SV) or
down- (negative SV) regulation level (Volke-Sepulveda et al., 2016).
Proteins with r2 < 0.75 and included in the 90th percentile of all the
proteins found in each type of culture were considered as abundant
and unregulated. Because of their low concentration, proteins
comprised in the 20th percentile, whose WSC values were less than
1.1  10
4 for SSF and 1.8  10
6 for SmF, were not considered in
the discussion. The complete set of proteins in the extracellular
extracts of A. brasiliensis obtained by SSF and SmF are shown in
Table S1.
726 D. Salgado-Bautista et al. / Fungal Biology 124 (2020) 723e734

2.13. Statistical analysis
The CO2 online measurements for the estimation of kinetic pa-
rameters were carried out at least eight times per condition. The
estimated kinetic data were tested by one-way ANOVA and the
comparison of the means was done using a Duncan’s multiple
range test (a ¼ 0.05). Statistical differences between means are
indicated with different letters. Analyses were performed using
SPSS software, version PASW 23 (IBM SPSS-IBM Corp).
3. Results
This study focuses on comparing the secretome of A. brasiliensis
obtained by SSF and SmF with increasing concentrations of glucose
as the sole carbon source. Glucose is the major carbon source used
in biotechnological processes, and it is responsible of catabolite
repression during the synthesis of a wide range of enzymes and
metabolites; therefore, in order to evaluate the effect of glucose on
the fungal metabolism without any interferences due to specific
inducers (i.e. sucrose, starch, pectin, cellulose) for a given enzyme
production, a defined medium with glucose as the sole carbon
source was used. Due to the inherent difference between both types
of culture and the need to compare secretomes in a well-defined
and homogeneous physiological state, the maximum CO2 produc-
tion rate (MCPR) was selected as a criterion to determine the
sampling time for the secretomic analysis.
3.1. Kinetic studies
Since the MCPR is sensitive to changes in glucose concentration
(Volke-Sepulveda et al., 2016), the time at which it is reached was
determined by the online analysis of CO2 concentration in the
exhaust gas in real time. The sampling time was stablished at
15e30 min after the MCPR was reached; this is, between 38.5 and
56.8 h for SSF and 53.2 and 62.1 h for SmF (Table 1). Values for the
Lag phase, MCPR, and specific CO2 production rate (mCO2) are higher
(up to 1.4-, 8.9-, and 2.0-fold, respectively) in SSF than in SmF;
however, their dependence of glucose concentration is different for
each parameter: whereas MCPR increases as glucose concentration
does, the mCO2 decreases in both types of cultures. The Lag phase
decreases in SmF (about 4 h) and increases (up to 6 h) under SSF
conditions as glucose concentration increases (Table 1).
3.2. Morphometric analysis
The branching level of hyphae (Figs. 1 and 2), expressed as the
number of tips per length of the hypha, is higher in SSF and
increases (up to 1.5-fold) as the glucose concentration increases,
reaching a maximum of 1.22 tips/100 mm with 180 g/L. The
branching level in SmF (~0.85 tips/100 mm) is lower than in SSF and
independent of glucose concentration.
3.3. Description of secretomes obtained by SSF and SmF
SSF increases the abundance and complexity of the secretome of
A. brasiliensis. Compared to SmF, the concentration of secreted
protein is from 3.6 to 5.9 times higher in SSF, and it increases (3.4
and 2.2-fold, respectively) as the glucose concentration augment
(Fig. 2). Of the 304 proteins identified, 242 correspond to SSF and 62
to SmF (Fig. 3), which results in a protein diversity 3.9 times higher
in SSF than in SmF. Noticeably, 33% and 1.6% of the identified pro-
teins in SSF and SmF, respectively, have no evidence of secretion by
conventional (SignalP) or non-conventional mechanisms (Secre-
tomeP). To obtain biological and biochemical insights into the
secretomes, the 304 proteins were classified at three levels: (i) all of
them were classified into unique, unregulated abundant and
regulated, according to their presence in one or more treatments
and their WSC; (ii) they were classified as enzymes (241), non-
enzymes (14), and unidentified proteins (49); (iii) proteins were
classified as follows, based on the main function/process in which
they are involved (SSF/SmF): carbohydrate metabolism (74/28),
redox/stress response (35/9), cellular processes/component (24/6),
proteins/proteolysis (21/5), amino acids (16/3), lipids (10/0), nu-
cleotides (8/0), energy (6/0), miscellaneous (28/4), and uncharac-
terized (20/7) (Fig. 3).
3.3.1. Unique proteins
One hundred and four proteins (87 of SSF and 17 of SmF) were
unique, since they are present in only one of the conditions tested
(Table S1); the highest number of unique proteins was detected at
the highest glucose concentration tested for each type of culture:
75 for SSF, and 13 for SmF. Most of the unique proteins are enzymes
(60 in SSF, and 13 in SmF) mainly involved in the metabolism of
carbohydrates (20 in SSF, and 7 in SmF), and amino acids (9 in SSF,
and 2 in SmF). Among the enzymes related to carbohydrate
metabolism, many participate in central pathways, such as glycol-
ysis (glucose 6-phosphate isomerase), pentose phosphate (PP)
pathway (6-phosphogluconate dehydrogenase, decarboxylating 1),
and tricarboxylic acid (TCA) cycle (citrate synthase). Among the
unique proteins, 44% in SSF and 6% in SmF have no evidence of any
secretory mechanism.

Table 1
Kinetic parameters estimated from the CO2 produced by A. brasiliensis grown under SSF and SmF conditions*.

Culture Glucose conc. (g/L) Lag phase (h) MCPR (mg CO2/mL media h) b
mCO2 (1/h) c ST (h) d
n e

a
Initial Final

SmF 20 1.00 ± 1.35B 28.9 ± 1.8B 0.50 ± 0.06E 0.137 ± 0.010D 52.95 ± 1.94C 25
40 8.75 ± 0.60A 24.5 ± 2.1C 0.68 ± 0.10DE 0.103 ± 0.007E 55.51 ± 2.67B 16
60 7.94 ± 0.50A 24.4 ± 2.9C 0.78 ± 0.11D 0.072 ± 0.005F 62.25 ± 5.43A 9
SSF 60 0.68 ± 0.57B 25.9 ± 1.3C 2.36 ± 0.24C 0.252 ± 0.026A 38.57 ± 1.12E 23
120 0.46 ± 0.28B 27.8 ± 1.2B 4.64 ± 0.27B 0.187 ± 0.014B 47.58 ± 1.98D 10
180 0.77 ± 0.43B 32.3 ± 2.9A 6.81 ± 0.61A 0.158 ± 0.007C 56.80 ± 2.82B 8

* Different letters indicate significant differences among glucose concentration (p < 0.0001, Duncan multiple range test; n  7). ANOVAs were performed considering data
from both types of culture.
a
Glucose concentration at the sampling time (measured in at least four independent replicates for each condition).
b
Maximum CO2 production rate.
c
Specific CO2 production rate.
d
Sampling time.
e
Number of experimental units analyzed in each condition.
 D. Salgado-Bautista et al. / Fungal Biology 124 (2020) 723e734
Fig. 1. Branching in hyphae of A. brasiliensis as function of the initial glucose concentration in SmF (up) and SSF (down); Images were filtered with Variance 3D in ImageJ.
Bar ¼ 100 mm.
3.3.2. Abundant unregulated proteins
From the pool of 29 unregulated proteins identified (17 in SSF
and 12 in SmF), five were considered as abundant, with WSC values
above the estimated value for the 90th percentile (1.6  10
3 for
SSF, and 3.0  10
5 for SmF) (Table 2). Such proteins are YkgB and a
protein with nitrilase activity (ASPBRDRAFT_40,942) for SSF, and
glucoamylase, glucan endo-1,3-b-glucosidase eglC, and a protein
with unknown function (ASPBRDRAFT_127,351) for SmF.
3.3.3. Regulated proteins
One hundred forty-six proteins from SSF (120 enzymes and 26
non-enzymes) and 23 from SmF (20 enzymes and three non-
enzymes) are regulated by the initial glucose concentration
(Tables 3 and 4). While all of them are upregulated in SSF extracts,
only six of those obtained by SmF are upregulated. Eleven of the 146
regulated proteins found in SSF account for the 50% of the WSC:
endo-1,4-b-xylanase A, transaldolase, peroxiredoxin pmp20, lipase,
protein ecm33, reductase-like protein, aldehyde reductase 1, ma-
late dehydrogenase, 1,3-b-glucanosyltransferase Gel1, glucoamy-
lase, and triosephosphate isomerase. For SmF, the WSC of three
proteins e cell wall protein PhiA, YkgB, and protein ecm33 e
Fig. 2. Number of hyphal tips in 100 mm of hyphae (closed symbols), and protein
concentration (open symbols) produced under solid or submerged fermentation
conditions. Data with different letter indicate significant differences between treat-
ments (p < 0.05; n  4 for protein; n > 30 for hyphal tips).
727
represents 64% of the total WSC of regulated proteins, the two later
being downregulated. Regulated proteins were further classified
according to the main function/process in which they participate.
3.3.3.1. Carbohydrate metabolism. Fifty of the 146 upregulated
proteins obtained in SSF are enzymes involved in carbohydrate
metabolism, 45 of them have prediction of a secretory mechanism
(Table 3). Hydrolases are the predominant enzymes (32), with 13
involved in starch and sucrose metabolism, six in endo-hydrolysis
of b-D-glucans and b-D-xylans, and four in amino sugar and
nucleotide sugar metabolism. The three most abundant enzymes
are transaldolase, endo-1,4-b-xylanase A, and malate dehydroge-
nase. Eight enzymes involved in carbohydrate metabolism are
present in the SmF secretome; two of them are upregulated, being
an acid-stable a-amylase and a pectin lyase F the most abundant
proteins in this group (Table 3).
3.3.3.2. Redox/stress-related response. This group of proteins rep-
resents the second in terms of the number of regulated proteins in
both types of culture. Twenty-six upregulated proteins in this
group were found in SSF (21 enzymes, and five non-enzymes)
(Tables 3 and 4); among them, two isoforms of aldehyde reduc-
tase 1, glutamate dehydrogenase, glutathione reductase, thio-
redoxin, a peroxiredoxin pmp20, a protein with peroxidase
function (ASPBRDRAFT_179,180), two heat shock proteins (HSP),
porphobilinogen deaminase, and an ADP-ribosylation factor have
no prediction of some secretion mechanism. The two most abun-
dant proteins in this group are peroxiredoxin pmp20, and a
reductase-like protein. The three regulated enzymes found in SmF
within this group e sulfhydryl oxidase, FAD-dependent oxygenase,
and FAD-binding domain protein e are downregulated.
3.3.3.3. Cellular processes/component. Regulated proteins that are
cellular components or are involved in cellular processes (20)
represent the third group in terms of the total number of the
regulated proteins. Fourteen upregulated proteins were obtained in
SSF (Tables 3 and 4); seven of them are enzymes, and seven have no
evidence of a secretion mechanism. The most abundant proteins in
this group are a 1,3-b-glucanosyltransferase Gel1 and the protein
NMT1. Six proteins of this group were found in SmF, all of them
identified as secreted. The four enzymes found in SmF are isoforms
728 D. Salgado-Bautista et al. / Fungal Biology 124 (2020) 723e734

Fig. 3. Venn diagrams and functional classification of proteins found in the secretomes of A. brasiliensis grown in SSF and SmF.

Table 2
Abundant unregulated proteins found in the secretomes of A. brasiliensis grown under SSF and SmF conditions.

Accession numbera Protein Nameb EC number Specific function/process SWSCix103 (Sp/mL) c

SignalPd

Solid-state fermentation (SSF)

Cellular processes/component
GAQ46904 YkgB (ext) - Lactonase superfamily 5.212 Y+
OJJ73275* ASPBRDRAFT_40942/nitrilase (mit) 3.5.5.1 Nitrogen metabolism (nitrilase) 1.753 N+
Submerged fermentation (SmF)
Carbohydrate metabolism
XP_001390530 Glucoamylase (ext) 3.2.1.3 Starch and sucrose metabolism 0.337 Y+
XP_001390410 Glucan endo-1,3-b-glucosidase eglC (ext) 3.2.1.39 Starch and sucrose metabolism 0.031 Y+
Cellular processes/component
OJJ71409* ASPBRDRAFT_127351 (cyt) - Unknown 0.038 N+
a
NCBI access numbers for A. niger CBS 513.88, except those indicated with * that correspond to A. brasiliensis CBS 101740;
b
letters in parentheses indicate the predicted subcellular localization of proteins (WoLF-PSORT): (cyt) cytosolic, (ext) extracellular, (mit) mitochondrial, (per) peroxisome;
c
summation of the weighted spectral counts (WSC) for the protein i in the three glucose concentrations tested;
d
proteins predicted with signal peptide (SignalP), proteins with + were predicted with a non-classical mechanism of secretion (SecretomeP).

of 1,3-b-glucanosyltransferases (Table 3), and the two non- is one of the most abundant proteins identified in this category in
enzymes are a YkgB and a cell wall protein PhiA (Table 4). The both types of culture.
latter (upregulated) is the most abundant protein found in SmF.

3.3.3.6. Lipid metabolism. Six upregulated proteins involved in

3.3.3.4. Amino acid metabolism. Seven regulated enzymes involved
in amino acid metabolism were found in SSF. The most abundant
protein in this group is spermidine synthase, and only two tyrosi-
nases have prediction of a secretory mechanism. The only protein
identified in SmF that participates in amino acid metabolism is an
amidase, which is downregulated and predicted as secreted.
3.3.3.5. Protein/proteolysis. Thirteen (12 enzymes and one non-
enzyme) and four (all enzymes) proteins related to protein meta-
bolism (biosynthesis and proteolysis) are present in SSF and SmF
extracts, respectively. All proteins found in SmF have evidence of
secretion, while only nine of those produced by SSF have it. All
these proteins are upregulated in SSF, and three are downregulated
in SmF. Of those produced by SSF, 11 are enzymes involved in
proteolysis (Tables 3 and S1), including an ubiquitin-activating
enzyme (ASPBRDRAFT_59,296) with function in the elimination
of defective proteins. The four enzymes found in SmF extracts
(Table 3) participate in proteolysis, and only an isoform of
tripeptidyl-peptidase-1 is upregulated. Tripeptidyl-peptidase sed2
lipid metabolism were identified in SSF; four of the five enzymes
identified are involved in both glycerolipid and glycer-
ophospholipid metabolism (Table 3), being a lipase the most
abundant protein in this group. Only one protein in this category,
acyl-CoA binding protein (Table 4), has no secretion mechanism
predicted. None regulated protein in this group was found in ex-
tracts produced by SmF.
3.3.3.7. Nucleotide and energy metabolism. Ten upregulated pro-
teins involved in nucleotide (five enzymes) and energy metabolism
(four enzymes and one non-enzyme) were identified in SSF. Nine of
them (except an inorganic pyrophosphatase) are found with 120
and 180 g/L of glucose. Five proteins e adenosine kinase, nucleoside
diphosphate kinase, ADP-ribose pyrophosphatase, orotidine 50 -
phosphate decarboxylase, and cyanate hydratase e have no secre-
tion signal. The most abundant proteins in these groups are cyanate
hydratase, adenosine kinase, and inorganic pyro-phosphatase
(partial). No proteins were identified within this category in SmF.
 D. Salgado-Bautista et al. / Fungal Biology 124 (2020) 723e734 729

Table 3
Regulated enzymes found in the secretomes of A. brasiliensis grown under SSF and SmF conditions.

Accession numbera Protein name EC number SignalPb Subcell. locationc SWSCix103 (Sp/mL)d SVe

Solid-state fermentation (SSF)

Carbohydrate metabolism
XP_001388522 Endo-1,4-b-xylanase A 3.2.1.8 Y ext 15.697 1.42
XP_001391467 Transaldolase 2.2.1.2 N cyt 14.568 1.48
XP_001391302 Malate dehydrogenase 1.1.1.37 N+ mit 4.442 1.47
XP_001390530 Glucoamylase 3.2.1.3 Y ext 3.730 1.17
XP_001401115 Triosephosphate isomerase 5.3.1.1 N per 3.608 1.48
XP_001389996 Endo-1,4-b-xylanase F1 3.2.1.8 Y ext 2.732 1.27
XP_001390410 Glucan endo-1,3-b-glucosidase eglc 3.2.1.39 Y ext 2.357 1.27
AGI04246 Glucose oxidase 1.1.3.4 Y nuc 2.080 1.33
XP_001400902 Endoglucanase A 3.2.1.4 Y ext 1.657 1.40
XP_001389926 Pectin lyase F 4.2.2.10 Y ext 1.583 1.23
XP_001395835 Glucosamine 6-phosphate N-acetyltransferase 2.3.1.4 N cysk 1.320 1.50
XP_001399898 Phosphoglucomutase 5.4.2.2 N+ cyt 1.276 1.50
XP_001390334 HAD superfamily hydrolase 5.4.2.- N cyt 0.992 1.50
XP_001389652 Exo-b-1,3-glucanase Exg0 3.2.1.58 Y ext 0.979 1.24
XP_001392475 Glucan 1,3-b-glucosidase 3.2.1.58 Y ext 0.903 1.34
XP_001398816 b-glucosidase A 3.2.1.21 Y ext 0.754 1.39
XP_001393626 a-amylase 3.2.1.1 Y ext 0.737 1.30
XP_001396893 Endo-arabinase 3.2.1.99 Y ext 0.684 1.50
XP_001397023 Endo-b-1,4-glucanase D 3.2.1.151 Y ext 0.661 1.18
XP_001389998 a-L-arabinofuranosidase axhA 3.2.1.55 N+ ext 0.651 0.85
XP_001395912 Glucokinase 2.7.1.2 N cyt 0.639 1.50
XP_001401742 Aldose 1-epimerase 5.1.3.15 N mit 0.623 1.50
XP_001394051 Ribose 5-phosphate isomerase A 5.3.1.6 N+ cyt 0.587 1.45
XP_001398868 Glucan 1,3-b-glucosidase A 3.2.1.58 Y ext 0.578 1.31
XP_001400489 Glycosyl hydrolase, family 18/chitinase 3.2.1.14 Y ext 0.559 1.40
OJJ67369 b-xylanase 3.2.1.8 Y ext 0.539 1.50
XP_001388594 Acid trehalase 3.2.1.28 Y ext 0.435 1.43
OJJ74329 ASPBRDRAFT_27362/probable mannosyl oligosaccharide a-1,2-mannosidase 1B 3.2.1.- Y ext 0.399 1.20
XP_001389510 a/b-glucosidase agdC 3.2.1.20 Y ext 0.324 1.46
XP_001392640 Endoglucanase-4 3.2.1.4 Y ext 0.320 1.38
OJJ66437 ASPBRDRAFT_49077/chitinase function (GO) Y ext 0.303 1.50
XP_001396769 a-N-arabinofuranosidase B 3.2.1.55 Y ext 0.270 1.50
GAQ42198 Acid-stable-a-amylase 3.2.1.1 N+ plas 0.241 1.29
XP_001399223 Endo-1,3(4)-b-glucanase 3.2.1.6 Y ext 0.226 1.18
GAQ44118 Extracellular cellulase celA/allergen Asp F7-like Y mit 0.222 1.25
XP_001395335 Endoglucanase 3.2.1.4 Y ext 0.213 1.50
XP_001401157 Cell wall glycosyl hydrolase YteR Y ext 0.173 1.50
OJJ75131 ASPBRDRAFT_53082 Y ext 0.172 1.06
XP_001396717 2-methylcitrate dehydratase 4.2.1.79 N+ mit 0.153 1.50
XP_001390419 Endoglucanase-1 precursor 3.2.1.4 Y ext 0.123 1.50
XP_001388482 a-N-arabinofuranosidase A 3.2.1.55 Y ext 0.119 1.50
OJJ68845 Glycosyl hydrolase family 71 protein/glucan endo-1,3-a-glucosidase 3.2.1.59 Y ext 0.106 0.78
OJJ72798 Glycosidase crf2 Enzyme N+ ext 0.097 1.26
OJJ75576 Exo-b-1,3-glucanase 3.2.1.58 N+ ext 0.094 1.50
OJJ66103 Glucan endo-1,6-b-glucosidase BGN16.3 3.2.1.75 Y ext 0.087 1.50
OJJ68479 N-acetylglucosaminidase 3.2.1.52 Y ext 0.084 1.31
OJJ66428 a-galactosidase C 3.2.1.22 Y ext 0.080 1.50
OJJ67021 a-amylase 3.2.1.1 Y ext 0.075 1.24
OJJ77986 b-mannosidase A 3.2.1.25 Y ext 0.060 1.26
OJJ67850 Cellobiose dehydrogenase 1.1.99.18 Y ext 0.034 1.50
Redox/stress response
XP_001395908 Peroxiredoxin pmp20 1.11.1.15 N cyt 8.711 1.46
XP_001389120 Reductase-like protein 1.1.1.- N+ cyt 8.483 1.46
XP_001394119 Aldehyde reductase 1 1.1.1.21 N cyt 5.098 1.47
XP_001391459 Superoxide dismutase [Cu-Zn] 1.15.1.1 N+ cyt 1.784 1.26
XP_001388687 Thioredoxin 1.8.4.10 N cyt 1.363 1.50
XP_001393905 Sulfhydryl oxidase 1.8.3.2 Y ext 1.165 1.24
XP_001398757 Porphobilinogen deaminase/hydroxymethylbilane synthase 4.2.1.24 N mit 0.844 1.50
XP_001388621 Catalase R 1.11.1.6 Y ext 0.703 1.31
XP_001388595 Catalase R 1.11.1.6 N+ ext 0.680 1.39
XP_001401851 Superoxide dismutase [Mn] 1.15.1.1 N+ mit 0.591 1.50
XP_001397405 Peroxiredoxin pmp20 1.11.1.15 N+ cyt 0.493 1.50
XP_001401464 NADP-specific glutamate dehydrogenase 1.4.1.2 N cyt 0.485 1.50
XP_001397790 NAD dependent epimerase/dehydratase family Enzyme N+ cyt 0.432 1.50
XP_001390247 Glutathione reductase 1.8.1.7 N mit 0.359 1.50
XP_001390337 Aldo-keto reductase 1.11.1.7 N+ cyt 0.327 1.41
OJJ71117 ASPBRDRAFT_179180/Peroxidase 1.11.1.- N mit 0.188 1.50
XP_001390822 Laccase-1 1.10.3.2 Y ext 0.186 1.50
XP_001389952 FAD-dependent oxygenase 1.5.3.6 Y ext 0.130 1.17
XP_001401635 Aldehyde reductase 1 1.1.1.184 N cyt 0.127 1.50
XP_001390256 Laccase-1 1.10.3.2 Y ext 0.119 1.50
(continued on next page)
730 D. Salgado-Bautista et al. / Fungal Biology 124 (2020) 723e734

Table 3 (continued )

Accession numbera Protein name EC number SignalPb Subcell. locationc SWSCix103 (Sp/mL)d SVe

OJJ65770 FAD-binding domain protein 1.-.-.- Y ext 0.103 1.17

Cellular process/component
XP_001402433 1,3-b-glucanoSolid-state fermentation (SSF)syltransferase gel1 2.4.1.- Y ext 4.222 1.34
OJJ74465 ASPBRDRAFT_27493 Y ext 0.511 1.50
XP_001393994 GTP-binding protein Ypt1 N+ cyt_nuc 0.487 1.50
XP_001392850 1,3-b-glucanosyltransferase gel2 2.4.1.- Y ext 0.345 1.33
XP_001398680 Ras-related protein Rab7/dGTPase 3.1.5.1 N mit 0.288 1.50
XP_001389030 Ras-related protein Rab-11A N cyt_nuc 0.275 1.50
XP_001390497 1,3-b-glucanosyltransferase gel3 2.4.1.- Y ext 0.179 1.33
Amino acid metabolism
XP_001392771 Spermidine synthase 2.5.1.16 N nuc 1.109 1.49
XP_001401543 5-methyltetrahydropteroyl triglutamate-b-homocysteine methyltransferase 2.1.1.14 N mit 0.875 1.46
XP_001389342 Tyrosinase 1.14.18.1 Y ext 0.844 1.37
XP_001397565 Threonine synthase 4.2.3.1 N mit 0.390 1.50
XP_001396639 1,2-dihydroxy-3-keto-5-methylthiopentene dioxygenase 1.13.11.54 N cyt 0.371 1.50
XP_001401406 Branched-chain-amino-acid aminotransferase 2.6.1.42 N mit 0.200 1.50
XP_001389935 Tyrosinase 1.14.18.1 Y ext 0.157 1.50
Protein/proteolysis
OJJ72478 ASPBRDRAFT_54314 /serine proteinase pepC 3.4.21.- Y ext 1.599 1.46
XP_001400873 Tripeptidyl-peptidase sed2 3.4.14- Y ext 1.562 1.30
XP_001401759 Aminopeptidase 2 3.4.11.- N per 0.735 1.50
XP_001401404 Dipeptidyl peptidase III 3.4.14.4 N cyt 0.641 1.50
XP_001398198 Carboxypeptidase S1 3.4.16.- Y ext 0.304 1.42
XP_001393995 Aminopeptidase 3.4.11.- N cyt 0.278 1.50
XP_001401653 Aminopeptidase C 3.4.22.40 N+ cyt 0.265 1.50
XP_001393937 Vacuolar aspartyl aminopeptidase Lap4 3.4.11.- N+ mit 0.220 1.50
XP_001401570 Peptidyl-prolyl cis-trans isomerase B 5.2.1.8 Y ext 0.208 1.50
OJJ66856 ASPBRDRAFT_59296/ubiquitin-activating enzyme E1 1 6.2.1.45 N cyt_nuc 0.118 1.43
OJJ68449 Aminopeptidase Y 3.4.11.15 Y ext 0.084 1.50
OJJ70197 Extracellular serine carboxypeptidase 3.4.14.2 Y ext 0.070 1.50
Lipid metabolism
XP_001397501 Lipase 3.1.1.3 Y ext 3.645 1.41
XP_001391356 SUN domain protein (Uth1)/triacylglycerol lipase 3.1.1.3 Y ext 1.801 1.47
XP_001398185 Triacylglycerol lipase 3.1.1.3 Y ext 0.166 1.43
XP_001393823 Cholinesterase 3.1.1.7 Y ext 0.131 1.26
OJJ65854 Lipase 1 precursor 3.1.1.3 Y ext 0.065 1.50
Nucleotide metabolism
XP_001398362 Adenosine kinase 2.7.1.20 N cyt 0.674 1.50
XP_001393898 Nucleoside diphosphate kinase 2.7.4.6 N cyt 0.542 1.50
OJJ67239 ASPBRDRAFT_136183/Orotate phosphoribosyl-transferase 2.4.2.10 N mit 0.462 1.50
XP_001395432 Orotidine 5’-phosphate decarboxylase 4.1.1.23 N mit 0.360 1.50
XP_001390332 ADP-ribose pyrophosphatase 3.6.1.13 N nuc 0.354 1.50
Energy metabolism
XP_001397234 Cyanate hydratase 4.2.1.104 N per 1.286 1.50
XP_001400329 Inorganic pyrophosphatase, partial 3.6.1.1 N+ cyt_mit 1.188 1.46
XP_001401521 Diphosphomevalonate decarboxylase 4.1.1.33 N+ cyt 0.245 1.50
XP_001391393 S-formylglutathione hydrolase 3.1.2.12 N+ mit 0.204 1.50
Miscellaneous
XP_001402471 Purine nuceoside permease Enzyme Y ext 1.856 1.27
OJJ72358 ASPBRDRAFT_42048/D-6-hydroxynicotine oxidase 1.5.3.6 N ext 0.800 1.45
XP_001397406 Thij/PfpI family protein 3.4.-.- N+ cyt 0.507 1.50
XP_001388959 Minor allergen Alt a 7 1.6.5.- Y ext 0.422 1.50
OJJ71705 ASPBRDRAFT_124942/Cell wall glucanase Y ext 0.399 1.50
OJJ67200 ASPBRDRAFT_59193 Y ext 0.346 1.21
XP_001395965 Dienelactone hydrolase family protein N+ cyt 0.283 1.50
XP_001400163 Geranyl transtransferase 2.5.1.1 N cyt 0.196 1.50
OJJ72740 Dihydrolipoyl dehydrogenase 1.8.1.4 N mit 0.068 1.50
Submerged fermentation (SmF)
Carbohydrate metabolism
GAQ42198 Acid-stable a-amylase 3.2.1.1 N+ plas 0.014 -0.89
OJJ70100 ASPBRDRAFT_45424 /Isopullulanase 3.2.1.57 N+ ext 0.010 -0.42
XP_001389926 Pectin lyase F 4.2.2.10 Y ext 0.010 1.50
OJJ67369 ASPBRDRAFT_69025 /b-xylanase 3.2.1.8 Y ext 0.009 1.50
OJJ74329 ASPBRDRAFT_27362/probable mannosyl-oligosaccharide a-1,2-mannosidase 1B 3.2.1.- Y ext 0.004 -1.06
XP_001388594 Acid trehalase 3.2.1.28 Y ext 0.002 -0.76
OJJ70876 a-glucosidase 3.2.1.20 Y ext 0.002 -1.00
OJJ77986 b-mannosidase A 3.2.1.25 Y ext 0.001 -0.62
Redox/Stress response
XP_001393905 Sulfhydryl oxidase 1.8.3.2 Y ext 0.020 -0.79
XP_001389952 FAD-dependent oxygenase 1.5.3.6 Y ext 0.004 -0.90
OJJ65770 FAD-binding domain protein 1.-.-.- Y ext 0.002 -0.96
Cellular process/component
XP_001402433 1,3-b-glucanosyltransferase gel1 2.4.1.- Y ext 0.024 -0.68
OJJ78207 ASPBRDRAFT_142667/1,3-b-glucanosyltransferase 2.4.1.- Y ext 0.004 1.17
XP_001392850 1,3-b-glucanosyltransferase gel2 2.4.1.- Y ext 0.003 0.17
 D. Salgado-Bautista et al. / Fungal Biology 124 (2020) 723e734 731

Table 3 (continued )

Accession numbera Protein name EC number SignalPb Subcell. locationc SWSCix103 (Sp/mL)d SVe

OJJ68065 1,3-b-glucanosyltransferase gel3 2.4.1.- Y ext 0.001 -1.50

Proteins/Proteolysis
XP_001400873 Tripeptidyl-peptidase sed2 3.4.14.10 Y ext 0.030 -1.04
XP_001401093 Aspergillopepsin 1 3.4.23.18 Y ext 0.007 -0.93
XP_001398198 Carboxypeptidase S1 3.4.16.6 Y ext 0.004 -0.65
XP_001392582 Tripeptidyl-peptidase 1 3.4.14.10 Y ext 0.002 0.78
Amino acid metabolism
XP_001399157 Amidase 3.5.1.4 Y ext 0.002 -0.47
a
NCBI access numbers for A. niger CBS 513.88, except those indicated with * and **, which correspond to A. brasiliensis CBS 101740 and A. niger, respectively;
b
proteins predicted with signal peptide, proteins with + were predicted with a non-classical mechanism of secretion;
c
predicted subcellular localization: (cyt) cytosolic, (ext) extracellular, (mit) mitchondrial, (per) peroxisome, (nuc) nucleus, (plas) plasma membrane, (cysk) cytoskeleton;
d
summation of the weighted spectral counts (WSC) for the protein i in the three glucose concentrations tested;
e
slope value.

Table 4
Regulated non-enzyme proteins found in the secretomes of A. brasiliensis grown under SSF and SmF conditions.

Accession numbera Protein Name SignalPb Subcell. locationc SWSCix103 (Sp/mL)d SVe

Solid-state fermentation (SSF)

Redox/stress response
XP_001392074 Heat shock protein N cyt 2.357 1.50
XP_001398917 Rho GDP-dissociation inhibitor N* cyt 0.530 1.50
XP_001392486 ADP-ribosylation factor N mit 0.358 1.50
XP_001401891 Aha1 domain family N* cyt 0.310 1.50
XP_001393974 Heat shock protein 90 N cyt_nuc 0.111 1.50
Cellular process/component
XP_001400160 Protein NMT1 N cyt 1.499 1.44
XP_001391851 14-3-3 protein N cyt 1.042 1.46
XP_001388668 Glial maturation factor N nuc 0.714 1.50
XP_001399080 14-3-3 protein ypt1 (exosomal) N nuc 0.321 1.50
OJJ68333 ASPBRDRAFT_58357 Y plas 0.200 1.50
XP_001400262 Cell surface spherulin 4-like protein N* ext 0.181 1.50
OJJ78318 Endocytosis protein end4 N nuc 0.048 1.50
Protein/proteolysis
OJJ69179 ASPBRDRAFT_132450/Elongation factor 1 gamma N cyt 0.161 1.50
Lipid metabolism
XP_001399436 Acyl CoA binding protein family N cyt 0.721 1.50
Energy metabolism
XP_001396780 nmrA-like family protein N mit 0.416 1.50
Miscellaneous
XP_001401488 Protein ecm33 Y ext 9.423 1.22
XP_001391915 Allergen Asp F4 Y ext 1.756 1.50
XP_001389020 Cyanovirin-N N cyt 1.748 1.43
XP_001398409 Eukaryotic translation initiation factor 6 N cyt 0.718 1.50
XP_001395113 Protein HMF1 N cyt 0.685 1.25
XP_001401709 Calcium homeostasis protein Regucalcin N cyt 0.235 1.50
Submerged fermentation (SmF)
Cellular process/Component
XP_001400808 Cell wall protein PhiA Y ext 0.125 1.16
GAQ46904 YkgB Y ext 0.084 -1.33
Miscellaneous
XP_001401488 Protein ecm33 Y ext 0.066 -1.29
a
NCBI access numbers for A. niger CBS 513.88, except those indicated with *, which correspond to A. brasiliensis CBS 101740;
b
proteins predicted with signal peptide, proteins with + were predicted with a non-classical mechanism of secretion;
c
predicted subcellular localization: (cyt) cytosolic, (ext) extracellular, (mit) mitchondrial, (nuc) nucleus;
d
summation of the weighted spectral counts (WSC) for the protein i in the three glucose concentrations tested per type of culture;
e
slope value.

3.3.3.8. Miscellaneous proteins. Thirteen upregulated proteins 4. Discussion

produced by SSF (seven enzymes and six non-enzymes) are
involved in several processes, such as nucleotide-binding, trans-
membrane transport, signaling, and metabolism of cofactors and
vitamins, among some others (Tables 3 and 4); six of them have no
evidence of secretion. The most abundant proteins in this category
are protein ecm33, allergen Asp F4 and cyanovirin-N (Table 4); due
to their WSC, they are also classified as abundant. The only protein
classified in this group for SmF is the protein ecm33, whose WSC is
also considered among the most abundant proteins found in SmF
extracts.
The culture conditions and increased glucose concentration
have a direct effect on the kinetic parameters associated with
growth, fungal morphology, and the secretome complexity of
A. brasiliensis. The online analysis of CO2 in the exhaust gas allowed
estimating kinetic parameters associated with fungal growth and
was crucial to establish a criterion for sampling the secretomes.
This approach allows the reproducibility of the physiological state
for each type of culture, regardless of the system and glucose
concentration (Carrillo-Sancen et al., 2016; Volke-Sepulveda et al.,
732 D. Salgado-Bautista et al. / Fungal Biology 124 (2020) 723e734
2016). Compared to SmF, all the estimated kinetic parameters are
improved in SSF, and their values are influenced by the glucose
concentration (Table 1). The MCPR in SSF conditions, increases
proportionally to the glucose concentration up to a maximum at
180 g/L. In SmF, glucose concentration greater than 60 g/L was not
explored because it favors fungal growth on the flask wall (data not
shown), resulting in heterogeneous physiological states hindering
an accurate secretomic analysis. The significantly higher values of
MCPR and mCO2 obtained in SSF can be attributed, in principle, to the
greater glucose concentrations (60e180 g/L) compared to SmF
(20e60 g/L). However, when comparing both types of culture at the
same glucose concentration (60 g/L), the values of these kinetic
parameters are much higher in SSF than in SmF, highlighting that
the value of mCO2 increases almost 3 times, and the sampling time is
reduced to almost half in SSF, indicating that the differences
depend only on the type of culture. The reduction in the specific
CO2 production rate (mCO2) and the increase in the Lag phase as
glucose increases is due to substrate inhibition and osmotic stress;
however, the MCPR values increase proportionally with the in-
crease in the initial glucose concentration from 60 to 180 g/L. The
higher metabolic rate in SSF than in SmF, in terms of kinetic pa-
rameters, may be the result of a more efficient oxygen transfer rate
(Viniegra-Gonza
lez et al., 2003), leading to a greater glucose
oxidation rate together with a lower substrate inhibition and car-
bon catabolite repression [CCR] (Maeda et al., 2004). These features
are intrinsic to SSF, and the higher rate of growth and branching of
hyphae have been proposed as responsible of the greater biomass
yields under SSF conditions (Gomes et al., 2018).
The results show that SSF enhances protein secretion compared
to SmF, both in terms of concentration (Fig. 2) and diversity (Fig. 3),
and evidence a differential regulation in protein synthesis
depending on glucose concentration. Differences among both types
of culture are evident even at the same glucose concentration (60 g/
L), where the extracellular protein produced by SSF is 1.6-fold
higher than the obtained in SmF. Other studies have also shown
that extracellular protein production, measured as enzymatic ac-
tivity (Acun ~ a-Argüelles et al., 1995; Nandakumar et al., 1999) or
total secreted protein (Oda et al., 2006; M et al., 2016; Zhao et al.,
2019), is higher in SSF than in SmF regardless of the carbon
source. Likewise, the number of isoforms of several hydrolytic en-
zymes increases in SSF (M et al., 2016), which means greater
complexity in the secretome, as observed in this work. The
increased protein secretion in SSF is attributed to the fungal
morphology (Cunha et al., 2012; Singhania et al., 2009); while in
SmF, fungal growth commonly occurs in the form of pellets that
may impair the metabolic efficiency (Gomes et al., 2018), SSF favors
the interaction between mycelium and solid substrates (M et al.,
2016), which consequently enhance the filamentous and apical
growth on or in the solid particles, providing growth conditions
that resemble the natural habitat of terrestrial fungi (Gomes et al.,
2018). It is known that protein secretion in filamentous fungi occurs
at the tips of hyphae (Gomes et al., 2018), so that in some studies
internal and external factors are manipulated as a strategy to
modify branching of hyphae and, therefore, increase protein
secretion (Ahamed and Vermette, 2009). In this study, the
morphology of A. brasiliensis changes depending on the type of
culture, resulting in a higher level of branching in SSF which,
together with the number of tips per hypha, increases with
increasing glucose concentrations (Figs. 1 and 2).
On the other hand, the lower water activity (aW) in SSF
compared to SmF is a key factor that modifies the secretion of
metabolites and proteins, as well as the well-known CCR observed
at high substrate concentration of a readily available carbon source,
such as glucose (Gomes et al., 2018). In filamentous fungi, CCR is
regulated by the transcription factor CreA, which in turn is
modulated to some extent by ubiquitination/deubiquitination and
phosphorylation (Adnan et al., 2018). The typical low aW of SSF
systems helps reduce the diffusion of readily metabolizable sugars
present in the medium, forming sugar gradients within the solid
support; this contributes to maintaining a localized sugar-limiting
condition regardless of the initial concentration, thus reducing
the CCR (Viniegra-Gonzalez and Favela Torres, 2006; Maeda et al.,
2004). Unlike SmF, overcoming the CCR in SSF has a direct impact
on the concentration and variety of enzymes whose expression is
controlled by this type of regulation, which may be the reason of
the upregulation of the enzymes involved in carbohydrate meta-
bolism obtained in SSF. In fact, most (62%) of the enzymes involved
in carbohydrate metabolism found in SSF participate in the break-
down of polysaccharides (cellulases, xylanases, arabinofur-
anosidases, and amylases), all of them influenced by CCR (Ries et al.,
2016) and upregulated by the increase in glucose, strongly sup-
porting the absence of CCR in SSF even at concentrations of 180 g/L
(Table 3). Among these, the most abundant protein identified in the
secretome of A. brasiliensis is endo-1,4-b-xylanase A. Similar to the
previous, the production of a-amylase and amyloglucosidase by
A. niger resulted much higher (~10-fold) in SSF than in SmF when
glucose was used as the sole carbon source (Nandakumar et al.,
1999). Similarly, an increase in the synthesis of endo- and exo-
polygalacturonase by A. niger was recorded in SSF with respect to
SmF as the sugar concentration increased, contrary to that obtained
in SmF (Solís-Pereira et al., 1993). Unexpectedly, although most of
the enzymes involved in carbohydrate metabolism are down-
regulated in SmF, a b-xylanase and a pectin lyase-F are upregulated
by increasing glucose (Table 3), indicating that the expression of
those genes is not affected by CCR in such a condition, and could
have a different regulation mechanism in this strain of
A. brasiliensis. Based on the previous results and the fact that the
regulation of the CCR in filamentous fungi depends on CreA, in
A. brasiliensis, the CCR could be independent of the expression of
CreA, particularly under SSF conditions. In fact, creA mutants have
been shown to exhibit a defective CCR, while CCR is ameliorated in
creB and creC mutants in Aspergillus nidulans, being a complex of
the CreB and CreC proteins that acts by eliminating the ubiquitin of
CreA (Ichinose et al., 2018). In SSF cultures of Aspergillus oryzae,
creA and creB deletion enhanced the production of b-glucosidase,
however, xylanase and endo-b-glucanase activities were not
affected by creA and creB deletion; in contrast, under SmF condi-
tions, xylanase and b-glucosidase activities were significantly
increased in single creA deletion mutants and, particularly in creA
and creB double deletion mutants (Ichinose et al., 2018). This re-
inforces the hypothesis that the regulation of the CCR in SSF could
be independent of CreA. Regarding the redox metabolism, the
number of regulated proteins produced by SSF is higher than the
produced by SmF (Tables 3 and 4); this may result from the in-
duction of antioxidant systems that maintain redox balance in
response to the increased availability of oxygen, as well as the in-
crease in branching level as a function of the initial glucose con-
centration in SSF (Barrios-Gonz
alez, 2018; Gomes et al., 2018). In
SSF cultures of Aspergillus terreus, a higher redox balance was found
compared to that in SmF; in the latter cultures, the ROS concen-
tration was about 10-fold higher during the exponential phase of
growth (Miranda et al., 2013). The higher number of antioxidant
enzymes is related to a more efficient antioxidant network during
early stages of fungal growth in SSF, and their upregulation could
also be linked to increased production of ROS as the concentration
of glucose increases.
On the other hand, unlike the SmF extracts, about 30% of the
proteins in SSF extracts lack evidence of a secretory mechanism
(Tables 2e4); a high proportion, even considering false negatives
derived from the fact that algorithms and databases used to predict
 D. Salgado-Bautista et al. / Fungal Biology 124 (2020) 723e734
protein secretion in microorganisms, have been constructed with
results obtained under SmF conditions. The presence of intracel-
lular proteins without secretory signals in fungal secretomes has
been previously reported (Lu et al., 2010; Oda et al., 2006; Volke-
Sepulveda et al., 2016), and evidence of secretion of these pro-
teins by non-conventional mechanisms is increasing for microor-
ganisms, plants and, animals (Miura and Ueda, 2018; Rodrigues
et al., 2014; Zamith-Miranda et al., 2018). Considering that the
sampling was performed as gently as possible, the high proportion
of such proteins in the SSF extracts could be due to two processes
that can act together: (i) they could be released on the cell mem-
brane surface of the hyphal tips, from where they can be carried
along with the flow of material from the cell wall (Gomes et al.,
2018), as the process of apical extension requires the constant
transport of proteins and cell wall components (Pakula et al., 2005);
(ii) they are secreted by non-conventional mechanisms, such as
extracellular vesicles (EVs), which are key components in the
secretion of fungal molecules. Fungal EVs contain a mixture of
proteins, with and without secretion signal, many of which are
involved in cell wall biosynthesis/remodeling, stress responses,
pathogenesis, transport, and signaling (Bleackley et al., 2019;
Rabouille, 2017). Proteins involved in both processes were identi-
fied as upregulated in the secretomes obtained by SSF: glucosi-
dases, glucanosyltransferases, glucanases, chitinases, and
peptidases (Table 3), supporting a close relationship with greater
branching and hyphal growth as the glucose concentration in-
creases (Fig. 1). The upregulation of three Ras-related proteins in
SSF (Rab7/dGTPase, Rab-11A, and GTP-binding Ypt1) could also be
related to increased protein secretion by non-conventional mech-
anisms, such as transport through EVs. These proteins play key
roles in membrane vesicular trafficking and, consequently, in pro-
tein secretion pathways in filamentous fungi: Ypt1 is involved in
the regulation of the exocytic pathway in yeasts, while Rab7 and
Rab-11A participate in endocytic transport processes in A. nidulans
(Segev, 2001; Zheng et al., 2016). The deletion of a Rab-GTPase
resulted in a decreased enzyme secretion in Magnaporthe oryzae
(Zheng et al., 2016).
The active biosynthesis and remodeling of the cell wall, as well
as the protein secretion by non-conventional mechanisms explain
the presence of typically intracellular proteins in the secretome, but
not their functions in the extracellular space. Proteomic analysis of
fungal EVs has shown a large number of proteins associated with
metabolic pathways, many of which also perform moonlighting
functions outside the cell (Huberts and van der Klei, 2010;
Nimrichter et al., 2016). Several of the intracellular proteins lack-
ing evidence of secretion found in this study (e.g. cytosolic enzymes
and chaperones) have been reported as moonlighting proteins,
exhibiting a second function in other locations (Amblee and Jeffery,
2015; Gancedo et al., 2016). Proteins lacking a secretory signal that
have been consistently found extracellularly with moonlighting
functions in fungi include: (i) enzymes involved in carbohydrate
metabolism (such as TPI, transaldolase and malate dehydrogenase),
(ii) stress- and redox-related proteins (such as peroxisomal catalase
and SOD1); (iii) chaperones (HSPs); and (iv) transcription elonga-
tion factors (Amblee and Jeffery, 2015; Gancedo et al., 2016;
Nimrichter et al., 2016; Serrano-Fujarte et al., 2016). Although
some proteins display moonlighting function as chaperones, such
as 14-3-3 and peroxiredoxins (Breitenbach et al., 2015; Sluchanko
and Gusev, 2017), most of the intracellular/cell surface moon-
lighting proteins act on the cell surface for binding to solid
matrices, as adhesins to attach to host cells, or as cell surface re-
ceptors for soluble proteins, as has been demonstrated for TPI and
SOD (Amblee and Jeffery, 2015). Transaldolase e a cytoplasmic
enzyme lacking a secretory signal e is the most abundant upre-
gulated protein found in the SSF secretome of A. brasiliensis
733
(Table 3), and has been consistently found in both EVs of patho-
genic fungi (Candida spp. and Aspergillus fumigatus) (Nimrichter
et al., 2016); as well as in secretomes of industrially important
Aspergillus species, such as oryzae (Oda et al., 2006), niger (Carrillo-
Sancen et al., 2016), and brasiliensis (Volke-Sepulveda et al., 2016),
and even in the extracellular medium or on the surface of several
Bifidobacterium species, acting as an important colonization factor
favoring adhesion to host intestinal tract (Westermann et al., 2016).
The abundance of extracellular transaldolase produced by
A. brasiliensis, as well as its upregulation in function of the increase
in glucose, could be related to a moonlighting function involved in
fungal adhesion to perlite in SSF.
In filamentous fungi, growth adhered to surfaces is a common
feature necessary for nutrient uptake, enzyme secretion and apical
growth of hyphae (Gamarra et al., 2010). Fungal growth on and
within solid substrates is fundamentally related to cell adhesion,
which is a complex and metabolically active process e mediated by
secreted molecules (carbohydrates, proteins, and lipids) that binds
to substrate e scarcely studied in species of industrial use (Gow
et al., 2017; Villena and Gutie
rrez-Correa, 2007). In most natural
situations, the cell wall is surrounded or linked to an outer layer of
proteins (many of which are cytoplasmic and perform moonlight
functions) associated with the cell wall (Gow et al., 2017). For
instance, under SSF conditions, the hyphae of A. niger were found
surrounded by an adhesive extracellular material forming a matrix
after 12 h, which triggered a differential metabolic behavior
compared to SmF (Gamarra et al., 2010). To date, the precise
composition of this matrix, as well as the function of each
component are not clear, but its role in adhesion and cellular pro-
tection to environmental changes has been demonstrated. Many of
the proteins identified as typically intracellular can be part of this
adhesive matrix performing moonlighting functions. Moreover, in
SSF extracts, some of the functionally unknown hypothetical pro-
teins without homology with known proteins, could also be related
to fungal adhesion to the solid support. Understanding the role of
extracellular proteins in fungal adhesion is important, since it has
been suggested that such a process e which involves signaling
cascades, and the differential expression of both genes and meta-
bolic behavior e can exert an effect on increasing productivity of
enzymes produced on solid surfaces (Villena and Gutie
rrez-Correa,
2007), and could help to further understand the physiological dif-
ferences between both culture systems.
5. Conclusions
This study shows morphological, physiological and secretory
differences in A. brasiliensis depending on culture conditions, SSF
and SmF. Kinetic parameters associated with growth are improved
in SSF. The results highlight the absence of catabolite repression in
SSF, as well as the increase in the level of hyphal branching,
oxidative metabolism, and the concentration and diversity of
secreted proteins. The SSF also favors the secretion of typically
intracellular proteins, which could be released during the extension
and branching of hyphae. Some of them have been recognized for
their moonlighting functions and could be associated with adhe-
sion processes of hyphae to solid supports.
Acknowledgements
The Mexican Council for Science and Technology (CONACyT)
supported this work (Project ID 254975). DSB is grateful for the
scholarship (number 300412) received from CONACyT.
734 D. Salgado-Bautista et al. / Fungal Biology 124 (2020) 723e734
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.funbio.2020.04.006.
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