G C A T
T A C G
G C A T
genes
Article
Fungal Screening on Olive Oil for Extracellular
Triacylglycerol Lipases: Selection of a
Trichoderma harzianum Strain and Genome Wide
Search for the Genes
Miguel Angel Canseco-Pérez 1 , Genny Margarita Castillo-Avila 1,2 , Bartolomé Chi-Manzanero 1
ID
, Ignacio Islas-Flores 3 , Max M. Apolinar-Hernández 1 , Gerardo Rivera-Muñoz 4 ,
Marcela Gamboa-Angulo 1 , Felipe Sanchez-Teyer 1 ID , Yeny Couoh-Uicab 5 and
Blondy Canto-Canché 1, * ID
1
2
3
4
5
*
Unidad de Biotecnología, Centro de Investigación Científica de Yucatán, A.C., Calle 43 No. 130 X 32 y 34,
Col. Chuburná de Hidalgo, C.P. 97205 Merida, Mexico; miguel.canseco@cicy.mx (M.A.C.-P.);
gmca1983@gmail.com (G.M.C.-A.); bchim@cicy.mx (B.C.-M.); mizramax@gmail.com (M.M.A.-H.);
mmarcela@cicy.mx (M.G.-A.); dirgen@cicy.mx (F.S.-T.)
Centro de Estudios Tecnológicos del Mar No. 17, km 1.5 Carr. Antigua Chelem,
C.P. 97320 Yucalpetén, Mexico
Unidad de Bioquímica y Biología Molecular de Plantas, Centro de Investigación Científica de Yucatán, A.C.,
Calle 43 No. 130 X 32 y 34, Col. Chuburná de Hidalgo, C.P. 97205 Merida, Mexico; islasign@cicy.mx
Departamento de Ingeniería Química y Bioquímica, Tecnológico Nacional de México, Campus Instituto
Tecnológico de Mérida, Km. 5 Carr. Mérida-Progreso S/N, C.P. 97118 Merida, Mexico; grivera@itmerida.mx
División de Ingeniería Bioquímica, Instituto Tecnológico Superior de Purísima del Rincón. Blvd del Valle
#2301, Guardarrayas, C.P. 36413 Purísima del Rincón, Guanajuato, Mexico; yeny.couoh@tecpurisima.edu.mx
Correspondence: cantocanche@cicy.mx; Tel.: +52-999-942-8330
Received: 7 November 2017; Accepted: 20 December 2017; Published: 25 January 2018
Abstract: A lipolytic screening with fungal strains isolated from lignocellulosic waste collected in
banana plantation dumps was carried out. A Trichoderma harzianum strain (B13-1) showed good
extracellular lipolytic activity (205 UmL−1 ). Subsequently, functional screening of the lipolytic activity
on Rhodamine B enriched with olive oil as the only carbon source was performed. The successful
growth of the strain allows us to suggest that a true lipase is responsible for the lipolytic activity in
the B13-1 strain. In order to identify the gene(s) encoding the protein responsible for the lipolytic
activity, in silico identification and characterization of triacylglycerol lipases from T. harzianum is
reported for the first time. A survey in the genome of this fungus retrieved 50 lipases; however,
bioinformatic analyses and putative functional descriptions in different databases allowed us to
choose seven lipases as candidates. Suitability of the bioinformatic screening to select the candidates
was confirmed by reverse transcription polymerase chain reaction (RT-PCR). The gene codifying
526309 was expressed when the fungus grew in a medium with olive oil as carbon source. This protein
shares homology with commercial lipases, making it a candidate for further applications. The success
in identifying a lipase gene inducible with olive oil and the suitability of the functional screening and
bioinformatic survey carried out herein, support the premise that the strategy can be used in other
microorganisms with sequenced genomes to search for true lipases, or other enzymes belonging to
large protein families.
Keywords: enzymes; lipases; triacylglycerol lipases; true lipases; olive oil induced lipases; protein
bioinformatic analyses
Genes 2018, 9, 62; doi:10.3390/genes9020062
www.mdpi.com/journal/genes
Genes 2018, 9, 62
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1. Introduction
Lipases are serine hydrolases defined as triacylglycerol acyl hydrolases (EC 3.1.1.3), which
hydrolyze glycerol esters of long chain fatty acids (water insoluble). Many enzymes have activity on
water soluble medium-chain triacylglycerols (<12 carbon atoms), long chain monoacyl glycerols or
long-chain nitrophenyl acyl esters but are not able to act against triacylglycerols (TAGs) with long-chain
fatty acids, such as vegetable oils. Those lipases are not considered true lipases but they are esterases
(EC 3.1.1.1), which hydrolyze esters of short chain fatty acids (water soluble). For lipase catalysis there
is a phenomenon called interfacial activation, which does not occur in esterases [1]. Substrate emulsion
generates a lipid interface that activates the lipase: a loop which covers the active site (lid) changes
conformation and the enzyme shifts to the open conformation, where the active site is accessible to the
substrates [2]. In addition to hydrolyses, lipases are involved in conversion reactions in non-aqueous
media: esterification, interesterification, transesterification, alcoholysis, acidolysis and aminolysis [1,3].
Lipases have been known since 1930 and have captured the interest of scientists and industrial
businessmen to the present day. These enzymes are used in the detergent industry, in the organic
synthesis of pharmaceuticals, pesticides and insecticides, the production of emollient for personal
care in cosmetics and more recently in transesterification reactions for biodiesel production and in the
emerging oil and fat industry, for example production of cocoa butter equivalent (chocolate) and human
milk fat substitute [1,4,5]. Recently, Daiha et al. [4] reviewed contemporary research publications and
patent applications dealing with lipases and concluded that these enzymes are of much relevance in
modern industry; however, further research is still needed to reach their full potential.
Lipases are ubiquitous in living beings. In particular, microbial lipases are of great importance
and have diverse commercial applications [6]. Bacteria, yeasts, filamentous fungi and a few protozoa
produce extracellular lipases for the digestion of lipid materials [1]. Screening for extracellular
lipases is currently performed on isolated microorganisms, by microbial genome-wide mining, or by
metagenomic mining. Lipolytic microorganisms have been isolated or identified in collections from
soils, plant matter, plant compost, oleaginous fruits, domestic vegetable oil wastes, pig manure, organic
dumps, garbage collectors and industrial effluents, among many other sources. Microbes are highly
successful in adapting and surviving in a wide range of environments, by exploiting their trophic
niche; thus, the ability to secrete enzymes is of great survival value.
There are many screening methods for lipolytic activity [7]. The most common is solid media
added with different lipid substrates and lipase activity can be detected as clear or turbid zones around
the colonies or by the production of crystals on the agar surface. Combination with Rhodamine B has
proved to be a fast method; Rhodamine B binds fatty acids, mono- and diglycerides and develops
fluorescence under ultra violet (UV) light [1,7]. Tributyrin-agar plate is a widely used method, but this
substrate cannot distinguish between true lipases and esterases. Olive oil-agar plates are a good test
for screening true lipase-positive colonies [1,8]. Lipase production is often induced by the presence of
vegetable oils and the best results have been obtained with olive oil [8–11].
In the present work, fungi isolated from banana plantation waste were screened for production of
extracellular lipases on olive oil-Rhodamine B plates. Trichoderma harzianum and Trichoderma longibrachiatum
were identified as the most prominent producers, the former being the best strain in our collection.
We hypothesize that the lipase responsible for the extracellular activity on olive oil is a true lipase.
Ülker et al. [12] performed the first characterization of the extracellular lipolytic activity of T. harzianum.
They found that the activity is optimum at 40 ◦ C and that it is thermostable, showing 55% of activity
when it was incubated at 50 ◦ C and 20% with incubation at 70 ◦ C. Coradi et al. [9] precipitated the
protein and found that the lipolytic enzyme worked on long-chain TAGs as triolein and olive oil, i.e.,
it is a true lipase.
To date there is no description regarding genes for lipases in T. harzianum. The goal of this work is
to identify the gene or genes that encode the protein responsible for the extracellular lipolytic activity
of this fungus on olive oil.
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2. Materials and Methods
2.1. Isolation of Lipolytic Microorganisms
Fungi were isolated from lignocellulolytic residues collected on (a) a garbage dump and (b) plant
debris from a banana plantation in Tabasco, Mexico. Decaying vegetal materials were sampled and
transported to the laboratory in paper bags and plastic boxes covered inside with sterile wet paper
(humid chamber). Fungi were isolated under a stereoscope (Motic, Richmond, BC, Canada) using
an inoculating teasing needle and plate (Sigma Aldrich, Saint Louis MO, USA) on Malt extract agar
medium (Sigma Aldrich) with 250 µg L−1 amikacin (Sigma Aldrich) subcultures [13]. Only fungal
cultures showing homogenous growth (i.e., pure strains) were considered for further analysis.
2.2. Screening of Lipolytic Fungi
Screening was performed on selective Potate Dextrose Agar (PDA) (DIBICO, Cuautitlan Izcalli,
Mexico) supplemented with 1% (v/v) olive oil (Selecto Choice, Spain) and 0.001% (w/v) of Rhodamine B
(Sigma Aldrich) [14]. The medium was emulsified by mixing with a polytron homogenizer Ultra-turrax
T25 (IKEA, Staufen, Germany) and poured into Petri dishes. A disk (1 cm diameter) of each fungus
grown on PDA medium (72 h) was inoculated on the center of the Petri dish containing the solid
medium. Each fungus was independently inoculated in triplicate and grown at 28 ◦ C. The presence
of free fatty acids was detected by UV Light [1,7]. The level of lipase production was evaluated by
monitoring on UV transilluminator (360 nm) (UVP Analytic Jena Company, Upland, CA, USA) every
12 h over a period of 48 h.
Four categories were defined, based on the fluorescence intensity and the area of the fluorescent
colony. Category 0 was assigned with “−” for all strains without fluorescent halo, category 1 (+)
includes strains with poor fluorescence and growth, category 2 (++) includes good growth and
moderate fluorescence and category 3 (+++) includes strains with high fluorescence and growth [8,15].
2.3. Lipase Production by Submerged Fermentation
Fungal strains which showed stronger fluorescence on Rhodamine B medium were grown in
liquid medium as described in Maia et al. [16] with 1% (v/v) olive oil as sole carbon source. Fungal
strains were grown on PDA for 72 h and two cylinders of 0.5 cm diameter each were macerated with
1 mL sterile water and inoculated in 125 mL of culture medium. Fungal cultures were incubated with
constant shaking at 180 rpm at 30 ◦ C in incubator shaker 3532 (LAB-LINE Instruments, Melrose Park,
IL, USA) Samples were harvested every 24 h over a period of 8 days.
Cell-free extracellular proteins were obtained by centrifuging the sample at 15,000 rpm for 15 min
at 4 ◦ C in centrifuge 5810R (Eppendorf, Hamburg, Germany). Supernatants were recovered to measure
lipase activity.
Lipolytic activity was determined spectrophotometrically on p-nitrophenyl palmitate (pNPP;
Sigma-Aldrich) as substrate. Buffer added with pNPP (1.2 mL) was prewarmed to 37 ◦ C. Reaction was
started by adding 50 µL of enzyme extract and incubation for 15 min at 37 ◦ C. The free p-nitrophenol
(pNP) was monitored at 410 nm in Genesys 10s UV-VIS (Thermo Spectronic, Madison, WI, USA) [17].
A blank sample was always used containing medium described in Maia et al. [16] instead of enzyme
solution. The molar extinction coefficient of ε = 15,000 M−1 cm−1 for pNP at 410 nm [17,18] was used
for calculus. One enzymatic unit was defined as the amount of the enzyme that releases one mmol of
pNP in one minute under the assay conditions.
2.4. Molecular Identification of Selected Lipolytic Fungi Isolated Here
To obtain DNA, 500 mg of mycelia were extracted following the procedure of Johanson et al. [19].
Internal transcribed spacer (ITS) regions were amplified using as template 10 ng of genomic DNA and
the ITS1 and ITS3 primers [20]. Amplicons were gel-purified and sent to LANBAMA (IPICyT, San Luis
Potosi, Mexico) for both strands sequencing. Bioedit software was used for sequence edition [21],
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removing the sequence ends to eliminate noise (approx. 20 bp in each end). Antisense sequences were
submitted to UGENE software to convert them into their respective reverse complement sequences [22].
To confirm ITS sequence, for each fungus the sense and the reverse complement were aligned with
MUSCLE [23] and manually checked. For each case the consensus sequence was used as query to
search by BLASTn [24] in the fungal non-redundant NCBI database [25] and TrichOkey2 [26] specific
database for identification of Hypocrea and Trichoderma species. In each case, the first hit in the list was
retrieved and data were manually analyzed.
2.5. Searching of Genes of True Lipases in Trichoderma harzianum Genome
The deduced proteome of T. harzianum in the JGI genome portal [27] was searched for lipases,
followed by refining the quest for true lipases with the keywords triglyceride lipase, triacylglycerol
lipase, and lipase class 3.
To support the quest of true lipases, BLASTp in the T. harzianum deduced filter models of
proteins was performed by using as queries the amino acid sequences of reported lipases with
activity on long-chain triacylglycerydes (vegetable oils): Fusarium graminearun FGL1 AAQ23181 [28],
Fusarium heterosporum AAB34680 [29]; and the long-chain triacylglyceryde lipases enlisted in the
US 9476008 B2 patent [30]: Rhizomucor miehei (P19515), Thermomyces lanigunosus (CAB58509),
Candida antarctica LipA (2VEO) and LipB (P41365) and Rhizopus oryzae (AER14043).
For each protein hit retrieved, the model name, the scaffold location of their genes in the
T. harzianum portal and their automatic annotations at portal were recorded.
2.6. In Silico Analysis of Trichoderma harzianum Putative True Lipases
Lipases found in T. harzianum with the keywords triglyceride lipase, and those annotated as
secreted lipases were selected for further bioinformatic analyses. Amino acid sequence in Fasta format
was downloaded for each of the retrieved hits. Lipase candidates were examined at different platforms
to search domains/motif information: the Common Domains database in the non-redundant fungal
database at NCBI [31], the Pfam databases using the sequence search tool [32], the HHpred (Homology
detection and structure prediction by HMM-HMM) [33] and the InterPro database [34].
For identification of extracellular lipases, amino acid sequences were analyzed with SignalP
4.1 server [35] to predict N-terminal secretion signal. Prediction of transmembrane helices was
performed with TMHMM 2.0 server [36]. The putative location in the cell was predicted with WoLF
PSORT [37]. Molecular size was in silico calculated with Protein Molecular Weight Calculator—Science
Gateway [38] and ProtParam tool [39].
BLASTp 2.6.0 was performed for rapid assignment [31,40]. Gene Ontology categories, i.e.,
Biological Process, Molecular Function and Cellular Component were retrieved from the different
bioinformatics tools used to search homology, conserved domains, motifs and signatures (Pfam,
InterPro, Superfamily, Protein Database, etc.). Each selected candidate was also examined in the
genomic portal (clicking on the ID number) to search automatic annotations.
The complete amino acid sequence of each candidate was submitted by BLASTp to the LED
(Lipase Engineering Database) [41] and ESTHER Database (ESTerases and alpha/beta-Hydrolase
Enzymes and Relatives) [42] to inference class, parent family and probable function through the use of
annotation transfer.
2.7. Phylogenetic Analysis
A list of representative fungal lipases was taken from Yadav et al. [6] and the amino acid sequence
from each was downloaded from the GenBank. The putative true lipases retrieved in this study from the
deduced proteome of T. harzianum and their characterized homologues or best hits in PSI-BLAST [43]:
Tolypocladium ophioglossoides (KND87948), Metarhizium anisopliae (KFG82987), Nectria haematococca
(CAC19602), Purpureocillium lilacinum (XP_018176823), N. haematococca (XP_003053404), Claviceps
purpurea (CCE34466), Fusarium graminearum (XP_01138453), Hirsutella minnesotensis (KJZ78693),
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Fusarium oxysporum (EWZ47945), Saccharomyces cerevisiae (AAA50367), T. ophioglossoides (KND93486),
Talaromyces cellulolyticus (GAM42581), Colletotrichum fioriniae (XP_007591165), T. ophioglossoides
(KND89897), S. cerevisiae (AJS39941), P. lilacinum (OAQ83707) and Drechmeria coniospora (ODA76916).
The known true lipases (from literature) mentioned above were included, as well as Lip1 (P20261) and
Lip5 (P32949) from Candida rugosa [44], the proteins Lip1 (EAA67628, ACE80261 and XP_001800960)
from the pathogenic fungi Gibberella zea [45], Blumeria graminis [46] and Stagonospora nodorum [47],
Malassezia restricta MrLip1 (ALG03641) [48], Malassezia globosa (XP_001732206) [49], Acremonium
alcalophilum LipA (P0CT91) [50], the S. cerevisiae’s Tgl3p (AJS99689) and Tgl5p (DAA10860) which
are the major TAG lipases in this yeast [51], the Ophiostoma piceae sterol esterase (4UPD) [52] and the
esterase Bjerkandera adusta (APW29213), the latter with no activity on long-chain glycerides [53] which
was included as one reference for non-true lipases. The lipases CAA00250 from R. miehei and Lip1 and
Lip5 from C. rugosa are fungal phylogenetic references [44].
A multi-alignment was performed in MAFFT [54] with default parameters. A phylogenetic tree
was constructed using the neighbor-joining method [55] with 500 bootstrap.
2.8. In Silico Modeling
Three-dimensional modeling was performed with SWISS-MODEL [56], HHpred [57] and
I-TASSER [58], using the complete amino sequence of each candidate. The retrieved hit with the highest
score was selected in each case as the best model. The catalytic site and the lid domain were identified
at IPBA web server [59] by 3D superposition with the best template. Amino acid composition in the
conserved motifs in the template were identified and labeled with PyMOL Molecular Graphics System
program [60] and then identified in the candidate by structure-based and sequence-based comparisons.
The PSI-BLAST (Position-Specific Iterated) at the NCBI [43] was conducted to search homologues
in the protein structure database.
For TAG lipase which was de novo expressed on olive oil, a structure-based multi-alignment was
conducted by ClustalW [61]. The alignment was represented using ESPript [62].
2.9. RT-PCR Amplification of Lipases
To validate the selection of putative true lipases and to identify the gene/protein responsible
for the extracellular lipolytic activity of T. harzianum strain B13-1 on olive oil, reverse transcription
polymerase chain reaction (RT-PCR) was performed on RNA obtained from the fungus grown in olive
oil-free and olive oil-containing medium (1% v/v).
The nucleotide coding regions of the selected lipases were downloaded from the T. harzianum
database [27] and sequence primers were designed with the Primer Quest Tool software [63]. The list
and expected sizes of the fragments is shown in Table S1.
Mycelium was harvested at 1, 3, 5 and 7 days. RNA was extracted with TRIzo Reagent (Invitrogen,
Carlsbad, CA, USA), pooled and cDNA was synthesized with Maxima First Strand cDNA Synthesis Kit
(Thermo Scientific, Vilnius, Lithuania) following supplier’s recommendations. Each reaction mixture
(total volume of 15 µL) contained 1× PCR buffer, 2.0 mM MgCl2, 1.6 µM dNTPs, 400 µM of each
primer and 1.0 U of GoTaq DNA polymerase (Promega, Madison, Wi, USA); 1 µL of cDNA was used
as template per reaction. The PCR parameters were 3 min of 95 ◦ C; followed by 30 cycles of 94 ◦ C for
30 s, 53 ◦ C for 30 s and 72 ◦ C for 30 s; and a final elongation at 72 ◦ C for 5 min. The PCR products were
purified from gel using QIAquick Gel Extraction Kit (Qiagen, Germantown, MD, USA); for sequencing
the samples were sent to LANBAMA. Sequences were edited as above and then used for BLAST in the
T. harzianum genome and NCBI non-redundant protein database.
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3. Results and Discussion
3.1. Screening of Lipolytic Fungi Using Olive Oil
A total of 18 fungal strains isolated from banana lignocellulosic wastes were tested on lipase
secretion assay (Table 1).
Table 1. Fungi isolated in this study from banana plant residue and used for screening of
lipolytic activity.
Strain
Morphology
Extracellular Lipase at 48 h *
A04-5
A06-6
B07(+)-1(3)N
B08-6
B09-4
B09-5
B09-8
B10-4(b1)Emmb
B10(+)-2(4)
B10-4(1)-2(1)
B11-6
B11-7
B13-1
B13-3
B13-4
B14-6
B17(+)-4(3)
B19-01-3(3)
Black, concentric rings
White, radial growth
White, radial growth
White, radial growth and Green spores
White, cottony mycelia
White, cottony mycelia
White, radial growth
Black, radial growth
White, radial growth
Black, radial growth
Fuchsia, cottony mycelia
Fuchsia, cottony mycelia
White with green concentric rings
White with green concentric rings
Yellow, compact colony
White, radial growth
Fuchsia, cottony mycelia
White, radial growth
+
+
+
++
++
++
++
+++
+++
++
++
++
+
* Visual observation according (Carissimi et al. [15]; Ortiz-Lechuga et al. [8]). Hours (h).
All fungal strains were able to grow on solid medium supplemented with olive oil. Thirteen of
them were positive on Rhodamine B plates (Table 1). The most active (category 3) were B13-1, B13-3
strains which were considered for further studies (Figure S1). The fluorescence was associated with
the mycelia and no clear halos were observed. Many reports on fungal lipolytic strains found no
halos around colonies but the mycelia were fluorescent [8,64–67]. The phenotype found here therefore,
is common in filamentous lipolytic fungi tested on olive oil-Rhodamine B medium. Observation of
hydrolysis halos is more common in bacteria [68,69].
ITS-sequence-based BLASTn in the non-redundant fungal database at NCBI, shows that fungal
strain B13-1 has 94% similarity with T. harzianum (98% coverage, Evalue 0.0) while B13-3 strain has
98% similarity with T. longibrachiatum (98% coverage, Evalue 0.0). Trichoderma species have been
previously identified in screening of lipolytic fungi from effluents collected in dumpsites with palm oil
mill residue [3], slaughterhouses and dairy industries [68], as well as soils contaminated with waste
vegetable oils [8]. In fact, Nwuche and Ogbonna [3] reported that the highest lipase producing strains
in their screening belong to the Trichoderma genus.
3.2. Lipolytic Activity Assay
In T. harzianum the extracellular lipase activity increased gradually reaching a maximum on the
8th day (205 UmL−1 ). T. longibrachiatum showed a similar pattern of extracellular lipolytic activity
with gradual increment but maximum activity at the 8th day (109 UmL−1 ) was half that produced by
T. harzianum B13-1 strain (Figure 1). Lipase activities in both fungi are in the range reported for good
lipase producing strains, such as Penicillium chrysogenum (205 UmL−1 ) [70] and R. oryzae from oil palm
fruit (120 UmL−1 ) [71]. To date, only one report with a stronger production of lipolytic activity from
Rhizopus sp. has been found. This fungus was isolated from contaminated oil and reported 870 UmL−1
of lipolytic activity [72].
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Figure 1. Extracellular lipolytic activity measured with p-nitrophenol palmitate. Black bars show the
activity in the strain B13-1; gray bars show the activity in the strain B13-3. The data show the standard
deviation of three independent samples.
Since the activity was higher in T. harzianum B13-1 than in T. longibrachiatum B13-3, we decided to
continue with the first strain. Based on the methodology followed in the present work, i.e., screening
of lipolytic fungi on olive oil-containing medium and the subsequent production of lipolytic activity
by using olive oil as carbon source, together with available information on T. harzianum lipase, it is
reasonable to propose that the lipolytic activity found in T. harzianum B13-1 corresponds to a true lipase.
3.3. Searching Genes of True Lipases in Trichoderma harzianum
A survey of the genome was conducted to explore the possibility of identifying the respective
gene among the T. harzianum TAG lipases. To date, there have been no reports on the lipase genes in
this microorganism. The quest in the T. harzianum deduced proteome at genomic portal [27] using
“lipase” as keyword resulted in 50 hits (Table S2), showing that the lipase family is large in this
fungus. The family comprises alpha-beta hydrolases, phospholipases, carbohydrate esterases, isoamyl
acetate-hydrolyzing esterase, among others. Four lipases (551811, 87496, 526309 and 514427) are
annotated as secreted in the T. harzianum genome portal and are therefore plausible candidates
to explain the extracellular lipolytic activity measured here in strain B13-1. To expand the list
of candidates, the search in the genome for true lipases was performed by using the keywords
“triglyceride lipase” and it retrieved 9 hits (IDs 510832, 79895, 92423, 135964, 492160, 77338, 78181,
502433 and 514252). The last four belong to lipase class 3, which is a lipase family of true lipases [25,28].
Lipases with activity on long-chain TAG were used as queries to search for more true lipases in
T. harzianum: F. graminearun FGL1 AAQ23181, F. heterosporum AAB34680, R. miehei P19515 and R. oryzae
AER14043 retrieved all of the T. harzianum proteins 78181 and 77338; T. lanigunosus CAB58509 retrieved
these two lipases and the protein 502433. These three lipases are in the list found previously.
Therefore, the list comprises 13 candidates in the search for the putative true lipase responsible
for the extracellular lipolytic activity on olive oil in T. harzianum.
3.4. In Silico Characterization of Trichoderma harzianum True Lipases
BLASTp 2.6.0 was performed for rapid assignment of lipase candidates but for most of them the
closest homologue was uncharacterized predicted lipase, or hypothetical protein, arising from genomic
sequencing projects. Therefore, this analysis was not of much help in supporting or eliminating
candidates. Genomic data is so abundant in the GenBank that usually 50–100 hits corresponded to
uncharacterized predicted lipase, although some of the lipase candidates under analysis were retrieved
Genes 2018, 9, 62
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by BLASTp from T. harzianum portal using a characterized TAG lipase as query. However, in the huge
sequencing data, those queries ranked so far in comparison with many genomic sequences.
PSI-BLAST increases the opportunities to predict function, especially when Trichoderma species
are excluded, because the searching result rules out the large list of predicted, hypothetical proteins
that come from genomic sequencing data from this genus. Table 2 shows a summary of sequence-based
function transfer based on PSI-BLAST.
Although BLASTp hits were not suitable to predict function by homology, the conserved domain
searching tool hosted in BLAST server enabled us to find motifs and conserved domains in the protein
candidates (Table 2). The analysis in the conserved domain database (CDD) confirms the lipase
3 domain in 77338, 78181, 514252 and 502433, consistent with the automatic annotation for these
proteins in the genomic portal of T. harzianum. The Lipase 3 domain contains the flip/lid domain
(TYITNTIIDLS in 77338, SNLRNFITDVV in 78181, TSTNDKVNDNL in 514252 and TLFEDVLADLT
in 502433), the catalytic triad (Ser-His-Asp) and the nucleophilic elbow (GXSXG consensus motif)
which is GHSLG in these four lipases. In addition to these domains, 502433 has one AF-4 domain.
This domain is present in AF4 and FMR2 nuclear proteins. In Drosophila AF4 protein homologue acts
in cytoskeleton regulation, segmentation and morphogenesis.
Since the survey performed in this work is for identifying the protein responsible for extracellular
activity in B13-1 strain on olive oil, molecular size and cellular localization were analyzed. One of the
putative lipase class 3 gave a negative result for signal peptide in SignalP program (502433) and its
size is larger (115 kDa) than expected for extracellular lipases, which ranks from molecular weights
less than 20 to 65 kDa [73,74]. The other three class 3 lipases were predicted with signal peptide and as
being extracellular, making them good candidates (Table 3).
The analysis in the CDD identified the alpha/beta hydrolase fold on the putative TAG lipases
510832, 92423 and 135964. Top hits at BLASTp retrieved sterol esterases for 510832 and GPI
inositol-deacylase PGAP1-like protein for 135964, the last one probably involved in vesicular traffic
from endoplasmatic reticulum.
92423 shares homology with lysosomal acid and other acid lipases. Consistent with intracellular
roles, any of these three lipases are predicted extracellular (Table 3). Regarding the other two putative
TAG lipases, 79895 and 492160, neither lipase nor hydrolase domains were identified but WD40 and Sec
domains in the former and Diaphanous FH2/FH3 domains in the later (Table 2), suggesting roles with
other functions than lipases. Deduced sizes are 134 and 198 kDa respectively; it is not prognosticated
secretion for these proteins and in agreement, signal peptide was not identified.
The four automatically annotated secreted lipases (514427, 526309, 551811 and 87496),
show Abhydrolase fcl21494 domain, containing the catalytic triad Ser-His-Asp and contain the Pfam
domain 03583 which is related with a lipase from C. albicans. Curiously, one of those proteins, 551811,
gave negative prediction of signal peptide with SignalP and WoLF PSORT predicts cytoplasmic
localization. A Rossmann-fold NAD(P)H/NAD(P)(+) binding (NADB) domain was identified in
551811, a domain found in numerous redox enzymes. 514427 shares homology with the acetyl xylan
esterase (AXE1) domain, related with acetyl xylan esterase. Signal P analysis supports prediction of
secretion for protein: 514427, 526309 and 87496 and in congruence WoLF PSORT deduced that they are
extracellular proteins (Table 3).
Rapid analyses and deduction of intracellular location and large size can exclude some of these
candidates, but we decided to perform further bioinformatics analyses on all these proteins in order to
achieve the most effective selection of final candidates. In accordance with the search criteria, the order
for extracellular TAG lipase in T. harzianum is 77338, 78181, 514427, 526309, 87496, 514252, 510832,
92423, 135964, 502433, 551811, 79895 and 492160.
Genes 2018, 9, 62
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Table 2. List of domains and family description of putative true lipases of Trichoderma harzianum *.
Protein ID
Superfamily
Domains
Domain Description
Active Site Domain
Substrate
Binding Pocket
BLAST Hits (Search Homologues
in the First 50 Hits)
PSI-BLAST
(Homologues
Characterized)
551811
Abhydrolase
family cl21494
LIP pfam03583
Mal_quin-oxido
TIGR01320
Secretory lipase: related with
lipases from Candida albicans.
Malate:quinone-oxidoreductase:
Membrane-associated enzyme as
part of the TCA cycle
Active site
Ser-His-Asp/Glu.
Nucleophilic attack on a
carbonyl carbon atom.
Substrate binding
pocket related
to pfam03583
Genome Sequence and Annotation
of different fungi (e.g., Trichoderma
sp., Metharhizium sp., Fusarium sp.)
as hypothetical protein and
Predicted lipases
Lipase 1 C. albicans
(KGR02689) Query
cover 94% Identity 33%
87496
Abhydrolase
family cl21494
LIP pfam03583
Secretory lipase: Related with
lipases from C. albicans
Active site
Ser-His-Asp/Glu.
Nucleophilic attack on a
carbonyl carbon atom.
Substrate binding
pocket related
to pfam03583
Genome Sequence and Annotation
of different fungi (e.g., Trichoderma
sp., Metharhizium sp., Fusarium sp.)
as hypothetical protein and
Predicted lipases
Lipase 1 C. albicans
(KGR02689) Query
cover 93% Identity 32%
Secretory lipase 5 C.
albicans (ADP21191.1)
Query cover 92%
Identity 39%
526309
514427
510832
92423
Abhydrolase
family cl21494
Abhydrolase
family cl21494
Alpha/Beta
hydrolase fold
cl26327
Alpha/Beta
hydrolase fold
cl26327
LIP pfam03583
Secretory lipase: Related with
lipases from C. albicans
Active site
Ser-His-Asp/Glu.
Nucleophilic attack on a
carbonyl carbon atom.
Substrate binding
pocket related
to pfam03583
GH16 protein [Trichoderma
guizhouense]. Genome Sequence
and Annotation of different fungi
(e.g., Trichoderma sp., Metharhizium
sp., Fusarium sp., Pochonia sp.,
Cordyceps sp., etc.) as hypothetical
protein. Secretory lipase. Probable
lipase precursor
LIP pfam03583 DAP2
Secretory lipase; Related with
lipases from
C. albicans Dipeptidyl
aminopeptidase/acylaminoacyl
peptidase:Amino acid transport
and metabolism Acetyl xylan
esterase (AXE1).
Active site
Ser-His-Asp/Glu.
Nucleophilic attack on a
carbonyl carbon atom.
Substrate binding
pocket related
to pfam03583
Genome Sequence and Annotation
of different fungi (e.g., Trichoderma
sp., Metharhizium sp., Fusarium sp.)
as hypothetical protein and prolyl
aminopeptidase (secreted protein).
No characterized
protein among the first
500 hits
-
Genome Sequence and Annotation
of different fungi (e.g., Trichoderma
sp., Metharhizium sp., Fusarium sp.,
Colletotrichum sp., etc.) as
hypothetical protein, triglyceride
lipase-cholesterol esterase,
alpha/beta-hydrolase, Sterol
esterase 2.
Yeh2p Saccharomyces
cerevisiae YJM1419
(AJV63174). Query
cover 58% Identity 35%
-
Genome Sequence and Annotation
of different fungi (e.g., Trichoderma
sp., Metharhizium sp., Fusarium sp.,
Colletotrichum sp., etc.) as steryl
ester lipase TPL1, lysosomal acid
lipase/cholesteryl ester hydrolase,
triglyceride lipase-cholesterol
esterase, hypothetical protein,
alpha/beta hydrolase fold-1
Tgl1p S. cerevisiae
YJM1083 (AJS39941)
Query cover 80%
Identity 43%
PLN02872
Abhydro_lipase
(pfam04083) Mhpc
(COG0596)
PLN02872
Abhydro_lipase
(pfam04083) MhpC
(Cpg0596)
Triacylglycerol lipase Partial
alpha/beta hydrolase lipase
region: Pimeloyl-ACP methyl
ester carboxylesterase
Triacylglycerol lipase Partial
alpha/beta-hydrolase lipase
region Pimeloyl-ACP methyl
ester carboxylesterase
-
-
Genes 2018, 9, 62
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Table 2. Cont.
Protein ID
79895
Superfamily
Domains
Domain Description
WD40 Cl25539
WD40 (cd00200) WD40
(COG2319) WD40
(smart00320) Wd40
(pfam00400) PHA03247
Atrophin-1 (pfam03154)
PLN00171 Amelogenin
(smart00818) PABP-1234
(TIGR01628)
WD40 domain, found in many
eukaryotic proteins with a wide
variety of functions. Ancestral
coatomer element 1 (ACE1) of
COPII with role in vesicular
traffic. Atrophin-1 family domain
Protein SPA1-related Cell
adhesion proteins Polyadenylate
binding protein:
135964
Abhydrolase
family cl21494.
492160
DRF_GBD
Superfamily
Cl05720
DRF_FH3
Superfamily
Cl05717 FH2
Superfamily
cl19758
77338
Abhydrolase
family cl21494.
EstA (COG1075) PGAP1
(pfam07819)
Triacylglycerol esterase/lipase
PGAP1-like protein
Drf_GBD (pfam06371)
Drf_FH3 (pfam06367)
FH2 (pfam02181) FH2
(smart00498) PHA03307
Diaphanous GTPase-binding
domain; Rho proteins, leading to
activation of the Drf protein.
Formin Homology 2 Domain
Involved in rearrangements of
the actin cytoskeleton.
Transcriptional
regulator ICP4-like
Lipase_3 (cd00519)
Lipase_3 (pfam001764)
PLN02310 Lip2
(COG3675)
Lipases (Class 3). Lipase that can
hydrolyze long-chain
acyl-triglycerides into di- and
monoglycerides, glycerol and free
fatty acids.
Active Site Domain
-
Active site
Ser-His-Asp/Glu.
Nucleophilic attack on a
carbonyl carbon atom.
-
Active site
Ser-His-Asp/Glu.
Nucleophilic elbow on
conserved domain
Lipase_3: GHSLG
Substrate
Binding Pocket
BLAST Hits (Search Homologues
in the First 50 Hits)
PSI-BLAST
(Homologues
Characterized)
-
Genome Sequence and Annotation
of different fungi (e.g., Trichoderma
sp., Metharhizium sp., Fusarium sp.,
Colletotrichum sp., etc.) as
hypothetical protein, Vesicle coat
complex COPII, Sec31, transport
protein sec31, transporter.
Web1p S. cevevisiae
(AAA50367) Query
cover 99% Identity 31%
-
Genome Sequence and Annotation
of different fungi (e.g., Trichoderma
sp., Hirsutella sp., Stachybotrys sp.,
Fusarium sp., Ophiocordyceps sp.,
Colletotrichum sp., etc.) as
hypothetical protein,
Triacylglycerol lipase, Lipase 2,
related to TGL2-triacylglycerol
lipase, GPI inositol-deacylase
PGAP1-like protein, PGAP1-like
protein lipase.
No characterized
protein among the first
500 hits
-
Genome Sequence and Annotation
of different fungi (e.g., Trichoderma
sp., Tolypocladium sp., Pochonia sp.,
Metharhizium sp., Fusarium sp.,
Colletotrichum sp., etc.) as
cytokinesis protein sepA,
Rho-GTPase effector BNI1 and
related formin, involved in mating,
karyogamy and meiosis,
hypothetical protein, SepA/Bni1
PSI-BLAST analysis did
not run
Active site flap/lid
on conserved
domain Lipase_3
(11 amino acids).
Genome Sequence and Annotation
of different fungi (e.g., Trichoderma
sp., Tolypocladium sp., Pochonia sp.,
Metharhizium sp., Fusarium sp.,
Colletotrichum sp., etc.) as lipase,
hypothetical protein, triacylglycerol
lipase, Mono- and diacylglycerol
lipase, Putative feruloyl esterase A,
Alpha/Beta hydrolase protein.
Triacylglycerol lipase
FGL2 Fusarium
graminearum
(ABW74155) Query
cover 89% Identity 62%
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Table 2. Cont.
Protein ID
514252
Superfamily
Lipase (class 3).
Alpha/beta
hydrolase
cd00519
502433
Predicted
triacylglycerol
lipase activity,
Lipase_3.
78181
Predicted
triacylglycerol
lipase activity,
Lipase_3. Lipid
transport and
metabolism.
Domains
Domain Description
Lipase_3 (pfam01764)
Lipase_3 (cd00519) CVT17
(COG5153) PRK11071
Lipase that can hydrolyze
long-chain acyl-triglycerides into
di- and monoglycerides, glycerol
and free fatty acids. Putative
lipase essential for disintegration
of autophagic bodies inside the
vacuole Esterase YqiA
Lipase 3 (cd00519)
Lipase_3 (pfam01764)
PLN02847 AF-4
(pfam05110)
Lipase that can hydrolyze
long-chain acyl-triglycerides into
di- and monoglycerides, glycerol
and free fatty acids. AF-4
Proto-oncoprotein; Nuclear
proteins linked to human disease
Lipase_3 (cd00519)
Lipase_3 (pfam01764)
PLN00413 Lip2
Lipase that can hydrolyze
long-chain acyl-triglycerides into
di- and monoglycerides, glycerol
and free fatty acids.
Active Site Domain
Active site
Ser-His-Asp/Glu.
Nucleophilic elbow on
conserved domain
Lipase_3: GHSLG
Active site
Ser-His-Asp/Glu.
Nucleophilic elbow on
conserved domain
Lipase_3: GHSLG
Active site
Ser-His-Asp/Glu.
Nucleophilic elbow on
conserved domain
Lipase_3: GHSLG
Substrate
Binding Pocket
BLAST Hits (Search Homologues
in the First 50 Hits)
PSI-BLAST
(Homologues
Characterized)
Active site flap/lid
on conserved
domain Lipase_3
(11 amino acids).
Genome Sequence and Annotation
of different fungi (e.g., Trichoderma
sp., Tolypocladium sp., Pochonia sp.,
Metharhizium sp., Fusarium sp.,
Colletotrichum sp., etc.) as
autophagy related lipase Atg15,
triacylglycerol lipase, hypothetical
protein, alpha/beta-hydrolase,
related to starvation induced
protein PSI-7, autophagy lipase.
Hypothetical protein
FGSG_02519 F.
graminearum PH-1
(XP_011318453) Query
cover 92% Identity 70%
Active site flap/lid
on conserved
domain Lipase_3
(11 amino acids).
Genome Sequence and Annotation
of different fungi (e.g., Trichoderma
sp., Tolypocladium sp., Pochonia sp.,
Metharhizium sp., Ophiocordyceps
sp., Fusarium sp., Colletotrichum sp.,
etc.) as esterase/lipase,
hypothetical protein, Sn1-specific
diacylglycerol lipase beta, Lipase,
Lipase, class 3,
No characterized
protein among the first
500 hits
Active site flap/lid
on conserved
domain Lipase_3
(11 amino acids).
Genome Sequence and Annotation
of different fungi (e.g., Trichoderma
sp., Purpureocillium sp., Fusarium
sp., Tolypocladium sp., etc.) as
extracellular lipase-like protein,
hypothetical protein, Lipase,
probable triacylglycerol lipase
precursor, Chain A, Crystal
Structure of Lipase
Lipase Fusarium
heterosporum
(AAB34680) Query
cover 98% Identity 52%
* Annotation is summary of information retrieved from Gene Ontology, Conserved Domain tool at NCBI, Pfam and Superfamily.
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Table 3. In silico analysis of putative true lipases of Trichoderma harzianum.
Protein ID
aa Residues
Molecular
Weight
(kDa)
SignalP
(Secreted)
TMHMM
Domains
Putative Cellular
Localization
Continue
for Next
Analysis
551811
87496
526309
514427
510832
379
430
452
454
722
41.185
46.62
47.99
49.25
82.80
No
Yes
Yes
Yes
No
No
No
No
No
No
Cytoplasmic
Extracellular
Extracellular
Extracellular
Cytoplasmic
Yes
Yes
Yes
Yes
No
Yes
No
no
No
Yes
Yes
Yes
Yes
92423
552
62.361
No
1
Ambiguous location
(plasmatic, endoplasmic
reticulum, Golgi
Apparatus)
79895
135964
492160
77338
514252
502433
78181
1254
339
1782
404
613
1059
340
134.85
37.51
198.07
44.5
65.4
115.6
36.2
No
No
No
Yes
Yes
No
Yes
No
No
No
1
No
No
No
Mitochondria
Endoplasmic reticulum
Nuclear
Extracellular
Extracellular
Cytosolic/nuclear
Extracellular
kDa: kilodalton; aa: amino acids.
3.5. Phylogenetic Analysis
In order to find a relationship with other fungal lipases, a phylogenetic tree was constructed.
We used 66 lipase sequences, 35 of which have been characterized, plus the 13 sequences of the
candidates. The characterized fungal (Ascomycete and Basidiomycete) lipases/esterases were included
as guides for functions. As far as we know, this is the first genome survey to retrieve true lipases
in T. harzianum and the first phylogeny analysis including its lipases. Seven clusters were defined
(Figure 2). Clusters 1A, 1B and II are largely consistent with the topology of the phylogenetic tree
published by Yadav et al. [6] where filamentous Ascomycete (cluster I) and Basidiomycete (cluster II)
are apart (Figure 2) but differs in the branching structure found by Feng et al. [47] and Barriuso
and Martínez [75] for Lip1 type lipases and Basidiomycete lipases. The reports presented above
placed Lip1-type and Basidiomycete lipases in sister clusters and here Lip1 type lipases placed in
the cluster of C. rugosa type lipases (cluster VI), apart from Basidiomycetes. We obtained the same
branching structure as those authors when a few amino acid sequences were aligned (data not shown);
many phylogenetic trees of fungal lipases have been constructed focusing on a narrow group of
lipases [45,47,48], which forces unrelated proteins to group together.
Our result is congruent with early phylogeny proposed by Schmidt-Dannert [44] by placing the
“Rhizomucor miehei lipase family” clearly apart (highlighted in the phylogenetic tree in purple letters)
and the “Candida rugosa lipase family” (highlighted in the phylogenetic tree in olive green letters and
corresponds to cluster VI; see Figure 2).
Cluster III comprises Tgl lipases and homologues; cluster IV is a branch comprising
uncharacterized proteins, probably related with intracellular protein traffic; cluster V contains B. adusta
and homologues. Cluster VII has two sub clusters where VIIA comprises C. antarctica lipase A
and homologues.
Genes 2018, 9, 62
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Figure 2. Phylogenetic tree of fungal lipases. The tree was constructed with 35 characterized
fungal lipases (accessions correspond to GenBank unless another source is specified) and 15 putative
triacylglycerol lipases from T. harzianum. (*) after the accession numbers are lipases which have
been characterized; unlabeled proteins correspond to hypothetical, uncharacterized, predicted lipases.
The tree was generated by MAFFT software using the neighbor-joining method [55] with 500 bootstrap
re-samplings. Clusters IA, IB and II as described by Yadav et al. [6]. Highlighted in purple letters,
the Rhizomocur miehei lipase-like group and in olive green letters, the Candida rugosa lipase-like group,
according Schmidt-Dannert [44]. Clade I and Clade II, are consistent with Gupta et al. [76]. T. harzianum
triacylglycerol lipases from this study, highlighted in bold red letters.
Genes 2018, 9, 62
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The topology of the phylogenetic tree reconstructed here is largely congruent with the
phylogenetic tree published recently by Gupta et al. [76]. These authors carried out a comprehensive
analysis on lipases available in the LED data base and then conducted the phylogenetic analysis of
61 representative lipases with different combinations of oxyanion and pentapeptide sequences in
fungal and yeast lipases; their tree defined two clades. From our phylogenetic tree, the clusters I,
III, V and VI belong to clade I and the cluster VII belongs to clade II. Clusters II and IV were not
represented in their phylogenetic tree. Predicted proteins in cluster IV share low homology with
known lipases and no member in this cluster has been characterized to date but reasons for exclusion
of Basidiomycetes (cluster II) are unclear. Other differences between both phylogenetic trees is that
they included 14 lipase sequences from Y. lipolytica (most of them placed in the clade I) and 10 lipase
sequences from C. albicans (most of them placed in the clade II, resulting in a larger clade II in their
phylogenetic tree, in comparison with the cluster II in the phylogenetic tree presented here). The most
important difference is that class 3 lipases comprise a single cluster in our phylogenetic tree but these
lipases split between clade I and clade II in theirs. This difference is because all T. harzianum class 3
lipases and their homologues, are lipases with the pentapeptide GHSLG, which is the most frequent
in this class of lipases. However, class 3 lipases can contain the pentapeptides GTSAG, GHSFG and
GHSYG [77]. Gupta et al. [76] included class 3 lipases with different pentapeptides. So, the differences
between both phylogenetic trees are apparent but they are actually quite consistent. The consistency in
the topology of our phylogenetic tree with, Yadav et al. [6], Schmidt-Dannert [44] and Gupta et al. [76]
validates it and supports the phylogeny of the candidate proteins.
The four lipases class 3 place in different branches in the large cluster I of “Filamentous fungi
lipase” (Figure 2). Class 3 family comprises true lipases [31,40] and they are characterized as having
an active site flap/lid and work on long-chain acyl-triglycerides. In consequence, these four class 3
lipases became strong candidates. The protein 78181 placed in the same cluster as F. graminearun FGL1
AAQ23181, F. heterosporum AAB34680 and T. lanigunosus CAB58509.
The protein 77338 clustered with P0CT91, LipA of Acremonium alcalophilum, a protein which is a
lipase/acetylxylan esterase activity that works on long-chain pNP esters and xylans [50]. The protein
502433 grouped with uncharacterized lipases, in a sister clade that grouped lipases involved in aflatoxin
B-producing in Aspergillus flavus, Aspergillus tamari and Aspergillus parasiticum. 502433 is probably
related with the production of toxic metabolites against T. harzianum antagonists.
T. harzianum 514252 grouped in the cluster II with lipases from C. purpurea and F. graminearum, in a
sister clade to lipases from Basidiomycetes (Cryptococcus gatti, Cryptococcus neoformans, Coprinus cinereus,
Melampsora larici-populina and Puccinia graminis); however functional information regarding these
lipases has not been elucidated.
Three of the four proteins automatically annotated as “secreted lipases” in the T. harzianum portal
(526309, 551811 and 87496), clustered with the C. antarctica Lipase A (CALA), a TAG lipase (cluster
VII). The fourth predicted secreted lipase (514427) grouped with B. adusta (cluster V) fungal esterase
APW29213, which has no activity on long-chain glycerides [53].
The protein 135964 grouped with Tgl3p and Tgl5p (cluster III) and 92423 with Tgl1 (cluster VIIB),
the major TAG lipases of the yeast S. cerevisiae [51]. 510832 also placed close to Tgl1.
The protein 492160 and 79895 grouped with F. oxysporum EWZ47945, S. cerevisiae Web1p
AAA50367, T. ophioglossoides KND93486 and M. anisopliae KFG85168 in cluster IV. All of which are
uncharacterized proteins originating from genomic sequencing projects and none predicted as lipase.
3.6. Structural Modeling
Currently there are a number of modeling programs available and practically all have pros and
cons. In this work Swiss model, HHpred and I-TASSER were simultaneously used for analyzing the
lipase candidates.
For most of the putative lipases under analysis, the protein database (PDB) accession for the best
model coincided for two of the three types of modeler software and the third software retrieved a PBD
Genes 2018, 9, 62
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accession which corresponded to another version of the same, or similar protein. For example, the
proteins 526309, 87496, 551811 and 514427 hit PDB-3GUU in Swiss model, corresponding to C. antarctica
Lipase A and in HHpred server retrieved top hit PDB-2VEO, which is the crystal structure of
C. antarctica lipase A in its closed state. BLAST searching in specialized α/β-hydrolases/lipase-esterase
databases was consistent to support homology-based and structure-based transfer annotation for most
of the T. harzianum lipases revised here. The summary is presented in Table 4.
Table 4. In silico localization of characteristic lipase domains in the candidate lipase proteins from
Trichoderma harzianum.
Protein &
Template
Lid Domain
Catalytic Triad
Oxyanion
551811
GYSGG
Gly160-Gly164
Asn195-Ser287
Ser162, Asp309,
His341
Asp92
Gly163
87496
GYSGG
Gly188-Gly192
Leu223-Glu313
Ser190, Asp334,
His368
Asp96
Gly191
526309
GYSGG
Gy208-Gly212
Asn243-Asp332
Ser210, Asp352,
His384
Asp116
Gly211
514427
GHSQG
Gly226-Gly230
Ala258-Phe349
Ser228, Asp373,
His405
Ile146
Gln229
CHSQG
Cys431-Gly435
Phe496-Glu550
Ser433, Asp634,
His667
Leu349
Gln434
GFSQG
Gly224-Gly228
Ile286-Ile321
Ser226, Asp396,
His422
Leu139
Gln227
3GUU
510832
1K8Q
92423
79895 #
135964
Reference Art
Ericsson et al. [78]
Roussel et al. [79] and
Selvan et al. [80]
No structural homologue of lipase was identified
4 X6U
492160 #
Dror et al. [81]
AHSMG
Ala-152-Gly156
Phe189-Pro200
Ser154, Asp275,
His297
Leu70
Met155
No structural homologue of lipase was identified
77338
3O0D
Bordes et al. [82]
514252
GHSLG
Gly214-Gly218
Thr137-Tyr154
Ser216, Asp282,
His343
Thr137
Leu217
GHSLG
Gly316-Gly320
Thr230-Trp262
Ser318, Asp379,
His457
Thr230
Leu319
Complete protein sequence does not model with lipase
502433 *
78181
Pentapeptide
3NGM
Lou et al. [83]
GHSLG
Gly174-Gly178
Asn115-Phe126
Ser176, Asp230,
His289
* Lipase domains restricted to a short fragment of the protein. # No modeling with lipases.
models are available as Figure S2.
&
Ser114
Leu177
Three-dimensional
General characteristics in lipases comprise α/β hydrolase fold, a catalytic triad (Ser-Asp/Glu-His)
and the pentapeptide motif GXSXG [84]. The catalytic triad found here was Ser-Asp-His. Most of
the predicted proteins under revision here have the pentapeptide GXSXG but two other versions
were identified, AHSMG and CHSQG. The pentapeptide AXSXG is in the lipase family 1.4 found in
many bacteria and it has been found in two yeasts, Trichospora asahii lipase TALipA, with activity on
p-NPC-18 [85] and S. cerevisiae TGL2, which prefers short chain-TAG but retains 20% of relative activity
on long chain C18:1 TAG [86]. CHSQG pentapeptide has not been identified in any characterized
lipase so far.
Three-dimensional superposition with IPBA software of each T. harzianum lipase model with its
respective best template model permitted the identification of putative lid domain, catalytic triad,
the pentapeptide sequence and the oxyanion hole in most of the candidate proteins. Specific position
of each domain and motif in each T. harzianum TAG lipase is described in Table 4.
Identity of lipase was not supported for two protein candidates: models for protein 79895 in
Swiss model and HHpred were SEC3-type transport proteins, involved in the structure of the COPII
coat transport-vesicles on membranes. I-TASSER identified an RNA-dependent RNA polymerase of
Cypovirus. The search for homologues in ESTHER and LED data bases retrieved no hits.
The other unsupported candidate was 492160. Swiss model resulted in hits with low coverage
(288 to 743 amino acids) over this large protein (which is 1782 amino acids). The first hit was PDB-1Y64
(30.38% identity), which corresponds to Bni1p formin from S. cerevisiae, complexed with ATP-actin.
Genes 2018, 9, 62
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This is a calcium-dependent and ATP-dependent protein binding, involved in actin filament bundle
assembly and cytoskeleton remodeling. HHpred showed homology with different proteins at N- and
C-ends. At N end, the first hit was 3EG5, a Rho-Diaphanus-binding protein involved in cytoskeleton
assembly and cell division. At C-end the first hit was PDB-2J1D, actin binding for actin assembly.
No hit was retrieved in any of the modeler servers belonging to lipase or alpha-beta hydrolase families.
I-TASSER was also unable to model 492160 with a non-lipase protein. BLASTp in ESTHER and LED
data bases retrieved no homologues for this candidate.
3.7. Functional Prediction of Candidates
Different strategies have been used to predict function. These strategies comprise sequence
homology (e.g., BLAST), phylogenetic relationship, structure homology (i.e., detection of classic domains
and motifs) and three-dimensional homology. Conducting a search with any one of them results in
satisfactory functional prediction for proteins with large conservation with characterized proteins;
however, more difficulties arise when proteins show low identity with known proteins, as in the
case of lipases. To overcome this problem, function prediction was based on the sum of those
function-transfer strategies.
The proteins 77338, 78181, are related with extracellular TAG lipases. 77338 modeled with
PDB-3O0d (Table 4), an olive oil- induced lipase from the non-pathogenic yeast Y. lipolitica [87],
with barely 35% identity and it placed closer to LipA (Figure 2), a lipase/acetylxylan esterase from
A. alcalophilum which works on long-chain pNP esters and xylans [50].
78181 is close to lipases involved in pathogenicity such as CAC19602 from N. haematococca [88],
F. heterosporum AAB34680 [29] and AAQ23181 (FGL1) from F. graminearium [28] which are able to work
on vegetable oils, including olive oil. The latter being identified as the best 3D model for 78181 (Table 4)
and FGL1 expression is induced by olive oil [28]. 77338 and 78181 therefore are plausible candidates
for what is being sought.
Information for 514252 is not consistent. Fragments of this protein share homology with lipases
class 3 and carboxyl esterases. 514252 has fragments of sequences with homology with domains
of lipases involved in the disintegration of autophagic bodies inside the vacuole at starvation
(Table S2) and the closest homologue (75% identity) is XP_011318453 and autophagy related lipase
from F. graminearum [89], reinforcing an intracellular role. However, the protein 514252 is predicted
extracellular, which is not congruent with the expected location of this protein in the cell.
502433 is a large protein (115 kDa); it shares homology with class 3 lipases, it is predicted
intracellular (Table 3) and probably function in relation to the binding of heat-shock proteins (Table S2).
This kind of putative lipases/calmodulin binding heat shock proteins are predicted in few genomic
data but so far, they have not been characterized at all.
The proteins 514427, 526309, 87496 and 551811 share homologies with CALA, a lipase with
interfacial activation. CALA is a secreted lipase from C. antarctica that catalyzes hydrolysis on
long-chain TAGs such as triolein and olive oil [9]. For 551811 Signal P and WoLF PSORT give
results that do not support its secretion (Table 3), decreasing its eligibility. The other three are predicted
extracellular, as expected for the activity in the fungal B13-1 strain. Gene Ontology relates 514427
with iron ion transport and searching by PSI-BLAST found homology of this protein with acetyl
xylan esterases.
92423 and 510832 are probably related to each other. Both share homology with acidic lipases
(Table 4), with cholesterol esterases and pimeloyl-ACP methyl ester carboxylesterases (Table 2).
Congruent with these probable functions, both are predicted intracellular (Table 3).
135964 has a small size but seems to be related with intracellular protein transport from
endoplasmic reticulum (Table S2, Tables 2 and 3).
Analyses of 79895 and 492160 do not support their association with lipases; only small fragments
of their sequences share homology with lipases. The former is related with proteins involved in the
traffic of proteins from endoplasmic reticulum to the Golgi apparatus and the latter with GTP-binding
Genes 2018, 9, 62
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and organization of actin in cytoskeleton (Table 2). Congruent with their putative roles, both of them
are predicted intracellular (Table 3).
3.8. Refining of Selection of Candidates
Based on sequence analysis (protein size and prediction of cellular location), structure analysis
(identification of domain and motifs), phylogenetic analysis (clustering with known true lipases or
known non-true lipases), 3D-modeling and function prediction, the list of initial 13 candidates was
reduced to a final list of candidates. Since the quest is for the gene/protein responsible for T. harzianum’s
extracellular olive oil-lipolytic activity and as we hypothesized that the responsible lipase(s) is or
are true lipases, the criteria for selecting the final list of candidates are: small size (<80 kDa) [73,74],
predicted secreted, preferably with lid domain (which means interfacial activation, common in true
lipases), preferably with homology with lipases active on long-chain acyl-triglycerides, including
vegetable oils, such as olive oil. The candidates that meet these criteria are five: 77338, 78181, 514427,
526309 and 87496. It was decided to include 514252 in the list, since it shows domains of lipase class 3
(this class has interfacial activation) and it is predicted extracellular, although its function could not be
proposed. However, sufficient information is not yet available to support its exclusion.
3.9. RT-PCR Analysis for Validation of Predicting Results
All primer pairs, except the primer for 87496, specifically amplified the expected PCR product
(Figure 3A). We were unable to screen the expression of 87496 because the primers failed to amplify
even on DNA template, despite multiple attempts.
Figure 3. Reverse transcription polymerase chain reaction (RT-PCR) analysis of selected putative
extracellular triacylglycerollipases from Trichoderma harzianum in medium without (B) or with 1%
(v/v) olive oil (C) as carbon source. Lane (M) 1 Kb plus DNA Ladder (ThermoFisher, Carlsbad, CA,
USA). The number of ID at genome portal of each candidate lipase corresponds to: (1) 77338; (2) 78181;
(3) 514252; (4) 526309; (5) 514427; and (6) 87496. Lane (7) Elongation factor 1 (400 bp), as positive control
of PCR. Panel (A) corresponds to PCR on genomic DNA, to test the primers. Base pair (bp).
RT-PCR analysis evidenced the olive oil-responding expression of 526309 in T. harzianum B13-1
strain (Figure 3C). Lipases 77338 and 514252 were also expressed but only 526309 was expressed de
novo (Figure 3B,C). Thus, the RT-PCR demonstrates that the bioinformatic analyses performed here
worked successfully to select candidates to explain the lipolytic activity in the strain B13-1. This result
gives support to the possibility that the gene estExt_fgenesh1_pg.C_1_t10183, located at scaffold
1:494922-496430 (-) and which codifies the protein 526309, is responsible for the extracellular lipolytic
activity of the T. harzianum B13-1 strain on olive oil. The closest homologue for 526309 is CALA,
with 36% identity. This low conservation between 526309 and CALA does not discard functional
relationship. Widmann et al. [90] reported 32 lipases from different sources phylogenetically related
Genes 2018, 9, 62
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with CALA and they share a similar tridimensional structure with this protein but low identity at
sequence level (as low as 15%), as found for homologues in T. harzianum. Conservation is usually low
in lipases [41,91]. Figure 4 shows the multi-alignment of 526309 and the other two predicted secreted
lipases in T. harzianum (this study), as well as CALA protein. They share conservation at catalytic triad
and pentapeptide sequences, while lids are composed of amino acids with similar characteristics.
Figure 4. Structural-based multi-alignment of T. harzianum 526309, 551811 and 87496 with 2VEO lipase
from C. antarctica (CALA). Blue line highlights the pentapeptide; orange line, the lid domain; green
triangles, the catalytic triad; and yellow triangles, the oxyanion. Alpha helices and beta sheets are
indicated at the top. Identical (bold white letters on red background) and similar amino acids (standard
red letters) are shown.
Genes 2018, 9, 62
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T. harzianum has different ecological niches, i.e., plant endophyte, antagonist of a wide range of
plant pathogens and it is also a cosmopolitan soil-borne fungus frequently found on decaying wood.
Different TAG lipases can be involved in the different ecological niches of the fungus. In the case of the
lipase 526309, which is induced by olive oil, it can play role in lipid-rich environments.
Interestingly, 526309 does not belong to class 3 of the lipases and is not even annotated as
TAG lipase in the genome database of T. harzianum but Brennes and Baeck [92] showed that CALA,
its homologue, works on long-chain TAGs. Therefore, transfer function based on structure-homology
predicts triacylglycerol lipolytic activity for 526309.
It is important to clarify that class 3 lipases are true lipases but not all true lipases belong
to class 3 [93]. The most abundant amino acids in 526309 are Alanine (10.8%), Leucine (11.5%),
Glycine (8.4%) and Serine (7.5%), congruent with those found in true lipases by Messaoudi et al. [93].
Three-dimensional modeling identified on 526309 the lid domain from Asn243 to Asp332 (Table 4),
which is compatible with the criteria we proposed in our search.
The survey emphasized proteins annotated in the genome database of T. harzianum as TAG lipases
but was not restricted to them and that was a success which permitted the identification of this lipase.
Cloning of the full-length cDNA of lipase 526309 is in progress in our laboratory for heterologous
expression and further characterization of this protein, to challenge this proposal.
This is the first report on the identification and in silico characterization of TAG lipases from
T. harzianum and expands the catalogue of potential enzymes available for industrial applications.
The combined strategy of functional screening of isolated microorganisms and bioinformatic analyses,
if the genome sequence is available, can be applied to identify lipases in other microorganisms, or to
identify other particular enzymes among a large family of protein candidates. To date, there are a large
number of sequenced genomes of fungi and bacteria.
4. Conclusions
In this work, we reported one T. harzianum strain as the best producer of extracellular lipolytic
activity among the fungal strains analyzed here. A search in the deduce proteome at T. harzianum
genomic portal retrieved 50 lipases. Bioinformatics survey managed to reduce the list to seven
candidate proteins and RT-PCR analysis identified de novo expression of one of those candidates when
the fungus grew on olive oil-containing medium, supporting the suitability of the procedure followed
here. 526309 shares homology with CALA lipase and has potential for further application in industry.
Supplementary Materials: The following are available online at http://www.mdpi.com/2073-4425/9/2/62/s1.
Table S1: List of primers used in this study, Table S2: List of all lipase proteins identified in the T. harzianum genome
homepage, Figure S1: Fungal strains B13-1 (A) and B13-3 (B) selected in screening for extracellular lipolytic activity
using olive oil as carbon source in Rhodamine B test, Figure S2: Three-dimensional model proposed for (A) 551811;
(B) 87496; (C) 526309; (D) 514427; (E) 510832; (F) 92423; (G) 135964; (H) 77338; (I) 514252; (J) 78181. In all protein
models: (1) Red, α-helixes; yellow, β-strands. (2) Close up showing the lid (yellow), the catalytic triad (in red)
and the oxyanion (in blue). (3) Superposition with best template protein model (see Table 4). (4) Superposition
of catalytic triad in both proteins. All models were generated by I-TASSER and visualizations were performed
in PyMOL.
Acknowledgments: M.A.C.P. acknowledges a scholarship granted by CONACyT-Mexico (242995). This work
was partially supported by the CONACYT Grants 220957 and 269833 and FOMIX 247355. Authors thank Miguel
Tzec-Sima for formatting the figures.
Author Contributions: Miguel Canseco and Blondy Canto conceived, designed and wrote the paper;
Miguel Canseco performed the experiments and analyzed the data; Marcela Gamboa contributed with support in
fungal isolation and manipulation; Ignacio Islas and Gerardo Rivera contributed by assisting Miguel Canseco in
lipolytic screening and measurement of the lipolytic activity; Bartolome Chi contributed with the gene expression
analysis; Max Apolinar contributes with support in phylogenetic analysis and the 3D-modeling; Blondy Canto
supervised all the work. All authors participated in the data analysis and writing of the paper.
Conflicts of Interest: The authors declare no conflict of interest.
Genes 2018, 9, 62
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